Analysis of Primary Aromatic Amines in Cosmetics and Personal Care Products

Primary aromatic amines (PAAs) are broadly used as raw materials in the manufacture of chemical materials. Many PAAs have either a proven or suspected carcinogenic nature and are rated as highly toxic, so there are a range of potential health risks, which have led to worldwide regulations. Despite the toxic and carcinogenic nature of PAAs, they are an important feedstock used in the production of many commodity products such as pharmaceuticals, pesticides, explosives, epoxy polymers, rubber, aromatic polyurethane products, and azo dyes.

While not desirable in final products, the presence of PAAs may be due to incomplete reactions, impurities, by-products, or as degradation products.  For example, PAAs can be produced as by-products of azo dyes, which are a diverse and extensively used group of organic dyes.  Azo dyes are used in special paints, printing inks, varnishes and adhesives, and can be found in many products such as textiles, cosmetics, personal care products, plastics, and also in food contact material.

In order to ensure public safety and product efficacy, the cosmetics and personal care industry is highly regulated. Therefore, manufacturers who use feedstock materials such as PAAs in the production of their products must monitor and quantify various regulated parameters, such as the presence or absence of PAAs.

Many previously used methods for PAA analysis lack robustness, selectivity and sensitivity, and require lengthy, costly, and time-consuming pre-treatments (derivatization, SPE).

This application note describes an accurate, fast, and robust alternative method for the rapid analysis of PAAs in cosmetic and personal care products, using the ACQUITY UPLC H-Class System coupled with the ACQUITY QDa Detector, and controlled by Empower 3 Software.

Benefits

ACQUITY QDa linked to the ACQUITY UPLC H-Class System provides improved confidence in the identification and quantification of Primary Aromatic Amines (PAAs) in cosmetics and personal care products offering:

• The ultimate in chromatographic resolution and sensitivity.
• Increased sample throughput and a reduction of solvent usage due to reduced run times.
• Improved sensitivity, selectivity, and robustness, compared with existing methodologies.
• Cost-effective, reliable mass confirmation.

Primary aromatic amines (PAAs) have been broadly used in large amounts as a chemical feedstock within the chemical industry. Many PAAs have either a proven or suspected carcinogenic nature and are rated as highly toxic,^1,2,3 so there are a range of potential health risks, which have led to worldwide regulations. In the  EU Cosmetic Regulations (EC) No 1223/2009,⁴ many PAAs are prohibited for  use in cosmetic products. 

Despite the toxic and carcinogenic nature of PAAs, they are an important feedstock used in the production of many commodity products such as pharmaceuticals, pesticides, explosives, epoxy polymers, rubber, aromatic polyurethane products, and azo dyes. While not desirable in final products, the presence of PAAs may be due to incomplete reactions, impurities, by-products,  or as degradation products. For example PAAs can be produced as by-products  of azo dyes which are a diverse and extensively used group of organic dyes. Azo dyes are used in special paints, printing inks, varnishes and adhesives, and can be found in many products such as textiles, cosmetics, personal care products, plastics, and also in food contact material.

In order to ensure public safety and product efficacy, the cosmetics and personal care industry is highly legislated. Hence, manufacturers who use feedstock materials such as PAAs in the production of their products must monitor and quantify various regulated parameters, such as the presence or absence of PAAs. Previous example methodologies for the analysis of PAAs include: 

• GC-MS analysis following ion-pair extraction with bis-2-ethyl phosphate followed by derivatization with isobutyl chloroformate;^5,6
• UPLC analysis following a solid phase extraction (SPE) using cation-exchange cartridges;⁷
• reduction by liquid phase sorbent trapping followed by thermal desorption GC-MS analysis.⁸

However, many previously used methods for PAA analysis lack robustness, selectivity and sensitivity, and require lengthy, costly, and time-consuming pre-treatments (derivatization, SPE). 

An ideal solution for the cosmetic and personal care industry for the analysis of PAAs, would overcome the limitations of prior methodologies, while ensuring confidence and versatility in order  to meet the regulatory requirement.

This application note describes an accurate, fast, and robust alternative method for the rapid analysis of PAAs in cosmetic and personal care products, using Waters ACQUITY UPLC H-Class System coupled with the ACQUITY QDa Detector, and controlled by Empower 3 Software.

An ideal solution for the cosmetic and personal care industry for the analysis of PAAs, would overcome the limitations of prior methodologies, while ensuring confidence and versatility in order to meet the regulatory requirement.

This application note describes an accurate, fast, and robust alternative method for the rapid analysis of PAAs in cosmetic and personal care products, using Waters ACQUITY UPLC H-Class System coupled with the ACQUITY QDa Detector, and controlled by Empower 3 Software.

Instrument control, data acquisition, and result processing

Empower 3 Software was used to control the ACQUITY UPLC H-Class System and the ACQUITY QDa Detector, as well as for data acquisition and quantitation.

Sample preparation

Cosmetic and personal care product sample analysis (eyeshadow, blush, shampoo)

• 0.5 g (solid samples) or 0.5 mL (liquid samples), add 8 mL water and 2 mL methanol. Vortex mixture for 2 min (1600 rpm).
• Centerfuge approximately 1 mL extract for 5 min (10,000 rpm).
• Centrifuge extract diluted with methanol in LC vials ready for analysis (250 μL extract plus 750 μL methanol).

Optimum UPLC and SIR conditions were developed, with the elution of all compounds occuring within a 10 minute run. The speed of method development was markedly improved using the ACQUITY QDa Detector instead of UV detection. 

Typically during method development, different conditions/parameters are considered such as  choice of columns, mobile phases, and gradients. These choices could potentially result in changes  to the elution order of the compounds being considered. The peak tracking when using UV detection only would require the analysis of the individual authentic standards in order to confirm  the elution order (Rt). However, with mass detection, the movement of chromatographic peaks can  easily be followed, and the presence of co-eluting peaks can also be easily identified. 

An illustration of the identification of the co-eluting peaks is shown in Figure 1 which shows two PAAs (4,4'-Methylene-Dianiline and 2-Methoxy-5-Methylaniline) that have similar optimum wavelengths.

Mixed calibration standards, over the range of  0.001 µg/mL to 1.0 µg/mL were prepared and analyzed for all the PAAs considered (equivalent range of 0.08 to 80 mg/Kg in the extracted sample, using the developed method, greater with extract dilution). The SIR chromatograms for each PAA are shown in Figure 2.

The SIR mass detection conditions detailed in Table 2 were used after appropriate sample preparation to screen for PAAs in cosmetic and personal care samples. 

Cosmetic and personal care sample analysis

Samples were fortified at various levels with selected PAAs, then prepared for analysis as described in the Experimental section. The results obtained for shampoo, blush, and eyeshadow are detailed in Tables 3, 4, and 5, and a selection of SIR chromatograms achieved are shown in Figure 3.

• A fast, robust, and sensitive method has been developed for the analysis of PAAs in cosmetic and personal care product samples.
• The ACQUITY QDa Detector provides more cost-effective and reliable mass confirmation, demonstrating improved experimental confidence over UV detection, during both method development and routine analysis.
• Combining the ACQUITY UPLC H-Class System with the ACQUITY QDa Detector offers accurate and reproducible quantification.
• Empower 3 Chromatography Data Software provides assurance in data management, data processing, and reporting.
• Business benefits compared to previous methodology include:
• Increased sample throughput
• Reduction of solvent usage due to no time-consuming derivatization or pre-concentration steps.
• Reduced run times.
• The ACQUITY H-Class System, a quarternary system based on UPLC Technology, offers the best in chromatographic resolution and sensitivity.

1. Benigni R, Passerini L. Carcinogenicity of the aromatic amines: from structure-activity relationships to mechanisms of action and risk assessment. Mutation Research. 511: 191–206; 2002.
2. Anirban M.P, Cote R.J. Molecular Pathogenesis and Diagnosis of Bladder Cancer. Annual Review of Pathology. 4: 501–506; 2009.
3. Ward E, Carpenter A, Markowitz S, et al. Excess Cancers in Workers Exposed to Ortho-Toluidine and Aniline. National Cancer Institute. 83(7): 501–506; 1991.
4. The European Parliament and the Council of the European Union. Regulations (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on Cosmetic Products. Official Journal of the European Union. L 342/59: 59–209, 22nd Dec 2009.[cited 2015 January 15]. Available from: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0 059:0209:en:PDF
5. Akyuz M, Ata S. Determination of aromatic amines in hair dye and henna samples by ion-pair extraction and gas chromatography-mass spectrometry. J Pharm Biomed Anal. 47, 68; 2008.
6. Ekladius L, King H K. A colorimetric method for the determination of aliphatic amines in the presence of ammonia. J Chrom A. 1129(1). Epub 2006 Jul 14.
7. Aznar M, Canallas E, Nerin J. Quantitative determination of 22 primary aromatic amines by cation-exchange solid-phase extraction and liquid chromatography-mass spectrometry. J Chrom A. 2009; 1216: 5176–5181; 2009.
8. Zhang Q, Wang C, et al. Determination of aromatic amines from azo dyes reduction by liquid-phase sorbent trapping and thermal desorption-gas chromatography-mass spectrometry. J Sep Sci. 32: 2434–2441; 2009.