A Fast Efficient Method to Determine the Presence of Nitrosamines

in Cosmetics, Personal Care, and Consumer Products

Michael Jones1 and Christopher Glover2
1
Waters Corporation, Manchester, UK
2
Northumbria University, Newcastle upon Tyne, UK

A P P L I C AT I O N B E N E F I T S INT RODUC T ION

This application note describes a method for the N-nitrosamines are a class of compounds that have been shown to exhibit
identification and quantitation of carcinogenic carcinogenic and mutagenic effects in animal models at several different tissue
nitrosamines in consumer products offering: sites and by several different routes of exposure.1-3 Nitrosamines can potentially
■■ Increased sample throughput and a reduction be formed in consumer products, cosmetics and personal care formulations, either
of solvent usage due to reduced run times. during manufacture or product storage.4 N-Nitrosamine formation occurs when
secondary amines are present in addition to a nitrating agent such as nitrous
■■ A single, direct LC-MS method to analyze
acid, nitrites, or nitrogen oxides, generally under acidic conditions. Analysis
both non-volatile and also volatile
of cosmetic, personal care and consumer products for nitrosamine content
nitrosamines with no requirements
should be included in Product Safety Assessments to ensure compliance with
for derivitization.
the new Cosmetic and Consumer Products Regulations.5 It has also been noted
■■ Quantitation of N-nitrosamines below that cosmetics over five years old had higher concentrations of N-nitrosamines
the regulatory limit of 50 µg/kg. than new samples of the same products.6 This indicates that the formation of
■■ The ability to mine for additional unknowns N-nitrosamines limits the shelf life of cosmetics and should be assessed as part
utilizing RADAR™ data acquisition. of a cosmetic product’s routine safety evaluation.

In both the United States and the European Union, consumer products
manufacturers ensure product safety prior to commercialization, list all
ingredients on the product label, and comply with any restrictions that are
WAT E R S S O LU T I O N S established for ingredients and products. Any potential risk from a product is
assessed as part of its safety evaluation. In the European Union, EU Directive
ACQUITY UPLC® I-Class System
1223/2009 has been in force since 2013.5 It strengthens the safety of cosmetic
ACQUITY UPLC HSS T3 Column products and streamlines the framework for all operators in the sector. It provides
a robust, internationally recognized regime, which reinforces product safety while
Xevo ® TQ-S micro
taking into consideration the latest technological developments. EU 1223/2009
MassLynx ® MS Software specifies in Annexes II and III that N-nitrosamines (along with other contaminants)
must not be present in cosmetic products above levels of 50 µg/kg. In the United
Oasis ® HLB SPE Cartridges
States, the U.S. FDA, under the auspices of The Food, Drug, and Cosmetics Act
(1938) and The Fair Packaging and Labelling Act (1966) considers any cosmetic
product containing N-nitrosamines as adulterated, and as such, has the power
KEY WORDS
to apply for those products to be seized and removed from the market.7,8 In Asia,
Nitrosamines, personal care products,
the ASEAN nations have modeled the ASEAN Cosmetics Directive 9 on the
cosmetics, consumer products, regulated
above European legislation. China, India, and Japan also regulate cosmetics
substances, RADAR, PICS
in a similar fashion.

1
EU Directive 2009/48/EC pertains to the safety of toys, finger paints, and elastomeric materials likely to be
placed in the mouth of children.10 This legislation also limits the cumulative presence of N-nitrosamines to less
than 50 µg/kg in total. The legislation applies to any toys or equipment containing elastomeric materials such
as soothers and bottle teats aimed at children. The applicability of this method was also tested against analysis
of infant feeding bottle teats for the presence of N-nitrosamines utilising sample preparation procedures
set out in draft European Standard prEN 71 – 12:2012:E. Previously utilised methods for the analysis of
N-nitrosamines in both cosmetics formulations and elastomeric materials include GC-TEA, GC-MS, and HPLC
coupled to colorimetric detectors and LC-ESI-MS.11-15 Run times ranged from 12 to 35 mins for LC methods and
20 to 40 mins for GC methods. TEA analysis was reported to be very time consuming, which limited sample
throughput. It was also generally only applicable to analysis of volatile nitrosamines.

In this application note, we describe the analysis of eight volatile and non-volatile N-nitrosamines previously
detected in cosmetics and personal care formulations using Waters ® ACQUITY UPLC I-Class System coupled
to Xevo TQ-S micro. The described method offers several benefits over previous methodologies including
enhanced selectivity and sensitivity over colorimetric and ESI-MS methods. Quantitation is possible below the
regulatory limits of 50 µg/kg. Runtimes are reduced offering associated time and cost savings over HPLC and
GC methods. The RADAR acquisition method used allows the ability to mine simultaneously acquired full scan
data to search for other contaminants.

A Fast Efficient Method to Determine the Presence of Nitrosamines in Cosmetics, Personal Care, and Consumer Products 2
E X P E R IM E N TA L
Cosmetic and personal care samples were prepared for analysis by an extraction protocol developed and validated by Qiang et al.11 The
samples underwent sonic extraction followed by sample cleanup utilizing Oasis HLB SPE Cartridges. Liquid and gel samples were prepared
using 1:1 MEOH: acetone as a sonic extraction medium. Solid or semi-solid cosmetic samples were prepared using ethanol: acetone 4:1.

Sonic extraction protocol Time Flow rate

%A %B Curve
■■ 1g aliquot of a personal care formulation was dissolved (min) (mL/min)
in a 5.0 mL appropriate extraction medium with vortex 1 Initial 0.30 97.5 2.5 –
mixing and sonicated for 30 min. 2 1.0 0.30 97.5 2.5 6
■■ The resulting extraction mixture was centrifuged at 3 2.0 0.30 50.0 50.0 6
6000 rcf for 15 min. 4 3.0 0.30 50.0 50.0 6
5 3.5 0.30 2.5 97.5 6
■■ 2-mL supernatant was evaporated to dryness under
6 6.0 0.30 2.5 97.5 6
a constant stream of nitrogen at 40 °C, and reconstituted in
7 6.1 0.30 97.5 2.5 6
2 mL 30% aqueous methanol ready for solid phase extraction.
Table 1. ACQUITY I-Class mobile phase gradient.
SPE protocol
■■ A 6 cc 3 Oasis HLB SPE Cartridge (Part no. WAT106202) MS conditions
containing 200 mg sorbent was primed with 6 mL methanol, MS system: Xevo TQ S-micro
followed by 6 mL ultrapure water. Ionization mode: APCI positive
■■ The sample was loaded onto the SPE cartridge and washed Corona pin voltage: 15 V
with 3 mL 30% aqueous methanol. The sample was then Source temp.: 150 °C
extracted from the cartridge with 6 mL MeOH. Probe temp.: 600 °C
■■ The sample was prepared for injection by evaporating to Desolvation gas: 1200 L/hr
dryness under N2, reconstituting in 1 mL 2.5% aqueous Cone gas: 200 L/hr
methanol and filtration through a 0.2 µm membrane filter. Acquisition: MRM with RADAR
Ionization parameters and transition pairs were optimized
LC conditions
automatically using the IntelliStart™ function of MassLynx
LC system: ACQUITY UPLC I-Class
Software. IntelliStart is a feature within MassLynx that can be used
Run time: 9.1 min
to monitor system health, perform QC checks, and aid the method
Column: ACQUITY UPLC HSS T3
development process by increasing ease of use and decreasing user
1.7 µm, 2.1 x 100 mm to user variation. In this instance the automatic tuning parameters
Column temp.: 40 °C feature of IntelliStart was used to determine optimum ionization
Sample temp.: 5 °C parameters such as cone voltages for parent ions and collision gas
Mobile phase A: Water (0.1% formic acid) energies for MRM transitions. Table 2 summarizes the optimum
Mobile phase B: Methanol (0.1% formic acid) parameters for the analytes tested, along with information about
the analyte names, chemical formulas, and CAS numbers.
Flow rate: 0.3 mL/min
Injection volume: 20 µL
Mobile phase gradient is detailed in Table 1.

A Fast Efficient Method to Determine the Presence of Nitrosamines in Cosmetics, Personal Care, and Consumer Products 3
 Retention time Cone voltage
Name, formula, CAS no. Structure Transition Collision energy
(min) (V)
N-Nitroso-dimethylamine, O
N
2.1 30 75.0→58.0* 13
C 2H6N2O, 62-75-9 N
O
N-Nitroso-diethylmine, N 103.1→47.0 15
N 3.1 24
C 4 H12N2O, 55-18-5 103.1→75.0* 9
N-Nitroso-dipropylamine, N
O 131.1→43.1 11
4.2 26
C 6H14N2O, 621-64-7 N 131.1→89.1* 8
N-Nitroso-diethanolamine, N
O 135.0→74.0 8
N
1.4 10
C 4 H10N2O3, 1116-54-7 HO OH 135.0→104.0* 4
N-Nitroso-dibutylamine, N
O 159.1→57.1* 12
4.5 26
C 8 H18 N2O, 924-16-3 N
159.1→103.0 9
N-Nitroso-pyrrolidine, N
O
101.0→40.5 19
N 2.8 44
C 4 H8 N2O, 930-55-2 101.0→55.0* 13
N-Nitroso-piperidine, N
O
115.1→40.9 25
N
3.2 36
C 5H10N2O, 100-75-4 115.1→69.1* 17
O
N-Nitroso-morpholine, N
117.0→45.0* 13
N
2.6 28
C 4 H8 N2O2, 59-89-2 O 117.0→85.9 9

Table 2. Aliphatic, cyclic, and heterocyclic N-nitrosamines previously found in consumer product formulations, structures, associated CAS numbers and empirical
formulas, expected retention times, cone voltages, MRM transitions, and associated collision energy values.
*Refers to the quantitation transition.

Instrument control, data acquisition, and results processing

MassLynx Software was used to control the ACQUITY UPLC I-Class System and the Xevo TQS-micro, and also for data acquisition.
Data quantitation was achieved using TargetLynx™ Application Manager.

A Fast Efficient Method to Determine the Presence of Nitrosamines in Cosmetics, Personal Care, and Consumer Products 4
R E S U LT S A N D D I S C U S S I O N
The analysis of eight N-nitrosamines historically found in cosmetic formulations was achieved using the Xevo TQ S-micro in MRM mode with
APCI ionization in positive mode, coupled to an ACQUITY I-Class System.

The MRM transitions for each of the analytes were optimized using the IntelliStart function of MassLynx Software as previously discussed.
LC conditions (column chemistry, gradient profile, injection volume, etc.) were based on previous work by Al-Kaseem et al16, and optimized
to improve run time, elution order, and signal intensity.

Example chromatograms of standards prepared in the cosmetic matrix at a concentration equivalent to the regulatory limit of 50 µg/kg
are shown in Figure 1.

N-Nitrosodipropylamine
N-Nitrosodiethanolamine N- Nitrosopyrrolidine

N-Nitrosodimethylamine
N- Nitrosodiethylamine N-Nitrosodibutylamine

N-Nitrosomorpholine N-Nitrosopiperidine

Figure 1. Example chromatograms of the eight analyzed N-nitrosamines in matrix spiked standards at the regulatory limit of 50 µg/kg.

Mixed calibration standards from 0.001 ppm to 0.5 ppm were prepared and analyzed for all of the compounds considered. All nitrosamines
were found to give a linear response over concentration ranges from 0.01 ppm to 0.5 ppm (10 times the regulatory limit of 0.05 ppm).
R 2 values for all curves were >0.99, and matrix effects were found to be <8%.

A Fast Efficient Method to Determine the Presence of Nitrosamines in Cosmetics, Personal Care, and Consumer Products 5
Analysis of cosmetics and personal care samples
Samples were prepared from a range of cosmetic and personal care formulations including shampoo, hand wash, and lipstick. All samples
were tested for the presence of the N-nitrosamines as detailed in the Experimental section, and were found to be free of N-nitrosamines. In
order to test the suitability of the method, blank matrix samples were spiked with nitrosamines in amounts at or around the regulatory limit.
In the example shown below in Figure 2, a shampoo sample was prepared, and spiked with N-nitrosodipropylamine at a concentration of
0.06 ppm (ng/mL). After sonic extraction and SPE the sample was analyzed alongside the matrix standards using a TargetLynx method to
determine the N-nitrosodipropylamine concentration.

The data was quantified using the TargetLynx method to determine the amount of N-nitrosodipropylamine present in the spiked sample.
Figure 1 shows the results generated in TargetLynx for the spiked shampoo sample, along with the calibration curve generated
from the matrix standards to quantify the spiked sample. Figure 1 also shows the MRM trace for the confirmatory transition for
N-nitrosodipropylamine. All of the results generated from the method for spiked samples were accurate in the range of 98%
to 102% expected result for tested formulations including shampoo, hand wash, and lipstick.

Figure 2. TargetLynx Result Summary for a shampoo sample spiked with N-nitrosodipropylamine.

A Fast Efficient Method to Determine the Presence of Nitrosamines in Cosmetics, Personal Care, and Consumer Products 6
Analysis of baby feeding bottle teats
Silicon-free feeding teats from a leading manufacturer were tested according to a draft protocol set out in European Standard prEN 71 –
12:2012:E. 10 cm2 portions of the elastomeric material used to form the teat were submerged in synthetic saliva containing a source of
nitrite. Nitrite has been proven to convert secondary amines to N-nitrosamines under acidic conditions such as those found in the human
stomach.17 The teat samples were submerged for 30 minutes before the resultant supernatant was analyzed according to the method with
no further sample preparation.

Unadulterated submerged samples were analyzed to determine the presence of nitrosamines. The samples were found to contain trace
amounts of N-nitrosodibutylamine at levels approximately 10 times below the regulatory limits. In order to further test the efficacy of the
method, additional teat samples were prepared and spiked with N-nitrosodiethanolamine at a concentration 1.4 times the regulatory limit.
Samples were also prepared spiked with a mixture of three different N-nitrosamines, each below the regulatory limit, but with a cumulative
concentration above the regulatory limit of 50 µg/kg. Figure 3 shows The TargetLynx Result Summary for the N-nitrosodiethanolamine
spiked sample, comprised of the calculated injections, a chromatogram of the spiked sample, and the N-nitrosodiethanolamine calibration
curve generated for the calculation.

Figure 3. TargetLynx Result Summary for NDELA spiked baby bottle feeding teat sample.

A Fast Efficient Method to Determine the Presence of Nitrosamines in Cosmetics, Personal Care, and Consumer Products 7
The TargetLynx Sample Quantification reports generated from the NDELA spiked samples and the low level spiked samples are shown
in Figure 4.

Figure 4. TargetLynx Summary reports detailing the presence of N-nitrosamines in baby bottle feeding teats.

RADAR analysis of aged consumer goods samples

As mentioned previously, it has been noted that N-nitrosamine levels can increase in personal care formulations upon storage.6 The ability
to determine the emergence of any N-nitrosamines over time would allow manufacturers to determine safe shelf lives for their products.
To this end the method was used to test for the presence of nitrosamines in a new lipstick formulation and an old sample of the same brand.
In addition to the targeted searching of the eight nitrosamines outlined in the method, utilizing the RADAR functionality available on the
Xevo TQ-S micro instrument allows for simultaneous recording of multiple MRM transitions, along with scanning experiments across a
defined m/z range. Figure 5 shows the TIC chromatograms comparing the new and aged lipstick samples. There appears to be an additional
peak in the TIC with a retention time of approximately 4.55 minutes that does not correspond to N-nitrosodibutylamine, as shown by the
absence of any peak at 4.55 minutes in the MRM transition chromatogram for that compound.

New lipstick sample

Aged lipstick sample

N-nitrosodibutyl MRM trace, aged lipstick

Figure 5. TIC chromatograms of new and
aged lipstick formulation samples and
an MRM transition chromatogram of
N-nitrosodibutylamine showing its
absence in the aged sample.

A Fast Efficient Method to Determine the Presence of Nitrosamines in Cosmetics, Personal Care, and Consumer Products 8
When analyses are conducted using RADAR data acquisition mode, expected analytes can be accurately quantified while simultaneously
probing the sample for the presence of unknowns – without any loss of data integrity. Figure 6 again shows the TIC chromatograms for
both the new and aged lipstick samples. When run in RADAR mode, the data acquired allows for examination of the relevant mass spectra
to determine any differences at the observed peak time. The magnified MS spectra views in Figure 6 show the significant presence of an
ion at m/z 199 in the aged sample over the new sample. Extracting this ion gives the extracted ion chromatogram shown in Figure 6 with a
corresponding retention time of 4.55 minutes. A search of the literature suggests that m/z 199 possibly corresponds to the [M+H] + ion for
N-nitrosodiphenylamine if the peak appearing in aged samples is due to N-nitrosamine formation. Further work is planned utilizing high
resolution mass spectrometry (HRMS) to confirm the identity of the unknown ion present in the aged lipstick samples.

A.

New lipstick
sample

B.

Aged lipstick
sample

C.

XIC m/z 199 aged lipstick sample

Figure 6. Probing the presence of unknowns using RADAR data acquisition: A. TIC of new lipstick sample and averaged MS spectra at 4.5 to 4.55 min.; B. TIC of new
lipstick sample and averaged MS spectra at 4.5 to 4.55 min.; C. XIC of ion at m/z 199 in the aged lipstick sample.

A Fast Efficient Method to Determine the Presence of Nitrosamines in Cosmetics, Personal Care, and Consumer Products 9
C O N C LU S I O N S 12. Flower C, Carter S, Earls A, Fowler R, Hewlins S, Lalljie S,
Lefebvre M, Mavro J, Small D, Volpe N. A method for the
An LC-APCI-MS method has been developed to rapidly detect and quantify determination of N-nitrosodiethanolamine in personal
care products – collaboratively evaluated by the CTPA
eight commonly found nitrosamines in consumer product formulations.
Nitrosamines Working Group. Intl J Cosmetic Sci. 28 (1):
All analytes are quantifiable at or below the regulatory limits of 50 µg/kg for 21–33, 2009.
cosmetics formulations and elastomeric toys. Three cosmetic formulations and 13. Schothorst R, Somers H. Determination of
one elastomeric material sample were tested for the presence of nitrosamines. N-nitrosodiethanolamine in cosmetic products
by LC–MS/MS. Analytical and bioanalytical chemistry.
The shampoo and hand wash formulations tested were found to be free of 381 (3): 681–685, 2005.
nitrosamines. The tested baby feeding teat sample was found to contain trace
14. Schothorst R C, Stephany R W. Occurrence of
amounts of an N-nitrosamine below the regulatory limit. The incorporation of N-nitrosodiethanolamine (NDELA) in cosmetics from the
Dutch market. Intl J Cosmetic Sci. 23 (2): 109–114, 2001.
RADAR data acquisition into the method allows for the mining of full scan
MS data for the potential presence of additional nitrosamines and suggested 15. Stepanov I, Carmella S G, Hecht S S, Duca G. Analysis of
Tobacco-Specific Nitrosamines in Moldovan Cigarette
the presence of an additional N-Nitrosamine in the aged cosmetic formulation. Tobacco. J Agricultural and Food Chemistry. 50 (10):
2793–2797, 2002.

References 16. Al-Kaseem M, Al-Assaf Z, Karabeet F O. A Rapid, Validated

RP-HPLC Method for the Determination of Seven Volatile
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2. Barnes J M, Magee P N. Some Toxic Properties of Dimethylnitrosamine. 17. Krul C A, Zeilmaker M J, Schothorst R C, Havenaar R.
British Journal of Industrial Medicine. 11 (3): 167–174, 1954. Intragastric formation and modulation of
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3. Jakszyn P, González C A. Nitrosamine and related food intake and gastric and oesophageal
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5. Regulation (EC) No 1223/2009 of the European Parliament.

Official Journal of the European Union, L342 (22.12.2009), 59–209, 2009.

6. Matyska M T, Pesek J J, Yang L. Screening method for determining the presence of

N-nitrosodiethanolamine in cosmetics by open-tubular capillary electrochromatography.
Journal of Chromatography A. 887 (1): 497–503, 2000.

7. Administration, U. S. F. D., Fed Reg 44. 1979, 21365.

8. Administration, U. S. F. D., Fed Reg 58. 1983, 28288–28292.

9. http://aseancosmetics.org/docdocs/directive.pdf

10. Directive 2009/48/EC of the European Parliament and of the Council of 18 June 2009 on the
safety of toys. Official Journal Journal Of The European Union. L170 (30.6.2009): 1–37, 2009.

11. Ma Q, Xi H W, Wang C, Bai H, Xi G C, Su N, Xu L Y, Wang J B. Determination of Ten Volatile

Nitrosamines in Cosmetics by Gas Chromatography Tandem Mass Spectrometry.
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