NDELA.pdf

A method for the determination of

N-nitrosodiethanolamine in personal care

products – collaboratively evaluated by the CTPA

Nitrosamines Working Group

C. Flower*, S. Carter�, A. Earls�, R. Fowler�, S. Hewlins§, S. Lalljie–, M. Lefebvre**, J. Mavro**,

D. Small�� and N. Volpe��*Cosmetic Toiletry and Perfumery Association, London, U.K., �Consumer Safety Department, LGC Limited, Teddington,

Middlesex, U.K., �Quality Department, Boots Manufacturing, Nottingham, U.K., §Procter and Gamble Technical Centre,

U.K. Limited, Egham, U.K.,–Safety and Environmental Assurance Centre, Unilever Colworth, Sharnbrook, Bedford, U.K.,

**Advanced Research – Analytical Chemistry – Physical & Chemical Sciences, L’Oreal Recherche, Aulnay sous Bois,

France, ��STIEFEL Laboratories, High Wycombe, Buckinghamshire, U.K. and ��Christian Dior, Technical R & D,

Laboratoire Chimie Fine, St Jean de Braye Cedex, France

Received 3 June 2005, Accepted 23 September 2005

Keywords: cosmetics, Griess reaction, N-nitrosodiethanolamine, post-column photolysis, reverse-phase

liquid chromatography, solid-phase extraction

Synopsis

A procedure for the determination of N-nitrosodi-

ethanolamine (NDELA) in personal care products

was evaluated in collaborative studies by member

organizations of the United Kingdom’s Cosmetic

Toiletry and Perfumery Association (CTPA) and

LGC Limited, formerly known as the Laboratory of

the Government Chemist (LGC). Samples were pre-

pared depending on the matrix of the cosmetic

product: aqueous samples were prepared by dilu-

ting in water followed by solid-phase extraction;

emulsions, oils and solid materials were dissolved

in dichloromethane and extracted with water.

NDELA was separated from the sample matrix

using reverse-phase liquid chromatography. The

N-nitroso bond was cleaved by photolysis to give

nitrite, which was colorimetrically quantified. The

nitrite functional group reacted with sulphanila-

mide in an acid medium to form a diazonium ion

which was then coupled with N-(1-naphthyl)ethy-

lenediamine dihydrochloride according to the Gri-

ess reaction to give a purple-coloured azo dye that

absorbed at 540 nm. Compared with other pub-

lished methods for NDELA, the method described

here is quick and easy to use. It has the required

sensitivity and specificity, and can accurately and

reliably quantify NDELA in a wide range of perso-

nal care product matrices.

Resume

Une methode de dosage de la N-nitrosodiethanol-

amine (NDELA) dans les produits cosmetiques a

ete evaluee, au cours d’etudes menees en collabor-

ation avec les representants du CTPA (United

Kingdom’s Cosmetic Toiletry and Perfumery

Association, et du LGC (Laboratory of the Govern-

ment Chemist, maintenant denomme LGC Lim-

ited). Les echantillons sont prepares en fonction de

la matrice du produit cosmetique a analyser. Les

produits dispersables dans l’eau sont prepares par

dilution dans l’eau, suivie d’une extraction sur

phase solide. Les produits non dispersables dans

Correspondence: Chris Flower, Cosmetic Toiletry and Per-

fumery Association, Josaron House, 5-7 John Princes

Street, London W1G 0JN, U.K. Tel.: +44 20 7491 8891;

fax: +44 20 7493 8061; e-mail: [email protected]

International Journal of Cosmetic Science, 2006, 28, 21–33

ª 2006 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie 21

l’eau ; emulsions, huiles ou echantillons solides sont

dissous dans le dichloromethane, la NDELA est en-

suite extraite par l’eau. La NDELA est separee des

autres constituants de la matrice par chromatograp-

hie liquide en phase inverse. La liaison N-nitroso en

sortie de colonne chromatographique est coupee

par photolyse a 254 nm pour donner du nitrite. Le

nitrite est ensuite quantifie colorimetriquement. Le

nitrite forme reagit avec la sulfanilamide en milieu

acide pour donner un ion diazonium. Celui-ci est

ensuite couple avec le N-(1-naphthyl)ethylene-

diamine dichlorhydrate selon la reaction de Griess

pour donner un colorant azoıque pourpre detecte

a 540 nm. Compare aux autres methodes de dos-

age publiees pour la NDELA, la methode decrite

est rapide et facile a mettre en oeuvre. Cette meth-

ode est sensible et specifique, elle est capable de

quantifier la NDELA de facon reproductible et

fiable dans une large gamme de produits cosme-

tiques.

Introduction

N-nitrosamines such as N-nitrosodiethanolamine

(NDELA) are potent animal carcinogens that

induce cancer by a ‘genotoxic’ mechanism. Indeed,

it has been demonstrated that nitrosamines are

carcinogenic in more animal species than any

other category of chemical carcinogen [1].

Although there are no adequate data from epi-

demiology studies to confirm such effects in

humans, it is prudent to regard nitrosamines as

potential human carcinogens that act by a geno-

toxic mechanism. When assessing the risks from

genotoxic carcinogens it is the widely adopted pol-

icy of regulatory authorities to make the assump-

tion that such compounds have the potential to

damage DNA at any level of exposure, and that it

is impossible to identify a ‘safe’ level. However, it

is recognized that exposure to low levels of nitrosa-

mines is unavoidable. Cosmetics represent a relat-

ively minor route of exposure, the main routes

being food and the environment.

Nitrosamine contamination of cosmetics may

result through the use of nitrosamine-contaminated

cosmetic ingredients or through the nitrosation of

precursors (principally secondary amines) present

in finished cosmetic products by nitrosating agents

such as nitrite or oxides of nitrogen. Under certain

conditions, nitrosation of diethanolamine yields the

nitrosamine, NDELA, a polar, non-volatile, N-nitro-

samine compound.

Within the European Community, the Fifteenth

Commission Directive relating to cosmetic products

(92/86/EEC) [2] formally prohibits the marketing of

cosmetic products that contain nitrosamines. An

allowance is made for trace levels if they are techni-

cally unavoidable, so long as the product cannot

cause damage to human health when applied

under normal or reasonably foreseeable conditions

of use. Essentially, this requires nitrosamine levels

to be kept as low as reasonably practicable,

although no specific level has been set for finished

cosmetic products. However, this Directive also set

a limit of 50 lg kg)1 (ppb) for the N-nitrosodialka-

nolamine content of fatty-acid dialkanolamides,

monoalkanolamines and trialkanolamines used as

raw materials in the manufacture of cosmetics.

A similar limit (50 lg kg)1) has been set for the

N-nitrosodialkylamine content of fatty-acid dialkyl-

amides, monoalkylamines and trialkalyamines and

their salts because the properties of these com-

pounds are similar to their respective alkanolamine

analogues with respect to their potential as precur-

sors of nitrosamine formation (2003/83/EC) [3].

Cosmetics manufacturers are also required not

to use the above-mentioned raw materials with ni-

trosating systems. Essentially, this means that

ingredients that contain or release nitrite ions,

should not be used in conjunction with these raw

materials, unless effective inhibition systems are

employed. In addition, the use of ‘secondary dialk-

anolamines’, e.g. diethanolamine, that have been

shown to be a major source of nitrosamine con-

tamination in cosmetics, has also been banned.

Because of their perceived carcinogenic potential

in several animal species, minimization of exposure

to N-nitrosamines from cosmetic products is import-

ant for the protection of public health. It is therefore

essential to have methods of analysis that can deter-

mine N-nitrosamines with the required sensitivity

and specificity in cosmetic matrices.

Screening methods for apparent total nitrosa-

mines have been published [4–6] but they suffer

from false-positive results. Specific analytical meth-

ods for each possible N-nitrosamines should not be so

prone to false-positive results but performing a num-

ber of analyses on each sample would not be prac-

ticable, even if such methods had been developed.

In practice, the most likely source of N-nitrosa-

mines and their precursors for cosmetic products

are the dialkanolamines used in the production of

dialkanolamides, of which diethanolamide is the

most widely used in cosmetic products. The corres-

ª 2006 International Journal of Cosmetic Science, 28, 21–3322

Determination of NDELA in personal care products C. Flower et al.

ponding N-nitrosamine, NDELA, is also generally

regarded as one of the more potent carcinogens

amongst the N-nitrosamine family of chemicals.

For that reason, there is a requirement for analyt-

ical procedures that are capable of reliably detect-

ing and quantifying NDELA in cosmetic products

and their associated raw material.

Analytical method

Existing methodologies for the determination

of NDELA

To date, two generic approaches have been used

for the determination of NDELA.

• The first involves extraction of NDELA from the

sample matrix, followed by derivatization. The

extracted NDELA is derivatizated, the separation

of the analyte from matrix components is

achieved by gas chromatography then the deri-

vatizated NDELA is detected by using a range of

detectors [electron capture, electrolytic conduc-

tivity, mass spectrometry or thermal energy ana-

lyser (TEA)] [7–9].

• The second approach involves the extraction of

NDELA from the sample matrix, followed by

separation by liquid chromatography (LC) and

detection using a TEA [10].

Both of these approaches involve lengthy sample

preparation steps and have potential for false posi-

tives, including in-situ formation of NDELA.

A commonly used procedure for the determin-

ation of N-nitrosodialkanolamines in cosmetics is

based on the method described by Sommer and

Eisenbrand [7]. The analysis involves the extrac-

tion of NDELA and other N-nitrosodialkanolamines

from the sample matrix by solid-phase extraction

on kieselguhr and further clean-up on a silica gel

column, followed by a derivatization of the N-nitr-

osodialkanolamine (silylation of the dried eluate

from the column). After, the trimethylsilyl deriva-

tives of N-nitrosodialkanolamines are determined

by gas chromatography – TEA. Like the other

methods, a major drawback with this procedure is

its complexity and the potential it presents for

in-situ formation of N-nitrosodialkanolamine. Analy-

sis of nitrosamines in cosmetic products containing

the corresponding precursors involves the risk of

artefactual nitrosamine formation during the

clean-up procedure. Particularly, the solid-phase

extraction is a critical step because traces of NOx

adsorbed on the kieselguhr column might cause

substantial nitrosamine formation. Losses during

clean-up are possible and recoveries should be

checked by the addition of an internal standard

with properties similar to NDELA. Suitable com-

pounds are N-nitrosodiisopropanolamine and

N-nitroso(2-hydroxyethyl)(2-hydroxypropyl)amine.

Screening for N-nitroso compounds in cosmetic

products

Several procedures for the determination of ‘total’

N-nitroso compounds have been described [4, 5].

Most are based on the denitrosation of N-nitroso

compounds and subsequent measurement of the

liberated NO using a chemiluminescence detector.

The analytical procedure currently employed was

published in the International Journal of Cosmetic

Science in 1995 [6]. This procedure was chosen

because it is highly sensitive and responds to a

wide range of N-nitroso compounds. Furthermore,

it is the method recommended by the United King-

dom’s Cosmetic Toiletry and Perfumery Associ-

ation for the determination of ‘total’ nitrosamines

in personal care products. In this method, all

N-nitrosamines, irrespective of their volatility, are

determined as a group to yield a ‘total’ nitrosa-

mine content calculated with respect to an

authentic nitrosamine calibration standard.

The procedure is a validated screening tech-

nique that provides an estimate of the ‘total’ con-

centration of all N-nitroso compounds present in a

cosmetic sample. Nitrosamines are determined

quantitatively, and the method is not prone to

false-negative results. However, there is some

potential for interference from non-N-nitroso

compounds including, for example, nitrite salts

and esters, nitrate salts, thionitrites, thionitrates,

nitrolic acids, peroxides and iron oxides, as well as

nitro-substituted hair dyes. Because the absence of

potential interferences cannot easily be shown, the

result obtained in the determination of ‘total’

N-nitroso compounds is commonly referred to as

the ‘apparent total N-nitroso compounds’ content.

Apart from N-nitrosamines, N-nitrosamides will

also be determined, but most other nitroso and

nitro species should give only a weak response.

These include C-nitroso and C-nitro compounds,

N-nitro compounds (nitramines) and nitrites, of

which the latter are decomposed prior to

introduction into the denitrosation agent. It is

strongly recommended that confirmatory analysis

of ‘positive’ results is undertaken.

ª 2006 International Journal of Cosmetic Science, 28, 21–33 23


Proposed method for the determination

of N-nitrosodiethanolamine

Shuker and Tannenbaun described a method that

utilizes liquid chromatography with colorimetric

detection of the nitrite generated by cleavage of

the nitroso compounds through post-column pho-

tolysis detection for nonvolatile N-nitroso com-

pounds in biological fluids [11]. Light from a high

intensity discharge lamp photolyses N-nitroso com-

pounds in aqueous solution to give nitrite ion,

which is determined colorimetrically with Griess

reagent in a post-column reactor. Methods by Pig-

natelli et al. [12] and Bellec et al. [13] also rely on

LC separation and subsequent photohydrolysis of

NDELA to form nitrite. The liberated nitrite can

then be reacted with sulphanilamide to give a

diazo intermediate which can be coupled with

N-(1-naphthyl)ethylenediamine dihydrochloride

(NED) to form a purple azo dye that can be deter-

mined spectrophotometrically.

As N-nitrosamines can easily undergo photolyti-

cally induced de-nitrosation, this three-step chem-

ical reaction process of photolysis, diazotization and

the coupling reaction (Fig. 1) has the potential to

form the basis of an appropriate method for the ana-

lysis and detection of N-nitroso compounds such as

NDELA in cosmetic products and raw materials.

An alternative procedure based on the Shuker

and Tannenbaun method and utilizing the three

steps described above was developed by a member

organization of the United Kingdom’s Cosmetic Toil-

etry and Perfumery Association (CTPA) Nitrosa-

mines Working Group for the quantitative

determination of NDELA in cosmetic matrices and

cosmetic raw materials. In this procedure, NDELA

and other N-nitrosodialkanolamines are separated

from the cosmetic matrix by reverse-phase liquid

chromatography. The N-nitroso bond is cleaved by

UV photolysis with the formation of nitrite ion.

According to the Griess reaction, the nitrite is diazo-

tized with sulfanilamide in an acid medium and

then coupled with NED to form a purple azo dye

that is quantitatively determined spectrophotomet-

rically at 540 nm.

The presence of NDELA can be confirmed by

repeating the analysis without photolysis (no nitrite

ion is then produced because the N-nitroso bond is

not cleaved). The absence of a chromatographic

peak at the retention time of NDELA in the chroma-

togram confirms that the peak observed in the first

analysis is NDELA (Fig. 2a and b). In order to avoid

any possible doubt in the identification of the peak

at the retention time of NDELA, an irradiation at

254 nm followed by one at 366 nm gives a very

specific determination for N-nitrosamines (constant

ratio).

This paper presents the findings of two studies

organized by the United Kingdom’s CTPA Nitrosa-

mines Working Group to evaluate collaboratively

this alternative method for the determination of

NDELA in personal care products. The method

was assessed for accuracy (recovery of NDELA

from spiked samples at different concentration lev-

els) and precision (the inter-laboratory variability).

Experimental

Test samples

In the first collaborative trial, the test samples con-

sisted of aliquots of a shampoo spiked at four

different levels (0, 20, 50, 100 ng mL)1 [ppb])

and a sample of cocodiethanolamide (unspiked).

NNO

C2H4OH

C2H4OH

+ H2Ohv

254nmHN

C2H4OH

C2H4OH

+ HNO2

H2NO2S NH2 + 2H+ + NO2-

H+

H2NO2S N N + 2H2O

H2NO2S N N +

NHC2C4NH2

H2NO2S NN NHC2H4NH2

Photolysis

Diazotization Reaction

Coupling Reaction

Azo dye

Figure 1 Reaction of nitrite with Griess Reagent to form an azo dye.



Additionally, a solution containing only NDELA

was prepared at three concentration levels [20,

50, 100 lg mL)1 (ppm)] and sent blindly to parti-

cipants.

In the second collaborative trial, six different

types of personal care products were prepared and

fortified with NDELA at a range of levels against

which the method could be evaluated, viz. a sham-

poo (0, 20, 50, 100 lg kg)1), a make-up remover

milk (0, 20, 50, 100 lg kg)1), a moisturizing

lotion (0, 50 lg kg)1), a cucumber cleansing

lotion (0, 50 lg kg)1), a wheat germ hair condi-

tioner (0, 20, 50, 100 lg kg)1) and a hair dye (0,

50 lg kg)1). In addition, test samples of a com-

monly used cosmetic ingredient, cocodiethanola-

mide (unspiked), and standard NDELA solutions

(20, 50, 100 lg mL)1) were prepared and tested.

Each sample was well mixed to ensure homo-

geneity, divided into portions, placed in amber-col-

oured (to protect against light) sample bottles and

add as in the first collaborative trial, was sent

blindly to participating laboratories for assay. After

distribution, the samples were refrigerated by the

participants prior to NDELA analysis.

Reagents and reactants

High-performance liquid chromatography (HPLC)-

grade dichloromethane, methanol, orthophosphoric

acid 85%, ammonium acetate, potassium chloride,

sulfanilamide and N-(1-naphthyl)ethylenediamine

dihydrochloride (components of the Griess reagent)

were obtained from Fisher Scientific (Loughbor-

ough, U.K.) and Sigma-Aldrich (Gillingham, U.K.).

N-nitrosodiethanolamine was supplied by Sigma-

Aldrich. Sep-Pak� 386 C18 cartridges were

supplied by Waters Ltd (Elstree, U.K.). The C18

Spherisorb ODS II column was supplied by Waters

Ltd and the C18 Aqua phase column was supplied

by Phenomenex (Macclesfield, U.K.).

Griess reagent

N-(1-naphthyl) ethylenediamine dihydrochloride

(0.25 g) was dissolved in deionized water and

made up to 250 mL in a volumetric flask; 4.0 g

sulfanilamide was dissolved in 250 mL of a 5%

aqueous solution of 85% orthophosphoric acid.

The reagents were mixed together in an amber

Photolysis at 254 nm

Without photolysis

100.0

100.5

101.0

101.5

102.5

103.0

Response

0 5 10 15 20 Retention time

NDELA

100.0

100.5

101.0

101.5

102.0

102.5

103.0

0 5 10 15 20 Retention time

(a)

(b)

Figure 2 (a) Chromatogram of a finished product spiked with 60 ppb of NDELA in water and with photolysis. (b)

Chromatogram of a finished product spiked with 60 ppb of NDELA in water and without photolysis.



glass bottle, stoppered and refrigerated when not

in use. Maximum shelf life was 5 days.

Preparation of N-nitrosodiethanolamine standard

solutions

A stock standard solution containing 1 mg mL)1

of NDELA was prepared. This solution was diluted

serially with deionized water to prepare working

standard solutions covering the concentration

range of 0–20 ng mL)1 (ppb). Working standard

solutions were freshly prepared.

Sample preparation

The clean-up of cosmetic products for the NDELA

analysis depends on the dispersibility of the

samples in water. If the cosmetic product is

dispersible in water, solid-phase extraction must be

applied to the sample. If the cosmetic product is

not dispersible in water but in dichloromethane,

then the dichloromethane clean-up must be

applied. Therefore, the dispersibility of the cosmetic

matrix in water or dichloromethane must be

established before a clean-up procedure is chosen.

Solid-phase extraction clean-up for samples that

are dispersible in water (e.g. lotions, bath foams,

shampoos)

Exactly 2.0 g of sample was weighed and dispersed

in 20 mL water and centrifuged for 10 min at

3600 g. The Sep-Pak� 386 cartridge was condi-

tioned with 2 mL methanol followed by 2 mL of

water. The cartridge was not allowed to dry out;

5 mL of the sample solution was loaded on to the

Sep-Pak� 386 cartridge and the first 2 mL of

eluant was discarded. The following 3 mL was

collected for chromatographic analysis. When

necessary, the collected solution was filtered

through an appropriate filter.

Dichloromethane clean-up for samples that are

not dispersible in water (e.g. oils, lipsticks)

Exactly 0.4 g sample was weighed and dispersed

in 4.0 mL dichloromethane; 4 mL water was

added, shaken and then centrifuged at 19 400 g

for 10 min. The upper aqueous layer was retained

for chromatographic analysis. With both type of

sample matrices, the final extract was analysed for

NDELA by HPLC with UV photolysis and Griess

reaction under the conditions specified in Table I.

Apparatus

The instrumental apparatus for the analysis com-

prises of five principal components:

• Chromatographic system

• Post-column photolysis reactor

• Chemical reactor

• Detector UV–Vis

• Data processing station.

Table I Chromatographic condi-

tions and equipment for the deter-

mination of NDELA in personal care

products

LC pump (1) Pumped equipped with a pulse damper

Column type 150 cm · 4.6 mm, packed with 5 lm Spherisorb ODS II

Pre-column type 1 cm · 4.6 mm packed with 5 lm Spherisorb ODS II

Injection volume 50 or 100 lL sample loop

Mobile phase 0.02 m aqueous ammonium acetate (pH 6.8)

Mobile phase flow rate 0.5 mL min)1

Griess Reagent flow rate 0.5 mL min)1

Temperature of

post-column reactor

50�C

Detection wavelength 540 nm

Photolysis unit (2) Photochemical reactor comprising a ‘high-pressure‘

knitted Teflon reactor coil, 5 or 6 m · 0.3 mm i.d.

placed around a low-pressure UV lamp emitting

at 254 nm (Beam Boost and Knauer Photoreactors)

Griess Reagent

delivery (3)

Low-pressure pump without pulsation, a second LC

pump with the same characteristics as the first may be used

Post-column reactor (4) Column oven (or water bath) containing a low dead-volume

mixing Tee connected to a 1 mL knitted

reactor coil, 5 m · 0.3 mm i.d.

Detector (5) UV/visible detector equipped with a tungsten lamp,

sensitivity 0.001 or 0.0005 AUFS (Shimadzu SPD-10AV).



A schematic illustration of the instrumental con-

figuration for the analysis of NDELA is shown in

Fig. 3. The chromatographic conditions and equip-

ment for the determination of NDELA in personal

care products are summarized in Table I.

Calibration and calculation

The external standard calibration method was

used to quantify the concentration of NDELA pre-

sent in a test sample. A least-squares linear regres-

sion of peak areas versus concentration of the

NDELA reference standard (express in ppb) was

constructed. The linear relationship of the increas-

ing concentrations of the NDELA reference stand-

ard (expressed in ppb) versus their corresponding

peak areas is expressed as y ¼ ax + b, where the

slope of the regression line is represented by a and

b is the intercept. From the chromatogram

obtained with the formulation under investigation

SF is the peak area of NDELA.

The concentration, C (lg kg)1) (ppb) of NDELA

in the formulation under investigation is calcula-

ted from the following equation:

Cðlg kg�1Þ ¼ SF � b

a� V

m

where SF is the peak area of NDELA in the

formulation under investigation, b the y-intercept

of the calibration curve, a the slope of the

calibration curve, m the weight of the formulation

under investigation (g), V is the volume of the

tested solution (mL).

Statistical treatment of data

Summary statistics were calculated for each sample

and spike combination in order to assess the variab-

ility across the laboratories. The mean, the standard

deviation and the coefficient of variation (CV) were

calculated. The CV is a statistical measure of the

deviation of the values from the mean. The smaller

the CV value, the less is variation from the mean.

Grubb’s test was used to identify whether there

were any outliers across the laboratories for each

sample and spike combination. Grubb’s test identi-

fies observations that may be considered outliers

because they are significantly higher than the oth-

ers in the data set. Where outliers were identified,

these results were excluded from the statistical

analysis.

In the cases where the level of NDELA found

was quantified as less than a threshold value, i.e.

limit of quantification, ‘nd’ is recorded in the data

tables. These results were excluded from the statis-

tical analysis.

Remarks concerning the method

Before the collaborative trial, some laboratories

investigated various method parameters. The fol-

lowing are some of the key elements of the

method that were assessed for effect on perform-

ance.

In the ring-trialled method we used a 0.02 m

aqueous acetate buffer at pH 6.8 as mobile phase,

a Spherisorb C18 LC column and an NED concen-

tration of 1 g L)1. Detection of the azo complex is

made at 540 nm. The concentration of NED was

not found to be critical.

The use of sulfamic acid (nitrite scavenger) was

investigated in samples which produced nitrite-

generating species. After treatment with acid, the

pHs of the solutions were re-adjusted to that of the

mobile phase with an ammonia solution before

HPLC analysis.

Pump(Griess Reagent)

Pump(HPLC)

Photolysis unit 254 nm

Heater 50 °C

Detector540 nmLow volume

mixing T

Figure 3 Schematic illustration of the instrumental configuration for the analysis of NDELA.



A limit of detection of 10 ng mL)1 (10 ppb) was

achieved initially by optimizing parameters such

as flow rate and composition of mobile phase, resi-

dence time of NDELA in photolysis unit and post-

column reactor, and flow rate of NED reagent. It

became obvious that the tungsten lamp source in

the UV–Vis detector gave better performance, sta-

bility and sensitivity at 540 nm than a deuterium

lamp. A limit of detection of 1 ng mL)1 (1 ppb) for

a standard NDELA solution was achieved with the

tungsten lamp. Figure 4(a) and (b) shows chroma-

tograms of 2.34 and 11.71 ppb of NDELA stand-

ard in water.

An improvement in the separation of NDELA

from nitrite was obtained using either a longer

Spherisorb column or a column with an ‘Aqua’

C18 stationary phase. Compared with the condi-

tions already mentioned above, both of these col-

umns increased the retention and separation of

NDELA from nitrite (Fig. 5a–c).

Under aqueous conditions, the octadecyl side-

chains of conventional C18 columns can collapse.

This can result in loss of retention and poor col-

umn performance. The C18 ‘Aqua’ phase, how-

ever, is end capped with a hydrophilic (polar)

reagent, which allows water in the eluent to ‘wet’

the silica and prevents the C18 side-chains from

collapsing. The ‘Aqua’ packed column was also

found to show greater stability over a longer time

period than a conventional C18 column. Prior to

analysis, the LC column must be allowed to equili-

brate.

Figure 5 (a) Spherisorb 5 lm ODS2 (150 · 4.60) mm with 5 lm ODS2 (10 · 4.60) mm guard column, (b) aqua

5 lmC18 (150 · 4.60) mm with Aqua 5 lmC18 (30 · 4.60) mm guard column, (c) Spherisorb 5 lmODS2 (250 · 4.60)

mm.

100.00

100.05

100.10

100.15

Response

0 2 4 6 8 10 12 Retention time

100.00

100.05

100.10

100.15

Response

0 2 4 6 8 10 12 Retention time

NDELA

NDELA (a)

(b)

Figure 4 (a) Chromatogram of a 2.34 ppb NDELA standard in water. (b) Chromatogram of 11.71 ppb NDELA standard

in water.



It should be noted that through out the analyt-

ical procedures, all solutions must be protected

from light. Only amber-coloured vials or alumin-

ium foil-coated volumetric flasks should be used.

In the discussion that follows, the two collaborat-

ive studies are referred to as Study 1 and Study 2.

Results and discussion

Validation of analytical method

As part of their in-house validation, one of the

participating laboratories investigated the repeata-

bility and recovery of the method. The repeatabili-

ty for this method describes the precision expected

from a set of replicate measurements made by a

single laboratory with the same analyst and the

same equipment. The standard deviation is used to

calculate a repeatability value r.

Repeatability

Repeatability (Table II) was determined for both

the Sep-Pak� and dichloromethane extraction pro-

cedures by analysing shampoo samples containing

known amounts of NDELA. A CV of 9.2% was

obtained using Sep-Pak� cartridges and 15.1%

after dichloromethane extraction. Repeatability of

the chromatographic system and post-column:

photolysis and Griess reaction technique was

calculated with measurements at 2 ng mL)1 (CV

10.7%), 4 ng mL)1 (CV 5.8%), 8 ng mL)1 (CV

3.7%) and 20 ng mL)1 (CV 3.7%).

Recovery

Recoveries at 10 ng mL)1 were also determined for

both the Sep-Pak� and dichloromethane extraction

procedures (Table II). Good agreement was obtained

for both procedures. The small differences between

the recoveries, 80–92% for Sep-Pak� Sep-cartridges

and 93–98% for dichloromethane extractions, can

be attributed to variations in the fractions collected

for analysis from the C18 Sep-Pak� cartridge.

Linearity

In order to quantify the concentrations present, a

least-squares linear regression of peak area against

Table II Repeatability of the chro-

matographic system, extraction

methods and method recovery

Repeatability of the chromatographic

system (standard solutions)*

Mean (ng mL)1) CV (%)

NDELA

20 ng mL)1 20.3 3.7

8 ng mL)1 7.89 3.7

4 ng mL)1 3.85 5.8

2 ng mL)1 1.91 10.7

Repeatability of extraction methods shampoo� Mean (lg kg)1) CV (%)

Sep-Pak� cartridges, NDELA (100 lg kg)1) 91.1 9.2

Dichloromethane extraction, NDELA (20 lg kg)1) 13.9 15.1

Method recovery� Recovery (%) CV (%)

Sep-Pak� cartridges, NDELA recovery (10 ng mL)1) 82.4 11.9

Dichloromethane extraction, NDELA recovery (10 ng mL)1) 96.1 1.9

*Repeatability was determined at four different concentrations by analysing six replicate

injections at each concentration.

�The repeatability (n ¼ 6) of the extraction method was also determined with shampoos of

known concentration of NDELA (100 and 20 lg kg)1).

�The recovery of both extraction methods was determined at 10 ng mL)1.

0

200

400

600

800

1000

1200

0 5 10 15 20 25NDELA concentration (ppb)

Pea

k ar

ea (

mV

)

Figure 6 Calibration graph showing good linearity of

response of NDELA over the 1–20 ng mL)1 (ppb) range.



concentration was constructed using the external

standard calibration method. Good linearity of

response was achieved over the 1–20 ng mL)1

(ppb) range with coefficient of correlation (r2)

>0.999 (Fig. 6).

Limit of detection and limit of quantification

The limit of detection for a standard solution of

NDELA using the method is 1 ng mL)1 (1 ppb).

Results of a validation exercise indicates a spread

in the limit of quantification in a range of prod-

ucts, including lipstick, sunscreens and hair sham-

poos, between 10 and 40 lg kg)1 (10–40 ppb).

Evaluation of personal care product matrices

Additional investigations were undertaken on dif-

ferent sample matrices including sunscreens,

shampoos, lipsticks and hair conditioners; some of

them were more difficult to analyse than others.

Recoveries from these products were determined

after spiking samples with NDELA with the follow-

ing results: sunscreens (73%), hair shampoos

(83%) and lipsticks (80%). Emulsion formation

required centrifugation of the sample solution

before analysis by liquid chromatography. Sulpha-

mic acid was added to remove nitrite.

The sample preparation of hand and face lotions

and bubble baths was performed by aqueous disso-

lution. Lipsticks and lip-gloss products were extrac-

ted with dichloromethane by water. An additional

clean-up procedure was applied to sunscreen prod-

ucts where persistent emulsions formed. Addition

of methanol and potassium chloride to the water/

dichloromethane solution followed by vigorous

agitation for 30 s removed the emulsion. For per-

sistent emulsions, solutions were centrifuged at

19 400 g for 10 min.

Less interference was encountered with oil-based

samples, presumably because much of the organic

content is removed with dichloromethane. One

sample, a shampoo, was found to contain a meas-

urable amount of NDELA (>1000 lg kg)1). This

result was confirmed by analysing without photo-

hydrolysis (cleavage of the N-nitroso bond to give

nitrite). No response was observed at the retention

time of NDELA in the chromatogram.

Collaborative trials

In Study 1, to explore the potential of the method,

six participating laboratories analysed a shampoo

and the same shampoo spiked at three levels [20,

50, 100 lg kg)1 (ppb)]. Also analysed were a cos-

metic raw material, cocodiethanolamide and

standard solutions of NDELA at three different con-

centration levels [20, 50, 100 lg mL)1 (ppm)].

Table III shows the results of Study 1. Consider-

ing the low level of measurement, acceptable

Table III Results of the first colla-

borative study (Study 1) for the

determination of NDELA in sham-

poos spiked at four levels, a cos-

metic raw material and standard

solutions of NDELA at three different

concentration levels

Sample

description

NDELA spike

level, lg kg)1

(ppb)

NDELA measured, lg kg)1 (ppb)

Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6

Shampoo 0 <25 <10 38 <10 nd <10

20 49 20 nd nd 20 20

50 47 54 51 67 31 47

100 141 103 94 102 88 103

Cocodiethanolamide 0 <20 17 <40 nd 28 18

NDELA solution 20 000 16 900 18 900 18 700 21 600 20 600 18 400

50 000 39 300 44 300 50 300 39 500 39 600 43 600

100 000 82 500 94 800 92 700 80 500 100 400 94 800

Table IV Inter-laboratory variability (precision) for the

samples tested in the first collaborative trial

Sample

NDELA spike

level, lg kg)1

(ppb) Mean SD CV %

Shampoo 20 27.2 14.5 53

50 49.5 11.7 24

100 105.2 18.6 18

Cocodiethanolamide 0 21.0 6.1 29

NDELA solution 20 000 19 300 1800 9

50 000 42 600 4700 11

100 000 90 100 8400 9



agreement was obtained between the laboratories

for both the spiked shampoo and the standard

solutions. CV at all spike levels is shown in

Table IV. The highest CV (53%) was observed with

the shampoo spiked at the 20 ppb level. However,

this 20 ppb spike was below the limit of quantifi-

cation for two of the six laboratories. The CVs for

the NDELA solution were about 10% for all three

spiked levels, in line with the reproducibility stand-

ard deviation predicted by the Horwitz [14] func-

tion at these levels.

In general, these results indicated that the

method has the potential to recover NDELA at

very low concentrations from cosmetic matrices

and the CV also indicates the robustness of the

method. The raw material proved more difficult.

However, it should be noted that fatty-acid dialka-

nolamide as a sample matrix is difficult to analyse

by the other approaches described earlier.

As a result of these promising initial findings, it

was decided to carry out a second collaborative

study with a wider range of matrices. In the sec-

ond study, five laboratories analysed a total of six

personal care products (shampoo, make-up

remover milk, moisture lotion, cucumber cleansing

lotion, wheatgerm hair conditioner, hair dye) that

were spiked with NDELA at 20, 50 and

100 lg kg)1 (ppb). A raw material (cocodiethanol-

amide) was also analysed in duplicate for NDELA.

In addition, three separate standard solutions con-

taining 20 000, 50 000 and 100 000 lg kg)1

(ppb) of NDELA were also analysed. Each sample

was analysed in duplicate.

Grubb’s test was used to identify whether there

were any outliers across the laboratories for each

sample and spiked combination. In view of the fact

that the data set was very small and the outliers

identified by the Grubb’s test were all from the

same laboratory, the ranking test was used to con-

firm the exclusion of all results from this laboratory.

Results of Study 2 are shown in Table V. The

data indicate that the method satisfactorily deter-

mined NDELA from five of the six spiked formula-

tions. Excellent results were obtained by four of

the five laboratories1 for the five spiked formula-

tions. Acceptable results were obtained for the

three standard solutions (at relatively much higher

concentrations). Recoveries of NDELA obtained by

the five laboratories at the different spike levels are

shown in Table VI and the CV for all matrices at

the different spike levels and the standard solutions

are shown in Table VII.

Table V Results of the second colla-

borative study (Study 2) for the

determination of NDELA in sham-

poos, make-up remover, cosmetic

raw material, moisture lotion,

cucumber cleansing lotion, wheat-

germ hair conditioner, hair dye, and

standard solutions of NDELA at

three different concentration levels

Sample description

NDELA spike

level, lg kg)1

(ppb)

NDELA measured, lg kg)1 (ppb)

Lab 1 Lab 2 Lab 3 Lab 4 Lab 5

Shampoo 0 <10 nd 38 <10 nd

20 26 20 100 15 23

50 51 50 298 58 56

100 93 101 85 104 115

Make-up remover milk 0 <10 nd nd <20 nd

50 44 52 123 <20 46

Cocodiethanolamide 66 52 nd <50 50

Moisture lotion 0 <10 nd 0 <10 nd

50 73 55 99 70 66

Cucumber cleansing lotion 0 <10 nd 0 <10 nd

50 72 56 109 61 74

Wheatgerm hair conditioner 0 <10 nd 0 <10 nd

20 30 27 83 21 34

50 63 61 71 47 79

100 114 135 189 104 143

Hair dye 0 130 nd nd nd –

50 163 90 nd nd –

NDELA solution 20 150 16 300 20 100 18 000 18 800 20 000

50 370 45 000 50 600 62 000 47 700 53 000

100 750 68 500 100 600 132 000 93 400 110 000

1The results of laboratory 3 were excluded fromthe statistical analysis on the basis of the rankingtest.



Again, the data confirms the good accuracy, pre-

cision (agreement between laboratories) and robust-

ness of the method observed in Study 1. However,

the hair dye sample proved to be a difficult matrix to

analyse because of matrix interference. Each of the

laboratories had difficulty with this matrix. The

results from the collaborative trial are not in agree-

ment with the expected amount of 50 lg kg)1.

Sample clean-up is the most critical step in the

analytical procedure for NDELA. This study shows

that not all types of personal care products can be

analysed using the same sample preparation,

chiefly because of varying matrices: for example,

lotions, shampoo, conditioner and hair dye are

water-soluble and were therefore mixed with water

before extracting through a Sep-Pak� 386 cart-

ridge. The presence of NDELA in cocodiethanol-

amide was determined by dissolving the material in

dichloromethane followed by extraction into water

and centrifuging at 19 400 g.

The results of these collaborative trials indicate

that NDELA was satisfactorily recovered, identified

and quantified by all but one of the participating

laboratories. It should be noted that prior to the

first ring trial most of the participating laborator-

ies were not familiar with the method and had to

purchase the instrumentation and set up the

method in the laboratory in order to participate

in the collaborative trial.

Yet, in general, there was broad agreement in

the results obtained by the laboratories. This

clearly demonstrated the relative simplicity and

potential of the method. The different analytical

equipment used by the participating laboratories,

lack of experience with the method and detector

sensitivity can explain the differences in the results

obtained.

Conclusions

The good inter-laboratory agreement between par-

ticipating laboratories confirmed that the method

is sensitive, robust and gives good accuracy and

Table VI Results of the recoveries

of NDELA obtained by the five

laboratories during the second colla-

borative study (Study 2)Sample description

NDELA spike

level, lg kg)1

(ppb)

% NDELA recovered

Lab 1 Lab 2 Lab 3 Lab 4 Lab 5

Shampoo 20 130 100 (500) 75 115

50 102 100 (596) 116 112

100 93 101 85 104 115

Make-up remover milk 50 88 104 (246) nd 92

Moisture lotion 50 146 110 198 140 132

Cucumber cleansing lotion 50 144 112 218 122 148

Wheatgerm hair conditioner 20 150 135 (415) 105 170

50 126 122 142 94 158

100 114 135 189 104 143

NDELA solution 20 150 81 100 89 93 99

50 440 89 100 123 95 105

100 700 68 105 131 93 99

Results in parenthesis are highly suspect.

Table VII Inter-laboratory variability (precision) for the

samples tested in the second collaborative trial (Study 2)

Sample

NDELA spike

level, lg kg)1

(ppb)

Mean,

lg kg)1

(ppb) SD

CV

(%)

Shampoo 20 21.0 4.7 22

50 53.8 3.9 7

100 99.6 9.1 9

Make-up remover milk 50 47.3 4.2 9

Cocodiethanolamide ) 56.0 8.7 16

Moisture lotion 50 66.0 7.9 12

Cucumber

cleansing lotion

50 59.7 9.3 16

Wheatgerm hair

conditioner

20 24.5 4.8 20

50 55.2 7.9 14

100 113.2 15.6 14

NDELA solution 20 150 18 800 1390 10

50 440 49 100 2800 16

100 700 93 100 1680 18

The hair dye sample was excluded because of the difficulty the

laboratories had in analysing this matrix.



precision. It has been shown to be applicable to a

range of different cosmetic formulation matrices

and has the ability to confirm the presence or

absence of NDELA without the sophistication of

mass spectrometry, or the technical expertise

required for mass spectrometric detection. It is

therefore acceptable for determining NDELA in a

broad range of personal care products. The

method described employs a relatively rapid sam-

ple preparation providing faster analysis than is

possible with existing methodologies, and can be

adapted for routine analysis of large numbers of

samples. Further work is required to extend the

method to complex formulations such as hair dyes

and lipstick.

Acknowledgements

The authors wish to express their sincere appreci-

ation to the analysts from the participating Labor-

atories: Unilever (U.K.), Boots Contract

Manufacturing (U.K.), L’Oreal (France), Christian

Dior (France), and LGC Limited (U.K).

The authors also wish to express their appreci-

ation to LGC Limited for providing the data on

their in-house validation of the method.

References

1. International Agency for Research on Cancer. Mono-

graphs on the Evaluation of the Carcinogenic Risk of

Chemicals to Human Beings, Vol. 17. IARC, Lyon

(1978).

2. European Commission Directive (92/86/EEC). Official

J. Eur. Communities, L325. 18–22 (1992).

3. European Commission Directive (2003/83/EC).

Official J. Eur. Communities, L 238. 23–27 (2003).

4. Waters, C.L., Downes, M.J., Edwards, M.W. and

Smith, P.L.R. Determination of non-volatile nitrosa-

mines on a food matrix. Analyst 103, 1127–1133

(1978).

5. Chou, H.J., Yates, R.L. and Wenninger, J.A. Screen-

ing cosmetics products for N-nitroso compounds by

chemiluminescence determination of nitric oxide.

JOAC 70, 960–963 (1987).

6. Challis, B.C., Colling, J., Cromie, D.D.O. et al. A

screening procedure for total N-nitroso contaminants

in personal care products: results of collaborative

studies undertaken by a CTPA Working Group. Int. J.

Cosmet. Sci. 17, 219–231 (1995).

7. Sommer, H. and Eisenbrand, G. A method for the

determination of N-nitrosoalkanolamines in cosmetics.

Z. Lebensm. Unters. Forsch. 186, 235–238 (1988).

8. Rollman, B., Lombart, P. and Mercier, J. Determin-

ation of N-nitrosodiethanolamine in cosmetics by gas

chromatography with electron capture detection.

J. Chromatogr. 206, 158–163 (1981).

9. Collier, S.W., Milstein, S.R. and Orth, D. Quantitative

assay of volatile and non-volatile N-nitrosamines

by gas chromatography with an electrolytic con-

ductivity detector. 1. Method development and

assay of N-nitrodiethanolamine (NDELA) in creams

and lotions. J. Soc. Cosmet. Chem. 39, 329–346

(1988).

10. Ikeda, K. and Migiorese, K. Analysis of nitrosamines

in cosmetics. J. Soc. Cosmet. Chem. 41, 308 (1990).

11. Shuker, D.E.G. and Tannenbaum, S.R. Determination

of nonvolatile N-nitroso compounds in biological flu-

ids by liquid chromatography with postcolumn pho-

tohydrolysis detection. Anal. Chem. 55, 2152–2155

(1983).

12. Pignatelli, B., Malaveille, C., Thuillier, P., Hautefeuil-

le, A. and Bartsch, H. Improved methods for analysis

of N-nitroso compounds and application in human

biomonitoring in Nitrosamine and related Com-

pounds, Chemistry and Biochemistry, eds R.N.

Loeppley & C.J. Micheda. ACS Symp. Ser. 553, ACS,

Washington DC, 102–118 (1992).

13. Bellec, G., Cauvin, J.M., Salaun, M.C. et al. Analysis

of N-nitrosamines by high-performance liquid chro-

matography with post-column photohydrolysis and

colorimetric detection. J. Chromatogr. A 727, 83–92

(1996).

14. Horwitz W., Kamps, L.R. and Boyer K.W. Quality

assurance in the analysis of foods for trace constitu-

ents. JAOAC 63, 1344–1453 (1980).



Documents

NDELA.pdf