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Mastering MicrosamplingAdvances in microsampling for
pharmacokinetic studies
2 Advances in Microsampling for In Vivo Pharmacokinetic Studies Stuart Kushon1, Daniel Kassel2, Hong Xin3, and Nurith Amitai3, 1Neoteryx LLC, 2SciAnalytical Strategies, 3Explora BioLabs Volumetric absorptive microsampling (VAMS) is gaining traction because it delivers the benef ts of dried blood spots (DBS) and overcomes its limitations while generating comparable PK data to conventional sampling methods. This article explains more.
Cover Story
Features
21 Recent Developments in Pharmaceutical Analysis (RDPA 2015) A look to the upcoming symposium on the Recent Developments in Pharmaceutical Analysis (RDPA 2015), which will be held 28 June to 1 July 2015 in Perugia, Italy.
15 Using GPC/SEC for Excipient Characterization Stephen Ball, Malvern Instruments This article looks at how gel permeation/size-exclusion chromatography (GPC/SEC) can be applied to measure characteristics such as molecular weight (MW), MW distribution and structure, and degree of branching for polymeric excipients.
Regulars8 News
Analysis of ink samples using microdestructive CE, detecting parabens in plastic teething toys, and identifying fracking contamination of drinking water using GC×GC–TOF-MS are featured this week.
11 Tips & Tricks GPC/SEC Answering Common Questions About GPC/SEC Columns
Daniela Held and Wolfgang Radke, PSS Polymer Standards Service GmbH A selection of commonly asked questions about GPC/SEC from users.
22 CHROMacademy
Find out what’s new on the professional learning site for chromatographers.
23 Training Courses and Events
25 Staff
5 June 2015 Volume 11 Issue 10
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Advances in Microsampling for In Vivo Pharmacokinetic Studies
Pharmacokinetic (PK) studies are performed throughout the drug discovery process. In general, 250 µL of whole blood is retrieved at each time point, processed to plasma, and stored at -80 °C prior to bioanalysis. Microsampling with dried blood spots (DBS) is an attractive alternative to the conventional whole blood plasma workup. DBS reduces the need to collect large volumes of blood and therefore the number of animals required. However, DBS technology has not been fully embraced as a result of its well-documented hematocrit bias and the labour-intensive sample manipulation required. A new approach — volumetric absorptive microsampling (VAMS) — is gaining traction because it delivers the benef ts of DBS and overcomes its limitations while generating comparable PK data to conventional sampling methods.
Stuart Kushon1, Daniel Kassel2, Hong Xin3, and Nurith Amitai3, 1Neoteryx LLC, Torrance, California, USA, 2SciAnalytical Strategies, La Jolla,
California, USA, 3Explora BioLabs, San Diego, California, USA.
Pharmacokinetic (PK) studies are routinely
performed throughout the drug discovery
process, from screening during the lead
generation phase to comprehensive PK
studies in candidate selection. During hit and
lead generation, compounds are synthesized
at the milligram level for intravenous (IV)
and oral (PO) dosing in a rat or mouse (for
example 1 mg/kg for IV and 5 mg/kg for
PO). Blood samples are typically taken over
a def ned time course and three animals are
used to obtain an average drug exposure
at each time point. A typical study design
for discovery PK is shown in Table 1. In
general, for rodent PK studies, 250 µL of
whole blood is retrieved at each time point,
processed to plasma (yielding approximately
100 µL), and stored at -80 °C prior to
bioanalysis. Liquid chromatography coupled
to tandem mass spectrometry (LC–MS–MS)
bioanalysis is performed to assess the
pharmacokinetic properties of the molecules
and their potential as lead candidates.
In general, compounds exhibiting >20%
oral bioavailability, moderate to low
clearance, and moderate to long terminal
half-life are prioritized for further prof ling
in pharmacodynamics (PD) and eff cacy
studies. With a plethora of high-sensitivity
LC–MS–MS systems available today, blood
volume is no longer a critical determinant
of bioanalytical success, especially for
discovery PK studies. Despite these analytical
advances, the “one mouse, one time point”
study design described above remains
common practice.
The Promise of Dried Blood Spotting
Microsampling and, more specif cally,
the dried blood spot (DBS) technique Ph
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Standard curve for acetaminophen (mouse plasma)(a)
5.3
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200 400 600 800 1000 1200 1400 1600 1800
Analyte Conc./IS Conc. (ng/mL)
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Standard curve for acetaminophen (mouse whole blood)(b)
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500 1000 1500 2000 2500 3000 3500 4000 4500
Analyte Conc./IS Conc. (ng/mL)
50000
Figure 1: Standard curves for acetaminophen: Standards met the criteria of the analytical assay, all measured to within +/- 30% of their target values. R values for plasma and whole blood were 0.9975 and 0.9986, respectively. Standard curves were generated at the front-end and back-end of the study samples.
Kushon et al.
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GPC/SECTheory or practice?If there is one thing we can do, it’s both.
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have gained considerable attention as an
alternative to the conventional whole-blood
plasma workup used for PK analysis. There
are several compelling reasons for the
interest in DBS. First, with improvements
in LC–MS–MS technology, the blood
volume requirements for bioanalysis are
signif cantly smaller. It is somewhat ironic
that in the last 10 years, the sensitivity
of triple quadrupole mass spectrometers
(the “gold standard” for PK bioanalysis)
has increased 100× to 1000× relative to
earlier generation instruments while blood
sampling protocols have stayed the same
for many in vivo DMPK and toxicology
groups. Second, DBS, relative to plasma,
offers a simplif ed and cost-effective sample
collection, storage, and shipping process.
Third, and perhaps most importantly, DBS
offers the very real opportunity to reduce
the number of animals required for PK,
toxicokinetic (TK), PD, and eff cacy studies.
For example, many groups still perform the
conventional “one mouse, one time point”
PK study design. As shown in Table 1, for a
standard mouse PK study design, a total of
27 mice are required for each dosing group.
Microsampling blood collection enables the
use of far fewer animals because multiple
blood samples can be collected from the
same animal. The use of fewer animals also
eliminates inter-animal variability and is
therefore likely to produce more consistent
data. For PD and eff cacy studies, PK data
generally comes from satellite PK groups.
This is because the traditional larger
volume blood collections preclude the
ability to acquire this information from the
main study animals. The fact remains that
large volumes of whole blood continue to
be used out of habit rather than out of
necessity and this has translated into the
use and sacrif ce of more animals than
necessary.
With the clear benef ts of DBS and
microsampling, the legitimate question to
ask is why haven’t more groups in drug
discovery fully embraced the technology?
There are several reasons for this. One of
the major reasons is the well-documented
hematocrit bias of DBS cards. The blood
hematocrit (HCT), or the volume percentage
of the whole blood that is comprised of red
blood cells, inf uences the viscosity of blood,
and therefore dramatically inf uences the
extent to which a given volume of blood
spreads out onto a DBS card. High-HCT
blood will be viscous and tend to not
spread out across the card, while low-HCT
blood will be more f uid and will spread
out farther. Therefore, a given volume of
blood will generate different diameter spots
on the DBS card depending on its HCT.
In a typical DBS workf ow a small circular
Kushon et al.
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Kushon et al.2 News8 Tips & Tricks: GPC/SEC11 Ball1588 11RDPA 2015 Event Preview21 CHROMacademy22 Training and Events23 Staff252222 2323
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punch is removed from the overall spot for
analysis. The effective volume contained in
this subpunch is not uniform from sample to
sample because it is a function of the overall
spot size (which is controlled by the HCT of
the blood sample).
To reduce this HCT bias, whole-spot
collections can be made from a DBS
card. This approach provides a uniform
volume; however, it is diffi cult to automate
because of the varied spot sizes and the
fact that liquid handlers commonly found
in laboratories are not amenable to the
card format. Another reason for the slow
adoption of DBS is that procedures and
techniques for preparing animals for
microsampling must be developed
and routinized through training. In
Microsampling Group 2
Microsampling Group 1Plasma Group 1
2000
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800
600
400
200
0
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0 1 20.5 1.5 2.5
Figure 2: Overlay of concentration versus time profi le for microsampling group 1 (cardiac puncture, whole blood wicked onto device tips) and 2 (saphenous vein whole blood wicked onto device tips) and plasma group 1 (cardiac puncture, whole blood processed to plasma). The 5 min (0.08 h) microsampling group 2 time point shows slightly higher concentration than plasma and whole blood for study 1 animals.
Kushon et al.
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addition, sample manipulation, including
card punching, sample extraction, and
handling prior to bioanalysis is more
labour-intensive when using DBS cards
compared to the simple plasma crash
approach that is widely adopted and
effectively used by discovery groups to
generate PK in discovery.
Recently, a novel volumetric absorptive
microsampling (VAMS) approach has been
developed to simplify the blood collection
process and offer other advantages. An
inert, porous, hydrophilic material is used
to absorb a set volume of blood, which
rapidly “wicks” up the volume independent
of hematocrit. Extensive trials have shown
that over a wide range of HCT values (20%
to 70%) there is no HCT bias associated
with the 10 µL volume capture.1,2 Another
important differentiating feature of this
approach to dried matrix microsampling is
that it simplif es the post-sample collection
handling and sample extraction protocol.
Simple sample handling and extraction is
critical in the drug discovery environment
because time is of the essence when
screening compounds. In the following case
study we show how microsampling using
the VAMS approach can be applied to
simplify blood collection.
Case Study: Volumetric Absorptive
Microsampling (VAMS)
In the past, because of the HCT bias
issue, there were major concerns that a
dried matrix sampling technique could
not produce data leading to the same
decisions as those resulting from a standard
plasma PK data set. To investigate if the
volumetric absorptive microsampling (VAMS
approach could deliver comparable data,
PK prof les were generated and compared
for acetaminophen following intravenous
dosing in mice using a conventional design
(standard plasma processing, n = 21
animals) versus a dried whole-blood design
(VAMS microsampling, n = 3 animals for
entire time course).
Table 1: Standard PK study design.
Group No. Route Time Points No. of Rats No. of Mice
1 IVpre-dose, 0.08 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 8 h
N = 3 N = 27
2 POpre-dose, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 8 h, 24 h
N = 3 N = 27
Volume of blood drawn at each time-point = 250 µL, processed to plasma, stored at -80 °C prior to bioanalysis.
Method: Acetaminophen was formulated
in saline to a concentration of 5 mg/mL and
a 2 mg/kg dose was administered into the
tail vein of the mouse. In group 1, three
animals were sacrif ced at each time point.
A total of 21 mice were required for the
entire time course (predose, 0.08 h, 0.25 h,
0.5 h, 1 h, 2 h, and 4 h). Time points past
4 h were not necessary because of the
short half-life of acetaminophen in mice.
Group 2 animals received acetaminophen
through the same route of administration.
However, just three mice were used
in this study, with each animal bled at
every time point across the entire time
course. For group 1, at the specif c time
point, blood was harvested by cardiac
puncture. A Mitra microsampling device
(Neoteryx LLC) using VAMS technology
was dipped into the collected blood and
set aside to dry. The remaining blood
was processed to plasma and stored at
-80 °C until analysis. LC–MS–MS analysis
was performed on a Symbiosis HPLC
(Spark Holland) coupled to an API4000
QTRAP triple quadrupole ion trap mass
spectrometer (Sciex). Chromatographic
separations were performed using a 2 mm
× 50 mm, 5-μm Kinetex C18 column
(Phenomenex). D4-acetaminophen was
used as the internal standard. Mobile phase
A was water containing 0.1% formic acid.
Mobile phase B was acetonitrile containing
0.1% formic acid. The gradient was 1% B
to 70% B in 2.5 min following an initial
hold at 1% B for 0.5 min. For group 2,
blood was retrieved at the specif c time
point by saphenous vein sampling. Plasma
and dried whole-blood standard curves
were generated for acetaminophen over
the concentration range of 5–5000 ng/
mL, as shown in Figure 1. The r-value for
the mouse plasma and mouse whole blood
was 0.9975 and 0.9986, respectively. These
standard curves were then used to quantify
the plasma and whole blood exposures,
respectively, for acetaminophen following
2-mg/kg intravenous dosing.
Shown in Table 2 are the PK parameters
for group 1 plasma (conventional) and
Table 2: Standard PK study design.
Group No.AUC (0-t)(ng*h/mL)
CLs(mL/min/kg)
T½(h)
Vd(mL)
Plasma Group 1 481 70 0.62 1270
Microsampling Group 1 432 77 0.2 983
Microsampling Group 2 739 45 0.4 835
Kushon et al.
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t = 0.08 h was 1830 ng/mL, and at
t = 0.25 h was 1000 ng/mL. In the case
of group 1 animals, the concentration
of acetaminophen at the t = 0.08 h
time point was 1320 ng/mL. The 0.5 h,
1 h, and 2 h time points showed similar
whole-blood concentrations between the
two groups. The differences are likely not
attributable to the bioanalytical assay;
rather, they may more likely be explained
by the source of whole-blood retrieval
(saphenous vein versus cardiac puncture)
and/or the fact that the group 2 study
was conducted on a different day with a
different (and fresh) preparation of test
article. The PK parameters for group 2
are shown in Table 2. Consistent with the
higher exposure at the early time points,
the AUC was higher relative to group
1 and the clearance lower. Importantly,
the conclusions from this group 2 are
consistent with group 1 — that is, the
compound exhibits moderate to high
clearance, high volume of distribution,
short half-life and moderate exposure after
intravenous dosing of acetaminophen at
2 mg/kg.
Conclusion
The data presented in this article suggest
that volumetric absorptive microsampling
technology is a very useful collection
dried whole blood (VAMS). The area
under the curve (AUC), systemic clearance
(CLs), and volume of distribution (Vd)
were comparable. Only the terminal
(elimination) half-life was significantly
different (T½ = 0.6 h for plasma versus
0.2 h for dried whole blood). Importantly,
in the context of drug discovery, the
interpretation of data would be the same
— that is, the compound exhibits high
clearance, high volume of distribution,
and short half-life in the mouse. Given the
concordance between the mouse plasma
and mouse whole-blood pharmacokinetics
incorporating a conventional “one mouse,
one time point” paradigm, a group 2
study was performed. For group 2, the
pharmacokinetics of acetaminophen using
only three mice was evaluated by doing
serial bleeding via saphenous vein sampling
directly onto the microsampling devices.
Shown in Figure 2 is the VAMS
microsampling whole blood versus time
profile for acetaminophen incorporating
an n = 3 study design as compared
to group 1. The mouse group 2 dried
whole-blood concentration versus
time profile was similar to the group 1
result, the primary difference being the
measured concentration at the 0.08 h and
0.25 h time points. The acetaminophen
dried whole-blood concentration at
technique for pharmacokinetic studies
involving mice, greatly reducing the
animal number requirement and opening
the door to the possibility of combining
PK and PD and efficacy assessments in
the same study animals. This advance in
microsampling could quite possibly change
the “one mouse, one time point” paradigm
commonly found in early discovery
pharmaceutical development. Ultimately,
pharmaceutical companies will need to
decide whether the potential differences
in quantitative bioanalytical data are
truly meaningful and might present any
legitimate risk to the drug discovery and
development process as well as if those
differences outweigh the benefits that
dried matrix microsampling provide.
References
1. Neil Spooner, Philip Denniff, Luc Michielsen, et al.,
Bioanalysis 7(6), 653–659 (2015).
2. Philip Dennif and Neil Spooner, Anal. Chem.
86(16), 8489–8495 (2014).
Stuart Kushon currently holds the position
of senior research scientist at Neoteryx
LLC, a company dedicated to developing
novel microsampling solutions for the
pharmaceutical and clinical markets.
Kushon is a physical organic chemist with
over 10 years of experience developing
products for the direct detection and
analysis of targets including: viral, bacterial,
and protein pathogens as well as small
molecules that serve as diagnostic markers
for disease states.
Daniel Kassel founded SciAnalytical
Strategies in 2013, a bioanalytical,
biomarker discovery, and consulting
firm that serves the pharmaceutical,
biotechnology, and clinical industries.
Kassel also maintains a half-time
appointment as director of research and
development for InSource Diagnostics,
a research-driven clinical diagnostics
organization.
Hong Xin is a biotechnology entrepreneur
specializing in preclinical research with
extensive experience in contract research
and operations, with academic knowledge
across multiple disciplines including cancer
biology, cell biology, molecular biology
and pharmacology. Xin currently works at
Explora BioLabs as COO.
Nurith Amitai is a biomedical scientist
with 10 years of predoctoral and
postdoctoral training, who previously held
the position of Scientist I at Explora BioLabs
in San Diego, California, USA.
E-mail: [email protected]: www.neoteryx.com
Kushon et al.
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Microdestructive Capillary Electrophoresis Analysis of
Ink Samples
A novel microdestructive capillary electrophoresis (CE) method
for the analysis of blue pen ink strokes has been published in the
Journal of Chromatography A.1 The analysis of pen inks can be
challenging because ink compositions are usually under patent,
the samples are small, and inks can degrade over time.
CE has previously given promising results when applied to the
analysis of questionable documents, according to coauthor Matías
Calcerrada. He told The Column: “We believe that CE could be an
eff cient analytical technique for casework involving questioned
documents, due to advantages such as its minimal sample
consumption and ability to detect and quantify different analytes
found in ink formulations.”
The team developed a microdestructive sample treatment
method using a scalpel to scratch 0.3 mg of an ink stroke from
paper, prior to analysis using CE with a DAD detector. Samples
of ink from 34 blue pens from different technologies (ballpoint,
rollerball, marker) with different composition (gel, water-based,
oil-based) were analyzed to discriminate between different
technologies and inks. Calcerrada said: “We believe that the
results published demonstrate that the method could be applied
in forensic casework after applying other non-destructive
methodologies, which are always recommended before destroying
the sample.” — B.D.
Reference
1. Matías Calcerrada, Journal of Chromatography A 1400, 140–148 (2015).
Parabens Detected in Plastic Teething ToysParabens are commonly used in cosmetics and personal care products to protect against microbial growth, but a new study
published in the Journal of Applied Toxicology suggests that manufacturers may also be using parabens in plastic teething
products designed for infants.1 Scientists applied an effect-directed approach (to determine endocrine disrupting ability) to
analyze teething products prior to chemical analysis using gas chromatography coupled to mass spectrometry (GC–MS). Of
10 products were tested, two were found to contain endocrine disrupting chemicals (EDCs).
EDCs are natural or synthetic compounds that disrupt the normal functioning of the human endocrine system, often
imitating natural hormones, and are recognized as a contributing factor to a range of diseases. In light of this, the use of
phthalates in toys is restricted in the European Union to reduce childhood exposure. However, there is a huge variety of
possible EDCs used in plastics, meaning that exposure is likely to be underestimated because analysis focuses on common
culprits such as Bisphenol A and phthalates. Corresponding author Martin Wagner from Goethe University Frankfurt am
Main, Germany, told The Column: “Our research is about the EDCs we do not know yet: Instead of analyzing well known
compounds, we use in vitro bioassays to screen all types of samples for hormonal activity and try to identify the causative
chemicals using non-target chemical analysis afterwards (effect-directed analysis).” He added: “In that sense, our research
demonstrates that there are far more EDCs out there (which we currently overlook) and that they come
from unexpected sources (who would have suspected a plastic to contain parabens).”
Samples were taken from 10 plastic and one natural rubber teething soothers purchased
in Germany in 2012. Methanol extracts and water eluates from the samples were screened
using a Yeast Estrogen Screen (YES) and a Yeast Antiandrogen Screen (YAAS). One sample
was shown to possess both estrogenic and antiandrogenic activity, and another was
found to be antiandrogenic. GC–MS analysis showed the presence of ethyl-, methyl-, and
propylparaben in one product. Wagner told The Column: “In [the] case of one product we
found three different parabens. These are well-known EDCs, which are normally used
as preservatives in cosmetics.” He added: “In [the] case of the second product, we
demonstrated that this teether leached six different EDCs, all of which remain so
far unidentif ed. In a broader sense, our study demonstrates that babies will be
exposed to EDCs when chewing on plastic teethers. Because they are especially
susceptible, we should aim at minimizing their exposure to EDCs.” — B.D.
Reference1. E. Berger et al., Journal of Applied Toxicology DOI 10.1002/jat.3159 (2015).
8
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Identifying Fracking Contamination of Drinking Water Using GC×GC–TOF-MSFracking activity in the northeast of the
USA has rapidly increased over recent years,
with over 8000 Marcellus wells drilled in the
Marcellus Formation in Pennsylvania, USA,
alone.1 Fracking is attracting more media
attention because it opens up more natural
resources but has also been associated with
environmental contamination. Scientists
in Pennsylvania investigating an isolated
contamination incident have published
data from their study, demonstrating
the application of comprehensive 2D gas
chromatography coupled with time-of-f ight
mass spectrometry (GC×GC–TOF-MS) to
investigate fracking contamination in a
drinking water source.1
The study published in the journal PNAS
reports that in 2011 the Pennsylvania
Department of Environmental Protection
(PADEP) documented the contamination of
an aquifer used as a source of drinking water
with natural gas from Marcellus Shale gas
wells. In the year prior, three households
discovered white foam in their drinking water
wells drawn from an aquifer, and subsequent
analyses performed by regulators detected
natural gas contamination, thought to be
attributable to the drilling of new gas well
pads. The regulators were not, however,
able to determine the cause of the white
foaming. In 2012, the company responsible
for the gas well leak acquired the three
households affected by the drinking water
contamination.
Co-author Frank Dorman from Pennsylvania
State University told The Column: “We did
the work because we were initially interested
in the potential for source identif cation and
apportionment in the event of an accidental
discharge of the f uids used in the drilling
industry. Our opinion was that this needed
to be done in a ‘true discovery’ approach,
because there is really little to no disclosure
as to what exact chemicals are used at the
potential sites. This was especially true when
we started this work ~5 years ago.”
The team performed GC×GC–TOF-MS
analyses on water samples collected from
one of the originally contaminated wells, two
of the replacement wells that were drilled
as a replacement for the homeowners, a
natural spring, and water wells near the
contaminated area. GC×GC–TOF-MS analyses
detected an “unresolved complex mixture”
(UCM) of over 1000 different hydrocarbons
in the contaminated samples that was
similar to the UCM of hydrocarbons prof led
in f owback/production water provided by
other gas fracking companies in the area.
The commercially available shale gas-drilling
additive, 2–BE, was also detected at low levels
in the contaminated water samples.
Dorman told The Column that the UCM
“signature” detected in the f owback/
production and well samples could be used as
a diagnostic to detect the impact of shale-gas
operations on surface or groundwater. He
said: “We are continuing to work on being
able to quantitatively describe the hydrocarbon
signatures using GC×GC–HRTOF-MS
through the development of mass-defect
plots (Kendrick diagrams), and then
hopefully developing the ability to determine
goodness-of-f t of the hydrocarbon signature
of reference samples to f eld samples.”
In an FAQ document published by the
authors, they state that it is not possible to
“unambiguously” determine the source of the
2-BE or UCM in the drinking water, and that
the gas company responsible for the leak has
since made changes to gas well construction
practices.2
In terms of future work, Dorman explains
that the team are exploring the possibility of
bioremediation to remove organic content prior
to recycling of water at the well sites. — B.D.
References1. G.T. Llewellyn et al., PNAS 112(20), 6325–6330
(2015).
2. FAQ Document: http://www.
appalachiaconsulting.com/home/whats_new/
pnasarticlefaqs
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News In Brief
Like us Join us Follow Us
Sciex and New Objective Partner UpSciex (Massachusetts, USA) has announced a
partnership with New Objective (Massachusetts,
USA) that will enable Sciex to offer high
performance nanospray ionization technology
with their LC–MS systems. Gary Valaskovic, Ph.D.,
President and co-founder of New Objective, said:
“Partnering with Sciex is a great opportunity
for us to forge new relationships through their
market leading position in quantitative analysis.”
www.sciex.com
One-Step QuEChERS Sample PreparationThe sample preparation method Quick, Easy,
Cheap, Effective, Rugged, and Safe (QuEChERS)
offers high recovery, reproducibility, and lower
costs. A new study published in the Journal of
Chromatography A proposes a new strategy to
combine the two steps involved — extraction
and purif cation — into one magnetic solid-phase
extraction step.
DOI:10.1016/j.chroma.2015.04.021
Thermo Fisher Scientif c Expands in Middle EastThermo Fisher Scientif c (California, USA) has
opened a new Customer Experience Center
(CEC) at the life sciences hub DuBiotech in
Dubai. The 7000-square-foot facility will expand
the company’s ability to offer demonstrations
and training in the region, as well as enable
collaborations with universities in the region.
news.thermof sher.com
LCGC TV HighlightsPrinciples of In-Tube Extraction for
Headspace Sampling of Beer
Torsten C. Schmidt of the University Duisburg-Essen in Germany, recently developed an in-tube extraction (ITEX) method for headspace sampling of beer prior to gas chromatography–mass
spectrometry (GC–MS). In this short video, he explains the principles of the technique and its advantages over other methods. Watch Here>>
Luigi Mondello on the Fundamentals of 2D LCComprehensive liquid chromatography (2D LC) has the potential to increase peak capacity resolution when separating complex mixtures, especially in food analysis. Luigi Mondello from the University
of Messina, Italy, describes the fundamental principles of 2D LC, and explains the advantages over 1D LC.
Watch Here>>
Peaks of the WeekCurrent Trends in Mass Spectrometry Supplement: An Accurate-Mass Database for Screening
Pesticide Residues in Fruits and Vegetables by Gas Chromatography–Time-of-Flight Mass
Spectrometry — The main objective of this study was to evaluate the capabilities of gas
chromatography (GC) with time-of-f ight mass spectrometry (MS) for screening pesticides in fruits and
vegetables using a purpose-built accurate-mass database. Read Here>>
The LCGC Blog: Troubleshooting Retention Time Issues in Reversed Phase HPLC — While some
of the relationships between parameters in gradient HPLC are complex, others are reasonably
straightforward. Here, Tony Taylor explains, through an example, how adjusting the f ow rate can
improve resolution between critical peak pairs. Read Here>>
LC Troubleshooting: Calibration Problems — LCGC Europe columnist John W. Dolan discusses a case
study to show how to troubleshoot calibration problems in the laboratory. Data is presented from a
routine liquid chromatography method in a clinical laboratory. Read Here>>
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Tips & Tricks GPC/SEC: Answering Common Questions About GPC/SEC ColumnsDaniela Held and Wolfgang Radke, PSS Polymer Standards Service GmbH, Mainz, Germany.
The hardest part of any gel permeation chromatography/size-exclusion chromatography (GPC/SEC) separation is selecting the right
columns and developing a robust method. Here, we present a selection of commonly asked questions from users together with our
answers based on our experiences.
Gel permeation chromatography/
size-exclusion chromatography (GPC/SEC)
is performed to determine the complete
molar mass distribution. It can be applied
over a wide range of molar masses for
different types of natural and synthetic
macromolecules soluble in mobile phases
of very different polarities. GPC/SEC is
often used in quality control (QC), but
developing a robust and high-resolution
method that delivers precise results, which
are reproducible in the long-term, is a
challenging task. It is therefore of no surprise
that many users need expert advice when
making the choice of the optimum column
(set) from the large selection available. At a
recent event we were asked a lot of good
questions that we want to share here.
Q. When avoiding high backpressure
and shear is it suff cient to run the
GPC/SEC at a lower f ow-rate (so long
as you have enough time) or is larger
particle size the better choice?
A: Macromolecules can be very sensitive,
so forcing high molar mass or stiff polymer
chains through a liquid chromatography (LC)
system at a very high pressure can result in
chain degradation and generate results only
for the fragments. The overall pressure in
a system depends mainly on the f ow-rate,
mobile-phase viscosity, temperature, inner
diameter, number of columns applied, and
particle size of the column stationary phase.
Small particles should be avoided when
analyzing high molecular weights.1 It is
recommended to use larger particle sizes
when running very high molar mass samples
because this will reduce shear. Also note
that in this case column frits with larger
porosity are used and this further reduces
shear stress. For really high molar masses a
combination of both large particle sizes and
low f ow-rate is ideal, if time permits. If you
are using highly viscous solvents, running at
higher temperatures (to reduce mobile phase
viscosity) is also recommended.
For lower molar mass samples, where
high resolution is required (for example to
separate oligomers) the application of larger
particles to overcome back pressure issues
is not recommended. Larger particles result
in lower plate counts, thereby reducing
resolution. Nevertheless most small molar
mass samples will also prof t from higher
temperatures and lower f ow rates for highly Ph
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The Column www.chromatographyonline.com
viscous solvents because both approaches
increase resolution as a result of better mass
transport.
Q. Could you please comment on the
pros and cons of mixed bed column vs.
individual pore size columns?
A: This is a tough question because
philosophy plays a part here and there are
many aspects to consider.
Let us start with the often-mentioned
expectation of a “linear calibration
curve”. Many people seem to feel more
comfortable with linear relations, even
though the requirements for simple
mathematics has diminished with the
widespread use of computers in the
laboratory that can handle more complex
algorithms.
Unfortunately, the relation between the
logarithm of the molar mass and the elution
volume is not linear. GPC/SEC calibration
curves are typically sigmoidal in shape,
where the logarithm of the molar mass is
plotted versus the elution volume. Most of
the time polynomial functions of 3rd (cubic),
5th, or 7th order are used to f t the data.
This is a good approach as long as the slope
of the calibration curve is also reviewed to
avoid overf tting.2 The approach also allows
the full use of the complete separation
range of the column. Please note that linear
columns are also non-linear at the high and
low molar mass end, therefore a different
f tting approach is required if samples elute
in that region.
Linear or mixed-bed columns are the result
of intense work by column manufacturers.
The production involves either a special
synthesis route or, much more often, the
careful blending of individual pore sizes. The
main advantage of linear columns is that
they can separate over a wide molar mass
range with a constant resolution, and are
ideal for routine QC or as screening columns
if users have to deal with very different
molar masses. You can easily increase the
resolution by adding other linear columns
of the same type. However, it is very
diff cult to alter the molar mass separation
range when higher or lower molar masses
need to be separated. The risk of porosity
mismatch is extremely high, for example
when combining linear columns with
individual columns ideally suited for oligomer
separations.3
The main advantage of individual pore-size
columns is that they provide a highly
eff cient separation but in a limited molar
mass range. Individual pore size columns
are therefore often combined in column
banks. Columns can be added and removed
to alter the molecular weight to tailor it to
the application and the time requirement.
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When following this approach an additional
advantage is that the column with the
largest porosity can be used f rst to separate
the high molar mass (and therefore high
viscous) chains to avoid viscous f ngering.
The best column type is therefore very
dependent on the requirements of the
laboratory.
Q. Do you expect polymeric columns
to be stable and tolerant to switching
eluents? Or do you like to have a
column set devoted to a particular
solvent and then switch columns
when switching solvents? What if
you repeatedly change between pure
tetrahydrofuran (THF) and THF with
small amounts of additives, such as
acids or amines?
A: In general, with solvents that are of a
similar polarity to the packing material —
such as THF, chloroform, dichloromethane, or
toluene for styrene-divinylbenzene columns —
exchanging the solvents should not harm the
columns. However, it is advisable to exchange
solvents slowly at reduced f ow-rates of
0.3–0.5 mL/min. The solvent leaving the
column should go directly to the waste and
the detectors should be disconnected. There
is no reason in principle not to exchange
solvents; however, time might be an issue
because completely re-establishing swelling
equilibrium after going from one solvent to
another often takes longer than reaching a
stable RI-baseline.
For solvents that differ substantially in
solvent polarity from the column material,
we recommend using different columns
because of the different swelling of the gel.
We even recommend ordering such columns
in the solvent of use. Please note that in many
of these cases a different stationary phase
polarity might be the better choice to avoid
interactions.4
For the exchange between pure solvent and
solvent with additives (amines, acids, salts)
we do not see any problems with switching
solvents back and forth.
In any case, columns should be stored in
pure solvents without additives (no salts,
amines, acids). The exception is columns for
aqueous applications where a small amount of
methanol or NaN3 (0.05 g/L) should be added
to avoid growth of algae.
Q. Does changing solvent composition
by addition of salts or other co-solvents
require recalibration of the detectors?
Should the standards be run with the
same modif er as you use for your
sample?
A: Calibration is always an issue in GPC/SEC.
There are two types of calibrations that can
be applied:
Tips & Tricks: GPC/SEC
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Conference Office: Symporg SA - Rue Rousseau 301201 Geneva / SwitzerlandTel. +41 22 839 84 [email protected]
Venue: International Conference Center in Geneva (CICG) - www.cicg.ch
Conference Chair: Prof. Gérard Hopfgartner, University of Geneva
www.hplc2015-geneva.org
HPLC 2015Geneva, Switzerland
High Performance
Liquid Phase Separations
& Related Techniques
21 - 25 June 2015
The program will be built around three main themes.A) Core Separation Technology, understanding the fundamental aspects to drive innovationB) Multidimensional separations, mass spectrometry, pushing the limits in separation, detection, identification and data processingC) High Impact Sample Preparation, Separation and Detection, on the edge of current and future applications
ES625647_LCTC060515_013.pgs 06.01.2015 21:23 ADV blackyellowmagentacyan
The Column www.chromatographyonline.com
E-mail: [email protected]: www.pss-polymer.com
1. Nearly all users perform a column
calibration where they measure the
elution volumes of calibration standards
with different molar masses and plot
the logarithm of the molar mass (or size)
against the elution volume to construct
a calibration curve (see also question
above).
2. In some cases, users need to also
calibrate their detectors in a high
performance liquid chromatography
(HPLC) type of detector calibration.
This is often required when
doing multi-detection GPC/SEC
(triple-detection, light-scattering, or
viscometry) or when they want to
determine concentrations or perform
copolymer analysis. In these cases
different concentrations are measured to
determine the detector responses.
In both cases we always recommend to
apply the same conditions for calibration
as for operation. So the standards should
be run with the same modif ers or with
the same co-solvents, and standards and
samples should be prepared from the solvent
bottle that also supplies the pump.
Q. Do you f nd some salts more/less
corrosive to the instrument than others?
For example, dimethylformamide (DMF)
with LiBr is hard on the instrument, but
gives good results.
A: Halides are usually more corrosive than
other salts, and chlorides are more aggressive
than bromides. Unfortunately, LiCl and LiBr
have better solubilities than other lithium
salts in commonly applied organic solvents;
in addition, because lithium is more superior
in breaking down aggregates than other
counter ions, there are not too many options.
In aqueous solvents, neutral salts like
nitrates or sulphates can be applied instead
of NaCl to reduce the danger of corrosion.
The main reason for salt addition is to shield
electrostatic interaction and reduce the
breakdown of aggregates.
An important factor when using salt
solutions is to ensure that the system always
runs with fresh solutions and that it is not
left to stand for a long time. Always apply a
low f ow-rate to avoid salt crystallizing out
and heavy corrosion. If the system is not
in use for a long period of time it should
be transferred into pure solvent before
switching it off.
Summary
As with other analytical techniques, small
details can have a big impact in GPC/SEC.
Selecting the best column option and
performing proper method development
is def nitely time-consuming, but can save
much more time afterwards. A stable,
robust method is much cheaper in the long
run than choosing the wrong settings in
the beginning. It is therefore important to
always ask questions and so we encourage
all readers to share their questions with us
because there are a lot more answers that
can help users make educated choices.
References
1. D. Held, The Column 10(10), 12–15 (2014).
2. D. Held, The Column 4(6), 18–21 (2008).
3. T. Hofe, The Column 4(4), 20–23 (2008).
4. T. Hofe and G. Reinhold, The Column 12, 30–33
(2007).
Daniela Held studied polymer chemistry
in Mainz, Germany, and works in the
PSS solutions department. She is also
responsible for PSS webinars, education
programmes, and customer training.
Wolfgang Radke studied polymer
chemistry in Mainz, Germany, and
Amherst, Massachusetts, USA, and is
head of the PSS application development
department. He is also responsible for the
PSS customer-training programmes, and for
customized trainings.
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Using GPC/SEC for Excipient Characterization
The properties of polymeric excipients can directly affect the clinical eff cacy, safety, and quality of a f nished pharmaceutical product and are routinely identif ed as critical quality attributes (CQAs). This article looks at how gel permeation/size-exclusion chromatography (GPC/SEC) can be applied to measure characteristics such as molecular weight (MW), MW distribution and structure, and degree of branching. Case study data for the measurement of poly-lactic acid (PLA) and poly-lactic glycolic acid (PLGA) highlights the detailed information that can be accessed.
Stephen Ball, Malvern Instruments, Malvern, UK.
Polymeric excipients are an important
addition to the sophisticated tableting
blends of today. Ingredients such as
poly-lactic acid (PLA), poly-lactic glycolic acid
(PLGA), and hydroxyl methyl cellulose and
its derivatives enable formulators to achieve
closely controlled drug release prof les
using a growing range of manufacturing
techniques that includes spray drying,
hot-melt extrusion, and lipid-based drug
delivery. The properties of these polymers
can directly affect the clinical eff cacy, safety,
and quality of the f nished pharmaceutical
product and are therefore often identif ed
as critical quality attributes (CQAs). CQAs
for polymer excipients typically include
molecular weight (MW), MW distribution,
and structural characteristics such as degree
of branching.
Gel permeation/size-exclusion
chromatography (GPC/SEC) is a powerful
technique for the characterization of
polymers and other macromolecules. In this
article we examine its application in the
analysis of polymeric excipients. Case study
data for the measurement of PLA/PLGA
highlights the detailed information that can
be accessed.
The Vital Role of Excipients
The workf ows associated with the
development of oral solid dosage forms,
whether innovator or generic, are
increasingly well established and are rooted
in a Quality by Design (QbD) approach.1
These workf ows emphasize the need for
detailed characterization of the excipient, as
well as the active ingredient, both alone and
within the blend. The resulting information
supports the development of a detailed
understanding of how the drug product
will behave and of a specif cation for each
component that will ensure successful drug
delivery and the necessary quality control.
Traditionally excipients have been used
simply as “bulking agents” — in this case
the impact of excipient properties on the
safety, eff cacy, and quality characteristics of
the drug product may be relatively limited.
However, in modern formulations polymeric
excipients often play a far more active role
controlling the drug delivery prof le and
other aspects of drug product performance.
Polymers are now routinely used to:2
• Formulate coatings that control the
rate of dissolution of the tablet in the
stomach.
• Develop spray-dried solid dispersions for
the delivery of sparingly soluble drugs.
• Improve blend ý ow properties.
• Tailor the taste and texture of tablet for
improved patient acceptability.
• Improve the stability of tablets
containing moisture-sensitive active
ingredients.
Precisely differentiating between grades
of excipient, choosing an optimal candidate,
and ensuring the consistency of supply
is critical to the manufacturing process;
however, it can be complicated by two
factors. Firstly, a number of pharmaceutical
excipients are derived from natural polymers,
such as naturally occurring celluloses, which
can limit the manufacturer’s ability to control
polymer properties. There is a choice of
different grades and sources but only a
limited ability to precisely tailor features
such as branching and MW distribution. The
second factor stems from the way in which
the pharmaceutical industry is structured. An
active ingredient often undergoes substantial
development in-house, including the
rigorous investigation of all CQAs, resulting Ph
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in a complete understanding of how the
drug substance behaves and, ideally, a
detailed def nition of the structure-function
relationships that def ne its performance.
In contrast an excipient tends to be bought
in, sometimes from multiple suppliers,
making it diff cult to secure supplies that
enable a rigorous investigation of the
effect of excipient properties. For example,
assessing the impact of branching relies
on sourcing excipients with different
degrees of branching. Furthermore, once an
excipient has been identif ed it can be quite
challenging to scope and control the degree
of variability associated with the supply.
Therefore, although a polymer excipient
may be a more straightforward chemical
entity than the active ingredient, and indeed
have a less direct impact on drug product
performance, there are unique challenges
associated with its characterization.
An Introduction to GPC/SEC
GPC/SEC is a two-step analytical technique
where samples are f rst separated into
fractions on the basis of hydrodynamic size
(by passing through a packed chromatography
column), which are subsequently characterized
using one or more detectors. Measuring
the amount of sample in each sized fraction
50
1
0
10 20
106
0.01
0.1
1
105
10-3
10-4
3.0039E-5
104
1000
100
10
0.2227
1
0
30
0
1
100
0
-3.4617 -0.5150
5.7551
-1.0494 -110.4509
40
30
20
Vis
com
ete
r -
DP (
mV
)
Refr
act
ive in
dex
(mV
)
Mo
lecu
lar
weig
ht
(Da)
Intr
insi
c vi
sco
sity
(d
L/g
)
Low
an
gle
lig
ht
scatt
eri
ng
(m
V)
Rig
ht
an
gle
lig
ht
scatt
eri
ng
(m
V)
10
Figure 1: A chromatogram and derived data for a PLA sample dissolved in THF demonstrating the excellent data quality achieved.
Ball
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detailed certificates of analysis • acknowledged by regulatory authorities world
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Pharmaceutical impurities
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enables determination of the size distribution,
and more importantly MW and MW
distribution data.
Traditional GPC/SEC systems use a
single-concentration detector, typically
a refractive index (RI) detector. With this
set-up, column calibration with appropriate
standards provides the correlation needed
to estimate a relative (to the standard)
MW distribution. These MW data are
only accurate if the relationship between
molecular size and weight is the same for
the sample as it is for the standard. This is a
crucial limitation especially when gathering
precise data for the detailed comparison of
relatively similar excipients, and when the
calibration standards are sub-optimal for the
polymers of interest.
The use of multiple detectors can directly
address this limitation — for example, a
light-scattering detector in combination
with a concentration detector enables
the direct measurement of absolute MW
with minimal, non-specif c calibration.
Further complementary additions include
a viscometer to enable the measurement
of structural features such as branching or
conformation.
GPC/SEC systems with sensitive
multi-detector characterization
capabilities are consequently useful for
QbD applications. The following case
study demonstrates the application of
multi-detection GPC/SEC for the analysis
of PLGA and PLA, polymers that are used
routinely in pharmaceutical formulation.
Case Study: Analyzing PLA/PLGA
Samples
Produced by polymerizing lactic acid or
lactic and glycolic acid respectively, PLA
and PLGA are used to formulate controlled
drug release systems and to manufacture
medical components such as absorbable
surgical thread and implants.3 Derived from
renewable and natural resources, they are
commonly classif ed as “green polymers”
because of their biodegradability and
biocompatibility. The properties of PLGA
can be controlled by varying the ratio of
lactic to glycolic acid used in its production
to manufacture polymers with different
MW, MW distribution, and structure.
Multi-detection GPC/SEC can be applied
to measure and sensitively compare the
properties of the resulting materials.
Method: Samples of PLA and of PLGA
were analyzed using an OMNISEC GPC/
SEC system with triple detector array (RI,
light-scattering, and viscometer detectors) (all
Malvern Instruments). Tetrahydrofuran (THF)
was used as the solvent for the samples
and as the mobile phase. The analyses were
performed at 30 oC using samples prepared
Ball
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LIVE WEBCAST: Wednesday, July 15, 2015 at 8 am PDT/ 11 am EDT/ 4 pm BST/ 5 pm CEST
Register free at www.chromatographyonline.com/lcgc/pesticide_residue_analysis_series
Series Part 4 Event Overview:Safeguarding the environment and the global food supply requires continuous monitoring of more and more compounds. Pesticide residues analysts are chal-lenged to detect, identify, and quantify hundreds of diferent pesticides with a fast turnaround time (often within 24 hours of receipt). Data processing and analysis can become a bottleneck. Method development, data analysis and pro-cessing can be simplifed with the use of comprehensive compound databases, pre-determined methodologies, and intuitive software. This webinar will provide pesticides residue analysts with valuable information on software method development and data processing for the analysis of pesti-cide residues in food for both LC–MS and GC–MS. Technical experts will review the latest in software advances to help with data interpretation and reporting. They will guide you through industry-relevant trends and new techniques that allow you to see more, do more, and be more productive. Example workfows will be shown, along with data analysis using the latest in compound databases and spectral libraries.
Who Should Attend:
n Researchers and analysts in pesticide analysis
n Food scientists interested in learning the latest technologies for sample preparation of food matrices
n Anyone struggling with sample preparation challenges for pesticide residue analysis in food
Series Moderator
Richard Fussell, Ph.D.Global Vertical Marketing Manager, Food and Beverage, Chromatography & Mass Spectrometry Division, Thermo Fisher Scientifc
Presenter:
Charles YangSenior Marketing Specialist, Environmental and Food Safety, Thermo Fisher Scientifc
LCGC Moderator:
Laura Bush: Editorial Director, LCGC
Key Learning Objectives:
n Learn how to quickly set up acquisition methods with TraceFinder software
n Proceed to rapidly analyze data with integrated software workfows
n Learn about the latest in comprehensive compound databases and libraries for triple-quadrupole and HRAM LC and GC mass spectrometry
For questions, contact Kristen Moore at [email protected]
Register free at www.chromatographyonline.com/lcgc/pesticide_residue_analysis_series
Sponsored by
Presented by
Part 1: Sample Prep Tips and Tricks Using QuEChERS and Accelerated Solvent ExtractionON-DEMAND WEBCAST, originally aired March 24, 2015
Part 2: Workflow Guide for the use of LC-MSON-DEMAND WEBCAST, originally aired April 29, 2015
Part 3: Maximizing Analysis Efficiency through New GC-MS ApproachesON-DEMAND WEBCAST, originally aired June 17, 2015
Part 4: Latest Developments & Future Directions in Data Processing & Analysis Software for LC-MS/MS & GC-MS/MSWed., July 15, 2015 at 8 am PDT/ 11 am EDT/ 4 pm BST/ 5 pm CEST
Register for the Pesticide
Residue Analysis Webinar Series
Register for the Pesticide
Residue Analysis Webinar Series
Latest Developments and Future Directions in Data Processing & Analysis Software for LC–MS-MS and GC–MS-MS
ES625665_LCTC060515_017.pgs 06.01.2015 21:28 ADV blackyellowmagentacyan
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and measured at concentrations in the range
1–5 mg/mL.
THF is a common solvent for many GPC/
SEC applications that readily dissolves both
PLA and PLGA; however, the RI sensitivity (dn/
dc) for this sample/solvent combination is low
(approximately 0.045 mL/g to 0.051 mL/g).
This means that any change in polymer
concentration induces a relatively small change
in RI, requiring a very sensitive RI detector to
measure concentration changes under these
conditions. Likewise, dn/dc values also impact
the response of light-scattering detectors.
A compromise was therefore forced on
the analyst by this dn/dc issue. They would
either have to analyze the samples at a high
concentration, which overloads the SEC
columns and distorts the MW distribution
obtained, or have to switch to an alternative
solvent, normally acetone, which is far less
suitable for the proper dissolution of the
sample. However, the results gathered here
using a multi-detector GPC/SEC approach
demonstrate that it is suff ciently sensitive to
enable the use of the preferred THF solvent.
Results: Figure 1 shows typical
chromatograms for a PLA sample, with
traces from each detector in the array, and
derived values of MW and intrinsic viscosity
(IV) plotted as a function of retention time.
Excellent data quality is seen across all the
measurements made with each detector, as
3.162
Molecular Weight (Da)
PLGA 5050 low mid MW
PLGA 5050 low mid MW
PLGA 5050 high mid MW
PLGA 5050 high mid MW
PLGA 6535 mid MW
PLGA 6535 mid MW
PLGA 7525 high MW
PLGA 7525 high MW
Intr
insi
c vis
cosi
ty (
dL/
g)
0.3162
1.0569
0.1
104 3.164 105 3.165 106149.863
Figure 2: M-H plots for the four PLGA samples with different lactide:glycolide compositions show that changes in composition are associated with differences in the structural characteristics of the polymer.
Ball
18
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ES625664_LCTC060515_018.pgs 06.01.2015 21:27 ADV blackyellowmagentacyan
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evidenced by the clean stable baseline and the
smooth well-defi ned signal peaks. Similar data
quality is also observed in the measurement of
the PLGA samples (data not shown).
Using the MW and IV data generated by
these measurements it is possible to construct
a Mark—Houwink (M-H) plot, a logarithmic
plot of IV against MW, to investigate the
structural differences between samples. IV is
inversely proportional to the molecular density
in solution so both the gradient and intercept
of a M-H plot reveal information about the
structural characteristics of a polymer. Three
different types of PLGA sample were measured
to investigate the impact of composition on
structure. These included copolymers with
lactide:glycolide compositions of 50:50;
65:35; and 75:25, respectively. For the 50:50
copolymer, two samples were analyzed that
had different overall MW, a low-to-mid MW
sample, and a mid-to-high MW sample. This
gave four samples in total and for each one
duplicate injections were made on the GPC/
SEC system to check repeatability.
The results indicate that increasing the
amount of lactide in the polymer decreases
its coil density in solution. Samples with
a 75:25 lactide:glycolide ratio exhibit the
highest IV at any given MW while those
that have a 50:50 composition have a
much lower IV, at an equivalent MW. This
means that any change in the copolymer
composition will change the molecular
structure of the polymer. More practically,
the data provide insight as to why these
polymer samples will behave differently,
thereby supporting the manipulation of
polymer properties to control drug delivery
performance. It is also worth noting that
this easy differentiation of copolymer
content by the M-H plot is independent
of the molecular weight, as shown by the
consistent, overlapping plots of the two
50:50 samples with different molecular
weights. In other words, the exact excipient
MW distribution and structure (composition)
can be determined, or compared to a
reference, in a single analysis.
Table 1: Data from multiple injections of the same PLGA sample.
Parameter Average Value Unit RSD (%)
Mn 26,102 g/mol 2.32
Mw 44,722 g/mol 0.37
Mz 67,009 g/mol 1.22
(η)w 0.36 dL/g 1.37
Rh.w 6.07 nm 0.50
Ball
19
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Learn about a 9-min, sensitive (LOQ 1—50 ng/mL) method to quantitate 30 immunosuppressant
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ES625687_LCTC060515_V19.pgs 06.01.2015 21:29 ADV blackyellowmagentacyan
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can also allow formulators to gather the
information needed to fully scope excipient
performance, and to swiftly specify an
optimal excipient for any given drug product.
References
1. “Analytical techniques with a place in the oral
solid dosage formulation toolkit’ Whitepaper
available for download at: http://www.malvern.
com/en/support/resource-center/Whitepapers/
WP141223AnalTechOralToolkit.aspx
2. A. Siew, Pharmaceutical Technology Europe 39(1),
(2015).
3. M. Chaubal, Drug Delivery Technology 2(5), (2002).
Stephen Ball is Product Marketing Manager,
Nanoparticle and Molecular Characterization,
at Malvern Instruments. He holds a degree
in computer aided chemistry from the
University of Surrey, UK, which included
a year in industry working as a research
chemist for the Dow Chemical Company
in Horgen, Switzerland. Before joining
Malvern Instruments, he worked for Polymer
Laboratories as an applications chemist,
before taking on a marketing position as
a product manager for light scattering
instrumentation at Agilent Technologies.
In an extension of the study, multiple
injections of the same PLGA sample were
performed to directly assess reproducibility.
Table 1 shows data from 10 repeat injections,
each of 100 µL, for a PLGA sample
containing 50% lactic acid, measured at a
concentration of 2.132 mg/mL. A relative
standard deviation of 0.53% for the 10
adjacent samples demonstrates the excellent
repeatability obtained. Such repeatability with
automation substantially lightens workload
associated with the rigorous and extensive
experimentation needed to adopt QbD.
Conclusion
The performance of many sophisticated
pharmaceutical products relies on the use
of polymeric excipients. The MW, MW
distribution, and structural characteristics
of such excipients def ne their behaviour
and are therefore identif ed routinely as
CQAs for the product. GPC/SEC is an
established technique for the measurement
of these properties and as a result has an
important role to play in pharmaceutical
formulation. The technique when applied
with multiple detection systems can provide
high sensitivity, which enables the precise
differentiation of excipient grades and
greater f exibility in solvent choice, and
highly automated repeatable measurement,
for eff cient, high productivity analysis. It
E-mail: [email protected] Website: www.malvern.com
Ball
20
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ES625675_LCTC060515_020.pgs 06.01.2015 21:28 ADV blackyellowmagentacyan
Recent Developments in Pharmaceutical Analysis (RDPA 2015)
E-mail: [email protected]
Website: rdpa2015.chimfarm.unipg.it
Ph
oto
Cre
dit
: R
aq
ue
l Lo
na
s/G
ett
y I
ma
ge
s
A symposium on the Recent
Developments in Pharmaceutical
Analysis (RDPA 2015) will be held at
the University of Perugia, Perugia,
Italy, from 28 June to the 1 July 2015.
The Scientific and Organizing
Committees invite you to RDPA 2015,
which will be held in Perugia, Italy,
a beautiful city with an outstanding
architectural heritage.
The programme will include plenary
and keynote lectures, as well as oral and
poster presentations on a wide range
of topics including: advanced methods
and instrumentation; hyphenated
techniques; fundamentals (theories,
retention models, chemometrics); (bio)
pharmaceutical analysis; food analysis,
nutraceuticals, functional food, natural
products; proteomics, glycomics,
metabolomics; biomarker discovery;
and sample preparation, validation,
quality by design, and data processing.
Participation of young researchers,
both from industry and university,
will be facilitated by low registration
fees.
Great opportunities to meet colleagues
in informal discussions will be made
easy by an attractive location, taking
advantage of a city that offers a
multitude of cultural, historical, and
artistic attractions, all at walking
distance from the symposium venue.
A look to the upcoming symposium on the Recent Developments in Pharmaceutical Analysis
(RDPA 2015), which will be held 28 June to 1 July 2015 in Perugia, Italy.
21
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ES625494_LCTC060515_021.pgs 06.01.2015 20:24 ADV blackyellowmagentacyan
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Website: http://anthias.co.uk/training-
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Gas Chromatography: Fundamentals, Troubleshooting, and Method Development8–11 September 2015
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Eu
rop
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