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Combined analytical techniques for the analysis of complex consumer productsand bio-samples
Chen, G.
Publication date2019Document VersionFinal published versionLicenseOther
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Citation for published version (APA):Chen, G. (2019). Combined analytical techniques for the analysis of complex consumerproducts and bio-samples.
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Download date:22 May 2021
Combined analytical techniques for the analysis of complex consumer products and bio-samples
Guoqiang (Leon) ChenC
ombined analytical techniques for the analysis of com
plex consumer products and bio-sam
ples G
uoqiang (Leon) Chen
Invitation
For attending the public defence of the thesis
Combined analytical techniques for the analysis
of complex consumer products and bio-samples
On Wednesday 5th June 2019
at 14.00
In theAgnietenkapel,
Oudezijds Voorburgwal 229,Amsterdam
Paranymphs
Randy ZhaoBoudewijn Hollebrands
Combined analytical techniques for the analysis of
complex consumer products and bio-samples
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. ir. K.I.J. Maex
ten overstaan van een door het College voor Promoties ingestelde
commissie,
in het openbaar te verdedigen in de Agnietenkapel
op woensdag 5 juni 2019, te 14:00 uur
door
Guoqiang Chen geboren te Shanghai
Promotiecommissie:
Promotor:
- prof. dr. ir. J.G.M. Janssen Universiteit van Amsterdam
Co-promotor:
- prof. dr. ir. P.J. Schoenmakers Universiteit van Amsterdam
Overige leden:
- prof. dr. A.C. van Asten Universiteit van Amsterdam
- prof. dr. W.P. de Voogt Universiteit van Amsterdam
- prof. dr. R.A.H. Peters Universiteit van Amsterdam
- prof. dr. J.P.M. van Duynhoven Wageningen University & Research
- dr. J.G.J. Mol Wageningen University & Research
Faculteit der Natuurwetenschappen, Wiskunde en Informatica
3
Table of Contents Chapter 1 ................................................................................................................................. 5 General Introduction
1.1 Needs and challenges of analytical sciences in the industry of foods and HPC ...... 6
1.2 Analytical techniques in the industry of foods and HPC ...................................... 8
1.3 Scope of the thesis ......................................................................................... 10
References .......................................................................................................... 14 Chapter 2 ............................................................................................................................... 19 A multi-residue method for fast determination of pesticides in tea
2.1 Introduction ................................................................................................... 20
2.2 Experimental ................................................................................................. 21
2.3 Results and discussion .................................................................................... 24
2.4 Conclusions ................................................................................................... 29
References .......................................................................................................... 34 Chapter 3 ............................................................................................................................... 37 Rapid and selective quantification of L-theanine in ready-to-drink teas from Chinese market
3.1 Introduction ................................................................................................... 38
3.2 Materials and methods .................................................................................... 39
3.3 Results and discussion .................................................................................... 43
3.4 Conclusions ................................................................................................... 48
References .......................................................................................................... 49 Chapter 4 ............................................................................................................................... 53 A method for measuring the noncovalent interaction between EGCG and β-CD
4.1 Introduction ................................................................................................... 54
4.2 Materials and methods .................................................................................... 55
4.3 Results and discussion .................................................................................... 57
4.4 Conclusions ................................................................................................... 63
References .......................................................................................................... 64
Appendix ............................................................................................................ 68 Chapter 5 ............................................................................................................................... 71 Quantification of climbazole deposition from shampoo onto artificial skin and human scalp
5.1 Introduction ................................................................................................... 72
5.2 Experiments .................................................................................................. 73
5.3 Results and discussion .................................................................................... 77
4
5.4 Conclusions ................................................................................................... 81
References .......................................................................................................... 82
Chapter 6 ............................................................................................................................... 85 Sensitive and simultaneous quantification of zinc pyrithione and climbazole in scalp buffer scrub samples
6.1 Introduction ................................................................................................... 86
6.2 Materials and methods .................................................................................... 87
6.3 Results and discussion .................................................................................... 90
6.4 Conclusion .................................................................................................... 95
References ......................................................................................................... 96
Chapter 7 ................................................................................................................... 99 Ex-vivo measurement of scalp follicular delivery of zinc pyrithione and climbazole from hair care products
7.1 Introduction ............................................................................................... 100
7.2 Materials and methods .............................................................................. 101
7.3 Results and discussion .............................................................................. 105
7.4 Conclusions ............................................................................................... 110
References ....................................................................................................... 111
Chapter 8 ................................................................................................................. 113 Visualization of zinc pyrithione particles deposited on the scalp from hair care products
8.1 Introduction ............................................................................................... 114
8.2 Materials and Methods .............................................................................. 115
8.3 Results and discussion .............................................................................. 116
8.4 Conclusion ................................................................................................ 118
References ....................................................................................................... 119
List of abbreviations ............................................................................................... 121 Summary ................................................................................................................. 123 Samenvatting........................................................................................................... 127 总结......................................................................................................................... 132 List of Publications ................................................................................................. 135 Overview of author’s contributions ........................................................................ 136 Acknowledgements ................................................................................................. 139
5
Chapter 1
General Introduction
Analytical chemistry is essentially the branch of chemistry that looks into molecular
compositions of complex mixtures. It involves sampling, sample treatment, instrumental
analysis, data processing and interpretation, and has the end goal of identifying and
quantifying specific matter or compounds. The science of analytical chemistry has
continuously evolved with the sustaining innovations in selective materials, computer science,
laser technology and instrumentation. In addition to these ‘technology push’ drivers, the
enormous need for analytical chemistry from new challenges in various areas has generated
a strong ‘market pull’ as well. To date there is a huge variety of analytical techniques
including titrimetry, gravimetry, potentiometry, voltammetry, spectroscopy, microscopy,
chromatography, etc. Combinations of two or more of these techniques enable the analysis of
ever more complex samples at ever increasing levels of detail, sensitivity and reliability.
The importance of analytical chemistry is without any doubt. Analytical measurements have
a tremendous impact everywhere. Not only in fundamental research, but also in industrial
settings and social applications. Without analytical sciences, other science fields including
life sciences, material sciences, space technology, etc. would come to a standstill. In industry,
quality control of raw materials and product development rely heavily on analytical sciences,
techniques and measurements. For monitoring and safeguarding public health and safety,
analytical chemistry plays a critical role in food safety assessment, environment protection
issues, forensic analysis and clinical diagnosis. In hospitals clinical analyses form the basis
of treatment strategies and in the pharmaceutical field analytical methods are indispensable
in the development and production of new drugs.
In this thesis we will demonstrate the large versatility of analytical chemistry by applying it
to two different application fields within the broader area of the life sciences. The thesis
focusses on the development and application of analytical tools and technologies for the
characterization of functional foods, and on methods to assess and underpin the efficacy of
6
personal care products, including the interaction of the functional actives with the human
body.
In this introductory chapter, the needs and challenges of analytical sciences in the industry of
foods and home and personal care (HPC) are summarized. Next, the analytical techniques
most widely used in the development and manufacturing of foods and HPC products are
introduced. Finally, the scope of this thesis is discussed by briefly introducing the successive
chapters of the thesis.
Figure 1.1. Generic applications of analytical sciences in new product development: from
raw materials to finished products on the market. MOA = mode of action.
1.1 Needs and challenges of analytical sciences in the industry of foods and HPC
High quality and safety demands are placed on products that people consume (foods) or apply
onto the body for body care or cosmetic reasons. This explains the large need for analytical
measurements from industries active in these areas. Analytical chemistry is crucial in all
phases of product development and production. As shown in Figure 1.1, analytical science is
vital at many places in the production and life-cycle of the finished products. Without high
quality analytical support, it is nearly impossible to perform the required detailed
characterization of raw materials, establish insights into the mode of action of key actives in
7
products, perform evaluation of prototypes, control the quality of products, build evidence for
claim substantiation, support patents and detect patent infringement, and eventually develop
new, improved products.
The analytical challenges in the foods and HPC industry are largely comparable to those in
other industries and in academic research. In some cases, the challenges are even more severe
because of the complicated matrix of foods and HPC products, in particular related to the high
levels of fats, salts and proteins in the products. A high sensitivity and a high degree of
selectivity are always demanded since the contents of many analytes are very low, and the
sample matrix is complex. As an example, the analysis of contaminants in foods, or of
biomarkers in in vitro and in vivo samples, requires methods with detection limits at the ppb
level that are tolerant to significant matrix variations. Sample treatment techniques like
ultrasound-assisted extraction, microwave-assisted extraction and solid phase extraction
(SPE), can be used to achieve clean-up and pre-concentration, and meet such challenges at
least to some extent in food analysis [1, 2]. However, most of the sample pretreatment
methods are time consuming and rendering automation necessary. Consumables for sample
treatment methods like solid phase extraction (SPE) are expensive and large volumes of toxic
and expensive solvents are often needed. Cost efficiency and sustainability have become
increasingly important, especially for routine analysis of large numbers of samples such as in
bioavailability studies or efficacy tests. A final important factor is speed. The world of foods
and HPC is a rapidly changing world where consumer wishes can quickly come and go.
Analysis time, manual labor and cost of ownership and operation are key features in the
development of methods for use in this industry.
In addition to having to deal with the qualitative and quantitative determination of ever more
complex chemical compositions, analytical scientists are now more than ever also requested
to determine where the components are and how they interact with each other in foods, HPC
products and even on human substrates like hair, scalp, skin, teeth, etc. There is a growing
need for methods that enable localized, spatially resolved analysis and visualization, in the
steady state or in dynamic situations, especially in in vivo studies aiming to understand the
functions and actions of key actives in personal care products.
8
To demonstrate the product benefits in the foods and HPC areas, there is a growing need to
develop methods that provide undisputable evidence on levels of actives at specific target
locations. Simple and easy-to-understand analytical methods are preferred over those that rely
on complicated processes and instrumentation. This is especially the case when the product
evaluations are used to engage consumers.
1.2 Analytical techniques in the industry of foods and HPC
To meet the above challenges and address the articulated needs, there is a massive number of
analytical techniques available to select from. Moreover, every year new analytical
instruments are launched with impressive innovations in performance. The analytical scientist
responsible for the development of novel methods will require excellent knowledge of all
analytical core capabilities including sampling, sample treatment, instrumental analysis, data
processing and interpretation. Next to this, he or she will require sufficient product knowledge
and application-understanding to be able to generate correct data and interpret these in a way
they help in building knowledge and understanding.
In clinical studies, reliable and efficient sampling methods are demanded for the analysis of
actives deposited from personal care products onto human substrates (e.g. hair, scalp, skin,
etc.) as well as for measuring their conversion products and biomarkers of efficacy in- or on
human substrates. A variety of methods for this purpose has been published in scientific
literature, each with its own application fields. To collect biomarkers or actives from human
skin, for example, various sampling methods have been proposed including buffer scrub
extraction [3], tape strip sampling [4], cyanoacrylate biopsy [5], Sebutape [6], and sorptive
tape extraction [7].
Sample treatment prior to instrumental analysis is a crucial part of the analytical process,
contributing to target enrichment and background depletion, and it strongly affects
reproducibility and accuracy of the final results. In sample preparation for food analysis, for
example, procedures as simple as weighing, liquid-liquid extraction, vortexing, filtration,
centrifugation, concentration, etc. are used, next to complicated methods like SPE [8], static
and dynamic headspace [9], solid phase microextraction [10], derivatization [11], matrix solid
9
phase dispersion [12, 13], gel permeation chromatography clean-up [14], etc. Derivatization
is necessary for stabilizing the target compounds in the analytical process, improving the
separation of target compounds from interfering species through an enhanced selectivity and
for increasing the sensitivity. Automation of sample treatment is becoming more important
because of the increasing need for a higher sample throughput. On-line extraction and analysis
is the ultimate in automation that has proven its usefulness in many applications [15-21].
Chromatographic separation techniques like high performance liquid chromatography (HPLC)
and gas chromatography (GC) coupled to a wide range of detection technologies are widely
applied for sensitive and selective quantitative analysis of compounds of interest in raw
materials and finished products. With continuous evolution, comprehensive chromatography
(GC×GC, LC×LC) has been successfully applied in both academic and industrial research
and development (R&D) laboratories [22-26].
Mass spectrometry is now a mature analytical tool that is increasingly being used, not only in
industrial applications but also in fundamental research [27-34]. In combination with HPLC
or GC, it is the most powerful method for the identification and quantitative determination of
compounds in raw materials, formulated systems, finished products, in vitro assays and
clinical samples. Especially in clinical research studies it is widely used. Matrix-assisted laser
desorption/ionization mass spectrometry imaging (MALDI MSI) has emerged as a chemical
imaging technology [35], enabling the visualization of the spatial distribution of target
molecules in various sample matrix like tea leaves, human tissues, etc.
Compared with MALDI MSI, Raman and Laser Confocal Scanning can offer higher spatial
resolution when taking chemical images. They are powerful tools in skin research [36,37], for
example to map spatial delivery of actives onto the skin, to study skin permeability, or to
monitor temporal or spatial changes in biomarkers in skin.
As an optical imaging technology, optical coherence tomography (OCT) has been
successfully applied in diagnostic medicine studies as well as in several industrial fields and
academic research projects [38-40]. In efficacy studies for skin care products, it enables in
vivo visualization of the internal microstructure of human tissues like skin and enamel.
10
Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) are
used to provide detailed microstructural information on human samples or other solid samples
at the nm to sub-mm range. They can be applied to bulk, sectioned and thin film systems,
spanning from solids to liquids through application of a wide range of preparatory techniques.
Both SEM and TEM have been employed to investigate the microstructure and nanostructure
of foods [41, 42].
Molecular spectroscopy including Infra-red (IR), Raman, UV and fluorescence is widely used
for molecular characterization of raw materials, interactions of ingredients in complex
formulations as well as for quantification of components of interest in many types of samples.
Surface enhanced Raman scattering and near Infra-red spectroscopy have been successfully
applied for the detection of adulterants and contaminants in foods and numerous other
samples [43-45].
Nuclear magnetic resonance (NMR) spectroscopy is used to provide structural information
on unknown molecules and characteristic fingerprints of complex mixtures [46]. Additionally,
it can provide quantitative information [47] or it can be used to study interactions between
different components [48].
From the above it is clear that a wide diversity of analytical techniques exists. Selection of
the most appropriate method for a given application is generally a tradeoff between
performance on the one hand, and complexity, cost and speed on the other. In the R&D
environment in the foods and HPC industry, short analysis times, a high data quality, low
costs and meeting all externally dictated safety compliance requirements are the key factors
to consider when selecting an analytical technique for a specific application.
1.3 Scope of the thesis
Foods and HPC companies must continuously launch new products to keep up with the
continuously changing demands of their customers. This high rate of innovation requires very
fast product innovation trajectories. The aim of this thesis is to develop new analytical
methods to meet the business needs for safety and quality control, performance evaluation,
11
claim substantiation and mode-of-action understanding in the case of consumer preferred tea
products and shampoos.
Following water, tea is the most widely consumed drink in the world. Pesticide residues in
tea can be a concern for tea drinkers. For reliable identification and quantification of the target
pesticide residues at trace levels, analytical laboratories are increasingly interested in finding
new analytical methods with shorter analysis times, improved sensitivities and higher sample
throughputs. In Chapter 2, a multi-residue method was developed and validated for rapid
determination of pesticide residues in tea using ultra-high-performance liquid
chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS) combined with a
modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation
procedure. In order to minimize the matrix effects from tea, an SPE cartridge layered with
graphite carbon/aminopropylsilanized silica gel was applied to complement the QuEChERS
method. Representative matrix-matched calibration curves were applied for quantification to
compensate for matrix effects. The efficiency and reliability of this method were investigated
by the analysis of both fermented and unfermented Chinese tea samples.
Ready-to-drink (RTD) teas are becoming increasingly popular as a healthier alternative to
carbonated drinks. One of the nutritionally-relevant compounds in tea is the amino acid L-
theanine. This amino acid is almost solely found in tea plants. In tea it only exists in the free
(non-protein) form and it is the predominant free amino acid. In Chapter 3, a UHPLC-UV
method combined with SPE sample pre-treatment was developed and validated for the rapid
quantification of L-theanine in RTD teas. The method was applied to determine the L-
theanine content in twenty-seven RTD teas from the Chinese market. The ratio of total
polyphenols content to L-theanine content was studied as a parameter for differentiating RTD
teas.
Reducing the bitter and astringent taste of green tea will engage the consumers who are used
to the mild taste of black tea. β-cyclodextrin (CD) is used as an effective bitterness and
astringency masker for catechins in green tea, especially for epigallocatechin gallate (EGCG).
Chapter 4 investigates the noncovalent interaction between β-CD and EGCG by electrospray
ionization mass spectrometry (ESI-MS) and NMR. The stoichiometry of the β-CD-EGCG
12
complexation product was determined using Job’s method. NMR experiments are performed
to provide independent evidence on the formation of an inclusion complex of β-CD and
EGCG. The topology of the complex was derived from ROESY spectra and from chemical
shift differences of the various protons of β-CD and EGCG in the free versus complexed state.
A mechanism explaining the β-CD reduction of the bitterness and astringency of green tea
EGCG was proposed.
The efficacy of anti-dandruff (AD) shampoos depends on the deposition properties of the AD
actives and the amount retained on the human scalp in the process of shampoo application and
rinse-off. To support in vitro and in vivo studies for the performance evaluation of AD
shampoos, robust and sensitive analytical methods for in vitro and ex vivo measurement of AD
active deposition on artificial skin and human scalp are required. In Chapter 5, a sensitive and
specific UHPLC-MS/MS method was developed and validated for the measurement of
climbazole (CBZ) deposition from hair care products onto artificial skin and human scalp. A
buffer scrub method using a surfactant-modified phosphate buffered saline (PBS) solution was
selected for the sampling of CBZ from human scalp. Deuterated CBZ was used as the internal
standard. Atmospheric pressure chemical ionization (APCI) in positive mode was applied for
the detection of CBZ. Using this method, CBZ deposition from several shampoos was
compared.
Chapter 6 proposed a sensitive UHPLC-MS/MS method for the simultaneous quantification
of two AD actives, zinc pyrithione (ZPT) and CBZ, deposited onto the human scalp from AD
shampoos. Scrubbing with a buffer solution was used as the sampling method for the
extraction of ZPT and CBZ from scalp. A method for ZPT derivatization prior to UHPLC-
MS/MS analysis was developed. The identification of ZPT and CBZ was performed by
examining ratios of selected MRM transitions in combination with UHPLC retention times.
The method was applied for the analysis of scalp buffer scrub samples from an in vivo study.
The levels of ZPT and CBZ deposited on the scalp at different time points after application
of the AD shampoo were measured and the efficacy of different shampoos was compared.
In Chapter 7, several new methods for studying the location- and depth specific deposition
of AD actives were developed. A method involving scalp cyanoacrylate biopsy sampling, a
13
tailor-made cutting device, and methods for UHPLC-MS/MS analysis, SEM measurement
and Raman imaging were developed for the measurement of delivery of ZPT and CBZ from
an AD shampoo into the scalp follicular infundibulum. Using this method, ZPT and CBZ
were simultaneously quantified and visualized within the scalp follicular infundibulum, after
scalp washing with a dual-active AD shampoo.
Finally, Chapter 8 proposed an ex vivo method that combines tape strip sampling and SEM
and energy dispersive X-ray spectroscopy (EDX) for measuring and visualizing the particle
size, morphology and composition of ZPT deposited on the scalp from an AD shampoo
containing ZPT and zinc carbonate. The possibilities for distinguishing ZPT from zinc
carbonate particles were evaluated. Moreover, the ability of the new method to study the
microstructures of ZPT and other zinc particles deposited onto the scalp was assessed.
14
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18
19
Chapter 2
A multi-residue method for fast determination of pesticides in tea
Abstract
A multi-residue method was developed for rapid determination of pesticide residues in tea by
ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry
(UHPLC-MS/MS). The quick, easy, cheap, effective, rugged and safe (QuEChERS) method
was used for sample preparation. In order to minimize the matrix effects from tea, a solid phase
extraction (SPE) cartridge layered with graphite carbon/aminopropylsilanized silica gel was
applied as complementary to the QuEChERS method. For accurate quantification,
representative matrix-matched calibration curves were applied for quantification to
compensate matrix effects. Limits of quantification varied with different pesticides, but all can
be measured at 0.01 mg/kg level in a 5 g tea sample except dichlorvos (0.02 mg/kg).
Recoveries ranged from 70% to 120% and relative standard deviations (RSD) met the
European United Quality Control guideline. Efficiency and reliability of this method were
investigated by the analysis of both fermented and unfermented Chinese tea samples.
This chapter was originally published as:
G. Chen, P. Cao, R. Liu, A multi-residue method for fast determination of pesticides in tea by ultra
performance liquid chromatography-electrospray tandem mass spectrometry combined with
modified QuEChERS sample preparation procedure, Food Chem. 125 (2011) 1406-1411.
20
2.1 Introduction
Tea farming is vulnerable to a great multitude of pests, especially mites, leaf-eating beetles
and caterpillars. Weeds and diseases can also be a problem. To minimize these problems, the
most common practice in tea crop production is to use pesticides. However, similar to other
raw agriculture commodities (RACs), unsafe pesticide residues in tea have been associated
with neurological dysfunction and disease [1]. Consequently, determination of pesticide
residues is at the forefront among preventive measures in public health safety. Furthermore,
there are potential international trade barriers due to maximum residue limits (MRLs) in tea
established by most countries and several international organizations, e.g. United States
Environmental Protection Agency, Food and Agriculture organization of the United Nations,
and European United, etc. The existing MRLs for some pesticides in many RACs including
tea are periodically revised and become stricter and more comprehensive. Moreover, there is
a trend for regulators to temporarily reduce the MRLs if new data unexpectedly indicate
certain risk to human or animal health. For reliable identification and confirmation of the
target pesticide residues at trace levels, food analytical laboratories are increasingly interested
in finding new analytical methods with shorter analysis time and higher sample throughput
[2].
Gas chromatography (GC) seems to be the technical choice for analysis of pesticides in food
commodities. However, many pesticides which are thermally unstable or non-volatile such as
carbamates and benzimidazoles are difficult to be analysed with GC. High performance liquid
chromatography (HPLC) offers an alternative and powerful tool for the determination of such
compounds, as complementary to GC [3]. Moreover, ultra-high-performance liquid
chromatography (UHPLC) with columns packed with small particles (1.7 μm) and high linear
velocities (accompanied by maximum back pressures up to 15,000 psi) have been shown to
give superior chromatographic resolution, reduce analysis time, consume less solvent and
increase sensitivity [3-7].
For pesticide residue analysis in tea, there are published methods available in literature
including official methods [8-13]. Traditional analytical methods cannot provide effective
solutions to minimizing matrix effects [11]. Solid phase extraction (SPE) is a common choice
21
for clean-up. Japanese official methods for residual compositional substances of agricultural
chemicals, feed additives and veterinary drugs in food apply SPE cartridges packed with
graphite carbon/aminopropylsilanized silica gel to clean-up tea matrix [14]. Gel permeation
chromatography (GPC) is also an effective clean-up method which has been widely applied
[15]. There is a trend to shift from labour intensive traditional methods to fast and simple
approaches, such as the quick, easy, cheap, effective, rugged and safe (QuEChERS) method
[16], which symbolizes a new milestone for pesticide residue analysis. Some laboratories [17,
18] have applied this clean-up method in fruits and vegetables.
Pesticide multi-residue methods applying gas chromatography-mass spectrometry
(GC/MS/MS or GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS)
are increasingly popular, which enable us to analyse more pesticides in one injection and with
higher sensitivity. For the confirmation of legal substances and banned substances, a
minimum of 3 and 4 identification points, respectively, are required according to EU Directive
(2002/657/EC, 2002) [19]. UHPLC coupled with triple quadruple MS/MS can improve the
speed of analysis and provide higher sensitivity and accuracy. To the best of our knowledge,
until now, there has been no literature or publications reporting about multi-residue methods
for the determination of pesticides in tea by UHPLC-MS/MS.
The purpose of this paper is to develop a multi-residue method based on the application of
UHPLC-MS/MS combined with a modified QuEChERS sample preparation procedure for
rapid determination of 65 selected pesticide residues in tea.
2.2 Experimental
2.2.1 Reagents, chemicals and materials
Pesticide reference standards, all 95% or higher purity, were obtained from Dr. Ehrenstorfer
(Augsburg, Germany), Chemservice (USA) and Accustandard (USA). Stock solutions of
mixture pesticides were prepared in acetonitrile or acetone stored in the freezer (-18 ℃). The
working solutions were prepared daily.
22
All chemicals used in the experiment were analytical grade or better: HPLC grade acetonitrile,
HPLC grade formic acid and acetic acid. A.R. Anhydrous CH3COONa and anhydrous MgSO4
were baked at 650 ℃ for 3 hours to remove phthalates and any residual water. Graphite
carbon/aminopropylsilanized silica gel layered SPE cartridges (Sep-Pak Carbon NH2, 6 cc)
were purchased from Waters Corporation (P.N. 186003369).
Analytically confirmed pesticides-free green tea from Shiru Tea Company in Guangxi
province and black tea from Unilever UK were used as blank samples for matrix-matched
calibration and recovery evaluations.
2.2.2 Apparatus
A Waters ACQUITY UPLC System (Waters, UK) was employed. An AQUITY UPLC BEH
Shield RP18, 2.1 mm (I.D.) × 150 mm, 1.7 μm column (Waters, Ireland) was applied in this
method. The mobile phase was constituted by 0.02% formic acid in acetonitrile (A) and 0.02%
formic acid in water (B) in a gradient mode [time 0 min, 10% A; 12 min, 98% A; 12.5 min,
10% A] and total analysis time of 18 min. The flow rate was 0.3 mL/min and injection volume
was 2 μL. The temperatures of column and sample room were set at 30 ℃ and 8 ℃,
respectively.
A Waters Quattro Micro API mass spectrometer (Waters, UK) equipped with electrospray
source was used for all experiments. MassLynx software (version 4.1) was used for
instrument control and data acquisition. The capillary voltage was 3.50 kV and the source
temperature was 100 ℃. The desolvation gas temperature was set at 350 ℃ with a nitrogen
flow of 300 L/hr. The collision gas (argon) pressure was set at 3×10-3 mbar. The multiple
reaction monitoring (MRM) mode was operated for each pesticide. All the parameters for
MRM transitions, cone voltage and collision energy were optimized in order to obtain highest
sensitivity and resolution (Table 2.1).
23
2.2.3 Sample preparation
For both unfermented and fermented tea, weigh about 50 g and comminute in a small dis-
integrator for 1 min. Transfer 5 g comminuted sample to a 50 mL centrifugal tube. Add 10
mL H2O and 10 mL acetonitrile (containing 1% acetic acid), vortex for 3 min and then allow
to settle for 1 h. Add 1.5 g anhydrous CH3COONa and 4 g anhydrous MgSO4, vortex for 1
min. Cool the tube in an ice-water bath immediately, for 5 min. Centrifuge for 5 min at 5000
r/min. The samples were then subjected to SPE clean-up. The SPE column was conditioned
with 10 mL acetonitrile/toluene (3:1, 1% acetic acid). Transfer 1 mL extracted solution to the
column. Elute the column with 20 mL acetonitrile/toluene (3:1, 1% acetic acid). Concentrate
the effluent to 1 mL or less by evaporating under a weak nitrogen stream at 40 ℃. The residue
was reconstituted in 1 mL acetonitrile (1% acetic acid) and filtered over a 0.2 μm organic
filter (Millipore), ready for injection into UHPLC-MS/MS.
2.2.4 Method performance
The sensitivity and precision of the method were evaluated by analyzing spiked blank tea
samples. Recoveries and RSD were determined for five replicates at two concentration levels
(0.050 and 0.010 mg/kg).
The accuracy of this method was evaluated externally by participating in a 31- laboratories-
proficiency-test for pesticide residue analysis in tea which was organized by FAPAS
(http://www.fapas.com) in 2008. All the target compounds (Acetamiprid, Bifenthrin,
Cypermethrin, DDT [four homologues], Ethion, Fenpropathrin, Fenvalerate, lamda-
Cyhalothrin, Propargite, S-421) covered by the proficiency test were properly identified and
the respective z-score values obtained were satisfactory (|z|<2).
24
2.3 Results and discussion
2.3.1 Optimization of UHPLC-MS/MS conditions
Application of UHPLC in this method provides superior chromatographic resolution, shorter
analysis time and higher sensitivity. The total analytical time for instrumentation was only 18
min including 6 min for column equilibrium. At the initial developmental stage, 5 mM
ammonium acetate was used in LC mobile phase. However, with this mobile phase the
UHPLC column was found to get clogged rapidly. It was proven that 0.02% formic acid
provided the same sensitivity and resolution as 5 mM acetic ammonium. Formic acid was
then selected as the replacement for ammonium acetate.
Some pesticides including fenxoycarb, indoxacarb,clethodim and flufenoxuron showed poor
peak shapes when they were dissolved into the initial gradient of mobile phase
(acetonitrile/H2O = 1:9). For other pesticides, there were no differences in sensitivity when
acetonitrile/H2O (1:9 & 5:5) or pure acetonitrile was used as solvent. Consequently, the
residue was reconstituted in 1 mL acetonitrile (1% acetic acid) for better peak formation and
sensitivity.
The tea matrix is very “dirty”, containing high levels of caffeine, sugars, organic acids and
other interferences. A tandem mass detector, which has high selectivity and sensitivity,
provides an effective solution. MRM parameters including ion transition, collision energy and
cone voltage of UHPLC-MS/MS were listed in Table 2.1 Each pesticide was tuned using a
single standard solution at 1 μg/mL which was infused into the MS detector at a flow rate of
0.3 mL/ min. Product ion mass spectra for the pesticides were obtained in electrospray
ionization using collision induced dissociation (CID). Variations in collision energy influence
both sensitivity and fragmentation. The collision energy was optimized for two selective ion
transitions for every pesticide. Both pairs of the MRM transitions were used for confirmation
analysis, which can meet the EU Decision (2002/657/EC, 2002) [19], and the most sensitive
transitions were selected for quantification analysis. Dwell times for different transitions were
optimized to achieve higher sensitivities, as well. Some compounds like bromoxynil and
ioxynil were analyzed in negative ESI mode ([M-H]-) while other pesticides were determined
25
in positive ESI mode ([M+H]+). In this investigation, a total of 65 pesticides were determined
in tea. A combined MRM chromatogram of fortified tea sample by 15 representative LC
amenable pesticides at 0.1 mg/kg is shown in Figure 2.1.
Figure 2.1. Combined UHPLC-MS/MS chromatogram of fortified green teas at 0.1 mg/kg
1. Propamocarb; 2. Pirimicarb; 3. Carbofuran-3-hydroxy; 4. Mevinphos; 5. Acetamiprid; 6.
Thiofanox-sulfon; 7. Spiroxamine; 8. Triasulfuron; 9. Bromoxynil; 10. Promecarb; 11.
Triadimefon; 12. Fenhexamid; 13. Fenoxycarb; 14. Clethodim; 15. Flufenoxuron.
2.3.2 Sample preparation
In the process of pesticide residue analysis, sample pretreatment and preparation are the most
time-consuming, labor intensive and complicated procedures. According to the characteristics
of pesticides, several solvents can be selected as the extraction solvent, e.g. acetone [9, 18],
ethyl acetate [2] and acetonitrile [20]. In comparison to other solvents, acetonitrile shows
more advantages such as higher recoveries, less interference from lipids and proteins, better
compatibility with LC and GC, and less co-extracted matrix components. For these reasons,
acetonitrile was chosen as the extraction solvent in the QuEChERS method [16]. Application
of MgSO4 for partitioning could yield a significant volume of the upper layer and give high
recoveries. Acetic acid with CH3COONa makes up of a buffer (pH 4-5), which could give
26
adequately high recoveries for acephate and imazalil etc. Moreover, the usage of buffer could
improve stability of the base-sensitive pesticides for their analysis [21].
Several sorbents can be used in the clean-up method for pesticide residue analysis, e.g.
primary secondary amine (PSA), -NH2, graphitized carbon black (GCB) and ODS SPE
cartridges. The use of PSA+GCB SPE [14] could remove more matrix materials. The
mechanism of PSA (or -NH2) sorbent is based on the weak ion exchange. It removes fatty
acids, sugars and other components that form hydrogen bonds. The use of GCB is attractive
to remove pigment especially chlorophyll. However, GCB strongly retains planar pesticides,
as well. In this research, QuEChERS method (dispersive-SPE) was proven to be unable to
remove the pigment from tea effectively. An SPE step (NH2+ GCB) was used for further
clean-up as complementary to the QuEChERS method. Acetic acid (1%) in the elution solvent
was proven to be able to reduce the absorption of planar pesticides in GCB and thus improve
the recoveries of pesticides residues, with the exception of pymetrozin, diflubenzuron and
thiabendazole (Figure 2.2).
Figure 2.2. Comparison of the recoveries of sulphonyl and urea pesticides with and without
1% acetic acid in SPE elution (n= 5, spiked concentration= 0.05 mg/kg). Each value is
expressed as mean ± standard deviation.
27
2.3.3 Matrix effects
The matrix effects may differ for different teas, e.g. green, black, Oolong and Puer.
Consequently, it is required to compensate the matrix effects by a matrix-matched calibration
[22], or by use of an isotope labeled standard, or ECHO technique [23] in LC/MS system.
In this study, analytically confirmed pesticides-free organic green tea from Shiru Tea
Company and black tea from Unilever UK were used as blank matrix. Organic green tea was
selected as the representative matrix for green and Puer tea samples while organic black tea
was selected as the representative matrix for black and Oolong tea samples. For the
determination of matrix effects, the responses of the standard solutions prepared in solvent
were compared with the responses of the standard solutions prepared in pesticides-free blank
tea sample. Matrix enhancement or suppression effects were observed for many tea samples.
The consequences of these abnormalities were considerable, causing significant and variable
errors in the quantification of the different pesticides. Hence, the matrix-matched calibration
method was applied in the quantitative analyses.
In addition, the complex matrix from tea could have a negative impact on the separation
performance of the UHPLC column. Moreover, the UHPLC column was prone to being
blocked if the clean-up of tea samples was not complete.
2.3.4 Validation of the method
Linearity, sensitivity, accuracy and precision of the multi-residue method were validated. The
mixed matrix-matched pesticides standard solutions of 5, 10, 20, 50, 100 μg/L were injected
into the UHPLC-MS/MS system. Relative coefficients (r) are listed in Table 2.1. Limits of
quantification (LOQ) of mixed pesticides standards were determined by injecting a series of
different matrix-matched pesticides standard solutions. Parameters are listed in Table 2.1
Although the LOQ of the method varies with different pesticides, all can be measured at 0.01
mg/kg level in a 5 g tea sample except for dichlorvos (0.02 mg/kg). The mixed standard
solution was added into the pesticides-free blank tea samples to make up the concentrations
of 10 and 50 μg/kg, and then the method was carried out as described in Part 2.3. The majority
28
of recoveries for these pesticides were in the range from 70% to 120%. But some recoveries
(60% - 70% or 120% - 130%) could also be accepted (European Council N°
SANCO/2007/3131, 2007). Reproducibility of this method is shown in Table 2.1 as RSD.
2.3.5 Pesticide residues in tea samples
In order to obtain accurate results, a two-step analytical strategy was applied. The first step
was a screening method, which monitored only one MRM transition for each compound. In
this way, negative and positive samples were separated. The second step is a confirmation
method, in which at least two MRM transitions for each compound were monitored. The most
sensitive MRM transition was selected for quantification.
This strategy was applied to pesticide residue analysis of 18 tea samples from different regions.
Among the pesticide residues detected in these 18 tea samples, acetamiprid had the highest
detection frequency (61.1%), followed by imidacloprid (56.8%), carbendazim (56.6%),
triazophos (44.4%), dimethoate & methomyl & uprofezin (33.3%), and triadimenol (22.2%).
For some pesticide residues, the detected levels varied greatly. The minimum value of
acetamiprid, for example, was 0.02 mg/kg in a green tea from Anhui province while the
maximum value of 1.03 mg/kg was found in an Oolong tea from Fujian province. However,
all the residue levels of these pesticides in these 18 tea samples were below the MRL required
by Chinese government, EU and Japanese government, except for dimethoate and methomyl
in 5 tea samples resulted higher than the Chinese and EU MRL.
The patterns of pesticide usage are different from one tea plantation to another because they
have different pest problems. The fact that we found multiple residues in a number of samples
may be due to the fact that some teas are custom blend to produce distinct finished products.
For instance, there were 5 tea samples contaminated by 8 pesticides simultaneously and there
were 6 tea samples in which dimethoate, methomyl and uprofezin were detected
simultaneously. The detected frequencies and detected levels of the pesticide residues in these
18 tea samples are shown in Figure 2.3.
29
In literature, some GC amenable pesticides like DDT, HCH and some pyrethroid could be
detected easily in tea. In light of the results of tea samples investigated in this paper, easily-
detectable LC amenable pesticides in tea were found to be acetamiprid, imidacloprid,
carbendazim, triazophos, dimethoate, methomyl, uprofezin, and triadimenol.
Figure 2.3. Detection frequencies and levels of the pesticide residues in 18 tea samples. The
error bars indicated the ranges of pesticide residue levels detected in tea samples.
2.4 Conclusions
A very quick, easy, effective, rugged, reliable and accurate multi-residue method based on
modified QuEChERS method was developed for determination of pesticides in tea by ultra-
performance liquid chromatography with tandem mass spectrometry. The performance of the
method was very satisfactory with results meeting validation criteria. The method has been
successfully applied for the determination of tea samples and ostensibly has further
application opportunities, including the analysis of e.g. dry vegetable and herb extracts.
30
Tabl
e 2.
1. P
aram
eter
s for
65
pesti
cide
resid
ue a
naly
sis b
y U
HPL
C-M
S/M
S.
Nam
e R
T (m
in)
MR
M T
rans
ition
*
Con
e
(v)
CE
(e
v)
LOQ
(μg/kg
) Li
near
ity
r
Rec
over
y /%
**
50 μ
g/kg
RS
D
10 μ
g/kg
RS
D
Carb
enda
zim
1.
76
192.
1>16
0.2
192.
1>13
2.1
25
18
30
5 0.
9918
85
.06
19.2
10
9.57
9.
1
Prop
amoc
arb
1.78
18
9.3>
102.
1 18
9.3>
144.
2 25
16
12
5
0.99
92
71.2
3 9.
2 81
.49
3.7
Met
ham
idop
hos
2.07
14
2.1>
94.1
142.
1>11
2.1
21
11
11
5 0.
9959
80
.86
7.5
61.8
6 23
.9
Ace
phat
e 2.
31
184.
1>14
3.1
18
4.1>
125.
1 15
7
17
10
0.
9932
84
.96
17.8
77
.57
28.9
Om
etho
ate
2.56
21
4.0>
155.
1
214.
0>18
3.2
20
15
10
5 0.
9979
76
.73
9.8
78.4
9 11
.4
Ald
oxyc
arb
3.56
22
3.1>
148.
1
223.
1>16
6.2
23
8
6 5
0.99
92
95.1
2 5.
9 78
.49
17.7
Mon
ocro
toph
os
3.63
22
4.1>
127.
0
224.
1>19
3.1
17
16
8 5
0.99
98
78.7
9 6.
1 70
.94
8.3
Pirim
icar
b 3.
86
239.
2>72
23
9.2>
182.
3 25
18
16
5
0.99
99
91.6
4 8.
6 76
.60
5.7
Met
hom
yl
3.88
16
2.9>
105.
9
162.
9>87
.8
15
10
8
5 0.
9997
70
.64
10.3
81
.91
20.1
Mev
inph
os
4.80
22
5.1>
127.
1
225.
1>67
.0
17
18
20
5 0.
9996
10
0.17
8.
4 82
.60
14.4
Met
amitr
on
4.84
20
3>17
5.1
2
03>1
45.1
28
16
14
5
0.99
77
78.5
5 18
.2
78.4
9 30
.2
Carb
ofur
an-3
-hyd
roxy
5.
06
220.
1>16
3.1
22
0.1>
107.
0 25
10
23
5
0.99
88
98.5
5 16
.6
60.8
1 17
.5
Imid
aclo
prid
5.
16
256.
1>17
5.1
25
6.1>
209.
1 22
20
16
5
0.99
56
84.7
7 15
.4
73.7
0 10
.2
Thio
fano
x-su
lfon
5.31
26
8.1>
76.0
26
8.1>
161.
2 10
10
16
5
0.99
85
84.3
2 14
.3
64.9
8 18
.2
Dim
etho
ate
5.33
23
0.0>
199.
1
230.
0>12
5 11
9
20
5
0.99
97
88.4
2 11
.5
62.1
7 15
.1
Ace
tam
iprid
5.
49
223.
1>12
6.1
22
3.1>
55.7
23
20
16
5
0.99
92
90.2
7 17
.9
70.9
8 12
.1
Imaz
alil
5.70
29
7.0>
159.
1
297.
0>69
.0
30
19
17
5 0.
9993
80
.41
6.4
64.3
4 45
31
Nam
e R
T (m
in)
MR
M T
rans
ition
*
Con
e
(v)
CE
(e
v)
LOQ
(μg/kg
) Li
near
ity
r
Rec
over
y /%
**
50 μ
g/kg
RS
D
10 μ
g/kg
RS
D
Buto
carb
oxim
5.
88
191.
2>75
.0
191.
2>11
6.0
10
20
3
5 0.
9993
91
.45
23
66.1
8 12
.4
Phos
pham
idon
5.
97
300.
0>12
7.1
30
0.0>
174.
2 24
24
10
5
0.99
94
87.4
9 9.
9 82
.01
15.5
Nic
osul
furo
n 6.
12
411.
0>19
2.2
41
1.0>
213.
2 22
18
26
5
0.99
99
67.7
3 6.
7 72
.25
15.1
Ald
icar
b 6.
21
191.
1>11
6.0
19
1.1>
88.8
14
3 12
5
0.99
68
85.9
5 11
.3
122.
67
17.6
Thia
clop
rid
6.23
25
3.0>
126.
1
253.
0>98
.9
25
23
39
5 0.
9985
10
6.09
8.
3 90
.40
10.7
Spiro
xam
ine
6.36
29
8.3>
144.
3
298.
3>10
0.2
28
18
31
5 0.
9999
83
.12
7.7
74.7
3 3.
4
Dic
hlor
vos
6.67
22
1.1>
109.
0
221.
1>12
7.1
25
9 9 20
0.
9966
96
.77
17.5
<L
OQ
-
Pyrim
etha
nil
6.79
20
0.2>
107.
2
200.
2>16
8.3
35
23
27
5 0.
9988
89
.64
3.5
106.
61
8.3
Thife
nsul
furo
n-m
ethy
l 6.
83
388.
0>16
7.3
38
8.0>
141.
3 20
25
20
5
0.99
92
76.5
4 12
.6
77.1
3 12
.1
Thio
dica
rb
7.01
35
5.2>
88.1
35
5.2>
108.
1 15
12
11
5
0.99
89
97.1
7 8.
9 70
.08
11.1
Prop
oxur
7.
13
210.
1>11
1.0
21
0.1>
168.
2 14
15
14
5
0.99
91
84.3
1 11
.2
81.5
2 8.
3
Bend
ioca
rb
7.21
22
4.1>
167.
1
224.
1>10
9.0
18
9
18
5 0.
9990
85
.95
18.8
62
.23
21.9
Carb
ofur
an
7.25
22
2.1>
165.
2
222.
1>12
3.1
20
12
20
5 0.
9980
73
.35
16.2
69
.40
5.0
Tria
sulfu
ron
7.30
40
2.0>
167.
2 40
2.0>
141.
2 25
14
19
5
0.99
86
69.9
7 15
.7
89.6
2 12
.0
Carb
aryl
7.
73
202.
2>14
5.2
20
2.2>
127.
2 15
13
2
5 5
0.99
75
103.
78
8.5
103.
90
12.0
Isop
rotu
ron
7.73
20
7.2>
72.0
20
7.2>
134.
1 20
15
24
5
0.99
92
90.5
9 12
.6
95.8
2 8.
2
Ethi
ofen
carb
7.
84
226.
1>10
7.1
22
6.1>
164.
2 15
15
8
5 0.
9952
80
.55
12.1
75
.11
19.9
Proc
hlor
az
7.90
37
6.0>
308.
1
376.
0>26
6.1
17
10
16
5 0.
9997
82
.39
10.9
81
.55
31.6
32
Nam
e R
T (m
in)
MR
M T
rans
ition
*
Con
e
(v)
CE
(e
v)
LOQ
(μg/kg
) Li
near
ity
r
Rec
over
y /%
**
50 μ
g/kg
RS
D
10 μ
g/kg
RS
D
Atra
zine
7.
93
216.
2>17
4.2
21
6.2>
146.
2 30
16
19
5
0.99
93
99.0
6 9.
6 86
.00
9.2
Mon
olin
uron
7.
98
215.
1>99
.0
215.
1>12
6.1
19
27
15
5 0.
9976
11
1.21
16
.2
104.
17
23.1
Cypr
odin
il 8.
03
226.
2>93
.1
22
6.2>
108.
2 38
30
24
5
0.99
98
69.2
8 9.
5 74
.15
12.8
Bens
ulfu
ron-
met
hyl
8.16
41
1.0>
149.
2
411.
0>18
2.2
18
18
19
5 0.
999
89.7
1 8.
1 85
.26
12.0
Diu
ron
8.26
23
3.1>
72.0
23
3.1>
160.
1 21
14
24
5
0.99
87
91.0
6 13
.1
103.
92
11.0
Tria
dim
enol
8.
37
296.
1>70
.0
296.
1>99
.1
14
10
11
5 0.
9997
90
.28
6.0
80.8
3 33
.7
Brom
oxyn
il 8.
59
273.
9>19
4.0
27
3.9>
78.9
38
30
26
5
0.99
75
83.1
2 17
.7
72.3
9 27
.3
Met
hioc
arb
8.84
22
6.1>
169.
2
226.
1>12
1.1
17
10
17
5 0.
9993
75
.81
12.2
87
.45
30.4
Ipro
valic
arb
8.94
32
1.2>
119.
2
321.
2>14
4.2
15
19
14
5 0.
9997
87
.77
6.9
94.5
8 3.
3
Azo
xystr
obin
8.
97
404.
1>37
2.3
40
4.1>
329.
2 19
12
28
5
0.99
88
93.2
8 10
.5
109.
45
11.8
Azi
npho
s-m
ethy
l 8.
98
318.
0>13
2.1
31
8.0>
160.
1 14
13
7 5
0.99
95
83.8
1 1.
7 84
.26
9.7
Prom
ecar
b 9.
06
208.
2>10
9.1
20
8.2>
107.
1 17
20
26
5
0.99
99
80.8
3 9.
6 97
.01
7.8
Bupr
ofez
in
9.09
30
6.1>
201.
3
306.
1>10
6.1
17
12
30
5
0.99
95
80.1
1 4.
7 65
.85
16.8
Trifl
usul
furo
n-m
ethy
l 9.
14
493.
1>26
4.2
49
3.1>
238.
3 25
22
26
5
0.99
99
91.3
7 8.
0 71
.65
14.3
Tria
dim
efon
9.
15
294.
1>19
7.3
29
4.1>
155.
2 21
13
20
5
0.99
98
96.1
4 11
.3
79.4
5 10
.7
Tebu
cona
zole
9.
31
308.
2>70
.1
308.
2>15
1.1
28
18
21
5
0.99
93
96.9
7 11
.9
85.4
9 6.
2
Ioxy
nil
9.38
36
9.8>
127.
0
369.
8>21
4.9
35
29
34
5 0.
9994
77
.24
8.9
90.9
1 9.
3
Met
olac
hlor
9.
47
284.
1>25
2.1
28
4.1>
176.
3 20
15
26
5
0.99
98
87.2
7 17
.8
66.5
3 5.
0
33
Nam
e R
T (m
in)
MR
M T
rans
ition
*
Con
e
(v)
CE
(e
v)
LOQ
(μg/kg
) Li
near
ity
r
Rec
over
y /%
**
50 μ
g/kg
RS
D
10 μ
g/kg
RS
D
Fenh
exam
id
9.57
30
2.1>
97.1
30
2.1>
55.0
31
21
33
5
0.99
97
88.6
6 7.
7 87
.13
10.2
Tria
zoph
os
9.60
31
4.1>
162.
2
314.
1>11
9.1
21
15
33
5
0.99
97
88.2
1 8.
3 12
4.53
9.
5
Feno
xyca
rb
9.73
30
2.2>
116.
1
302.
2>25
6.2
20
11
14
5
0.99
96
89.0
7 9.
8 89
.36
7.6
Azi
npho
s-et
hyl
9.80
34
6.0>
132.
0
346.
0>16
0.1
16
15
6
5 0.
9996
82
.64
11.3
11
4.55
22
.7
Difl
uben
zuro
n 9.
82
311.
0>15
8.1
30
14
5 0.
9888
67
.33
9.0
64.1
7 8.
1
Tebu
feno
zid
10.0
0 35
3.2>
133.
2
353.
2>29
7.3
13
19
7
5 0.
9999
92
.77
7.9
103.
64
12.9
Indo
xaca
rb
10.7
6 52
8.1>
150.
2
528.
1>21
8.2
24
25
23
5 0.
9976
10
7.33
14
.1
88.0
7 7.
6
Qui
zalo
fop-
ethy
l 10
.94
373.
0>29
9.2
37
3.0>
271.
2 28
18
18
5
0.99
99
88.3
5 8.
9 93
.31
10.5
Clet
hodi
m
11.1
3 36
0.2>
164.
1
360.
2>20
6.3
19
19
18
5
0.99
98
72.2
3 13
.6
60.0
8 16
.1
Fura
thio
carb
11
.19
383.
1>19
5.2
38
3.1>
252.
2 20
17
11
5 0.
9992
10
1.66
5.
5 11
5.80
1.
5
Flua
zifo
p-p-
buty
l 11
.31
384.
1>28
2.2
384.
1>32
8.2
24
20
17
5
0.99
99
86.2
2 8.
6 76
.11
11.8
Fluf
enox
uron
11
.73
488.
9>15
8.2
48
8.9>
141.
2 25
22
32
5
0.99
98
81.8
4 11
.2
78.1
8 6.
2
* Th
e un
derli
ned
MRM
tran
sitio
ns a
re u
sed
for q
ualit
ativ
e an
alys
is.
** n
=5
34
References
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Doi.org/10.1289/ehp.7135. [2] A.G. Frenich, M.J. Gonzalez-Rodrıguez, F.J. Arrebola, J.M. Vidal, Potentiality of Gas Chromatography-
Triple Quadrupole Mass Spectrometry in Vanguard and Rearguard Methods of Pesticide Residues in Vegetables. Anal. Chem., 77 (2005) 4640-4648.
Doi: 10.1021/ac050252o. [3] G. Gervais, S. Brosillon, A. Laplanche, C. Helen, Ultra-pressure liquid chromatography-electrospray
tandem mass spectrometry for multiresidue determination of pesticides in water. J. Chromatogr. A, 1202 (2008) 163-172.
Doi.org/10.1016/j.chroma.2008.07.006. [4] C.C. Leandro, P. Hancock, R.J. Fussell, B.J. Keely, Comparison of ultra-performance liquid
chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry. J. Chromatogr. A, 1103 (2006) 94-101.
Doi.org/10.1016/j.chroma.2005.10.077. [5] C.C. Leandro, P. Hancock, R.J. Fussell, B.J. Keely, Ultra-performance liquid chromatography for the
determination of pesticide residues in foods by tandem quadrupole mass spectrometry with polarity switching. J. Chromatogr. A, 1144 (2007) 161-169.
Doi.org/10.1016/j.chroma.2007.01.030. [6] D.T.T. Nguyen, D. Guillarme, S. Rudaz, J.L. Veuthey, Fast analysis in liquid chromatography using small
particle size and high pressure. J. Sep. Sci., 29 (2006) 1836-1848. Doi.org/10.1002/jssc.200600189. [7] Y. Picó, M. Farré, C. Soler, D. Barceló, Identification of unknown pesticides in fruits using ultra-
performance liquid chromatography-quadrupole time-of-flight mass spectrometry Imazalil as a case study of quantification. J. Chromatogr. A, 1176 (2007) 123-134.
Doi.org/10.1016/j.chroma.2007.10.071. [8] CIQ (custom, immigration and quarantine) People’s Republic of China SN/T 1747-2006 (2006).
Inspection of carbamate insecticide multi-residues in tea for export-Gas Chromatographic method. [9] Germany DFG method S 19 (1999). Modular Multiple Analytical Method for the Determination of
Pesticide. L 0-34. [10] B. Hu, W. Song, L. Xie, T. Shao, Determination of 33 pesticides in tea using accelerated solvent
extraction/gel permeation chromatography and solid phase extraction/gas chromatography-mass spectrometry. Chin. J. Chromatogr., 26 (2008) 22-28.
Doi.org/10.1016/S1872-2059(08)60009-7. [11] Z. Huang, Y. Li, B. Chen, S. Yao, Simultaneous determination of 102 pesticide residues in Chinese teas
by gas chromatography-mass spectrometry. J. Chromatogr. B, 853 (2007) 154-162. Doi.org/10.1016/j.jchromb.2007.03.013. [12] J. Ji, C. Deng, H. Zhang, Y. Wu, X. Zhang, Microwave-assisted steam distillation for the determination
of organochlorine pesticides and pyrethroids in Chinese teas. Talanta, 71 (2007) 1068-1074. Doi.org/10.1016/j.talanta.2006.05.087.
35
[13] J. Schurek, T. Portolés, J. Hajslova, K. Riddellova, F. Hernández, Application of head-space solid-phase microextraction coupled to comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry for the determination of multiple pesticide residues in tea samples. Anal. Chim. Acta., 611 (2008) 163-172.
Doi.org/10.1016/j.aca.2008.01.007. [14] Department of Food Safety Ministry of Health, Labour and Welfare, Japan (2006). Analytical methods
for residual compositional substances of agricultural chemicals, feed additives, and veterinary drugs in food.
[15] H. Kerkdijk, H.G.J. Mol, B.V.D. Nagel, Volume overload cleanup: An approach for on-Line SPE-GC,
GPC-GC, and GPC-SPE-GC. Anal. Chem., 79 (2007) 7975-7983. Doi: 10.1021/ac0701536. [16] M. Anastassiades, S.J. Lehotay, D. Stajnbaher, Quick, easy, cheap, effective, rugged, and safe
(QuEChERS) approached for the determination of pesticide Residues. 18th Annual Waste Testing and Quality Assurance Symposium Proceedings. (2002) 231-241.
[17] C. Lesueur, P. Knittl, M. Gartner, A. Mentler, M. Fuerhacker, Analysis of 140 pesticides from
conventional farming foodstuff samples after extraction with the modified QuEChERS method. Food control, 19 (2008) 906-914.
Doi.org/10.1016/j.foodcont.2007.09.002. [18] P. Payá, M. Anastassiades, D. MackIrina, I. Sigalova, B. Tasdelen, J. Oliva, A. Barba, Analysis of
pesticide residues using the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) pesticide multiresidue method in combination with gas and liquid chromatography and tandem mass spectrometric detection. Anal. Bioanal. Chem., 389 (2007) 1697-1714.
Doi.org/10.1007/s00216-007-1610-7. [19] European Council 2002/657/EC (2002). Implementing Council Directive 96/23/EC concerning the
performance of analytical methods and the interpretation of results. [20] R.M.K. Hajou, F.U. Afifi, A.H. Battah, Comparative determination of multi-pesticide residues in
Pimpinella anisum using two different AOAC methods. Food Chem., 88 (2004) 469-478. Doi.org/10.1016/j.foodchem.2004.03.051. [21] M. Hiemstra, A. de Kok, Comprehensive multi-residue method for the target analysis of pesticides in
crops using liquid chromatography-tandem mass spectrometry. J. Chromatogr. A, 1154 (2007) 3-26. Doi.org/10.1016/j.chroma.2007.03.123. [22] European Council N° SANCO/2007/3131 (2007). Method validation and quanlity control procedure for
pesticide residues analysis in food and feed. [23] L. Aldera, S. Luderitz, K. Lindtner, H. Stan, The ECHO technique-the more effective way of data
evaluation in liquid chromatography-tandem mass spectrometry analysis. J. Chromatogr. A, 1058 (2004) 67-79.
Doi.org/10.1016/j.chroma.2004.08.120.
36
37
Chapter 3
Rapid and selective quantification of L-theanine in ready-to-drink teas
from Chinese market
Abstract
An ultra-high-performance liquid chromatography (UHPLC) method combined with solid
phase extraction (SPE) sample pre-treatment was developed and validated for the rapid
quantification of L-theanine in ready-to-drink (RTD) teas. UHPLC analysis of twenty-seven
RTD teas from the Chinese market revealed that the L-theanine levels in various types of RTD
teas were significantly different. RTD green teas were found to contain highest mean L-
theanine level (37.85 ± 20.54 mg/L), followed by jasmine teas (36.60 ± 12.08 mg/L),
Tieguanying teas (18.54 ± 3.46 mg/L), black teas (16.89 ± 6.56), Pu-erh teas (11.31 ± 0.90
mg/L) and Oolong teas (3.85 ± 2.27 mg/L). The ratio of total polyphenols content to L-
theanine content could be used as a featured parameter for differentiating RTD teas. L-theanine
in RTD teas could be a reliable quality parameter that is complementary to total polyphenols.
This chapter was originally published as:
G. Chen, Y. Wang, W. Song, B. Zhao, Y. Dou, Rapid and selective quantification of l-theanine in
ready-to-drink teas from Chinese market using SPE and UPLC-UV. Food Chem. 135 (2012) 402-
407.
38
3.1 Introduction
RTD teas are increasingly popular as a healthy alternative to carbonate drinks and bottled
water. In China, the RTD tea market has become the most dynamic category in the soft drinks
industry. It is required by Chinese national standards for tea beverages (GB/T 21733-2008)
that the content of total polyphenols in RTD black tea, green tea, Oolong tea, and other tea
should be no less than 300, 500, 400 and 300 mg/kg, respectively. Those containing total
polyphenols below this requirement are defined as tea flavoured beverages. However,
polyphenols in RTD teas are prone to oxidation during storage, which could result in
underestimation of tea extracts in RTD teas. The establishment of a reliable quality parameter
would help the RTD tea market in setting standards, creating objective price criteria, and
improving the image of RTD teas.
L-theanine is an amino acid almost solely found in tea plants [1]. It only exists in the free
(non-protein) form and is the predominant free amino acid in tea [2]. L-theanine in RTD teas
can be a reliable quality parameter for RTD teas. A number of analytical methods has been
developed to determine L-theanine individually, or with other amino acids both in tea
compositions and simultaneously in different matrices. These include capillary
electrophoretic [3-6] and chromatographic methods. L-theanine can be analysed
simultaneously with other amino acids by high performance liquid chromatographic (HPLC)
methods involving precolumn derivatization with o-phthaladehyde, phenylisothiocyanate or
dabsyl chloride, and fluorescence and diode array UV detection [7-12]. Other HPLC methods
employing different columns and detectors were reported for the quantitative and qualitative
analysis of L-theanine in different teas without derivatization [13-19].
In recent years, UHPLC has been shown to give superior chromatographic resolution, reduced
analysis time, reduced solvent consumption and increased sensitivity when employed for tea
related analyses [20-24]. However, to the best of our knowledge, there have been no
publications or literature reporting UHPLC methods for rapid analysis of L-theanine in RTD
teas until now.
The aim of this chapter is to develop a rapid method for the analysis of L-theanine in RTD
teas using UHPLC-UV and solid phase extraction (SPE). Furthermore, using this method will
39
enable us to assess the qualities of RTD teas from the China market by quantification of L-
theanine. It is the first time to report L-theanine level in RTD teas.
3.2 Materials and methods
3.2.1 Reagents and solvents
All reagents and solvents used in the experiments were analytical grade or above. L-theanine,
gallic acid and Folin-Ciocalteu phenol reagent were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Acetonitrile and methanol were purchased from Merck (Darmastadt, Germany).
Formic acid was from Fluka (Steinheim, Germany). Pure water used to prepare standard
solutions and UHPLC mobile phase was produced by a Milli-Q system (Bedford, MA, USA).
Oasis MCX 3cc (6 mg) extraction cartridges were purchased from Waters (Milford, MA,
USA). All other reagents and solvents were purchased from SCRC (Shanghai, China).
3.2.2 Ready-to-drink tea samples
A total of twenty-seven RTD tea samples of different types, flavours and producers (listed in
Table 3.1) were analysed, which were purchased from a supermarket in Shanghai and
estimated to cover at least 90% of the Chinese market for RTD teas.
3.2.3 UHPLC-UV method for the analysis of L-theanine in RTD teas
UHPLC analysis was performed on a Waters ACQUITY UPLC System equipped with
photodiode array (PDA) detector. Different ACQUITY UPLC columns were employed for
the separation of L-theanine, including a BEH phenyl column (2.1 x 100 mm, 1.7 µm particle
size), BEH C18 column (2.1 x 100 mm, 1.7 µm particle size), BEH shield RP 18 column (2.1
x 100 mm, 1.7 µm particle size), BEH HILIC column (2.1 x 100 mm, 1.7 µm particle size)
and HSS T3 column (2.1 x 100 mm, 1.8 µm particle size). Mobile phase A was pure water
with formic acid (v/v: 0.05%, 0.1%, 0.5%) and prepared freshly for every analysis series.
Mobile phase B was acetonitrile. The analytes were monitored by UV detection at 195 ± 2
40
nm. Injection volume was 2.5 µL. The optimised conditions were: BEH phenyl column, pure
water with 0.1% formic acid for mobile phase A and a flow rate of 0.4 mL/min.
3.2.4 Estimation of total polyphenols
Total polyphenol contents of RTD teas were determined by a colorimetric assay using Folin-
Ciocalteu phenol reagent with gallic acid as standard according to a method specified by the
International Standardisation Organisation (ISO 14502-1).
3.2.5 Sample pre-treatment
For UHPLC analysis of L-theanine, all RTD samples were acidified to pH=2 with formic acid
before an optimized sample pre-treatment using Waters Oasis MCX extraction cartridges. The
cartridge was conditioned with 3 mL methanol and equilibrated with 3 mL pure water, prior
to loading 1 mL acidified RTD tea sample. Then the cartridge was washed with 2 mL pure
water containing 2% formic acid and 2 mL methanol. After that, the cartridge was eluted with
2 mL of a 5% ammonia solution in water. Finally, the fraction was evaporated to dryness and
then reconstituted in 1 mL pure water with 0.1% formic acid.
41
Tabl
e 3.
1. In
form
atio
n of
27
anal
ysed
RTD
teas
: sam
ple
ID, t
ea ty
pe, p
acka
ging
mat
eria
l, sa
mpl
e siz
e, p
rodu
cer,
flavo
ur, c
onte
nts o
f L-
thea
nine
and
tota
l pol
yphe
nols
(TP)
.
Sam
ple
ID
Tea
type
Pa
ckag
ing
mat
eria
l
Sam
ple
size
(mL)
Pr
oduc
er
Flav
our
L-th
eani
ne (m
g/L)
TP
(mg/
L)
UH
PLC
-UV
a R
P-H
PLC
-UV
b
RTD
-1
Blac
k te
a PE
T 50
0 P-
4 O
rigin
al, l
ow e
nerg
y 14
.25
± 0.
16
14.5
0 42
0.57
RTD
-2
Blac
k te
a PE
T 50
0 P-
4 Le
mon
19
.27
± 0.
14
19.1
9 50
8.54
RTD
-3
Blac
k te
a PE
T 50
0 P-
7 Ch
erry
9.
79 ±
0.1
6 N
/A
387.
83
RTD
-4
Blac
k te
a PE
T 45
0 P-
8 Le
mon
14
.16
± 0.
19
N/A
32
6.03
RTD
-5
Blac
k te
a PE
T 45
0 P-
8 O
rigin
al
26.9
8 ±
0.20
N
/A
506.
18
RTD
-6
Gre
en te
a PE
T 55
0 P-
1 O
rigin
al, l
ow su
gar
30.9
1 ±
0.18
30
.81
674.
12
RTD
-7
Gre
en te
a PE
T 50
0 P-
1 O
rigin
al, n
o su
gar
39.9
2 ±
0.10
39
.99
503.
05
RTD
-8
Gre
en te
a
PET
500
P-2
Orig
inal
, low
suga
r 63
.42
± 0.
60
N/A
65
7.67
RTD
-9c
Gre
en te
a
PET
500
P-2
Orig
inal
, zer
o en
ergy
50
.76
± 0.
28
N/A
45
5.84
RTD
-10
Gre
en te
a
PET
560
P-2
Orig
inal
, no
suga
r 59
.69
± 0.
21
N/A
64
4.15
RTD
-11
Gre
en te
a
PET
480
P-3
Orig
inal
, low
suga
r 39
.03
± 0.
13
N/A
64
8.56
RTD
-12c
Gre
en te
a
PET
500
P-5
Lem
on
8.92
± 0
.16
N/A
18
4.65
RTD
-13c
Gre
en te
a
PET
500
P-5
Hon
ey a
nd p
umel
o 10
.16
± 0.
15
N/A
21
2.33
RTD
-14
Jasm
ine
tea
PET
500
P-1
Orig
inal
, low
suga
r 31
.09
± 0.
13
32.0
0 74
4.34
RTD
-15
Jasm
ine
tea
PET
500
P-2
Orig
inal
, low
suga
r 22
.53
± 0.
17
21.1
8 51
2.35
RTD
-16
Jasm
ine
tea
PET
480
P-3
Orig
inal
43
.44
± 0.
32
N/A
75
3.09
RTD
-17
Jasm
ine
tea
PET
480
P-3
Hon
ey
49.3
4 ±
0.45
N
/A
822.
15
RTD
-18
Ool
ong
tea
PET
500
P-1
Orig
inal
7.
91 ±
0.1
2 7.
12
619.
45
RTD
-19
Ool
ong
tea
PET
350
P-5
Orig
inal
, no
suga
r 4.
51 ±
0.3
1 4.
65
577.
02
42
Sam
ple
ID
Tea
type
Pa
ckag
ing
mat
eria
l
Sam
ple
size
(mL)
Pr
oduc
er
Flav
our
L-th
eani
ne (m
g/L)
TP
(mg/
L)
UH
PLC
-UV
a R
P-H
PLC
-UV
b
RTD
-20c
Ool
ong
tea
PET
350
P-6
Orig
inal
, no
suga
r 2.
52 ±
0.8
1 N
/A
382.
62
RTD
-21
Ool
ong
tea
PET
350
P-6
Orig
inal
, no
suga
r 2.
03 ±
1.2
4 N
/A
863.
63
RTD
-22
Ool
ong
tea
PET
500
P-6
Orig
inal
, zer
o en
ergy
1.
96 ±
1.0
1 N
/A
564.
37
RTD
-23
Ool
ong
tea
PET
500
P-6
Orig
inal
, low
suga
r 4.
18 ±
0.9
7 N
/A
466.
05
RTD
-24
Pu-e
rh te
a PE
T 50
0 P-
5 O
rigin
al, n
o su
gar
11.9
5 ±
0.36
11
.99
440.
56
RTD
-25
Pu-e
rh te
a PE
T 35
0 P-
5 O
rigin
al, n
o su
gar
10.6
8 ±
0.19
N
/A
642.
87
RTD
-26
Tieg
uany
ing
Tea
PET
500
P-1
Orig
inal
, low
suga
r 16
.10
± 0.
19
16.0
3 60
4.5
RTD
-27
Tieg
uany
ing
Tea
PET
350
P-5
Orig
inal
, no
suga
r 20
.99
± 0.
61
N/A
43
4.56
a Ave
rage
± S
D (n
=3).
b Afte
r SPE
pre
-trea
tmen
t, se
lect
ed R
TD te
as w
ere
sent
to a
con
tract
lab
for t
he a
naly
sis o
f L-th
eani
ne, u
sing
a pr
e-co
lum
n O
PA d
eriv
ativ
e
RP-H
PLC
met
hod.
c A
ccor
ding
to G
B/T
2173
3-20
08, t
hese
RTD
teas
wer
e de
fined
as t
ea fl
avou
red
beve
rage
s.
43
3.2.6 Reference solutions
L-theanine standard stock solution was prepared in a mixture of pure water and acetonitrile
(9/1, v/v) at a concentration of 1 mg/mL and stored at -20 °C. The shelf life of the stock
solution is suggested to be six months. L-theanine standard solutions at 0.2, 1, 2, 5, 10, 20,
50, 100, 150, and 200 mg/L were prepared by diluting suitable amounts of stock solution in
pure water with 0.1% formic acid and stored at 4 °C. The shelf life of the calibration solutions
was suggested to be one month.
3.2.7 Method validation for the analysis of L-theanine in RTD teas
Experiments to validate the method were carried out. The precision was evaluated by running
sample analysis including UHPLC analysis and SPE pre-treatment with three replicates. To
evaluate the accuracy, the results of the L-theanine analyses of the RTD teas obtained using
UHPLC-UV and RP-HPLC-UV following derivatisation with OPA were compared.
Moreover, recovery experiments were done. The specificity was evaluated by control samples
(L-theanine standard and spiked RTD tea samples). A calibration curve was constructed from
the results of nine different concentrations. The linearity of the L-theanine response was
accessed by regression of the peak area against the corresponding concentration. Limit of
detection (LOD) and limit of quantification (LOQ) were determined as the UHPLC-UV
giving a signal equal to three and ten times the noise, respectively.
3.3 Results and discussion
3.3.1 Method development and validation
Chromatographic conditions were optimized by selection of acidic mobile phase, column and
flow rate. The overall time required for chromatographic separation did not exceed 8 min,
including 5 min for column equilibration. The mean L-theanine retention time was 1.156 ±
0.015 min. Representative chromatograms for RTD tea samples are shown in Figure 3.1.
44
Figure 3.1. UHPLC-UV chromatograms of L-theanine standard, 100 mg/L (A) and L-
theanine in representative RTD teas, including Tieguanying without SPE pre-treatment (B),
green tea without SPE pre-treatment (C), jasmine tea without SPE pre-treatment (D), Pu-erh
tea without/after SPE pre-treatment (E/F), black tea without/after SPE pre-treatment (G/H),
Oolong tea without/after SPE pre-treatment (I/J).
thea
nine
- 1.
162
AU
0.00
0.50
1.00
Minutes0.00 1.00 2.00 3.00 4.00
thea
nine
- 1.
157
AU
0.00
0.50
1.00
Minutes0.00 1.00 2.00 3.00 4.00
thea
nine
- 1.1
54
AU
0.00
0.50
1.00
Minutes0.00 1.00 2.00 3.00 4.00
thea
nine
- 1.
152
AU
0.00
0.50
1.00
Minutes0.00 1.00 2.00 3.00 4.00
thea
nine
- 1.
145
AU
0.00
0.20
0.40
Minutes0.00 1.00 2.00 3.00 4.00
thea
nine
- 1.
170
AU
0.00
0.20
0.40
Minutes0.00 1.00 2.00 3.00 4.00
thea
nine
- 1.
152
AU
0.00
0.20
0.40
Minutes0.00 1.00 2.00 3.00 4.00
thea
nine
- 1.
168
AU
0.00
0.20
0.40
Minutes0.00 1.00 2.00 3.00 4.00
1.15
7AU
0.00
0.20
0.40
Minutes0.00 1.00 2.00 3.00 4.00
thea
nine
- 1.
170
AU
0.00
0.20
0.40
Minutes0.00 1.00 2.00 3.00 4.00
A B
C D
FE
G H
I J
45
Formic acid was selected to prepare acidic mobile phase in this study. A mobile phase of 0.1%
formic acid in water presented better retention and peak shape of L-theanine than those of
0.05% and 0.5% formic acid in water. Different columns (ACQUITY UPLC HSS T3, BEH
C18, C8, Shield RP 18, HILIC and phenyl) were investigated. The BEH phenyl column
offered the best separation efficiency. The phenyl stationary phase affords a higher level of
hydrophilicity than the C18 and C8 column, which makes L-theanine to have more retention.
Moreover, the pi-pi interaction between the phenyl stationary phase and analytes containing
pi electrons affords additional retention power. Optimization of flow rate was carried out to
shorten the analysis time while affording better separation efficiency. The optimum flow rate
was 0.4 mL/min based on the optimization of peak shape, separation and analysis time.
The method was validated with regard to precision, accuracy, specificity, linearity, LOD and
LOQ. Triplicate analyses for each sample were completed and the content of L-theanine was
expressed as the average ± SD (Table 3.1). The recovery results of L-theanine in different
RTD teas are shown in Table 3.2. Analysis of L-theanine in selected RTD teas was performed
using a pre-column OPA derivative RP-HPLC method. The L-theanine contents measured
using the UHPLC-UV method were consistent with those measured using the OPA/ derivative
RP-HPLC method (Table 3.1). The calibration curve was plotted using peak area versus
concentration of the L-theanine standard and it was fitted to a linear equation (Y = 1350 X -
242, R2=0.9999). The linear range covered from 1 to 200 mg/L, the LOD and LOQ were 0.1
and 1 mg/L respectively.
3.3.2 Minimization of matrix interference by SPE pre-treatment
MCX SPE cartridges were used for sample clean-up before UHPLC analysis. The effect of
MCX SPE clean-up on the L-theanine separation in different RTD teas varied for the different
RTD teas. For RTD black teas, Oolong teas and Pu-erh teas, MCX SPE clean-up significantly
minimized the matrix interference (Figure 3.1). For RTD green teas, jasmine teas and
Tieguanying teas, co-elution of L-theanine and matrix interference were not observed and
MCX SPE clean-up did not offer an additional benefit. The recovery study using spiked RTD
46
teas showed that MCX SPE clean-up increased the recovery of L-theanine and reduced the
variation (Table 3.2).
Table 3.2. Recovery of L-theanine in various RTD teas.
Spiked sample
ID* Tea type
Recovery (%), mean (n=5) ± RSD
Without SPE clean-up After SPE clean-up
RTD-1 Black tea 85.6 ± 20.1 95.5 ± 3.8
RTD-2 Black tea 89.3 ± 15.9 94.2 ± 2.1
RTD-6 Green tea 94.5 ± 3.2 95.1 ± 3.0
RTD-7 Green tea 96.7 ± 2.4 95.8 ± 3.1
RTD-14
RTD-18
Jasmine tea
Oolong tea
95.5 ± 4.2
88.5 ± 14.6
94.5 ± 3.0
97.0 ± 2.8
RTD-19
RTD-24
RTD-26
Oolong tea
Pu-erh tea
Tieguanying tea
85.5 ± 17.6
76.4 ± 30.3
93.6 ± 4.4
96.2 ±3.5
96.3 ± 3.9
94.2 ± 4.8
*The spiked L-theanine concentration was 50 mg/L.
3.3.3 L-theanine in various RTD teas
Many studies have reported the contents of L-theanine in different teas [4, 7-10, 12, 15-19,
25]. However, there are no literature reports for the L-theanine level in RTD teas.
In this study, twenty-seven RTD teas of various types from the Chinese market were analysed
using UHPLC-UV. The L-theanine contents of these RTD teas are shown in Table 3.1. L-
theanine was detected in all RTD teas, but the contents varied. The mean L-theanine levels in
RTD green tea, jasmine tea, black tea, Oolong tea, Pu-erh tea and Tieguanying tea were 37.85
± 20.54, 36.60 ± 12.08, 16.89 ± 6.56, 3.85 ± 2.27, 11.31 ± 0.90 and 18.54 ± 3.46 mg/L,
respectively. It was revealed that the highest L-theanine level was detected at 63.42 mg/L in
a green RTD tea and the lowest L-theanine level was detected at 1.96 mg/L in an Oolong
RTD tea.
47
3.3.4 Quality evaluation of RTD teas by contents of L-theanine and total polyphenols
The analysis of total polyphenols of twenty-seven RTD teas from the Chinese market revealed
that three of eight RTD green teas and one of six RTD Oolong teas, according to the official
GB/T 21733-2008 regulations, were tea flavoured beverages rather than RTD teas. The scatter
plot of L-theanine contents vs total polyphenols content in various RTD teas (Figure 3.2)
shows that as L-theanine content increased in RTD black and jasmine teas, total polyphenols
content increased as well. The ratio of total polyphenols content to L-theanine content (Figure
3.2) could be used as a featured parameter for differentiating RTD teas. The ratio for RTD
Oolong teas has both the biggest variation and was the highest (78- 425), followed by RTD
Pu-erh teas (37-60), RTD Tieguanying & black teas (19-40), RTD jasmine teas (16-24) and
RTD green teas (9-22).
Figure 3.2. Scatter plot of contents of L-theanine vs total polyphenols in various RTD teas: 8
green teas, 4 jasmine teas, 5 black teas, 6 Oolong teas, 2 Pu-erh teas and 2 Tieguanying teas
from Chinese market.
0
10
20
30
40
50
60
70
0 200 400 600 800 1000
Cont
ent o
f L-th
eani
ne, m
g/L
Content of Total polyphenols, mg/L
RTD balck teas RTD green teas
RTD jasmine teas RTD oolong teas
RTD Pu-erh teas RTD Tieguanying teas
48
Figure 3.3. The ratio of total polyphenols content to L-theanine content in different RTD
teas.
3.4 Conclusions
A UHPLC-UV method was proposed for the rapid quantification of L-theanine in RTD teas.
The applicability and reliability of this analytical approach was confirmed by method
validation and successful analysis of twenty-seven real samples of RTD teas. It was revealed
that the L-theanine levels in the different RTD teas were significantly different. The scatter
plot of L-theanine content vs total polyphenol content in various RTD teas suggested that
there was a positive correlation between L-theanine content and total polyphenol content in
RTD black and jasmine teas. The ratio of total polyphenols content to L-theanine content can
be used as a featured parameter for differentiating RTD teas. Quantification of L-theanine in
RTD teas can be a reliable quality parameter for RTD teas which is complementary to total
polyphenols.
49
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51
[24] Y. Zhao, P. Chen, L. Lin, J.M. Harnly, L. Yu, Z. Li, Tentative identification, quantitation, and principal component analysis of green pu-erh, green, and white teas using UPLC/DAD/MS. Food Chem., 126 (2011) 1269-1277.
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52
53
Chapter 4
A method for measuring the noncovalent interaction between EGCG
and β-CD
Abstract
Reducing the bitter and astringent taste of green tea will engage the consumers who are used
to the mild taste of black tea. β-cyclodextrin (CD) is used as an effective bitterness and
astringency masker for catechins in green tea, especially for epigallocatechin gallate (EGCG).
The present study aims to reveal the underlying mechanism of the noncovalent interaction
between β-CD and EGCG. The complex of β-CD and EGCG was directly studied by
combined electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic
resonance spectroscopy (NMR). The stoichiometry of the β-CD-EGCG complexation product
was determined using Job’s method, which showed a 1:1 stoichiometry of the β-CD-EGCG
complex. NMR experiments confirm the formation of an inclusion complex of β-CD and
EGCG. The topology of the complex was derived from ROESY spectra and chemical shift
differences of the various protons of β-CD and EGCG in the free versus complexed state. The
direct observation of noncovalent interactions using ESI-MS and NMR enables fast screening
of molecular maskers for reducing bitterness and astringency of catechins in green tea as a
potential alternative to a tasting panel.
This chapter was submitted for publication as:
G. Chen, H-G. Janssen, the use of ESI-MS & 2D NMR to reveal the reduction of bitterness and
astringency of EGCG by β-CD inclusive complexation. Food Res. Int. manuscript submitted.
54
4.1 Introduction
Consumption of green tea is associated with several health benefits like anti-oxidant effects,
anti-carcinogenity, and suppression of cardiovascular diseases and obesity [1-7]. Green tea is
rich in a series of flavanols, more specifically catechins including (+)-gallocatechin (GC), (-)-
epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (CA), (+)-gallocatechin gallate
(GCG), (-)-epigallocatechin gallate (EGCG), (+)-catechin gallate (CG) and (-)-epicatechin
gallate (ECG) [8]. These compounds are thought to be responsible for the above-mentioned
health benefits, yet unfortunately they also cause green tea to taste bitter and astringent [9-
12]. This bitterness and astringency can be a barrier to consumers who are used to the mild
taste of black tea. For reducing the undesired taste characteristics of green tea, β-cyclodextrin
(CD) was shown to be a very effective taste masker [13-17]. The natural, sweet taste of β-CD
might help to mask the bitterness of catechins: a 0.5% β-CD solution is as sweet as sucrose
[18]. However, it is unclear whether the sweet taste of β-CD is the only mechanism for this
bitterness reduction, or that complexation mechanisms also play a role in the masking of green
tea bitterness and astringency.
β-CD has been described many times as a scavenging molecule which is able to complex with
bitter molecules [17,19-20]. β-CD was even used to develop a detection system for sensing
the bitterness/astringency of green tea catechins [21]. However, the exact consequences of
this complexation for taste are unknown. Investigation of the interactions between β-CD and
catechins can be helpful to reveal to which extent and how β-CD masks the bitter/astringent
taste of green tea catechins.
A number of analytical techniques has been applied to study the noncovalent interactions
between β-CD and bitter variant molecules. The use of proton nuclear magnetic resonance
(NMR), UV, and circular dichroism spectroscopy were reported to examine the inclusion
complex of CA with CDs [22]. The probable structures of the inclusion complexes of β-CD
with CA and EC were investigated using NMR [23-25]. The complexation of EC and CA
with hydroxypropyl-β-CD was investigated by using isothermal titration calorimetry,
fluorescence and proton NMR [26]. Recently, Aree and his team reported the structure-
antioxidant property relationship of the CD inclusion complexes with tea non-epicatechins by
55
means of single-crystal X-ray diffraction, density functional theory calculation and radical
scavenging activity assay [27].
Green tea catechins are well known as antioxidant actives [28]. Inclusion complexes of
catechins with CDs were investigated for the enhancement of antioxidant activity [27, 29-32].
Meanwhile, EGCG is the most bitter and astringent catechin in green tea [10,33].
Understanding the noncovalent interaction between EGCG and β-CD at the molecular level
is crucial to reveal the mechanism of β-CD bitterness reduction. In addition, the use of mass
spectrometry (MS) to characterize the inclusion complexes of β-CD with catechins has not
been reported, although MS technologies have been extensively shown to be fast and reliable
tools to determine noncovalent binding interactions and complexation [34-35].
In this chapter, the noncovalent interactions between β-CD and green tea catechins were
studied by electrospray ionization-mass spectrometry (ESI-MS). The inclusion complex of β-
CD and EGCG was characterized by the combined use of ESI-MS and 2D NMR. A
mechanism of β-CD reducing the bitterness and astringency of EGCG was proposed based
on the understanding of the noncovalent interaction of β-CD and EGCG.
4.2 Materials and methods
4.2.1 Reagents and solvents
All reagents and solvents used in the experiment were analytical grade or better. β-CD was
purchased from SCRC (Shanghai, China). Milli-Q pure water (>18.2 MΩ, Bedford, MA, USA)
was used to prepare solutions. D2O and EGCG (>95%) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Formic acid was from Fluka (Steinheim, Germany). HPLC grade
acetonitrile was purchased from Merck (Darmstadt, Germany). Green tea powder (batch
number: xs090805-02) was supplied by Novanat (Shanghai, China). The contents of some
selected catechins in this batch of green tea powder are shown in Table 4.1.
56
Table 4.1. Contents of catechins in the green tea powder studied.
GC EGC CA EC EGCG GCG ECG CG
MW* 306 306 290 290 458 458 442 442
w/w %** 1.31 14.28 1.09 5.68 34.52 0.76 8.47 Not detected
* MW: molecular weight
** Details of the method (ISO14502-2) for the analysis of the catechins in the green tea powder
are given in Appendix.
4.2.2 ESI-MS analysis
The ESI-MS experiments were performed with a Micromass® Quattro MicroTM API
instrument (Waters, Wilmslow, UK) equipped with an ESI source. MassLynx software
(version 4.1) was used for instrument control and data acquisition. The sample solutions were
directly infused into the ESI source with a flow rate of 30 µL/min by a syringe pump and
analysed in negative ion mode. All MS parameters were optimized to obtain highest
sensitivity and resolution as well as to minimize disturbance of the complexes. Briefly, the
capillary voltage was 3.50 kV, the cone voltage was 25 V, the extractor voltage was 3 V and
the source temperature was 100 ℃. The desolvation gas temperature was 350 ℃ with a
nitrogen flow rate of 300 L/hr. The ESI-MS scan range was set from m/z 200 to 2000.
4.2.3 NMR analysis
All 1H NMR experiments (1D and 2D) were performed at 400 MHz on a Bruker Avance-
AV400 spectrometer (9.4 T) (Bruker, Rheinstetten, Germany) at 25 ℃. The resonance at 4.8
ppm, originating from residual H2O in the D2O, was used as internal reference. Complexation
was assessed by applying the rotating frame Overhauser effect spectroscopy (ROESY)
method using the ROESYPHPR pulse sequence. The parameters of the ROESY experiment
were set as follows: 16 scans, acquisition time 0.150 s, pulse delay 2.3 s and time domain size
1224.
57
4.2.4 Preparation of inclusive complexes
Stock solutions of β-CD (1 mM), EGCG (1 mM) and green tea powder (1 mg/mL) were
prepared in Milli-Q demineralised water. The inclusion complexes of β-CD and green tea
catechins were prepared by mixing 100 µL of each stock solution into 1 mL Milli-Q water at
room temperature. The work solutions of β-CD-EGCG inclusion complexes were prepared in
molar host-to-guest ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. All complex
solutions were placed at room temperature for 24 hr before ESI-MS analysis. For NMR
analysis, equimolar mixtures of β-CD and EGCG (10 mM) were prepared in D2O.
4.3 Results and discussion
4.3.1 Direct observation of complexation of β-CD and green tea catechins using ESI-
MS
To detect CD inclusion complexes, mixtures of β-CD and green tea powder in pure water
were analysed by ESI-MS in negative ion mode using parameter settings similar to those
employed by others in the study of CD complexation of other small organic molecules [36-
39]. As GC and EGC, CA and EC, EGCG and GCG, and EGC and GC are isomer pairs, they
have the same molecular weights (Table 4.1) and yield identical m/z fragments. A
representative ESI-MS spectrum obtained for a mixture of β-CD and green tea powder in
water is shown in Figure 4.1. Ions at m/z 289.1, 305.1, 441.1, 457.0, and 493.1 correspond to
[EC/CA-H]-, [EGC/GC-H]-, [ECG-H]-, [EGCG/GCG-H]- and [EGCG/GCG+Cl]-,
respectively. The ion at m/z 915.1 corresponds to [2EGCG-H]-, an EGCG dimer adduct ion
which possibly resulted from the high concentration of EGCG in the green tea powder. Ions
at m/z 1133.7 and 1169.7 correspond to [β-CD-H]- and [β-CD+Cl]-, respectively. Ions at m/z
1423.6, 1439.6, 1575.7, 1591.9 and 1609.0 originate from [β-CD+EC]-, [β-CD+EGC-H]-, [β-
CD+ECG]-, [β-CD+EGCG-H]- and [β-CD+EGCG+Cl]-, respectively. These observed ions
clearly demonstrate the extensive complexation of β-CD and green tea catechins.
58
Figure 4.1. ESI-MS spectrum of a mixture of β-CD (100 µL stock solution) and green tea
powder (100 µL stock solution) in 1 mL pure water.
4.3.2 Direct observation of complexation of β-CD and EGCG using 1H NMR
Mass spectrometry of non-covalent complexes holds a risk of incorrect conclusions because
complexes can either be formed, or destroyed, in the ion source. It is for this reason that MS
analyses of non-covalent complexes should, if possible, be confirmed by e.g. NMR or other
spectroscopic methods. The chemical shift differences of the protons of β-CD and EGCG in
the mixture versus that in the free solutions clearly indicate that complexes are present and
justify the conclusions from the MS noncovalent interaction studies. The exact chemical shifts
are listed in Table 4.2. Comparing the chemical shifts of specific protons allows to identify
which protons from both the β-CD and EGCG are involved in the complexation reaction.
Table 4.2 clearly shows the largest chemical shift differences for the EGCG protons a and b,
and the β-CD protons 2, 3 and 5 (Figure 4.2). This would mean that mainly the B & D rings
of the EGCG molecule are involved in the complexation process. Figure 4.3 shows 1H NMR
spectra of free β-CD, free EGCG and of a mixture of the two.
59
OHO
OH
O
OH
OH
OH
OOH
OH
OH
c
c
f;ge
d
b
b
a
a
A C
B
D
O
OHHO
OH
O
O
OH
HOOH
O
OOH
OH
OH
O
O
OHOH
OH
OO
OH
OH
HO
O
OOH
OHHO
O
OOH
HO
HO
O
12
34
5 6
Figure 4.2. Chemical structures and protons of EGCG and β-CD.
Table 4.2. 1H chemical shifts of EGCG and β-CD, in free and complex status in D2O
solution.
Protons δ Free δ Complex Δδ (PPM)
EGCG a 6.94 7.10 -0.16 b 6.52 6.79 -0.27 c 6.08 6.03 0.05 d 5.48 5.43 0.05 e 4.91 4.95 -0.04 f 2.91 2.98 -0.07 g 2.87 2.91 -0.04
β-CD 1 5.07 5.02 -0.05
2,3 3.87 3.73 -0.14 4 3.59 3.55 -0.04 5 3.97 3.81 -0.16 6 3.65 3.60 -0.05
60
Figure 4.3. 1H NMR spectra of free β-CD, free EGCG and a β-CD / EGCG mixture (mole
ratio =1:1, 10 mM in D2O).
4.3.3 Stoichiometry of the β-CD-EGCG complex
The stoichiometry of the β-CD-EGCG complex was determined using Job’s method [40]. In
this method, the total molar concentration of β-CD and EGCG is held constant while varying
their mole ratios. Information on the stoichiometry, i.e. the number of EGCG molecules
present in a complex, can then be obtained from the molar β-CD/EGCG ratio at which the
strongest complexation occurs. For the measurements, a quantitative parameter that is
proportional to complex formation has to be plotted against the mole fractions of β-CD and
EGCG. Here the absolute intensity of the β-CD-EGCG complex in the ESI MS measurements
was used. The MS absolute intensity of the β-CD-EGCG complex (m/z 1591.9) was obtained
by averaging the MS signals of 15 scans. The Job-plot showed a maximum at 0.5, indicating
a 1:1 stoichiometry of the β-CD-EGCG complex (Figure 4.4).
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
2.91
22.
980
3.27
43.
306
3.55
13.
603
3.67
73.
729
3.78
83.
815
4.79
84.
949
5.01
7
5.42
9
6.03
5
6.79
3
7.09
6
2.86
82.
913
4.79
24.
908
5.48
2
6.08
5
6.51
6
6.93
6
3.58
83.
652
3.87
23.
969
4.79
9
5.06
9
CD
CD+EGCG
EGCG
61
Figure 4.4. Job-plot for the β-CD-EGCG complex.
4.3.4 Suggested topology of the β-CD-EGCG complex
Advanced 2D NMR techniques (ROESY) were successfully used to provide unambiguous
assignment of the structures of complexes of β-CD with several small organic molecules,
including for example amino acids and peptides [41]. Figure 4.5 presents the ROESY
spectrum of the β-CD-EGCG complex with cross peaks between the hydrogen protons of β-
CD and EGCG. Intermolecular correlations between the internal H3 and H5 protons of β-CD
and aromatic hydrogen protons (Ha and Hb) of EGCG are seen and aligned with the findings
reported by Cai and his team [42]. These correlations show that parts of the EGCG molecule
enter the β-CD cavity resulting in an inclusion complex of β-CD and EGCG. Based on the
ROESY spectrum and the chemical shift differences of β-CD and EGCG protons in the free
and complexed state, the topology of β-CD-EGCG complex could be that suggested in Figure
4.6, showing inclusion of the B-ring of EGCG in the β-CD cavity.
0.0 0.2 0.4 0.6 0.8 1.01x105
2x105
3x105
4x105
5x105
6x105
Inte
nsity
Mole Fraction of EGCG/(EGCG+beta-CD)
62
Figure 4.5. Rotating frame Overhauser effect spectrum containing cross peaks between β-
CD and EGCG protons.
Figure 4.6. Suggested topology of β-CD-EGCG complex.
EGCGHa, b β-CD
H3, 5
OHO
OH
O
OH
OH
OH
OOH
OH
OH
c
c
f;ge
d
b
b
a
a
A C
B
D
OHO
OH
O
OH
OH
OH
OOH
OH
OH
c
c
f;ge
d
b
b
a
a
A C
B
D
OHO
OH
O
OH
OH
OH
OOH
OH
OH
c
c
f;ge
d
b
b
a
a
A C
B
D
+
Beta-CD
H3
H5
H3
H5
63
4.3.5 Mechanism of β-CD reducing the bitterness and astringency of EGCG
Several studies in literature have indicated that the bitter, astringent taste of green tea is caused
by catechins, with galloylated catechins being considerably more bitter than their non-
galloylated counterparts [43]. Especially the presence of a gallic acid group at the D ring of
the catechin increase bitterness significantly. Removal of the gallate moiety by means of the
enzyme tannase or the use of physical barriers to exclude the gallate moiety from the taste
receptors have been used to reduce bitterness [10,12,15,33]. Based on the ESI-MS and NMR
observations of the noncovalent interaction between β-CD and EGCG presented here, it can
be concluded that inclusion complexes of β-CD and EGCG are formed in aqueous solutions.
The D ring, representing the gallate moiety, and B ring are ‘trapped’ by the β-CD molecule
upon complexation. This molecular encapsulation could ‘hide’ the gallate from the taste
receptor, and therefore reduce the perception of bitterness and astringency of EGCG.
4.4 Conclusions
The noncovalent interaction between β-CD and EGCG was studied at the molecular level by
ESI-MS and NMR. Inclusion complexation of β-CD and green tea catechins was directly
observed by ESI-MS. The stoichiometry of the β-CD-EGCG complex was determined using
Job’s method, which showed a maximum at 0.5, indicating a 1:1 stoichiometry of the β-CD-
EGCG complex. NMR experiments indicated that inclusion complexes of β-CD and EGCG
were formed and that the D ring or B ring of EGCG were present inside the β-CD cavity. This
molecular encapsulation could prevent the gallate moiety from binding to the taste receptor,
in that way reducing the perception of a bitter-astringent taste of EGCG. The direct
observation of noncovalent interactions makes the combined deployment of ESI-MS and
NMR a valuable chemical vehicle for fast screening of molecular maskers for reducing
bitterness and astringency of green tea catechins as an alternative to a tasting panel.
64
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68
Appendix
Appendix 4.1. UHPLC conditions for green tea catechin analysis.
Column: ACQUITY UPLC@BEH phenyl 1.7 µm (2.1 mm×150 mm)
UV length: 278 nm
Mobile phase: ACN with 0.2% FA., water with 0.2% FA
Column temperature: 30 ℃
Gradient table:
Time(min) flow A(0.2%formatic/ACN) B(0.2%formatic/water)
0 0.4 10 90
2 0.4 15 85
5 0.4 15 85
6 0.4 20 80
8 0.4 20 80
10 0.4 70 30
10.2 0.4 10 90
12 0.4 10 90
69
Appendix 4.2. Representative chromatogram of catechins in green tea powder.
1. Gallic acid; 2. Theobromine; 3. EGC; 4. CA; 5. Caffeine; 6. EC; 7. EGCG; 8. GCG; 9. ECG.
1
2 3
5
6
7
8
9
Minutes 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
4
70
71
Chapter 5
Quantification of climbazole deposition from shampoo onto artificial
skin and human scalp
Abstract
A sensitive and specific ultra-high-performance liquid chromatography-tandem mass
spectrometry (UHPLC-MS/MS) method was developed and validated for the measurement of
climbazole deposition from hair care products onto artificial skin and human scalp. Deuterated
climbazole was used as the internal standard. Atmospheric pressure chemical ionization (APCI)
in positive mode was applied for the detection of climbazole. For quantification, multiple
reaction monitoring (MRM) transition 293.0 > 69.0 was monitored for climbazole, and MRM
transition 296.0 > 225.1 for the deuterated climbazole. The linear range ran from 4 to 2000
ng/mL. The limit of detection (LOD) and the limit of quantification (LOQ) were 1 ng/mL and
4 ng/mL, respectively, which enabled quantification of climbazole on artificial skin and human
scalp at ppb level (corresponding to 16 ng/cm2). For the sampling of climbazole from human
scalp the buffer scrub method using a surfactant-modified phosphate buffered saline (PBS)
solution was selected based on a performance comparison of tape stripping, the buffer scrub
method and solvent extraction in in vitro studies. Using this method, climbazole deposition in
in vitro and in vivo studies was successfully quantified.
This chapter was originally published as:
G. Chen, M. Hoptroff, X. Fei, Y. Su, H-G. Janssen, Ultra-high-performance liquid
chromatography-tandem mass spectrometry measurement of climbazole deposition from hair care
products onto artificial skin and human scalp. J. Chromatogr. A. 1317 (2013) 155-158.
72
5.1 Introduction
Climbazole (1-4-Chlorophenoxy)-1-(1H-Imidazonyl)-3, 3-Dimethylbutan-2-one) is an
imidazole anti-fungal agent widely used in marketed anti-dandruff (AD) shampoos. It claims
to offer benefits of inhibiting microbe growth and improving the skin barrier on the scalp.
Climbazole is either used as a single anti-fungal agent or applied in combination with other
AD actives like zinc pyrithione or piroctone olamine to enhance its efficacy [1, 2]. The
efficacy of climbazole as an AD agent depends on its deposition behaviour and the amount
retained on the human scalp in the process of shampoo application and rinse-off. To support
in vitro and in vivo studies for the performance evaluation of AD shampoos containing
climbazole, a robust and sensitive analytical method for the measurement of climbazole
deposition on artificial skin and human scalp is required.
Besides being used as AD active in shampoo, climbazole has been widely used in daily life
as fungicide in pharmaceutical products and personal care products like lotions, conditioners,
etc. A number of analytical methods using HPLC-UV have been published for the
determination of climbazole in shampoo [3, 4]. Although the high levels of surfactants in the
shampoo can cause problems in the analysis, these methods are not extremely complex
because of the rather high level of the active. With increasing concern on the potentially
negative impact of climbazole to the aquatic environment additional methods have been
developed for the determination of climbazole in environmental matrices [5-6]. To the best
of our knowledge, only one method appeared in the literature for the analysis of climbazole
in in vitro skin studies. Schmidt-Rose [2] proposed a reversed phase HPLC-MS method
following methanol extraction for the quantification of climbazole in an in vitro study. The
method proved to be successful for the determination of skin substantivity of climbazole using
pig skin as model substrate. However, the application of this method to human scalp samples
could be challenged by the more complicated sample matrix and lower climbazole levels.
In this chapter, an ultra-high-performance liquid chromatography-tandem mass spectrometry
(UHPLC-MS/MS) method was developed and validated for the measurement of climbazole
deposition on artificial skin (for in vitro studies) and human scalp (for in vivo studies).
Deuterium labelled climbazole (Figure 5.1) was applied as the internal standard for the
73
quantification. Tape stripping and buffer scrub extractions were evaluated and compared as
sampling methods for climbazole extraction from artificial skin and human scalp.
Figure 5.1. Chemical structure of (A) climbazole and (B) deuterated climbazole.
5.2 Experiments
5.2.1 Chemicals and reagents
All reagents and solvents used in this experiment were analytical grade or better. Climbazole
(99.9%) and PBS tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Ammonium acetate, glycerol, Triton X-100 and Tween 80 were purchased from SCRC
(Shanghai, China). HPLC grade methanol and acetonitrile were purchased from Merck
(Darmstadt, Germany). Milli-Q pure water (18.2 MΩ, Millipore, Bedford, MA, USA) was
used to prepare samples, standard solutions and UHPLC mobile phases. Deuterated
climbazole (>95%) was custom synthesized and supplied by Qinba Chemical (Shanghai,
China). A surfactant-modified phosphate buffered saline (PBS) solution for the buffer scrub
sampling method was prepared by adding four PBS tablets, 1.0 g of Triton X-100, and 5.0 g
of Tween 80 to 1 L of pure water.
A B
74
5.2.2 Test shampoos
Two different shampoos were used: AD shampoo and beauty shampoo. The AD shampoo
contained 0.5% (w/w) climbazole. The beauty shampoo was a commercially available
shampoo without climbazole. The application of beauty shampoo in in vitro and in vivo study
was to obtain blank samples.
5.2.3 In vitro study (Artificial skin samples)
Artificial skin (VITRO-SKIN®) was purchased from IMS testing group (ME, USA,
www.ims-usa.com). The sheet of artificial skin was cut into 6 × 6 cm pieces and laid over one
side of the smaller diameter X-ray fluorescence spectroscopy (XRF) ring. The bigger XRF
ring was then placed on top of the smaller one and pressed down to snugly combine together
in that way yielding an XRF ‘cup’ (5 cm diameter) with rough topography of the artificial
skin inside. Pure water (1.8 mL) and test shampoo (0.2 g) were added into the cup and mixed
on the surface of the artificial skin. The mixture was stirred for 30 s with a teflon stirring rod
which remained in contact with the surface of the artificial skin. The shampoo solution was
then removed with a pipette and the artificial skin was rinsed twice with 4 mL of pure water
with stirring for 30 s (as before). The rinsing water was removed with a pipette. After the cup
was allowed to dry overnight under ambient conditions, the artificial skin was subjected to
climbazole extraction.
Three sampling methods for climbazole extraction from artificial skin were investigated: tape
stripping, buffer scrub and solvent extraction. For tape stripping, tapes of Sellotape® (Se),
Leuoflex® (Le), and Standard D-Squame® (DS) were evaluated in terms of sampling
efficiency and matrix influence. After stripping climbazole from artificial skin, the tapes were
extracted by methanol in an ultrasonic bath. In the buffer scrub experiments, surfactant-
modified PBS solution and 50% aqueous glycerol solutions were evaluated as extraction
buffers. Two milliliter of extraction buffer was added into the XRF cups where the artificial
skin had been treated by test shampoos and dried naturally. A teflon stirring rod was employed
to scrub the artificial skin for 30 s. The buffer scrub sampling was repeated five times using
2 mL extraction buffer each time. All the extraction liquid was transferred into a 10 mL
75
volumetric flask using a plastic pipette and made to volume using methanol. For solvent
extraction, the artificial skin was cut out from the XRF rings and submersed in the methanol
for ultrasonic extraction. Every method for climbazole extraction from artificial skin
described above was performed in triplicate. In vitro blank samples were prepared by applying
climbazole-free beauty shampoo onto the artificial skin. As the internal standard 0.2 µg/L
deuterated climbazole was added to the extraction solution of every artificial skin sample. All
sample solutions were centrifuged prior to UHPLC-MS/MS analysis of the supernatant.
5.2.4 In vivo study (human scalp samples)
The in vivo study was designed as a single centre, whole head, and single gender (male) study.
Healthy male subjects were screened using the so-called Total Weighted Head Score (TWHS)
system [7]. Six samples were collected for each subject (three per each half head). The study
lasted for 3 days with 2 visits. The buffer scrub sampling method using modified PBS as the
extraction fluid was selected for extracting climbazole from human scalp. This selection was
based on the performance comparison of tape stripping, buffer scrub and solvent extraction
(see below) taking into consideration also the safety characteristics of the three methods. In
the buffer scrub method, a sterile plastic ring of 18 mm internal diameter and 6 cm height was
placed and held steady on the sample site of the human scalp where 2.0 mL of modified PBS
solution was applied. The scalp was gently massaged with a teflon rod for 1 min. This was
repeated with a further 2.0 mL buffer for a further 1 min. Suspensions were pooled in a single
vial using a sterile plastic pipette. The first visit was a screening session. Five subjects were
recruited for the next phase after this screening. During the second visit, subjects firstly had
their baseline sample (in vivo blank sample) collected using the buffer scrub sampling method
and then had their hair washed using a whole head hair wash procedure in which 8 gram of
AD shampoo was applied and the scalp was massaged. After rinse-off, their hair was blown-
dried and scalp samples were collected using the buffer scrub method.
As the internal standard 0.2 µg/L deuterated climbazole was added into the extraction solution
of every human scalp sample. Further analysis was performed as described for the in vitro
artificial skin samples.
76
5.2.5 Instrumentation
All sample analyses were carried out on a Waters ACQUITY UPLC System coupled to a
Quattro Micro API mass spectrometer (Waters, Manchester, UK). MassLynx software
(version 4.1) was used for instrument control and data acquisition. The samples were
separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm particle size) supplied
by Waters. The mobile phase was composed of 20 mM ammonium acetate in water (A) and
methanol (B) programmed in the linear gradient mode [time 0 min, 50% A; time 5 min,
decrease immediately to 10% A; time 8 min, increase immediately to 50% A]. Total analysis
time was 8 min including 3 min for re-equilibration. The flow rate was 0.2 mL/min and the
injection volume was 5 µL. The temperatures of the column and sample compartment were
set at 30 ℃ and 4 ℃, respectively. Based on the comparison of the performance of
atmospheric pressure chemical ionization (APCI) and electro spray ionization (ESI), APCI
under positive polarity was used for all experiments. The optimized APCI conditions were
achieved by a corona current of 3.6 µA, a cone voltage of 32 V, an extractor voltage of 3 V,
a source temperature of 100 °C, an APCI probe temperature of 300 °C, a desolvation gas flow
of 450 L/hr and a cone gas flow of 25 L/hr. The multiple reaction monitoring (MRM) mode
was used for the determination of climbazole. The collision gas (argon) pressure was set at 3
× 10-3 mbar. The dwell time for each MRM transition was 0.30 s. Climbazole was analysed
using the transitions of m/z 293.0 > 69.0 (collision energy 20 V) and 293.0 > 197.1 (collision
energy 18 V) signals while deuterated climbazole was measured using the transition of m/z
296.0 > 225.1 (collision energy 14 V). These transitions were found to be the optima in the
MRM optimization experiments.
5.2.6 Quantification of climbazole
Standard stock solutions of climbazole and deuterated climbazole were prepared in methanol
at a concentration of 2 mg/mL. The working solutions of different climbazole concentrations
with 0.2 µg/L deuterated climbazole as the internal standard were prepared freshly by the
appropriate dilution of stock solutions with a mixture of 50% of mobile phase A and 50%
mobile phase B. All stock and working solutions were stored at 4 ℃ in the dark. For
77
quantification, MRM transition 293.0 > 69.0 was monitored for climbazole, and MRM
transition 296.0 > 225.1 for the deuterated climbazole. The response ratio (climbazole to
deuterated climbazole) versus climbazole concentration was plotted as the calibration curve.
5.3 Results and discussion
5.3.1 Optimization of the UHPLC-MS/MS conditions
In order to shorten the analysis time, a UHPLC instrument equipped with a column packed
with small particles (1.7 µm, Acquity UPLC BEH C18 column) was applied. The mobile
phase composition, flow rate and gradient program were optimized to obtain both good peak
shape and high chromatographic resolution at a short total analysis time. The retention time
of climbazole was 3.36 ± 0.02 min (mean ± RSD, n=10). The total analysis time was 8 min
including 3 min for system re-equilibration. The application of MS/MS guaranteed the
method selectivity and allowed simple sample preparation. ESI and APCI were compared in
terms of signal intensity and signal suppression or enhancement. When the APCI source was
applied, no signal suppression or enhancement was observed neither within one run nor after
10 injections of artificial samples and human scalp samples. Signal suppression on the other
hand, was significant when the ESI source was applied. This phenomenon was found in many
other studies as well [e.g. 8-14]. Therefore, the APCI ionization mode was selected for the
analysis of all samples. Figure 5.2 shows the representative chromatogram obtained from
climbazole standard (20 ng/mL), an artificial skin sample and a human scalp sample.
78
Figure 5.2. Representative chromatogram of climbazole in standard solution of 20 ng/mL
(a), extraction solution of artificial skin using tape stripping and buffer scrub (b & c) and
extraction solution of human scalp using buffer scrub (d).
5.3.2 Method validation
The UHPLC-MS/MS method for climbazole analysis was validated with regard to precision,
accuracy, specificity, linearity, LOD and LOQ. In the precision study, triplicate analysis of
selected artificial skin samples and human scalp samples obtained using the buffer scrub
sampling method was carried out. The mean climbazole concentrations and standard
deviations of these two samples were 386.8 ± 33.2 and 246.3 ± 16.6 ng/cm2 for selected
artificial skin samples and human scalp samples, respectively. The recoveries of climbazole
in extraction solutions of artificial skin and human scalp samples are shown in Table 5.1. The
recoveries seen, and the repeatability found are good, meaning that the method can be applied
in studies with real hair washing. In the experiments climbazole was identified by the ratio of
a
b
c
d
79
the MRM transitions m/z 293.0 > 69.0 and 293.0 > 197.1 signals, which should be 1.4 ± 0.1,
as established from the analysis of the pure compound. The calibration curve was plotted
using the peak area ratio (climbazole to deuterated climbazole) versus concentrations of
climbazole. The linear range ran from 4 to 2000 ng/mL. The LOD and LOQ were 1 ng/mL
(signal-to-noise ratio > 3) and 4 ng/mL (signal-to-noise ratio >10), respectively. The LOQ
allowed the quantification of ppb level (corresponding to 16 ng/cm2) of climbazole on
artificial skin and human scalp.
Table. 5.1. Recovery of CBZ in extraction solutions of artificial skin samples and human
scalp samples.
Spiked sample Spiked CBZ concentration
(ng/mL)
Recovery (%)
mean (n=3) ± SD
Artificial skin 5 92.8 ± 6.0 100 96.7 ± 3.6
Human scalp 5 89.8 ± 5.5 100 94.5 ± 3.0
5.3.3 Comparison of sampling method for climbazole extraction from artificial skin
For climbazole extraction from artificial skin three sampling methods were investigated: tape
stripping, buffer scrub and solvent extraction. Figure 5.3 compares the performance of these
methods. Methanol extraction was the most efficient method for climbazole extraction from
artificial skin, followed by buffer scrub using the surfactant-modified PBS solution, buffer
scrub using 50% aqueous glycerol solution, tape stripping using DS, tape stripping using Le,
and tape stripping using Se in descending order. Compared with the tape stripping sampling
methods, buffer scrub sampling methods using the surfactant-modified PBS solution, or a 50%
aqueous glycerol solution were found to be much more efficient. Climbazole is a lipophilic
compound. Apparently, the surfactants (Triton X-100 and Tween 80) in the surfactant-
modified PBS solution improved the efficiency of extracting climbazole from (artificial) skin.
There are two possible causes to explain the extremely low efficiency of tape stripping method.
The first one can be the uneven topography of artificial skin which is designed to mimic the
80
surface properties of human skin. The second one can be the glue on the tapes which is not
able to efficiently extract climbazole from artificial skin.
Figure 5.3. Comparison of sampling methods for climbazole extraction from artificial skin.
5.3.4 Climbazole deposition levels on human scalp
Although methanol extraction showed the highest efficiency for climbazole extraction from
artificial skin, it is not allowed to apply this method to human scalps due to health and safety
reasons. Giving the second highest efficiency method, the buffer scrub method using the
surfactant-modified PBS solution was selected as the sampling method for human scalp
samples. Three samples from each side of the subject’s head were taken. The mean climbazole
deposition levels for each side of a subject’s head are presented in Figure 5.4. The error bars
(standard deviation) reflect the variability between the three samples from the same side of
the same subject’s head. Significant variations of climbazole deposition levels were observed
in the samples of the two sides of one subject as well as between the samples of different
subjects. These are most likely due to varying scalp status (e.g. sebum content) and the (local)
irreproducibility of the washing and rinsing process. Despite the rather large variability in the
81
results in real hair washing experiments, the new method presented here can be useful in
studies aimed at maximizing the deposition of the climbazole active from hair care products.
Although not studied here, our method most likely can also be applied in environmental
analysis.
Figure 5.4. Climbazole deposition on human scalp (subject # left/right side of the head).
5.4 Conclusions
A sensitive and specific UHPLC-MS/MS method was developed and validated for the
measurement of climbazole deposition on artificial skin (for in vitro studies) and human scalp
(for in vivo studies). APCI in the positive mode was applied for detection because this
ionization mode gave less signal enhancement or suppression than ESI. Deuterated
climbazole was used as the internal standard. For the extraction of climbazole from human
scalp, the buffer scrub method using a surfactant-modified PBS solution was selected based
on the performance comparison of tape stripping, buffer scrub and solvent extraction in in
vitro experiments. The sensitivity and selectivity of the developed method enabled the
quantification of ppb level (corresponding to 16 ng/cm2) of climbazole on artificial skin and
0
200
400
600
800
1000
1200
002#R 002#L 003#R 003#L 005#R 005#L 006#R 006#L 007#R 007#L
Dep
ositi
on le
vel,
ng/c
m2
scalp samples
82
human scalp. Using this method, climbazole deposition in vitro and in vivo studies was
successfully measured.
References
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Doi.org/10.1111/ics.12007. [2] T. Schmidt-Rose, S. Braren, H. Fölster, T. Hillemann, B. Pltrogge, P. Philipp, G. Weets, S. Fey, Efficacy
of a piroctone olamine/climbazol shampoo in comparison with a zinc pyrithione shampoo in subjects with moderate to severe dandruff. Int. J. Cosmet. Sci., 33 (2011) 276-282.
Doi.org/10.1111/j.1468-2494.2010.00623.x. [3] L. Chao, Simultaneous determination of four anti‐dandruff agents including octopirox in shampoo
products by reversed‐phase liquid chromatography. Int. J. Cosmet. Sci., 23 (2001) 183-188. Doi.org/10.1046/j.1467-2494.2001.00090.x.
[4] L. Gagliardi, L. Turchetto, A. Amato, Determination of climbazole in shampoos by reversed-phase liquid
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of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem., 404 (2012) 3175-3188.
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characterized by decreased levels of intercellular lipids in scalp stratum corneum and impaired barrier function. Arch. Dermatol. Res., 294 (2002) 221-230.
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MS/MS assays: Evaluation of matrix effects. J. Chromatogr. B, 877 (2009) 2198-2207. Doi.org/10.1016/j.jchromb.2009.01.003. [9] B.K. Matuszewski, M.L. Constanzer, C.M. Chaves-Eng, Strategies for the Assessment of Matrix Effect
in Quantitative Bioanalytical Methods Based on HPLC−MS/MS. Anal. Chem., 75 (2003) 3019-3030. Doi: 10.1021/ac020361s. [10] R. King, R. Bonfiglio, C. Fernandez-Metzler, C. Miller-Stein, T. Olah, Mechanistic investigation of
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83
[11] R. Dams, M.A. Huestis, W.E. Lambert, C.M. Murphy, Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: Influence of ionization type, sample preparation, and biofluid. J. Am. Soc. Mass Spectrom., 14 (2003) 1290-1294.
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84
85
Chapter 6
Sensitive and simultaneous quantification of zinc pyrithione and
climbazole in scalp buffer scrub samples
Abstract A sensitive ultrahigh performance liquid chromatography-tandem mass spectrometry
(UHPLC-MS/MS) method has been developed and validated for simultaneous quantification
of zinc pyrithione (ZPT) and climbazole (CBZ) deposited onto human scalp from anti-
dandruff (AD) shampoos. Scrubbing with a buffer solution was used as the sampling method
for the extraction of ZPT and CBZ from scalp. Derivatization of ZPT was carried out prior to
UHPLC-MS/MS analysis. The identification of ZPT and CBZ was performed by examining
ratios of selected multiple reaction monitoring (MRM) transitions in combination with
UHPLC retention times. The limit of detection for ZPT and CBZ was established to be 1 and
2 ng/mL, respectively. This sensitivity enables the quantification of ZPT and CBZ at
deposition levels in the low ng/cm2 range. The method was successfully applied for the
analysis of scalp buffer scrub samples from an in vivo study. The levels of ZPT and CBZ
deposited on the scalp at different time points after application of the AD shampoo were
measured. The results revealed that dual-active AD shampoo delivered more ZPT onto the
scalp in a single wash than single active shampoo did. The amount of ZPT and CBZ retained
on the scalp after AD shampoo application declined over 72 hours.
This chapter was originally published as:
G. Chen, M. Miao, M. Hoptroff, X. Fei, L.Z. Collins, A. Jones, H-G. Janssen, Sensitive and
simultaneous quantification of zinc pyrithione and climbazole deposition from anti-dandruff
shampoos onto human scalp. J. Chromatogr. B. 1003 (2015) 22-26.
86
6.1 Introduction
The use of an anti-dandruff (AD) shampoo is one of the most applied home remedies for the
treatment of dandruff. The active ingredients of AD shampoos are anti-fungals, the two most
common being zinc pyrithione (ZPT) and climbazole (CBZ). The combination of ZPT and
CBZ, as dual actives in a shampoo formulation has been proven to be able to deliver superior
antidandruff efficacy and desired end sensory benefits [1]. The amount of AD actives
deposited onto the human scalp in the process of shampoo application and rinse-off is
considered as one of the crucial factors which determine the AD shampoo efficacy [2, 3].
Hence, methods for the measurement of AD actives deposited onto the scalp are required.
There are many analytical methods for the determination of ZPT and CBZ in shampoos [4-
10]. The complicated matrices of shampoos can make the analysis challenging, but high
sensitivities for the detection of ZPT and CBZ are not required due to their rather high levels
in the AD shampoos. A number of more sensitive methods were developed for the
determination of ZPT and CBZ in environmental matrices [11-14]. For in vivo studies
monitoring ZPT and CBZ deposition onto the scalp, besides sensitive detection, efficient
sampling and sample pre-treatment techniques are required. In Chapter 5, we developed a
sensitive method for the determination of CBZ deposited on artificial skin and human scalp
from AD shampoos. For ZPT analysis, unfortunately, sensitive and easy-to-use methods are
still lacking. Due to the unwanted interactions of the compound with the silanol groups from
silica-based liquid chromatography (LC) stationary phases [13] and the trans-chelation with
other cations (e.g. Fe, Cu) [6, 13] during the analysis, poor peak shapes are often obtained,
especially at ppb levels. A liquid chromatography-mass spectrometry (LC-MS) method for
the direct analysis of ZPT was developed by Yamaguchi et al. in 2006 [15]. Unfortunately,
the sensitivity of this method was insufficient to ensure detection of ZPT at all relevant levels.
Derivatization of the ZPT with fluorescing groups was reported to stabilize the pyrithione
complex and improve the detection limits [16], but for the complex scalp samples the
selectivity of fluorescence detection is not sufficient. Mass spectral confirmation is needed,
while maintaining the excellent peak shape and sensitivity of the derivatization/fluorescence
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route. Moreover, simultaneous determination of ZPT and CBZ deposited on human scalp
would be needed, for which, however, no method has been reported till now.
In the present contribution, a method is described and validated for simultaneous
quantification of ZPT and CBZ deposited on human scalp. Scrubbing the scalp with a buffer
solution was applied as the sampling method for extraction of ZPT and CBZ from scalp and
an in vivo study was designed for method development and validation. An ultrahigh
performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method
was employed for the separation and detection of ZPT (after derivatization) and CBZ in scalp
buffer scrub samples.
6.2 Materials and methods
6.2.1 Chemicals and reagents
All reagents and solvents used in the experiment were analytical grade or better.
Demineralised water (>18.2 MΩ) prepared using a Milli-Q® Advantage A10 Water
Purification System (Millipore, Bedford, MA, USA) was used for preparation of solutions
and dilutions. ZPT (95%), CBZ (99%), EDTA-2Na, 2, 2-Dipyridyl disulfide (DPS) and
phosphate buffered saline (PBS) tablets were supplied by Sigma Aldrich (St. Louis, MO,
USA). HPLC grade dimethyl sulfoxide (DMSO), acetonitrile and methanol were supplied by
Merck (Darmstadt, Germany). Ammonium acetate, Triton X-100 and Tween 80 were
purchased from SCRC (Shanghai, China). A surfactant-modified PBS solution for the buffer
scrub sampling method was prepared by adding four PBS tablets, 1.0 g of Triton X-100 and
5.0 g of Tween 80 to 1 L of Milli-Q water. Saturated EDTA-2Na solution was prepared by
dissolving 25 g EDTA-2Na in 100 mL Milli-Q water, stirring at 60 ℃ for 1 hour. DPS
derivatization solution was prepared by dissolving 0.6 g DPS in 50 mL acetonitrile.
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6.2.2 Test Shampoos
The three test shampoos used in this study were purchased at a local supermarket in Shanghai,
China. They were one dual-active AD shampoo (1% ZPT, 0.5% CBZ), one single active AD
shampoo (1% ZPT) and one beauty shampoo (no AD actives).
6.2.3 In vivo study
The in vivo study was conducted in Shanghai, China at the Unilever internal facility. It was
cleared by the Joint Research Ethics Committees, Shanghai, and all subjects gave their
informed consent to participate. This was a single centre, randomized, half head, double-blind
and single gender study. Each subject tested two products. Healthy male subjects aged 18-65
years (inclusive) not using anti-dandruff shampoo during the past 3 months were recruited
and screened by the so-called Total Weighted Head Score (TWHS) system [17]. The study
lasted for one week with five visits. A total of 27 subjects completed the whole study. The
buffer scrub sampling method was applied for extraction of ZPT and CBZ during the whole
study. More details on this method can be found in Chapter 5. A surfactant-modified PBS
solution was used as extraction fluid for extracting ZPT and CBZ after the scalp was gently
massaged with a Teflon rod. For each sampling site, about 3.5 mL of the buffer solution was
collected for quantitative analysis of ZPT and CBZ.
During the first visit the subjects washed their hair using the commercial beauty shampoo and
they were reminded that no hair washing, or wetting was allowed except during the visits.
The second visit was three days later. During this visit the subjects washed their hair using
the commercial AD Shampoos. Sampling was carried out before and after shampoo
application to obtain baseline samples and ‘right after wash’ samples. During the third visit
(twenty-four hours after AD shampoo application), sampling was performed to obtain 24 h
after washing samples. During the fourth visit (forty-eight hours after AD shampoo
application), sampling was performed to obtain the 48 h after washing samples. During the
fifth visit (72 h after AD shampoo application), sampling was performed to obtain 72 h after
washing samples. All the scalp buffer scrub samples were collected in Nunc 15 mL centrifuge
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tubes (Thermo Scientific, Waltham, Massachusetts USA) and were stored at -20 ℃ prior to
analysis.
6.2.4 Sample treatment
The scalp buffer scrub samples (about 3.5 mL per sample) were first diluted with methanol
(two-fold) to break the emulsion and precipitate proteins. For the DPS derivatization, 5 mL
of the methanol extract of the sample was transferred into a 15-mL centrifuge tube. Next, 80
μL of a saturated solution of EDTA-2Na and 200 μL of DPS solution were added; the sample
was thoroughly mixed by vortex and then placed in the dark for 1 hour. After the
derivatization, the sample solutions were filtered over a 0.45 μm Nylon filter supplied by
SCRC (Shanghai, China) prior to UHPLC-MS/MS analysis.
6.2.5 Standard solutions
A CBZ and ZPT mixed stock solution was prepared in DMSO at a concentration of 1000
mg/L for each analyte. It was stored at 4 ℃ in the dark prior to use. The working solutions
were prepared freshly by the appropriate dilution of this stock solution with a mixture of 50%
of mobile phase A and 50% mobile phase B. Prior to UHPLC-MS/MS analysis, the working
solutions were subjected to the same DPS derivatization procedure as the buffer scrub samples.
6.2.6 UHPLC-MS/MS analysis
A Waters ACQUITY UPLC system coupled to a Quattro Micro API mass spectrometer
(Waters, Manchester, UK) was used for the sample analysis. Separation was carried out on a
Waters ACQUITY UPLC BEH C18 column (2.1 mm x 50 mm x 1.7 μm). The mobile phase
was composed of 20 mM ammonium acetate in water (A) and methanol (B) programmed in
the linear gradient mode [time 0 min, 80% A, maintain 1 min; time 3.5 min, decrease
immediately to 50% A; time 5.5 min, decrease immediately to 10% A; time 6.5 min, maintain
10% A; time 8 min, increase immediately to 80%]. Atmospheric pressure chemical ionization
(APCI) in positive mode was used for all experiments. Optimum APCI performance was
90
obtained at a corona current of 3.6 µA, a cone voltage of 32 V, an extractor voltage of 3 V, a
source temperature of 100 °C, an APCI probe temperature of 300 °C, a desolvation gas flow
(nitrogen) of 450 L/hr and a cone gas flow of 25 L/hr (nitrogen). The multiple reaction
monitoring (MRM) mode was used for the determination of CBZ and ZPT. The collision gas
(argon) pressure was set at 3 × 10-3 mbar. The dwell time for each MRM transition was 0.20
s. ZPT derivative was analysed using the transitions of m/z 237.0 > 126.0 (collision energy
17 V) and 237.0 > 111.0 (collision energy 17 V) signals while CBZ was measured using the
transition of m/z 293.0 > 69.0 (collision energy 20 V) and 293.0 > 197.1 (collision energy 18
V).
6.2.7 Method validation
The sensitivity and precision of the method were evaluated by analysing spiked blank samples
(analytically confirmed ZPT- and CBZ-free scalp buffer scrub samples). The specificity was
evaluated by the ratio of the MRM transitions. Calibration curves for ZPT and CBZ were
constructed using standards of five different concentrations. The linearity was assessed by the
regression of the peak area against the corresponding concentration. Limits of detection (LOD)
and limits of quantification (LOQ) were determined as the concentrations where the signal-
to-noise (S/N) ratios were 3:1 and 10:1, respectively.
6.3 Results and discussion
6.3.1 Optimization of DPS derivatization
To circumvent the problems with direct analysis of ZPT we opted for the use of a
derivatization step between extraction and UHPLC-MS/MS quantification. In the current
study, PDS was applied for the derivatization of ZPT. EDTA-2Na was used to chelate zinc
and dissociate ZPT into free pyrithione which reacts with the PDS as illustrated in Figure 6.1.
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Figure 6.1. Reaction of ZPT and PDS.
Table 6.1. Effect of dosages of statured EDTA-2Na and DPS on the derivatization of ZPT.
No. Dosage of
EDTA-2Na
Dosage of
DPS
Analytical results
Mean ± SD, n=3
Peak area of ZPT
derivative
Peak area of
CBZ
1 Low Low 2011 ± 14 1023 ± 10
2 Low Medium 1887 ± 21 1125 ± 24
3 Low High 1765 ± 37 956 ± 25
4 Medium Low 1971 ± 22 1170 ± 20
5* Medium Medium 1972 ± 9 1057 ± 12
6 Medium High 1798 ± 31 961 ± 12
7 High Low 1949 ± 23 1158 ± 38
8 High Medium 1833 ± 60 1173 ± 35
9 High High 1672 ± 10 979 ± 9
*The optimum reaction conditions for the derivatization of ZPT was using 80 μL of
EDTA-2Na saturated solution, 200 μL of DPS solution and a reaction time of 1 h in the
dark at room temperature. More details are given in the text.
The PDS derivatization was carried out at room temperature. The levels of EDTA-2Na and
PDS and the reaction conditions were optimized. The results of these experiments are
summarized in Table 6.1. To obtain complete derivation of ZPT, excess DPS reagent and
EDTA-2Na salt can be used. However, this can cause interferences during the
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chromatographic separation and the mass spectrometric detection. Three levels (low, medium
and high) were selected for the concentrations of DPS reagent and the saturated EDTA-2Na
solution, respectively: 40, 80 and 400 μL for the EDTA-2Na solution; and 100, 200 and 1000
μL for DPS reagent. Peak areas of the ZPT derivative and the repeatability were used as to
establish the derivatization efficiency under different conditions. It was proven that
derivatization was most efficient when using 80 μL of EDTA-2Na saturated solution, 200 μL
of DPS solution and a reaction time of 1 h in the dark. CBZ in standard solutions and samples
was not influenced by the derivatization, evidenced by the good recovery of this compound
under all conditions.
6.3.2 Quantification of ZPT derivative and CBZ
In order to simultaneously determine ZPT (as its derivative) and CBZ in one run, the mobile
phase gradient was optimized. Total analysis time was 8 min including 2.5 min for re-
equilibration. Figure 6.2 shows a representative chromatogram obtained for a standard
solution and two real samples. The retention times for the ZPT derivative and CBZ are 2.15
± 0.02 min and 4.40 ± 0.02 min, respectively. Quantification of the two analytes was
performed in MRM mode. For the ZPT derivative, [M+H]+ ion m/z 237.0 was selected as
parent ion and fragment ions m/z 111.0 and 126.0 were selected as daughter ions. For CBZ,
[M+H]+ ion m/z 293.0 was selected as parent ion and fragment ions m/z 69.0 and 197.1 were
selected as daughter ions. For quantification, MRM transition 237.0 > 111.0 was monitored
for the ZPT derivative and MRM transition 293.0 > 69.0 for CBZ.
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Figure 6.2. Representative chromatogram of ZPT derivative and CBZ detected in a standard
solution of 10 ng/mL (A), in a buffer scrub sample from a subject who had applied dual-
active AD shampoo (B) and in a buffer scrub sample from a subject who had applied single
active AD shampoo (C).
6.3.3 Validation of the method
The method was validated with regard to precision, accuracy, specificity, linearity, LOD and
LOQ. In the precision study, triplicate analyses of ZPT and CBZ standards of 100 ng/mL and
1000 ng/mL were performed. For ZPT, the mean concentration and standard deviation of
these standards were 98.9 ± 2.3 and 997.8 ± 3.4 ng/mL. For CBZ, the mean concentration and
standard deviation were 106.3 ± 1.8 and 1014.3 ± 2.7 ng/mL. In the specificity study, ZPT
was identified by the ratios of the MRM transitions m/z 237.0 > 111.0 and 237.0 > 126.0
signals, which should be 1.5 ± 0.1, as established from the analysis of the pure compound.
CBZ was identified by the ratio of the MRM transitions m/z 293.0 > 69.0 and 293.0 > 197.1
signals, which should be 1.4 ± 0.1. In the recovery study, 100 ng/mL spiked to analytically-
A
B
C
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confirmed ZPT- and CBZ-free scalp buffer scrub samples were used. The recoveries of ZPT
and CBZ in these samples were 100.0 ± 0.4% and 116.3± 3.5%, respectively (n=3). The
calibration curve was linear in the range from 5 to 5000 ng/mL. The LOD and LOQ were
found to be 1 ng/mL and 4 ng/mL for CBZ analysis, and 2 ng/mL and 5 ng/mL were found
for ZPT analysis, respectively. The LOQ allowed the quantification of ppb levels in the buffer
scrub solution, corresponding to approximately 5 ng/cm2 of CBZ and ZPT on the scalp.
6.3.4 ZPT and CBZ deposition on scalp from dual-active and single active shampoos
The mean levels of ZPT and CBZ deposited onto the scalp from dual-active and single active
AD shampoos are presented in Figure 6.3. The vertical bars reflect the variability resulting
from the analysis as well as inter-subject differences. Dual-active AD shampoo delivered
much more ZPT onto the scalp after one wash than the single active AD shampoo did. This
can most likely be attributed to the different compositions of the shampoos. The amount of
ZPT retained on scalp declined over 72 h, irrespective of whether dual-active AD shampoo
or single active AD shampoo was used. The amount of CBZ retained on the scalp declined
over 72 h as well. Because the study participants were not allowed to wash or wet their hair
in this 72-h time interval, the decrease of the actives is most likely caused by losses from the
skin during the normal process of skin desquamation and/or by Malassezia consumption [18].
The ZPT and CBZ delivered from dual-active AD shampoo can still be detected 72 h after
application. But in some samples in which single active AD shampoo was applied, ZPT could
no longer be detected already after some 48 h. This finding indicated that the dual-active AD
shampoo delivers longer lasting AD efficacy than single-active AD shampoo. Although not
studied here, this method can also be applied for other (in vitro or in vivo) matrices such as
for measuring ZPT and CBZ deposition on artificial skins.
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Figure 6.3. Deposition levels of ZPT (A) and CBZ (B) on scalp from dual-active and single
active shampoos.
6.4 Conclusion
A sensitive UHPLC-MS/MS method was developed and validated for simultaneous
quantification of ZPT and CBZ deposited on human scalp from AD shampoos. The method
demonstrated high sensitivity, enabling the quantification of ng to μg amounts of the two AD
actives on human scalp. It was successfully applied for the analysis of scalp buffer scrub
samples from an in vivo study. The levels of ZPT and CBZ deposited on scalp at different
96
time points after AD shampoo application were measured. The results revealed that dual-
active AD shampoo delivered more ZPT onto the scalp after one wash than single active
shampoo did. The amount of ZPT and CBZ retained on the scalp after AD shampoo
application declined over 72 hours. The method is also applicable in other studies e.g.
artificial skin studies to improve shampoo formulations to maximize ZPT and CBZ deposition.
References
[1] G.A. Turner, J.R. Matheson, G.-Z. Li, X.-Q. Fei, D. Zhu, F.L. Baines, Enhanced sensory properties of an anti-dandruff shampoo containing zinc pyrithione and climbazole, Int. J. Cosmet. Sci., 35 (2012) 78-83.
Doi.org/10.1111/ics.12007. [2] J.R. Schwartz, R. Shah, H. Krigbaum, J. Sacha, A. Vogt, U. Blume-Peytavi, New insights on
dandruff/seborrhoeic dermatitis: the role of the scalp follicular infundibulum in effective treatment strategies, Br. J. Dermatol., 165 (2011) 18-23.
Doi.org/10.1111/j.1365-2133.2011.10573.x. [3] J.R. Schwartz, R. A. Bacon, R. Shah, H. Mizoguchi, A. Tosti, Therapeutic efficacy of anti-dandruff
shampoos: A randomized clinical trial comparing products based on potentiated zinc pyrithione and zinc pyrithione/climbazole, Int. J. Cosmet. Sci., 35 (2013) 381-387. Doi.org/10.1111/ics.12055.
[4] L. Chao, Simultaneous determination of four anti-drandruff agents including octopirox in shampoo
products by reversed liquid chromatography, Int. J. Cosmet. Sci., 23 (2001) 183-188. Doi.org/10.1046/j.1467-2494.2001.00090.x. [5] L. Gagliardi, L. Turchetto, A. Amato, Determination of climbazol in shampoos by reversed-phase liquid
chromatography, Anal. Chim. Acta., 235 (1990) 465-468. Doi.org/10.1016/S0003-2670(00)82110-8. [6] R.J. Fenn, M.T. Alexander, Determination of zinc pyrithione in hair care products by normal phase liquid
chromatography, J. Liq. Chrom., 11 (1988) 3403-3413. Doi.org/10.1080/01483918808082263.
[7] Y. Shih, J. Zen, A.S. Kumar, P. Chen, Flow injection analysis of zinc pyrithione in hair care products on
a cobalt phthalocyanine modified screen-printed carbon electrode, Talanta, 62 (2004) 912-917. Doi.org/10.1016/j.talanta.2003.10.039. [8] H. Cheng, R.R. Gadde, Analysis of zinc pyrithione in shampoos by reversed-phase high-performance
liquid chromatography, J Chromatogr., 291 (1984) 434-438. Doi.org/10.1016/S0021-9673(00)95055-6. [9] E.M. Peña-Méndez, J. Havel, J. Malecek, High-performance capillary electrophoresis determination of
pyrithione in antidandruff preparations and shampoos, J. Capillary Electrophor., 4 (1997) 269-272. [10] K. Nakajima, T. Yasuda, H. Nakazawa, High-performance liquid chromatographic determination of zinc
pyrithione in antidandruff preparations based on copper chelate formation, J. Chromatogr. A, 502 (1990) 379-384. Doi.org/10.1016/S0021-9673(01)89602-3.
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[11] A. Wick, G. Fink, T.A. Ternes, Comparison of electrospray ionization and atmospheric pressure chemical ionization for multi-residue analysis of biocides, UV-filters and benzothiazoles in aqueous matrices and activated sludge by liquid chromatography-tandem mass spectrometry, J. Chromatogr. A, 1217 (2010) 2088-2103.
Doi.org/10.1016/j.chroma.2010.01.079. [12] Z.-F. Chen, G.-G. Ying, H.-J. Lai, F. Chen, H.-C. Su, Y.-S. Liu, F.-Q. Peng, J.-L. Zhao, Determination
of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography-tandem mass spectrometry, Anal. Bioanal. Chem., 404 (2012) 3175-3188.
Doi.org/10.1007/s00216-012-6444-2. [13] J. Bones, K.V. Thomas, B. Paull, Improved method for the determination of zinc pyrithione in
environmental water samples incorporating on-line extraction and preconcentration coupled with liquid chromatography atmospheric pressure chemical ionisation mass spectrometry, J. Chromatogr. A, 1132 (2006) 157-164.
Doi.org/10.1016/j.chroma.2006.07.068. [14] V.A. Sakkas, K. Shibata, Y. Yamaguchi, S. Sugasawa, T. Albanis, Aqueous phototransformation of zinc
pyrithione degradation kinetics and byproduct identification by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry, J. Chromatogr. A, 1144 (2007) 175-182.
Doi.org/10.1016/j.chroma.2007.01.049. [15] Y. Yamaguchi, A. Kumakura, S. Sugasawa, H. Harino, Y. Yamada, K. Shibata, T. Senda, Direct analysis
of zinc pyrithione using LC-MS, Intern. J. Environ. Anal. Chem., 86 (2006) 83-89. Doi.org/10.1080/03067310500249930. [16] N. Voulvoulis, M.D. Scrimshaw, J.N. Lester, Analytical methods for the detection of 9 antifouling paint
booster biocides in estuarine water samples, Chemosphere, 38 (1999) 3503-3516. Doi.org/10.1016/S0045-6535(98)00580-3. [17] C.R. Harding, A.E. Moore, J.S. Rogers, H. Meldrum, A.E. Scott, F.P. McGlone, Dandruff: a condition
characterized by decreased levels of intercellular lipids in stratum corneum and impaired barrier function, Arch. Dermatol. Res., 294 (2002) 221-230.
Doi.org/10.1007/s00403-002-0323-1. [18] R.J. Hay, Malassezia, dandruff and seborrhoeic dermatitis: an overview, Br. J. Dermatol., 165 (2011) 2-
8. Doi.org/10.1111/j.1365-2133.2011.10570.x.
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Chapter 7
Ex-vivo measurement of scalp follicular delivery of zinc pyrithione and
climbazole from hair care products
Abstract
Efficient delivery of anti-dandruff (AD) actives into the scalp follicular infundibulum as well
as onto the scalp surface is critical for the efficacy of AD shampoos. A method involving scalp
cyanoacrylate (CA) biopsy sampling, a tailor-made cutting device, ultra-high-performance
liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, scanning
electron microscopy (SEM) measurement and Raman imaging has been developed for the
measurement of delivery of zinc pyrithione (ZPT) and climbazole (CBZ) from an AD shampoo
into the scalp follicular infundibulum. Scalp CA biopsy enables the sampling of ZPT and CBZ
delivered into the scalp follicular infundibulum as well as onto the scalp surface. Raman
imaging of scalp CA biopsy samples allows the visualization of the spatial distribution of ZPT
and CBZ deposited on the scalp. A tailor-made cutting device enables the separation of the
scalp follicular infundibulum sample (20 µm below the scalp surface) from the scalp surface
samples (including top 20 μm of infundibula). UHPLC-MS/MS was used as a sensitive and
specific methodology enabling the quantification of ZPT and CBZ without interferences.
Using this method, ZPT and CBZ were successfully quantified and visualized within the scalp
follicular infundibulum, after the scalp was washed with an AD shampoo.
This chapter was originally published as:
G. Chen, C. Ji, M. Miao, K. Yang, Y. Luo, M. Hoptroff, L.Z. Collins, H.-G. Janssen, Ex-vivo
measurement of scalp follicular infundibulum delivery of zinc pyrithione and climbazole from an
anti-dandruff shampoo. J. Pharma. Biomed. Anal. 143 (2017) 26-31.
100
7.1 Introduction
Dandruff is a common scalp issue which affects approximately 50% of the global population
and is characterized by flakes and itch on the scalp without visible inflammation. [1-3].
Overgrowth of Malassezia, a genus of lipophilic yeasts was reported to be associated with
dandruff [4-7]. Hence, one of the most common treatments of dandruff is the use of an anti-
dandruff (AD) shampoo containing antifungal agents like zinc pyrithione (ZPT) and
climbazole (CBZ) [8, 9].
It is known that Malassezia yeasts live on human skin around the opening of the hair follicles
(also called infundibulum) as well as on the scalp surface [5, 10 and 11]. Schwartz and his
team claimed that efficient delivery of AD actives into the scalp follicular infundibulum as
well as onto the scalp surface is critical for the efficacy of AD shampoos [12, 13]. Reliable
analytical methods are required for the measurement of spatial delivery of AD actives on the
scalp. However, besides buffer scrub, cyanoacrylate (CA) biopsy, tape stripping and hair
plucking, very limited sampling methods have been developed for detecting AD actives on
the scalp after the application of AD shampoos.
In Chapter 6, we developed a method for the measurement of ZPT and CBZ deposition on
scalp from AD shampoo using buffer scrub as the sampling method. However, the method is
not able to distinguish between scalp surface delivery and follicular infundibulum delivery.
An in vivo imaging method using confocal microscopy was reported to enable the
measurement of ZPT spatial distribution in the scalp follicular infundibula [13]. Confocal
microscopy imaging can detect ZPT particles optically but not chemically. When other similar
particles are delivered together with ZPT, chemical imaging tools like Raman imaging are
demanded to offer chemical specificity, so as to eliminate the impact of other particles.
Since Marks and Dawber first used CA for skin surface biopsy in 1971 [14], CA based
sampling methods have been further developed and proven to enable both skin surface and
follicle biopsies. Combined with imaging technologies, cyanoacrylate skin surface stripping
(CSSS) has been widely applied in diagnostic dermatopathology and cosmetology, as well as
in experimental dermatology settings [15]. To investigate the penetration of topically applied
101
substances into hair follicles, Teichmann and his team developed a differential stripping
method which was based on CSSS [16, 17]. For measuring ZPT delivery into the scalp
follicular infundibula, a sampling method which combined CA biopsy and hair pluck was
reported [13]. In this method, a drop of CA glue was applied to the infundibulum of a hair
follicle. After drying, the CA glue together with hair(s) was removed, obtaining a follicular
cast containing cell debris, sebum, hair(s), ZPT etc. To make the analysis be representative
for the whole hair follicles, the sampling should cover multiple follicles.
The aim of this study was to develop a method to measure scalp follicular infundibulum
delivery of ZPT and CBZ are delivered onto the scalp from a dual-active AD shampoo. A CA
based in vivo sampling method was developed to harvest ZPT and CBZ delivered on both the
scalp surface and in follicular infundibula. After the sampling, scalp CA biopsy samples (casts)
were subjected to Raman and scanning electron microscopy (SEM) imaging. A tailor-made
cutting device was developed to efficiently and accurately separate the scalp surface casts
(SCs) from scalp follicular infundibulum casts (FICs) at a defined z-distance. After separation
of the layers, the contents of ZPT and CBZ in scalp SCs and FICs were determined by an
ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)
method.
7.2 Materials and methods
7.2.1 Chemicals and reagents
All reagents and solvents used in the experiment were analytical grade or above. CBZ (99.9%),
ZPT (95%), and 2, 2-dipyridyl disulfide (DPS), were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Ammonium acetate and EDTA-2Na were purchased from SCRC
(Shanghai, China). HPLC grade acetone, methanol and acetonitrile were purchased from
Merck (Darmstadt, Germany). Milli-Q pure water (>18.2 MΩ, Millipore, Bedford, MA, USA)
was used to prepare samples, standard solutions and UHPLC mobile phases. Model sebum
was prepared by mixing the lipid compounds purchased from Sigma-Aldrich (St. Louis, MO,
USA).
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7.2.2 Test shampoos
A commercial beauty shampoo without AD actives was used as run-in shampoo. The test
shampoo used in this study was a commercial dual-active AD shampoo containing ZPT and
CBZ.
7.2.3 In vivo clinical study and CA biopsy sampling
The in vivo clinical study was a randomized, double blind and controlled study. The study
was conducted in China and was reviewed and approved by the ethical committee of the
Shanghai Clinical Research Center. Informed consent was obtained from all subjects before
participation. Healthy subjects of both gender aged 18-60 years were screened and had their
scalp condition visually assessed using Unilever Total Weighted Head Score (TWHS) system
[3]. Subjects with scalp condition of whole head TWHS adherent flake (AF) ≤ 8, with no
flake grade B or above, were accepted. Subjects who had used shampoo containing anti-
dandruff actives within the last 4 weeks were excluded. Forty-five subjects were accepted and
43 subjects (24 females + 19 males) completed the whole study. Two subjects were withdrawn
for personal reason. No products or procedure related adverse event was reported.
All subjects had their hair washed using beauty shampoo 2 days before the test shampoo wash.
Then subjects had their hair washed using the same dose of the dual-active AD Shampoo and
blow-dried following standard hair wash operation. Square regions of 1 cm×1 cm were
identified as sampling sites for scalp CA biopsy. Hair in this region was carefully cut from
the root as close to the scalp skin as possible and operation contact with scalp skin was
minimum.
A small amount of CA glue (Loctite Medical grade CA glue 4061) was applied onto the rough
side of the Melinex® sampling strip and spread evenly to give a thin film. Afterwards, the
glue-coated strip was applied to the sampling area and pressed firmly onto the scalp. The strip
sample was peeled off when dry. All CA scalp biopsy samples were stored in an air-tight
container before further analysis. Figure 7.1 shows how CA scalp biopsy is used to obtain the
FICs and SCs consisting of cell detritus, lipids, microbes and AD actives.
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Figure.7.1. Principle of scalp CA biopsy sampling.
7.2.4 SEM measurement
The scalp CA biopsy samples before and after cutting were sputter-coated with platinum using
an ion sputter coater (E-1045, Hitachi, Tokyo, Japan) for 90 seconds with a current of 15 mA.
The SEM images were taken by using a SEM (Hitachi S-4800) to measure the size of FIC.
Imaging conditions were set as follows: high voltage = 5 or 10 kV; beam current = ~10 μA;
working distance = 5 – 10 mm; magnification = 30 × – 20,000 ×.
7.2.5 Raman measurement
A Raman microspectrometer (Horiba Jobin Yvon, HR Evolution, Kyoto Japan) was employed
to visualize the spatial distribution of CBZ and ZPT on scalp CA biopsy samples before
cutting. Pure materials of CBZ, ZPT, CA glue and model sebum were used as reference
104
materials to assign signals in the obtained Raman spectra. On each sample, one region in size
of ~ 2 mm × 2 mm (containing at least 3 follicles) was scanned under a 10x air objective with
a 532 nm laser source. The step size was 10 µm (~ 40, 000 pixels), and spectra in each pixel
were taken from 400 cm-1 to 2000 cm-1, with 0.5 second laser exposure with 2 times
accumulation to remove cosmic rays. The resultant spectra matrix was analysed by the built-
in direct classical least square (CLS) fitting algorithm to demonstrate the relative distribution
of each active.
7.2.6 Separation of scalp FICs from scalp SCs
After sampling, the scalp CA biopsy samples were cut by a tailor-made cutting device which
was developed in house. The principle of cutting down the follicular infundibulum cast is
shown in Figure 7.2. Before cutting, the blade height of the cutting device was carefully
adjusted to 20 µm, so as to only cut down the FICs without SCs. Scalp CA biopsy samples
are placed on a weighing paper. Then the cutting device was pushed forward horizontally to
sweep over the sampling area. This was repeated several times. The obtained FICs were so
tiny that they should be carefully collected from the tape as well as from the blade. After
cutting, the white ash was transferred into a tube pre-marked “scalp infundibulum sample”.
The residual CA biopsy sample was collected as “scalp surface sample”, which included the
top 20 µm infundibulum.
Figure.7.2. Principle of cutting down the follicular infundibulum cast.
105
7.2.7 Quantification of ZPT and CBZ by UHPLC-MS/MS analysis
A Waters ACQUITY UPLC system coupled to a Quattro Micro API mass spectrometer
(Waters, Manchester, UK) was used for the quantitative analysis of ZPT and CBZ. Both
“scalp infundibulum samples” and “scalp surface samples” were subjected to acetone
extraction. A standard stock solution of ZPT and CBZ was prepared in methanol at a
concentration of 2 mg/mL each. The working solutions were prepared freshly by the
appropriate dilution of the stock solution with a mixture of 50% of mobile phase A (20 mM
ammonium acetate in water) and 50% mobile phase B (methanol). Following the previously
developed method in Chapter 6, both sample extraction and standard solutions were subjected
to DPS derivatization prior to UHPLC-MS/MS analysis. The delivery levels of ZPT and CBZ
were expressed as ng/cm2.
7.3 Results and discussion
7.3.1 SEM imaging of scalp CA biopsy samples
The infundibulum is the upper segment of the hair follicle, extending from the surface to the
sebaceous gland. The total length of the infundibulum is approximately 500 µm [18]. SEM
images (Figure 7.3) of scalp CA biopsy samples visualize the morphology of a scalp FIC
which look like hollow cones without tips. The hollow cone was caused by residual hair shafts
in follicles, although hair was clipped to scalp level prior to sampling. The dimensions of the
FICs (height and diameter) were measured by the build-in ruler function of the SEM. The
FICs heights were in the 50-200 µm range, which indicates that scalp CA biopsy enables a
sampling depth of 200 µm in the follicular infundibula. The diameter at the base of FICs
ranged from 100 to 250 µm. All scalp CA biopsy samples were measured by SEM and images
were studied individually to verify that infundibulum casts had been successfully collected.
106
Figure.7.3. SEM images of scalp CA biopsy samples.
A and B: top view images of a representative scalp CA biopsy sample; C and D: cross-
sectional view of a representative scalp CA biopsy sample at different magnifications.
7.3.2 Spatial distribution of ZPT and CBZ on scalp CA biopsy samples
Pure CA glue, CBZ, ZPT and model sebum were used as reference materials to obtain
standard Raman spectra (Figure 7.4). A multivariate curve resolution (MCR) algorithm (CLS)
[19] was used for spectra interpretation and image reconstruction. For each image, at least
three FICs were measured. Figure 7.5 shows a typical Raman image of a scalp CA biopsy
sample, which indicates that both ZPT and CBZ were delivered more into the follicular
107
infundibula than onto the scalp surface when applying the dual-active AD shampoo. Most
likely during rinse-off, the ZPT and CBZ on the scalp surface were more easily rinsed-off
than that present in the follicular infundibula. Current Raman images enable the visualization
of ZPT and CBZ distribution on scalp.
Figure.7.4. Raman spectra of CA glue, CBZ, ZPT and sebum.
Figure. 7.5. Raman images of a representative scalp CA biopsy sample. (Scale bars indicate
40 µm). A: Mapping of CA glue (in grey scale); B: Mapping of CBZ; C: Mapping of ZPT
(Red colour shows higher intensity and blue colour shows lower intensity).
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7.3.3 Separation of scalp FICs from scalp SCs by a cutting device
A tailor-made cutting device was designed and made for cutting down the scalp FICs. It is
constructed of a vernier caliper and a blade. The vernier caliper enables easy tuning of the
blade height with a minimum tuning step size of 1 µm. The blade is circular, which ensures
the blade edge always faces the casts after adjusting the blade height. Efficiency of the follicle
cutting device was verified by SEM images of the scalp CA biopsy samples before and after
cutting (Figure 7.6). The blade height away from surface is critical for complete separation of
FICs from SCs. Based on the surface morphology of CA biopsy samples, the blade height
was set at 20 µm in this study. This setting ensured pure FICs were cut off without any SCs.
As a result, however, some FICs (less than 20 µm height) remained on the CA tape and were
treated as scalp surface samples. After cutting, the scalp CA biopsy samples were divided into
scalp infundibulum samples (deeper than 20 μm) and scalp surface samples (including top 20
μm of infundibula).
Fig.7.6. SEM images of scalp CA FICs before cutting (left) and after cutting (right).
7.3.4 Deposition levels of ZPT & CBZ into scalp follicular infundibula
The contents of ZPT and CBZ in scalp infundibulum samples and scalp surface samples
(shown in Table 7.1) were quantified by the UHPLC-MS/MS method. The method is sensitive
enough to detect ZPT and CBZ at ppb level (corresponding to 5 ng/cm2). More ZPT and CBZ
109
were detected in scalp surface samples (ZPT, 2770 ± 2540 ng/cm2 and CBZ, 550.1 ± 270.5
ng/cm2) than in scalp infundibulum samples (ZPT, 11.0 ± 9.0 ng/cm2 and CBZ, 10.3 ± 9.5
ng/cm2). This finding looks inconsistent with the Raman imaging results in which more ZPT
and CBZ were observed in the follicular infundibula than on scalp surface. A study on the
depth profile of scalp infundibular ZPT [13] observed a sharp drop-off of the amount of ZPT
as the depth increases with the majority of ZPT (>80%) being present in the upper part of the
infundibula (<20 µm). Due to the limitation of the cutting in the current method, the scalp
surface samples included the top 20 µm of infundibula. Consequently, analysis of the
harvested FIC underestimates the total ZPT delivery to the entire infundibulum. Another
reason for the difference in findings between Raman imaging results and deposition levels is
that the combined area of scalp follicular infundibula is dramatically smaller than that of the
scalp surface [20-22].
Table 7.1. Scalp surface and follicular infundibulum delivery of ZPT and CBZ from the dual-active shampoo.
AD active deposition from the dual-active AD shampoo
ZPT CBZ
Scalp surface samples, ng/cm2 (including top 20 µm infundibulum) *
2770.0 ± 2540.0 550.1 ± 270.5
Scalp infundibulum samples, ng/cm2 (20 µm deeper than surface) *
11.0 ± 9.0 10.3 ± 9.5
Total deposition, ng/cm2 2781 ± 2547.0 560.4 ± 277.0 Ratio of infundibulum delivery vs total deposition
0.40% 1.80%
* Mean ± Standard Deviation, n=60.
The dual-active AD shampoo delivered almost the same levels of ZPT (11.0 ± 9.0 ng/cm2)
and CBZ (10.3 ± 9.5 ng/cm2) into the infundibulum. However, a comparison of the ratio of
infundibulum delivery (20 µm deeper than surface) vs total deposition between CBZ (1.80%)
and ZPT (0.40%) suggests that CBZ penetrates deeper into follicular infundibulum than ZPT,
suggesting that CBZ may be up to 4x more efficient at “targeting” the follicular infundibulum.
The reason for this difference is likely due to CBZ having a higher solubility in sebum than
ZPT.
110
7.4 Conclusions
For the measurements of follicular infundibulum delivery of ZPT and CBZ from a dual-active
AD shampoo, a method involving scalp CA biopsy sampling, FICs cutting, UHPLC-MS/MS
analysis SEM measurement and Raman imaging has been developed. Scalp CA biopsy
enables the sampling of ZPT and CBZ delivered into the scalp follicular infundibulum. Raman
imaging of scalp CA biopsy samples allows the visualization of the spatial distribution of ZPT
and CBZ deposition on the scalp. The cutting device enables the separation of scalp FICs
from scalp SCs and the contents of ZPT and CBZ can be quantitated by the sensitive UHPLC-
MS/MS analysis. The method detection limit allows the quantification of ppb levels of CBZ
and ZPT delivered onto the scalp surface and into the follicular infundibulum (corresponding
to 5 ng/cm2).
Using this method, ZPT and CBZ delivered into the scalp follicular infundibulum (20 µm
lower than scalp surface) from the dual-active AD shampoo was successfully visualized and
quantified. Due to the lipophilic nature of CBZ and subsequent increased solubility in sebum,
CBZ possesses the ability to penetrate further into the sebum-rich infundibulum whereas ZPT
remains within the upper 20 µm of infundibula. This differential distribution of actives allows
for the effective targeting of Malassezia species throughout the depth of the scalp follicular
infundibulum.
111
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Kerr, J.P. Henry, R.C. Rust, M.K. Robinson. A comprehensive pathophysiology of dandruff and seborrheic dermatitis - towards a more precise definition of scalp health. Acta. Derm. Venereol., 93 (2013) 131-137.
Doi: 10.2340/00015555-1382. [5] R.J. Hay. Malassezia, dandruff and seborrhoeic dermatitis: an overview. Br J Dermatol., 165 (2011) 2-8. Doi.org/10.1111/j.1365-2133.2011.10570.x. [6] T.L. Dawson Jr. Malassezia globosa and restricta: breakthrough understanding of the etiology and
treatment of dandruff and seborrheic dermatitis through whole-genome analysis. J. Investig. Dermatol. Symp. Proc., 12 (2007) 15-19.
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etiologic facets of dandruff and seborrheic dermatitis: Malassezia fungi, sebaceous lipids, and individual sensitivity. J. Investig. Dermatol. Symp. Proc., 10 (2005) 295-297.
Doi.org/10.1111/j.1087-0024.2005.10119.x. [8] G.A. Turner, J.R. Matheson, G.Z. Li, X.Q. Fei, D. Zhu, F. Baines. Enhanced efficacy and sensory
properties of an anti-dandruff shampoo containing zinc pyrithione and climbazole. Int. J. Cosmet. Sci., 35 (2013) 78-83.
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Chapter 8
Visualization of zinc pyrithione particles deposited on the scalp from
hair care products
Abstract
An ex vivo method that combines tape strip sampling and SEM/EDX has been developed for
measuring and visualizing the particle size, morphology and composition of zinc pyrithione
(ZPT) deposited on the scalp from AD shampoos. Hair was washed with a commercially
available AD shampoo containing ZPT and zinc carbonate (ZnCO3). Tape strips were applied
to collect the deposited particles from the scalp after AD shampoo application and rinse-off.
The scalp tape strip samples were subjected to scanning electron microscopy/energy
dispersive X-ray spectroscopy (SEM/EDX) measurement. The morphology of the ZPT
particles was visualized by SEM imaging and identification of ZPT particles was confirmed
by EDX analysis. For the commercial shampoo studied it was observed that two zinc-
containing particulates with different morphologies and composition remained on the scalp
after shampoo application and rinse-off. As indicated by the EDX spectra, the ZPT particles
deposited onto the scalp surface had polygonal crystal structures. ZnCO3 was also deposited
onto the scalp surface, mainly presenting as aggregated particulates. The new method allows
the microstructures of both ZPT and other zinc particles on the scalp to be imaged.
This chapter was originally published as:
G. Chen, C. Ji, L.Z. Collins, M, Hoptroff, H-G. Janssen, Visualization of zinc pyrithione particles
deposited on the scalp from a shampoo by tape strip sampling and scanning electron
microscopy/energy dispersive X-ray spectroscopy measurement, Int. J. Cosmet. Sci. 40 (2018)
530-533.
114
8.1 Introduction
The anti-dandruff (AD) efficacy of an AD shampoo is highly dependent on the deposition of
the AD actives onto the human scalp during the process of shampoo application and the
amounts remaining after rinse-off. Zinc pyrithione (ZPT) is the most common anti-fungal
active formulated in commercially available (AD) shampoos [1, 2]. There are two types of
ZPT materials regularly used in commercially available AD shampoos, which are different in
particle size and morphology, and as a consequence of that, in deposition mechanism [3].
Robust and reliable methods for studying the ZPT deposition behaviour onto the scalp are
required for developing a better understanding of the difference in AD efficacy among
different formulations and products.
In Chapter 6 and 7, we developed methods for the quantification of ZPT deposition onto the
scalp surface and into the hair follicles. To measure the particle size and morphology of ZPT
deposited on the scalp, imaging methods are demanded. Schwartz et al. reported a reflectance
confocal microscopy (RCM) imaging method for in vivo optical detection of ZPT particles
deposited on the scalp [4]. Due to limitations in the resolution of the RCM method,
information on particle size and morphology of the ZPT particles could not be obtained. An
additional drawback of confocal microscopy is that when other similar particles are delivered
together with the ZPT, RCM imaging cannot distinguish between ZPT and these other
particles. Chemical imaging methods using stimulated Raman scattering (SRS) were
developed to eliminate this drawback and allow mapping the distribution of the ZPT active
on intact skin [5]. Several other imaging technologies have been developed, e.g. to understand
the effects of nanoparticles and microparticles in living tissues [6] and on human substrates
like zinc oxide and titanium dioxide on skin [7-10], and silica in dentine [11]. However,
methods that offer sufficient chemical selectivity, sensitivity and resolution to enable in vivo
or ex vivo visualization of ZPT particles deposited on the scalp so far have not been reported.
The aim of this chapter was to develop an ex vivo measurement method for the visualization
of the particle size and morphology of ZPT deposited on the scalp from AD shampoos. A
previously developed tape-strip sampling method [12] was evaluated for its ability to capture
the ZPT deposits remaining on the scalp after hair wash. Scanning electron microscopy (SEM)
115
was studied as a means to visualize particle size and morphology. Finally, energy dispersive
X-ray spectroscopy (EDX) was used to confirm the presence of the ZPT active.
8.2 Materials and Methods
8.2.1 AD Shampoo
A commercially-available AD shampoo was purchased in a local supermarket in Shanghai,
China and used in this study. The shampoo was a potentiated ZPT shampoo containing ZPT
and zinc carbonate (ZnCO3) [3, 13].
8.2.2 Shampoo wash and specimen collection
The study protocol was reviewed and approved by the Joint Research Ethics Committees
(Shanghai, China). All recruited subjects of both genders aged 18-60 years were medically
healthy and gave their informed consent prior to participating in this study. Forty-five subjects
were accepted and 43 subjects (24 females + 19 males) completed the whole study. Two
subjects were withdrawn for personal reason.
A hair wash procedure was applied to the participating subjects, as previously described in
Chapter 7. After hair wash, a standardised tape strip sampling method was used to collect
deposited actives. In brief, the sampling sites on the scalp of each subject were exposed by
parting the hair. The length of parting line was about 10 cm. A commercially-available sticky
tape (J-LAR, Permacel, Wisconsin, United States) was placed along the parting line. The tape
was pressed onto the scalp surface by applying a uniform pressure using a roller. This roller
was passed 20 timed, unidirectionally along the length of the tape. The tape was then removed
and mounted in the suitable holders for the SEM/EDX instruments (vide infra).
116
8.2.3 SEM/EDX measurement
The tape strip specimens were mounted on an aluminium sample holder, sputter-coated with
platinum (Hitachi, E-1045, Tokyo, Japan), and transferred into the SEM chamber for
visualisation (Hitachi, S-4800, Tokyo, Japan). The working distance was 15 mm, and a
voltage of 10 kV was applied. The deposition site was identified by EDX examination (Horiba,
X-max, Kyoto, Japan) through the detection of zinc (6 seconds X-ray exposure on each spot).
Molar ratios of sulphur and zinc (S/Zn) were calculated using the instrument’s built-in
software. Studies with blank samples showed that there was no detectable zinc and sulphur
on new tape nor in the scalp flakes that were captured on the tape from untreated skin.
Therefore, it can be confirmed that zinc and sulphur only came from the shampoo used.
8.3 Results and discussion
In the SEM images particles of different size and morphology were seen. EDX analysis of
these particles showed two main characteristic types of morphology for the zinc-containing
deposits on the scalp surface. As shown in Figure 8.1A, distinct ZPT crystalline particles with
an average size of approximately 2 µm (n=129) were dispersed across the SEM image. These
particles exhibited a well-recognised polygonal shape. Another characteristic zinc-containing
deposit, consisting of aggregated sub-micron particles, was also abundantly present (Figure
8.1B). These deposits were generally of an irregular shape.
EDX signals of sulphur and the S/Zn ratios were used to differentiate ZPT from other Zn
containing materials (e.g. ZnCO3) that had deposited on the scalp from the shampoo after hair
wash. The polygonal crystals showed a typical EDX spectrum (Figure 8.1C), indicating the
presence of both zinc and sulphur. The sulphur to zinc molar ratio for these crystals is close
to its theoretical ratio for ZPT (2:1). These results demonstrate that the polygonal crystals are
ZPT. On the other hand, there was no detectable sulphur signal in the EDX spectra for the
aggregated zinc deposits (Figure 8.1D). These results indicate that these aggregates were
mainly composed of ZnCO3.
117
Figure 8.1. Typical microstructures of zinc-containing deposits (A and B).
The corresponding EDX spectra are shown accordingly (C for panel A, and D for panel B).
The white asterisks indicate the positions of the EDX analysis.
Previous studies showed that deposited ZPT can be visualised using RCM [1, 4]. In these
studies, all bright white dots in RCM images of scalp skin were assumed to be ZPT deposits.
However, based on the findings in the current study it is concluded that it is not possible to
differentiate ZPT from other particles in RCM analysis. In addition, the low resolution of
RCM does not allow for differentiating ZPT from ZnCO3 on the basis of microstructural
features. RCM hence is not an appropriate tool assess ZPT deposition on the scalp if other
crystalline materials are present in the formulation at a significant concentration, such as e.g.
ZnCO3. Clearly, under these conditions, using RCM ZPT deposition would be overestimated.
The current method with EDX verification eliminates this drawback.
118
8.4 Conclusion
An ex vivo method combining tape strip sampling and SEM/EDX characterisation has been
developed for measuring and visualizing the particle size and morphology of ZPT deposited
on the scalp from a commercially available AD shampoo. This ex vivo measurement method
provides higher imaging resolution and more chemical specificity than RCM. To the best of
our knowledge, this is the first time that ZPT particles could be distinguished from other zinc-
containing particles deposited onto the scalp. The combined SEM/EDX method also enabled
us to characterise the microstructures of both ZPT and other zinc particles deposited onto the
scalp.
119
References
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Doi.org/10.1111/ics.12055. [2] G. Turner, J. Matheson, G. Li, X. Fei, D. Zhu, L. Baines, Enhanced efficacy and sensory properties of an
anti-dandruff shampoo containing zinc pyrithione and climbazole. Int. J. Cosmet. Sci., 35 (2013) 78-83. Doi.org/10.1111/ics.12007. [3] J.R. Schwartz, Zinc Pyrithione: A Topical Antimicrobial with Complex Pharmaceutics. J. Drugs Dermatol.,
15 (2016) 140-144. [4] J.R. Schwartz, R. Shah, H. Krigbaum, J. Sacha, A. Vogt, U. Blume-Peytavi, New insights on
dandruff/seborrhoeic dermatitis: the role of the scalp follicular infundibulum in effective treatment strategies. Br. J. Dermatol., 165 (2011), 18-23. Doi.org/10.1111/j.1365-2133.2011.10573.x.
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[12] R.R. Warner, J.R. Schwartz, Y. Boissy, T.L. Dawson, Dandruff has an altered stratum corneum ultrastructure that is improved with zinc pyrithione shampoo. J. Am. Acad. Dermatol., 45 (2001) 897-903.
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List of abbreviations
AD Anti-dandruff APCI Atmospheric pressure chemical ionization CA (+)-catechin (chapter 4)
Cyanoacrylate (chapter 7) CBZ Climbazole CD Cyclodextrin CG (+)-catechin gallate CID Collision induced dissociation CLS Classical least square CSSS Cyanoacrylate skin surface stripping DPS 2, 2-Dipyridyl disulfide EC (-)-epicatechin ECG (-)-epicatechin gallate EGC (-)-epigallocatechin EGCG (-)-epigallocatechin gallate ESI Electro spray ionization ESI-MS Electrospray ionization mass spectrometry FICs Follicular infundibulum casts GC Gas chromatography
(+)-gallocatechin (in chapter 4) GCB Graphitized carbon black GCG (+)-gallocatechin gallate GC/MS Gas chromatography-mass spectrometry GPC Gel permeation chromatography HPC Home and personal care HPLC High performance liquid chromatography IR Infra-red LC Liquid chromatography LC/MS/MS Liquid chromatography-tandem mass spectrometry LOD Limit of detection LOQ Limit of quantification MALDI MSI Matrix-assisted laser-desorption ionization mass
spectrometry imaging MRLs Maximum residue limits MRM Multiple reaction monitoring MS Mass spectrometry NMR Nuclear magnetic resonance spectroscopy OCT Optical coherence tomography PBS Phosphate buffered saline
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PDA Photodiode array PSA Primary secondary amine QuEChERS Quick, easy, cheap, effective, rugged and safe RACs Raw agriculture commodities RCM Reflectance confocal microscopy R&D Research and development RSD Relative standard deviation RTD Ready-to-drink SEM Scanning electron microscopy SEM/EDX Scanning electron microscopy/energy dispersive X-ray
spectroscopy SCs Surface casts S/N Signal-to-noise SPE Solid phase extraction SRS Stimulated Raman scattering TP Total polyphenols TEM Transmission electron microscopy TWHS Total weighted head score UHPLC-MS/MS Ultra-high-performance liquid chromatography-tandem
mass spectrometry UHPLC-UV Ultra-high-performance liquid chromatography-UV XRF X-ray fluorescence spectroscopy ZnCO3 Zinc carbonate ZPT Zinc pyrithione
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Summary
Analytical chemistry plays a critical role in many fields ranging from fundamental research
to life sciences, industrial analysis and social applications. The aim of this thesis is to develop
new analytical methods to meet the ever-increasing need for safety and quality control,
performance evaluation, claim substantiation and mode-of-action understanding in the area
of consumer products for foods, and home and personal care. In Chapter 1, the needs and
challenges of analytical sciences in the industry of foods and HPC are summarized. The most
widely used analytical techniques in these product fields are introduced. Finally, the scope of
this thesis is discussed with a brief introduction of the subsequent chapters.
In Chapter 2, a multi-residue method was developed for rapid determination of pesticide
residues in tea by UHPLC-MS/MS. An adapted QuEChERS method was used for sample
preparation. In order to minimize the matrix effects from tea, an SPE cartridge layered with
graphite carbon/aminopropylsilanized silica gel was applied as complementary to the original
QuEChERS method. Representative matrix-matched calibration curves were applied for
quantification to compensate for matrix effects. Limits of quantification varied for the
different pesticides. Except for dichlorvos, that has a quantification limit of 0.02 mg/kg, all
others can be measured at 0.01 mg/kg level or better in a 5 g tea sample. Recoveries ranged
from 70% to 120% and the method RSD met the European Union Quality Control guideline.
Efficiency and reliability of this method were investigated by the analysis of both fermented
and unfermented Chinese tea samples. The method has further application opportunities,
including the analysis of e.g. dried vegetables and herb extracts.
In Chapter 3, a UHPLC-UV method combined with SPE sample pre-treatment was
developed and validated for the rapid quantification of L-theanine in ready-to-drink (RTD)
teas. UHPLC-UV analysis of twenty-seven RTD teas from the Chinese market revealed that
the L-theanine levels in various types of RTD teas were significantly different. RTD green
teas were found to contain the highest mean L-theanine level (37.85 ± 20.54 mg/L), followed
by jasmine teas (36.60 ± 12.08 mg/L), Tieguanying teas (18.54 ± 3.46 mg/L) black teas (16.89
± 6.56), Pu-erh teas (11.31 ± 0.90 mg/L) and Oolong teas (3.85 ± 2.27 mg/L). The ratio of the
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total polyphenols to L-theanine content could be used as a characteristic parameter for
differentiating RTD teas. L-theanine in RTD teas could be a reliable quality parameter that is
complementary to total polyphenols.
In Chapter 4, the noncovalent interaction between β-CD and EGCG was studied at the
molecular level by ESI-MS and NMR. Inclusion complexation of β-CD and green tea
catechins was observed by ESI-MS. The stoichiometry of the β-CD-EGCG complex was
determined using Job’s method, which showed a maximum at 0.5, indicating a 1:1
stoichiometry of the β-CD-EGCG complex. NMR experiments indicated that inclusion
complexes of β-CD and EGCG were formed and that the D ring or B ring of the EGCG
molecule penetrated into the β-CD cavity. This molecular encapsulation could prevent the
gallate moiety from binding to the human taste receptors, in that way reducing the bitter,
astringent taste of EGCG. The direct observation of non-covalent interactions makes the
combined deployment of ESI-MS and NMR a valuable chemical vehicle for fast screening of
molecular maskers for reducing bitterness and astringency of green tea catechins as an
alternative to a tasting panel.
In Chapter 5, a sensitive and specific UHPLC-MS/MS method was developed and validated
for the measurement of climbazole (CBZ) deposition from hair care products onto artificial
skin and human scalp. Deuterated CBZ was used as the internal standard. APCI in positive
mode was applied for the detection of CBZ. For quantification, MRM transition 293.0 > 69.0
was monitored for CBZ, and MRM transition 296.0 > 225.1 for the deuterated CBZ. The linear
range ran from 4 to 2000 ng/mL. The LOD and the LOQ were 1 ng/mL and 4 ng/mL,
respectively, which enabled quantification of CBZ on artificial skin and human scalp at ppb
level (corresponding to 16 ng/cm2). For the sampling of CBZ from human scalp the buffer
scrub method using a surfactant-modified PBS solution was selected based on a performance
comparison of tape stripping, the buffer scrub method and solvent extraction in in vitro studies.
Using this method, CBZ deposition in in vitro and in vivo studies was successfully quantified.
In Chapter 6, a sensitive UHPLC-MS/MS method has been developed and validated for
simultaneous quantification of Zinc pyrithione (ZPT) and CBZ deposited onto human scalp
from AD shampoos. Scrubbing with a buffer solution was used as the sampling method for
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the extraction of ZPT and CBZ from scalp. Derivatization of ZPT was carried out prior to
UHPLC-MS/MS analysis. The identification of ZPT and CBZ was performed by examining
ratios of selected MRM transitions in combination with UHPLC retention times. The limit of
detection for ZPT and CBZ was established to be 1 and 2 ng/mL, respectively. This sensitivity
enables the quantification of ZPT and CBZ at deposition levels in the low ng/cm2 range. The
method was successfully applied for the analysis of scalp buffer scrub samples from an in
vivo study. The levels of ZPT and CBZ remaining on the scalp at different time intervals after
application of the AD shampoo were measured. The results revealed that dual-active AD
shampoo delivered more ZPT onto the scalp in a single wash than a single active shampoo
did. The amount of ZPT and CBZ remained on the scalp after AD shampoo application
declined over 72 hours. The method is also applicable in other studies, e.g. in artificial skin
studies to improve shampoo formulations to maximize ZPT and CBZ deposition.
In Chapter 7, a method involving scalp cyanoacrylate biopsy sampling, a tailor-made cutting
device, UHPLC-MS/MS analysis, SEM measurement and Raman imaging are described for
the measurement of delivery of ZPT and CBZ from an AD shampoo into the scalp follicular
infundibulum. Scalp cyanoacrylate biopsy enables the sampling of ZPT and CBZ delivered
into the scalp follicular infundibulum as well as onto the scalp surface. Raman imaging of
scalp cyanoacrylate biopsy samples allows the visualization of the spatial distribution of ZPT
and CBZ deposited on the scalp. A tailor-made cutting device enables the separation of the
scalp follicular infundibulum sample (20 µm below the scalp surface) from the scalp surface
samples (including the top 20 μm of the infundibula). UHPLC-MS/MS was used as a sensitive
and specific methodology enabling the quantification of ZPT and CBZ without interferences.
Using this method, ZPT and CBZ delivered into the scalp follicular infundibulum from the
dual-active AD shampoo was successfully visualized and quantified. Due to the lipophilic
nature of CBZ and hence the increased solubility in sebum, CBZ has the ability to penetrate
further into the sebum-rich infundibulum whereas ZPT remains within the upper 20 µm of
infundibula. This differential distribution of actives allows for the effective targeting of
Malassezia species throughout the depth of the scalp follicular infundibulum.
Finally, Chapter 8 proposes an ex vivo method that combines tape strip sampling and
scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) for
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measuring and visualizing the particle size, morphology and composition of ZPT deposited
onto the scalp from an AD shampoo containing ZPT and zinc carbonate. Hair was washed
with a commercially available AD shampoo containing ZPT and zinc carbonate (ZnCO3).
Tape strips were applied to collect the deposited particles from the scalp after AD shampoo
application and rinse-off. The scalp tape strip samples were subjected to scanning SEM/EDX
measurement. The morphology of the ZPT particles was visualized by SEM imaging and
identification of ZPT particles was confirmed by EDX analysis. For the commercial shampoo
studied it was observed that two types of zinc-containing particles with different
morphologies and composition remained on the scalp after shampoo application and rinse-off.
As indicated by the EDX spectra, the ZPT particles deposited onto the scalp surface had
polygonal crystal structures. ZnCO3 was also deposited onto the scalp surface. This material
was mainly present as aggregated particles. This ex vivo measurement method provides higher
imaging resolution and more chemical specificity than reflectance confocal microscopy. To
the best of our knowledge, this is the first time that ZPT particles could be distinguished from
other zinc-containing particles deposited onto the scalp. The new method allows the
microstructures of both ZPT and other zinc particles on the scalp to be imaged.
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Samenvatting
Analytische chemie speelt een cruciale rol op vele terreinen. De discipline wordt veelvuldig
ingezet in gebieden variërend van fundamenteel onderzoek in de levenswetenschappen tot
industriële analyse en sociale toepassingen. Het doel van dit proefschrift is om nieuwe
analysemethoden te ontwikkelen om tegemoet te komen aan de steeds toenemende behoefte
aan veiligheids- en kwaliteitscontrole, prestatie-evaluatie, claim-onderbouwing en de opbouw
van kennis van werkingsmechanismen op het gebied van consumentenproducten voor
voeding, en huishoudelijke- of persoonlijke verzorging. In Hoofdstuk 1 worden de behoeften
en uitdagingen van de analytische chemie in de voedingsmiddelenindustrie en bij de
ontwikkeling en productie van huishoudelijke- en persoonlijke verzorgingsproducten (HPC)
samengevat. De meest gebruikte analytische technieken in deze toepassingsgebieden worden
kort besproken. Ten slotte wordt de scope van dit proefschrift besproken met een korte
introductie van elk van de volgende hoofdstukken.
In Hoofdstuk 2 werd een multi-residu methode ontwikkeld voor snelle bepaling van
bestrijdingsmiddelen en residuen daarvan in thee met behulp van UHPLC-MS/MS. Een
aangepaste QuEChERS-methode werd gebruikt voor monstervoorbewerking. Om de
matrixeffecten van thee te minimaliseren, werd een SPE-cartridge gevuld met een
combinatiebed van grafiet-koolstof en aminopropyl gemodificeerde silicagel gebruikt als
aanvulling op de SPE stap in de originele QuEChERS-methode. Representatieve matrix-
gecorrigeerde kalibratiecurves werden gebruikt voor kwantificering om te compenseren voor
matrixeffecten. De kwantificeringslimieten varieerden voor de verschillende
bestrijdingsmiddelen. Met uitzondering van dichloorvos, waarvoor een kwantificeringslimiet
van 0,02 mg/kg gevonden werd, kunnen alle andere componenten gemeten worden op 0,01
mg/kg niveau of beter, in een 5 g thee monster. De recoveries varieerden van 70% tot 120%
en de RSD van de methode voldeed aan de kwaliteitsrichtlijnen van de Europese Unie. De
efficiëntie en betrouwbaarheid van de nieuw ontwikkelde methode werd onderzocht door de
analyse van zowel gefermenteerde, als niet-gefermenteerde Chinese thee monsters. De
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werkwijze heeft verdere toepassingsmogelijkheden, bijvoorbeeld in de analyse van
gedroogde groenten of kruidenextracten.
In Hoofdstuk 3 werd een UHPLC-UV-methode met SPE-monstervoorbehandeling
ontwikkeld en gevalideerd voor de snelle bepaling van L-theanine in ‘ready to drink’ (RTD)
thee. UHPLC-UV-analyse van zevenentwintig Chinese RTD-thee monsters gaven significant
verschillende L-theanine gehalten te zien in de diverse soorten RTD-thee. Groene RTD-thee
monsters bevatten het hoogste gemiddelde L-theanineniveau (37,85 ± 20,54 mg/L), gevolgd
door jasmijnthee (36,60 ± 12,08 mg/L), Tieguanying thee (18,54 ± 3,46 mg / L), zwarte thee
(16,89) ± 6,56), Pu erh thee (11,31 ± 0,90 mg / L) en Oolong-thee (3,85 ± 2,27 mg / L). De
verhouding van het totale gehalte aan polyfenolen versus L-theanine zou kunnen worden
gebruikt als een kenmerkende parameter voor het onderscheiden van de diverse RTD-
theesoorten. L-theanine in RTD-thee kan een betrouwbare kwaliteitsparameter zijn die
complementair is aan het gehalte totaal polyphenolen.
In Hoofdstuk 4 werd de niet-covalente interactie tussen β-cyclodextrine (β-CD) en
epigallocatechinegallaat (EGCG) op moleculair niveau bestudeerd met behulp van ESI-MS
en NMR. Inclusiecomplexatie van β-CD en groene thee catechines kon direct bestudeerd
worden met ESI-MS. De stoichiometrie van het β-CD/EGCG complex werd bepaald met
behulp van de Job’s-methode, die een maximum bij 0,5 vertoonde, hetgeen duidt op een 1:1
stoichiometrie van het β-CD/EGCG complex. NMR-experimenten bevestigden dat
inclusiecomplexen van β-CD en EGCG werden gevormd en dat de D-ring of B-ring van het
EGCG molecuul in de holte van het β-CD molecuul kon binnendringen. Deze moleculaire
inkapseling zou kunnen voorkomen dat de gallaten aan menselijke smaakreceptoren binden,
waardoor de bittere smaak van EGCG wordt verminderd. De directe waarneming van niet-
covalente interacties maakt de combinatie van ESI-MS en NMR een waardevol methode voor
snelle screening van moleculaire methoden voor het maskeren van bitterheid en astringentie
van groene thee catechines als een alternatief voor een menselijk testpanel.
In Hoofdstuk 5 werd een gevoelige en specifieke UHPLC-MS/MS-methode ontwikkeld en
gevalideerd voor het meten van de depositie van climbazole (CBZ) uit
haarverzorgingsproducten zoals anti-roos shampoos op kunsthuid en op echte hoofdhuid.
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APCI in positieve mode werd toegepast voor de detectie van CBZ. Gedeutereerd CBZ werd
gebruikt als de interne standaard. Voor kwantificering werd de MRM-overgang 293,0> 69,0
gevolgd voor CBZ, en de MRM-overgang 296,0> 225,1 voor het gedeutereerde CBZ. Het
lineaire bereik van de methode liep van 4 tot 2000 ng/mL. De LOD en de LOQ waren
respectievelijk 1 ng/mL en 4 ng/mL, wat kwantificering van CBZ op kunsthuid en op de
menselijke hoofdhuid op ppb-niveau mogelijk maakte (overeenkomend met 16 ng/cm2). Voor
de bemonstering van CBZ op de menselijke hoofdhuid werd de ’buffer scrub’-methode
gevolgd waarbij een PBS-oplossing met daarin opgenomen een oppervlakte actieve stof
gebruikt werd. Deze methode was gekozen op basis van een vergelijking met het strippen met
een plakstrip, de ‘buffer scrub’-methode en vloeistofextractie in in vitro studies. Met behulp
van deze methode werd de CBZ-depositie in in vitro en in vivo studies met succes gemeten.
Hoofdstuk 6 beschrijft de ontwikkeling en validatie van een gevoelige UHPLC-MS/MS-
methode voor de gelijktijdige kwantificering van zinkpyrithione (ZPT) en CBZ deposities op
menselijke hoofdhuid vanuit anti-roos shampoos. Wassen met een bufferoplossing werd
gebruikt als de bemonsteringsmethode voor de extractie van ZPT en CBZ van de hoofdhuid.
ZPT werd gederivatiseerd voorafgaand aan de UHPLC-MS/MS-analyse. De identificatie van
ZPT en CBZ werd uitgevoerd op basis van verhoudingen van geselecteerde MRM-
overgangen in combinatie met UHPLC retentietijden. De detectiegrens voor ZPT en CBZ
werd vastgesteld op respectievelijk 1 en 2 ng/mL. Deze gevoeligheid maakt de kwantificering
van ZPT en CBZ mogelijk op depositieniveaus in het lage ng/cm2 bereik. De methode werd
met succes toegepast voor de analyse van monsters uit een in vivo onderzoek. De
hoeveelheden van ZPT en CBZ die op de hoofdhuid achtergebleven waren op verschillende
tijden na toepassing van de anti-roos shampoo werden gemeten. De resultaten toonden aan
dat ‘dual-active’ anti-roos shampoo meer ZPT op de hoofdhuid afleverde in een enkele
wasbeurt dan een anti-roos shampoo met slechts één actief bestanddeel. De hoeveelheid ZPT
en CBZ die op de hoofdhuid terug gevonden werd na toepassing van de anti-roos shampoo
daalde in 72 uur naar nul. De methode is ook toepasbaar in andere studies, b.v. in studies ter
verbetering van shampooformuleringen met als doel de ZPT- en CBZ-depositie te
maximaliseren.
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In Hoofdstuk 7 wordt een methode beschreven voor het nemen van biopsiemonsters van de
hoofdhuid. Verder wordt een nieuwe snij-inrichting voor het snijden van zeer dunne plakjes,
een UHPLC-MS/MS-analyse methode, SEM-meting en tot slot een methode voor Raman-
beeldvorming voor het meten van de overdracht van ZPT en CBZ uit een anti-roos shampoo
naar het zogenaamde folliculaire infundibulum (het haarfollikel), van de hoofdhuid
beschreven. Toepassing van de cyanoacrylaat biopt methode maakt het mogelijk de opname
van ZPT en CBZ in het folliculaire infundibulum van de hoofdhuid en depositie op het
hoofdhuidoppervlak separaat te bestuderen. Met Raman-beeldvorming van de cyanoacrylaat
biopsiemonsters kunnen vervolgens de ruimtelijke verdeling van ZPT en CBZ op- en in de
hoofdhuid gevisualiseerd worden. Met behulp van een nieuw ontworpen snij-mechanisme
kunnen separaat monsters van het folliculaire infundibulum (20 μm onder het
hoofdhuidoppervlak) en van de totale hoofdhuid (inclusief de bovenste 20 μm van de
infundibula) gemaakt worden. UHPLC-MS/MS werd gebruikt als een gevoelige en specifieke
methodologie die de kwantificering van ZPT en CBZ zonder interferenties mogelijk maakt.
Met behulp van de hier beschreven methoden werd de opname van ZPT en CBZ in het
folliculaire infundibulum van de hoofdhuid vanuit een ‘dual-active’ anti-roos shampoo met
succes gevisualiseerd en gekwantificeerd. Door het lipofiele karakter van CBZ, en daardoor
de verhoogde oplosbaarheid in talg, kan CBZ verder doordringen in het talgrijke
infundibulum terwijl ZPT in de bovenste 20 μm van het infundibulum blijft. Dit verschil in
verdeling van actieve stoffen zorgt voor een effectieve aanpak van Malassezia bacteriën
dieper in het folliculaire infundibulum van de hoofdhuid.
Tot slot wordt in Hoofdstuk 8 een ex-vivo methode voorgesteld die de plakstrip-bemonstering
en elektronenmicroscopie/energiedispersieve röntgenspectroscopie (SEM/EDX) combineert
voor het meten en visualiseren van de deeltjesgrootte, morfologie en samenstelling van ZPT
op de hoofdhuid na wassen met een anti-roos shampoo met ZPT en zinkcarbonaat. Het haar
werd gewassen met een in de handel verkrijgbare anti-roos shampoo die ZPT en
zinkcarbonaat (ZnCO3) bevatte. Plakstrips werden toegepast om de neergeslagen deeltjes van
de hoofdhuid te verzamelen na het wassen van het haar met de anti-roos shampoo en
uitspoelen. De plakstrip met daarop de hoofdhuidmonsters werden bestudeerd met scanning
SEM/EDX-meting. De morfologie van de ZPT-deeltjes werd gevisualiseerd door SEM-
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beeldvorming en verdere identificatie van de ZPT-deeltjes werd bevestigd door EDX-analyse.
Voor de bestudeerde commerciële shampoo werd waargenomen dat twee soorten
zinkbevattende deeltjes met verschillende morfologieën en samenstelling op de hoofdhuid
achterbleven na het toepassen en uitspoelen van de shampoo. Zoals aangegeven door de EDX-
spectra, hadden de ZPT-deeltjes afgezet op het hoofdhuidoppervlak veelhoekige
kristalstructuren. ZnCO3 werd ook op het hoofdhuidoppervlak afgezet. Dit materiaal was
voornamelijk aanwezig als geaggregeerde deeltjes. Deze ex-vivo meetmethode biedt een
hogere beeldresolutie en meer chemische specificiteit dan reflectie confocale microscopie.
Voor zover ons bekend, is dit de eerste keer dat ZPT-deeltjes kunnen worden onderscheiden
van andere zinkbevattende deeltjes achtergebleven op de hoofdhuid. Met de nieuwe methode
kunnen de microstructuren van zowel ZPT als andere zinkdeeltjes op de hoofdhuid bepaald
worden.
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总结
分析化学在基础研究、生命科学、工业分析和社会应用等领域都发挥着重要作用。本
文的目的是开发新的分析方法和技术,以满足食品、家庭和个人护理领域中各种新产
品开发需求,包括安全和质量控制、功能性评估、功效证明、机理理解等。第一章概
述了分析科学在食品、家庭和个人护理行业的需求和挑战,介绍了该领域中应用最广
泛的分析技术。然后,简要介绍了本论文的研究范围。
第二章介绍了一个基于超高效液相色谱和串联质谱联用的分析方法, 用于快速分析茶
叶中农药残留。为了最小化茶叶的基体效应,作者使用石墨碳/氨基丙基硅烷化硅胶固
相萃取小柱结合 QuEChERS 方法的样品前处理技术,有效降低了基质干扰。另外,采
用了基质匹配的标准溶液,使得定量结果更加准确。除敌敌畏的定量限为 0.02 mg/kg,
其他所有农残的定量限皆在 0.01 mg/kg 水平或更高灵敏度。该方法的回收率在 70%-
120%之间,相对标准偏差符合欧盟质量控制准则。通过分析发酵茶和未发酵茶样品中
的农残,验证了该方法的有效性和可靠性。并且,该方法具有进一步应用的机会,比
如分析干蔬菜或草本提取物中的农残。
在第三章中,作者建立了一种固相萃取为预处理和超高效液相色谱紫外光检测器相结
合的分析方法,用于快速测定即饮茶中茶氨酸的含量。采用该方法,对中国市场上的
27 种即饮茶中茶氨酸进行了定量分析。结果表明,不同类型即饮茶中的茶氨酸水平存
在显著差异。即饮绿茶中茶氨酸含量最高(37.85 ± 20.54 mg/L),其次为即饮茉莉花茶
(36.60 ± 12.08 mg/L)、即饮铁即饮观音茶(18.54 ± 3.46 mg/L)、即饮黑茶(16.89 ± 6.56
mg/L)、即饮普洱茶(11.31 ± 0.90 mg/L)和即饮乌龙茶(3.85 ± 2.27 mg/L)。作者发现,总
多酚与茶氨酸含量的比值可以作为鉴别即饮茶的特征参数。并且,茶氨酸的含量可以
作为一种与总多酚互补的可靠质量参数。
第四章介绍了作者利用电喷雾质谱和核磁共振从分子水平上,研究了 β 环糊精 (β-
CD)与表没食子儿茶素没食子酸酯(EGCG)的非共价相互作用。β-CD 与绿茶儿茶素
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在水溶液中的相互作用可以直接被电喷雾质谱观察到。用 Job 法测定了 β-CD 和 EGCG
复合物的化学计量比,在 0.5 处达到最大值,表明 β-CD 和 EGCG 复合物的化学计量
比为 1:1。核磁共振实验表明,β-CD 与 EGCG 形成包合物,EGCG 分子的 D 环或 B 环
进入 β-CD 空腔。这种分子包埋可以防止没食子酸酯部分与味觉受体结合,从而减少
EGCG 的苦涩味道。采用电喷雾质谱和核磁共振研究分子间的非共价相互作用,可以
成为快速筛选分子掩蔽剂用于降低绿茶儿茶素的苦味和收敛性,并作为感官评测的替
代方案。
第五章,作者建立了一种灵敏的超高效液相色谱和串联质谱联用的分析方法,用于检
测使用洗发水后,在人工假皮和人的头皮上沉积的甘宝素。为了能有效的从人的头皮
上采集沉积的甘宝素,不同采样方法包括胶带剥离、缓冲液洗涤和溶剂萃取,在人工
假皮上进行的测试和比较,最后选定表面活性剂改良的磷酸盐缓冲液。大气压化学电
离阳离子模式用于甘宝素的检测。定量时,氘代甘宝素被用做内标。线性范围从 4 到
2000 ng/mL。检测限和定量限分别为 1 ng/mL 和 4 ng/mL,从而使得人工假皮和人的头
皮上,低至 16 ng/cm2的甘宝素都能被定量。使用这种方法,在人的头皮上沉积的甘宝
素成功的提取到和定量。
第六章,作者建立了一种灵敏的超高效液相色谱和串联质谱联用的分析方法,用于同
时测定使用去屑洗发香波后,沉积在人的头皮上的吡硫翁锌和甘宝素。采用表面活性
剂改良的磷酸盐缓冲液擦洗作为提取头皮中吡硫翁锌和甘宝素的采样方法。为了提高
灵敏度和稳定性,在仪器分析之前,吡硫翁锌被进行了衍生化。吡硫翁锌和甘宝素的
检测限分别为 1 和 2 ng/mL。这样的种灵敏度使得头皮上 ng/cm2范围内的吡硫翁锌和
甘宝素也能够被检测和定量。该方法已成功地应用于临床样品分析。测定了试用去屑
洗发水后,不同时间点,头皮上的吡硫翁锌和甘宝素的含量。结果表明,在一次洗涤
后,双活性去屑洗发香波比单活性去屑洗发香波在头皮上沉积更多的吡硫翁锌。使用
去屑洗发水后,头皮上残留的吡硫翁锌和甘宝素量下降随时间下降。该方法可应用于
测定使用其它个人护理产品后,皮肤上沉积的活性功效成分。
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在第七章中,作者描述了一种方法用于测量使用去屑洗发水后,沉积在头皮毛囊漏斗
中的的吡硫翁锌和甘宝素。该方法包含了头皮氰基丙烯酸盐粘合剂活检取样(CA
biopsy)、特制的切割装置、超高效液相色谱和串联质谱联用分析、扫描电镜测量和拉
曼成像的方法。头皮 CA biopsy 能够取到头皮毛囊漏斗中以及头皮表面的吡硫翁锌和
甘宝素。对 CA biopsy 样品的拉曼成像可以显示头皮上的吡硫翁锌和甘宝素的空间分
布。一种特制的切割装置能够将头皮毛囊漏斗样品(在头皮表面下 20 微米)与头皮表
面样品(包括漏斗的顶部 20 微米)分离。超高效液相色谱和串联质谱联用分析是一种
灵敏、特异的方法,能够不受干扰地定量吡硫翁锌和甘宝素。该方法成功地应用于临
床样品分析,在使用双活性去屑洗发水后,输送到头皮毛囊漏斗中的吡硫翁锌和甘宝
素被测定。由于甘宝素的亲脂性质和随后在皮脂中的高溶解度,甘宝素具有快速扩散
到富含皮脂的毛囊漏斗区的能力,并进到更深处。而吡硫翁锌则主要保留在漏斗的上
部 20 微米区域内。这种差异性的分布使得甘宝素对于处在头皮毛囊深处的马拉色菌有
更好的抑制效果。
最后的第八章中,作者提出了一种方法用于测量使用含有吡硫翁锌和其它锌盐的去屑
香波后,沉积到头皮上的含锌颗粒,并对相关颗粒的形态和元素组成进行分析。该方
法包括胶带取样与电子显微镜/ X 射线能谱检测。在使用市售的含有吡硫翁锌和碳酸锌
去屑洗发水后,用胶带从头皮上收集沉积的颗粒物。对头皮胶带样品进行扫描电镜成
像,观察了吡硫翁锌颗粒的形貌,并通过元素分析证实了吡硫翁锌颗粒的鉴定。对于
所研究的市售洗发水,观察到两种不同形态和组成的含锌颗粒。元素分析表明,沉积
在头皮表面的吡硫翁锌颗粒具有多边形晶体结构。碳酸锌也被沉积在头皮表面上,这
种材料主要以聚集颗粒的形式存在。该方法提供了比反射共聚焦显微镜更高的成像分
辨率和更高的化学特异性。据我们所知,这是首次将沉积在头皮上的吡硫翁锌颗粒与
其他含锌颗粒有效区分开来。并且该方法允许对头皮上的吡硫翁锌和其他锌颗粒的微
观结构进行成像分析。
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List of Publications
- van der Pijl, P.C., Chen, L., Mulder, T.P.J. (2010). Human disposition of L-theanine intea or aqueous solution. Journal of Functional Foods, 2, pp. 239-244.
- Chen, G., Cao, P., Liu, R. (2011). A multi-residue method for fast determination ofpesticides in tea by ultra performance liquid chromatography-electrospray tandem massspectrometry combined with modified QuEChERS sample preparation procedure. FoodChemistry, 125(4), pp. 1406-1411.
- Chen, G., Wang, Y., Song, W., Zhao, B., Dou, Y. (2012). Rapid and selectivequantification of l-theanine in ready-to-drink teas from Chinese market using SPE andUPLC-UV. Food Chemistry, 135(2), pp. 402-407.
- Chen, G., Hoptroff, M., Fei, X., Su, Y., Janssen, H.-G. (2013). Ultra-high-performanceliquid chromatography-tandem mass spectrometry measurement of climbazoledeposition from hair care products onto artificial skin and human scalp. Journal ofChromatography A, 1317, pp. 155-158.
- Chen, G., Miao, M., Hoptroff, M., Fei, X., Collins, L.Z., Jones, A., Janssen, H.-G. (2015).Sensitive and simultaneous quantification of zinc pyrithione and climbazole depositionfrom anti-dandruff shampoos onto human scalp. Journal of Chromatography B:Analytical Technologies in the Biomedical and Life Sciences, 1003, pp. 22-26.
- Wu, Y., Chen, G., Ji, C., Hoptroff, M., Jones, A., Collins, L.Z., Janssen, H.-G. (2016).Gas chromatography-mass spectrometry and Raman imaging measurement of squalenecontent and distribution in human hair. Analytical and Bioanalytical Chemistry, 408(9),pp. 2357-2362.
- Chen, G., Ji, C., Miao, M., Yang, K., Luo, Y., Hoptroff, M., Collins, L.Z., Janssen, H.-G. (2017). Ex-vivo measurement of scalp follicular infundibulum delivery of zincpyrithione and climbazole from an anti-dandruff shampoo. Journal of Pharmaceuticaland Biomedical Analysis, 143, pp. 26-31.
- Chen, G., Ji, C., Collins, L.Z., Hoptroff, M., Janssen, H.-G. (2018) Visualization of zincpyrithione particles deposited on the scalp from a shampoo by tape strip sampling andscanning electron microscopy/energy dispersive X-ray spectroscopy measurement.International Journal of Cosmetic Science, 40, pp.530-533.
- Chen, G., Janssen, H.-G. The use of ESI-MS & 2D NMR to reveal the reduction ofbitterness and astringency of EGCG by β-CD inclusive complexation. Food ResearchInternational, manuscript submitted.
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Overview of author’s contributions
Chapter 1: General introduction
Guoqiang Chen: wrote the manuscript.
Hans-Gerd Janssen: reviewed the manuscript and gave suggestions for improvement.
Chapter 2: A multi-residue method for fast determination of pesticides in tea Guoqiang Chen: developed the ideas and experimental set-up, conducted the method
development and validation, interpreted the data and wrote the manuscript.
Pengying Cao: performed the sample treatment and instrumental analysis.
Renjiang Liu: gave suggestions for the optimization of the sample treatment procedure.
Hans-Gerd Janssen: reviewed the manuscript and gave suggestions for improvement.
Chapter 3: Rapid and selective quantification of L-theanine in ready-to-drink
teas from Chinese market Guoqiang Chen: developed the idea and experiment design, conducted the method
development, optimisation and validation, interpreted the data and wrote the manuscript.
Yun Wang: performed the sample treatment and UHPLC analyses for part of the samples.
Weiqi Song: performed the sample preparation and UHPLC analyses for part of the
samples.
Bo Zhao: contributed to the experiment design and method validation.
Yuling Dou: contributed to the experiment design and method validation.
Hans-Gerd Janssen: reviewed the manuscript and gave suggestions for improvement.
Chapter 4: A method for measuring the noncovalent interaction between
EGCG and β-CD Guoqiang Chen: developed the idea, designed the experimental plan, performed the
experiments, interpreted the results and wrote the manuscript.
Hans-Gerd Janssen: supervised the project, reviewed the manuscript and gave suggestions
for improvement.
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Chapter 5: Quantification of climbazole deposition from shampoos onto
artificial skin and human scalp Guoqiang Chen: developed the idea and experimental set-up, conducted the method
validation, performed the sample analysis, interpreted the data and wrote the manuscript.
Michael Hoptroff: supervised the project, reviewed and revised the manuscript.
Xiaoqing Fei: contributed to the design of the in vivo study.
Ya Su: contributed to the design of the in vitro study.
Hans-Gerd Janssen: co-supervised the project, reviewed the manuscript and gave
suggestions for improvement.
Chapter 6: Sensitive and simultaneous quantification of zinc pyrithione and
climbazole in scalp buffer scrub samples Guoqing Chen: developed the idea and experimental set-up, conducted the method
validation, interpreted the data and wrote the manuscript.
Maio Miao: co-developed the idea, performed the sample analysis.
Michael Hoptroff: co-supervised the project, reviewed and revised the manuscript.
Xiaoqing Fei: contributed to the design of the in vivo study.
Luisa Z. Collins: co-supervised the project, reviewed the manuscript and gave suggestions
for improvement.
Andrew Jones: co-supervised the project, reviewed the manuscript and gave suggestions for
improvement.
Hans-Gerd Janssen: supervised the project, reviewed the manuscript and gave suggestions
for improvement.
138
Chapter 7: Ex-vivo measurement of scalp follicular delivery of zinc pyrithione
and climbazole from hair care products Guoqiang Chen: developed the idea, developed the experimental plan, interpreted the data
and wrote the manuscript.
Chengdong Ji: performed the Raman imaging analyses.
Miao Miao: performed the quantitative analysis of zinc pyrithione and climbazole by
UHPLC-MS/MS.
Kang Yang: co-developed the idea of the cutting device.
Yajun Luo: contributed to the design of in vivo study.
Michael Hoptroff: co-supervised the project, reviewed the manuscript and gave suggestions
for improvement.
Luisa Z. Collins: co-supervised the project, reviewed the manuscript and gave suggestions
for improvement.
Hans-Gerd Janssen: supervised the project, reviewed the manuscript and gave suggestions
for improvement.
Chapter 8: Visualization of zinc pyrithione particles deposited on the scalp
from hair care products Guoqiang Chen: developed the idea, compiled the experimental plan, interpreted the data
and wrote the manuscript.
Chengdong Ji: co-developed the idea, performed the SEM measurement
Luisa Z. Collins: co-supervised the project, reviewed the manuscript and gave suggestions
for improvement.
Michael Hoptroff: reviewed the manuscript and gave suggestions for improvement.
Hans-Gerd Janssen: supervised the project, reviewed the manuscript and gave suggestions
for improvement.
139
Acknowledgements
Throughout the whole procedure of my PhD study and the writing of this thesis, I have
received a great deal of assistance and support.
First and foremost, I would like to express my very great appreciation to my promotor,
Professor Hans-Gerd Janssen who gave me the invaluable opportunity to pursue the PhD
degree. His patient guidance, enthusiastic encouragement, constructive suggestions and
expertise are indispensable to my PhD study and thesis preparation.
I am particularly grateful to my co-promotor, Professor Peter Schoenmakers whose kind
support is critical for my application for the PhD study and the preparation of my defense.
The board of Unilever Research Shanghai, U66, in particular Mason Wang, Jin-Fang Wang
and Manfred Aben, I kindly acknowledge for giving me the opportunity to perform this
exciting research and allowing me to perform this PhD study.
I would also like to extend my thanks to my (former) colleagues in Unilever: Jin-Fang Wang
as my current line manage for his great mentoring, support and aid; Axel Ekani and Yumo
Zhang as my former line managers for their kind support and enthusiastic encouragement;
Renjiang Liu and Pengying Cao for their contributions to Chapter 2; Yun Wang, Yuling Dou,
and Weiqi Song for their contributions to Chapter 3; Bo Zhao for his contributions to Chapter
3 & 4; Michael Hoptroff for his contributions to Chapter 5-8; Xiaoqing Fei and Ya Su for
their contributions to Chapter 5; Andrew Jones for his contributions to Chapter 6; Luisa
Collins for contributions to Chapter 6-8; Chengdong Ji for his contributions to Chapter 7 &
8; Miao Miao for her contributions to Chapter 6 & 7; Yajun Luo and Kang Yang for their
contributions to Chapter 7.
Lastly but not least, I would like to thank my family for their unconditional support.
感谢所有帮助过我的人;感恩父母的养育;感激老婆的全力支持,尤其是为我的论文
设计了封面;希望我的博士学习对女儿的成长有所启发,活到老,学到老;我爱你们。
Combined analytical techniques for the analysis of complex consumer products and bio-samples
Guoqiang (Leon) Chen
Com
bined analytical techniques for the analysis of complex consum
er products and bio-samples
Guoqiang (Leon) C
hen
Invitation
For attending the public defence of the thesis
Combined analytical techniques for the analysis
of complex consumer products and bio-samples
On Wednesday 5th June 2019
at 14.00
In theAgnietenkapel,
Oudezijds Voorburgwal 229,Amsterdam
Paranymphs
Randy ZhaoBoudewijn Hollebrands