Determination of aromatic amines in hair dye and henna samples by ion-pair extraction and gas chromatography–mass spectrometry

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Abstract

A gas chromatography–mass spectrometry (GC–MS) method has been proposed for the determination of carcinogenic and toxic aromatic amines in hair dye, henna and dyed hair samples. The method includes ion-pair extraction of aromatic amines from aqueous samples with bis-2-ethylhexylphosphate (BEHPA) released after solving the samples in acidic solution followed by sonication, derivatisation of compounds with isobutyl chloroformate (IBCF) and their GC–MS analysis in both electron impact (EI) and positive and negative ion chemical ionisation (PNICI) mode as their isobutyloxycarbonyl (isoBOC) derivatives. The obtained recoveries of aromatic amines ranged from 92.2 to 98.4% and the precision of this method, as indicated by the relative standard deviations (RSDs) was within the range of 0.7–4.2%. The detection limits obtained from calculations by using GC–MS results based on signal-to-noise ratio (S/N)= 3 were within the range from 0.02 to 0.20 ng/g. In the present study, the commercially available 54 permanent hair dye, 35 modified or natural henna and 15 dyed hair samples were analysed for the aromatic amines by the proposed method and the method was shown to be suitable to determine the aromatic amine ingredients and metabolites of these commercial products.

Introduction

Aromatic amines like phenylenediamines, toluenediamines, aminophenols, toluidines, and chloroanilines are biologically active compounds widely used in the formulation of commercial oxidative (permanent) hair dyes as primary intermediates and/or couplers [1]. Aromatic amines in commercial hair dyes are absorbed percutaneously during normal use [2], [3] and they can be toxic [4], mutagenic in vitro [5] and carcinogenic in experimental animals [5], [6], [7]. In vitro and animal studies support the carcinogenic potential of certain hair dye ingredients [5], [8], [9], [10], [11]. These include 4-methoxy-m-phenylenediamine, 2,4-diaminoanisole (2,4-DAAS), 4-chloro-m-phenylenediamine (4-C-m-PDA), 2,4-toluenediamine (2,4-TDA), 2-nitro-p-phenylenediamine (2NPPD) and 4-amino-2-nitrophenol (4A2NP) [9]. As reported in literature [5], [6], [7], [8], [9], [10], [11], different isomers of aromatic amines have different toxic and/or carcinogenic effects.

The contents of intermediates of oxidative hair dyes in hair colouring formulations are restricted according to Annex III of the EU Cosmetic Directive. In addition, the use of certain substances is banned in these formulations according to Annex II of the Directive because of their toxicity and/or carcinogenicity [4]. Therefore, the monitoring of levels of individual isomers of aromatic amines in commercial hair dye and henna samples is important to protect human health because of human exposure to these compounds.

The most widely used techniques to determine aromatic amines in hair dyes are high-performance liquid chromatography (HPLC) coupled with different detectors [12], [13], [14], [15], [16], [17], [18], [19], gas chromatography (GC) [20], [21] and micellar electro kinetic capillary chromatography (MEKC) [12]. The other analytical techniques have been described in the literature for the determination of aromatic amines in environmental samples [6], [22], [23], [24], aqueous food simulants [25], [26], biological fluids [27], [28], [29] and aromatic amines released from azo colorants [6], [30], [31], [32], [33], [34]. GC–MS has been recognised as the method of choice for the analysis of aromatic amines in hair dye and henna samples, due to the high efficiency of GC in separation and superior capacity of MS in structural identification. A pre-concentration step [22], [26], [28], [35], [36] is necessary to obtain good sensitivity, and derivatisation step [37] is generally required to improve the gas chromatographic properties because of the polarity of the amines.

The aim of this study was to develop analytical technique that could be applied to hair dye, henna and dyed hair samples to determine aromatic amines by adapting our previous method for the analysis of amines in environmental samples [22], [23]. This paper presents an analytical procedure, proposed to enable the precise determination of individual isomers of aromatic amines in commercially available oxidative hair dye, henna and dyed hair samples. This procedure offers several significant advantages over the other techniques available, such as higher selectivity in isolation, resolution and sensitivity. In the proposed method, the compounds of interest were isolated from aqueous solution with chloroform containing bis-2-ethylhexylphosphate (BEHPA) as ion-pair reagent after dilution with acidic solution and derivatised with isobutyl chloroformate (IBCF). Aromatic amines were then analysed as their isobutyloxycarbonyl (isoBOC) derivatives by GC–MS.

Section snippets

Chemicals and reagents

All the reagents were of analytical grade. Bis(2-ethylhexyl)-phosphate, isobutyl chloroformate (IBCF), aniline (ANI), N-methylaniline (NMA), o-toluidine (OT), m-toluidine (MT), p-toluidine (PT), 4-chloroaniline (PCA), 4-ethylaniline (4-EA), 4-nitroaniline (4-NA), 2-aminophenol (2-AP), 3-aminophenol (3-AP), 4-aminophenol (4-AP), 2,4-toluenediamine (2,4-TDA), 2,6-toluenediamine (2,6-TDA), 3,4-toluenediamine (3,4-TDA), 2-nitro-p-phenylenediamine (2NPPD), diphenylamine (DPA), chloroform, toluene,

Interpretation of mass spectra of model compounds

Firstly, aromatic amine standards were derivatised with IBCF and their isoBOC derivatives were analysed by GC–MS. Total Ion Chromatogram (TIC) of derivatised amine standards are shown in Fig. 1.

Identification of the components was performed by interpreting their mass spectra obtained in the EI mode. All the other isoBOC derivatives of aromatic amines were interpreted in the same way and expected fragmentation patterns were obtained. For example, interpretation of mass spectrum of isoBOC

Conclusion

Consequently, the method described in this study has been shown to be suitable with satisfactory accuracy and good reproducibility for the qualitative and quantitative determination of aromatic amines at the levels of ng/g in hair dye, henna and dyed hair samples.

Through the obtained results, it may be concluded that, the method developed can be proposed as a procedure for the precise determination of aromatic amine ingredients of commercial oxidative hair dye and henna samples to check their

Acknowledgements

The authors wish to thank the State Planning Organization, Turkey, (2003K121120) and the Zonguldak Karaelmas University (2004-13-02-12) for the opportunity and support to carry out this research.

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