RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL.

11, 13291334 (1997)

Analaysis of Cosmetic Products by Gas Chromatography/Mass Spectrometry and Fast-atom Bombardment Mass Spectrometry: When a Combined Approach is Successful
R. Maffei Facino1*, M. Carini1, G. Aldini1, C. Marinello1, P. Traldi2 and R. Seraglia2
1 2

Istituto Chimico Farmaceutico Tossicologico, University of Milan, Viale Abruzzi 42, 20131, Milan, Italy CNR, Area della Ricerca, Corso Stati Uniti 4, 35020, Padua, Italy

Fast-atom bombardment mass spectrometry (FAB-MS, both in the positive- and negative-ion modes) and gas chromatography/mass spectrometry (GC/MS) were applied for the rapid identication of the main ingredients of two widely employed cosmetic products, a mascara and a semipermanent hair dye. FAB-MS, on the untreated product (hair dye) or on a crude methanol extract (mascara) gives an unequivocal ngerprint of involatile constituents, i.e. surfactants, anionic (cetyl/stearyl soaps) in mascara and cationic (polyethoxylated oleyl/linoleyl/ricinoleyl-methyl ammonium chloride) in the hair dye. GC/MS (n-hexane and chloroform extracts), detects hydrocarbons, volatile silicon compounds, perfume components, preservatives and antioxidants in mascara and fatty acids (C8C18), dye solvents and 4 colouring agents: 2-amino-5-nitrophenol, HC Yellow 2 (N-2-hydroxyethyl-o-nitroaniline); O-(2-hydroxyethyl)-4-amino-3-nitrophenol and HC Red3, N-(2-hydroxyethyl)-2-nitro-p-phenylendiamine in the hair dye. These results demonstrate that the combined FAB-MS and GC/MS approaches, enabling a complete picture of the constituents of the cosmetic matrix, provide an invaluable analytical tool for the quality control of cosmetics, a subject that has been rather neglected to date. 1997 by John Wiley & Sons, Ltd.
Received 13 May 1997; Revised 5 June 1997; Accepted 27 June 1997 Rapid. Commun. Mass Spectrom. 11, 13291334 (1997) No. of Figures: 10 No. of Tables: 2 No. of Refs: 13

The quality control of a cosmetic product is not only important from a technological point of view, to guarantee to the consumer the expected cosmetic performance and to verify that it satises the legislative requirements, but above all to avoid the risk of toxic reactions due to contaminants. The inspection becomes urgent and essential when a toxic adverse reaction (e.g. cosmetic dermatitis) is experienced by a consumer and/ or when fraudulence is suspected. This challenges the analytical chemist to a difcult task, because of the multi-ingredient composition of cosmetic products, the wide variety of cosmetic formulations on the market and the ever increasing availability of new raw materials (more than 8,000 raw materials and fragrances ingredients are available to the cosmetic chemist!). Continuing our interest in the eld of quality control of cosmetics, raw materials and nished formulations,14 in the present work we evaluated the potential of mass spectrometry for the rapid identication of the main components of two widely used stay-on products, viz a mascara and a hair dye, both chosen because of the well known problem of toxicity associated with colourants (and to their decomposition or by-products) since they are close to, or actually in contact with mucous membranes. The problem was approached by a combination of gas chromatography/mass spectrometry (GC/MS) and fast-atom bombardment mass spectrometry (FAB-MS) techniques, in an attempt to draw up a protocol/guideline for the rapid analysis of these potentially irritant formulations.

EXPERIMENTAL Chemicals and apparatus All the solvents used were analytical grade (Merck, Bracco, Milan, Italy). The mascara and the direct hair dye were from commercial sources. GC/MS determinations were performed under the following experimental conditions: (a) Mascara. C. Erba (Milan, Italy) Fractovap model gas chromatograph equipped with a capillary column (fused silica SE54, 25 m 0.32 mm i.d.); programming temperature: 70 C-300 C (7 C/min); injector temperature: 270 C; carrier gas helium (30 cm.s1). Mass spectrometer-ion trap detector (ITD 800, Finnigan MAT Bremen, Germany); electron energy: 70 eV; trap temperature 200 C; transfer line temperature 250 C; released software version n.3 IBM XT. (b) Hair dye. Shimadzu (Tokyo, Japan) GC/MS QP1000 instrument equipped with a capillary column (fused silica SPB5); programming temperature: 70 C-300 C (7 C/min); injector temperature: 270 C; carrier gas helium (30 cm s1); mass spectrometer quadrupole; electron energy 70 eV; source temperature 200 C; software MSPAC 200. Gas chromatography/Fourier Transform Infrared Spectroscopy studies (GC/FT-IR) were done on a (Hewlett Packard, Palo Alto, CA, USA) HP 5890 gas chromatograph linked to a HP5965A IRD model operating under the following conditions: capillary column HP1 (25 m 0.5 mm i.d.); programming temperature 70 C280 C (5 C/min); injector temperature 270 C; carrier gas helium (30 cm s1); transfer line 280 C; ow cell 280 C; computer HP 9000. FAB
1997 by John Wiley & Sons, Ltd.

Presented at the 15th Informal Meeting on Mass Spectrometry, Smolenice, Slovak Republic, 1216 May 1997 *Correspondence to: R.M. Facino CCC 09514198/97/12132906 $17.50

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mass spectra were obtained on a VG (Manchester, UK) 7070 EQ instrument, employing argon atoms with 7 kev kinetic energy; The FAB gun was manufactured by Ion-Tech (Teddington, UK). Recordings in the positiveand negative-ion modes (matrix thioglycerol) were taken at a resolution of 3000, with a run speed of 20 s/decade. Data were processed by a Digital PDP8/A computer system. Sample treatment (a) Mascara. A 0.1 g sample was extracted by vortexing with methanol (2 3 mL); the methanol extracts were collected, ltered, concentrated in vacuo to small volume and directly submitted to FAB-MS analysis. Another aliquot (0.1 g) was extracted by vortexing with 3 mL of n-hexane and centrifuged for 3 min at 5000 rpm to separate the inorganic material; the organic phase was recovered and the residue re-extracted with n-hexane and then with chloroform (2 3 mL). All the organic extracts were combined, concentrated in vacuo to dryness and taken up in n-hexane (0.5 mL): 1 L aliquots were injected into the GC/MS and GC/FT-IR systems. (b) Semipermanent hair dye. The product (0.1 g sample) was analysed by FAB-MS after methanol solubilization. For GC/MS experiments, 0.1 g samples were dispersed in water (10 mL), adjusted to pH 1 with 1 N HCl and extracted with n-hexane (3 volumes; 2 times); the organic phases were collected and concentrated in vacuo to small volume (0.5 mL). The aqueous residue was re-extracted with chloroform at different pH values (1, 7, 10) and the combined organic extracts processed in the same way. 1 L aliquots of the n-hexane and chloroform extracts were injected into the GC/MS system. Thin layer chromatographic (TLC) analysis of the chloroform extract was carried out by two-dimensional development using toluene + chloroform + acetone; 40:25:35 (solvent system 1) and methylethyl-ketone + butylacetate + water + methanol: 60:40:2:1 (solvent system 2) and colourants were detected under visible light. RESULTS AND DISCUSSION Mascara Mascara is a matrix in which the dyes (inorganic pigments) are nely dispersed in a complex oil/water emulsion. Since, as previously demonstrated,2 FAB-MS is a technique of choice for the characterization of different classes of surfactants, we analysed rst by FAB-MS (positive- and negative-ion modes), a crude methanol extract from a commercial mascara, to achieve an immediate indication of the emulsifying agent. The positive-ion FAB mass spectrum of the crude methanol extract does not show any diagnostic peak, relative to non ionic, cationic or amphoteric surfactants (data not shown); by contrast, in the negative-ion mass spectrum (Fig. 1) three peaks are clearly detectable at m/z 227, 255 and 283, which correspond to the carboxylate anions [M-H] of myristic (C14), palmitic (C16) and stearic (C18) acids. From this pattern, the emulsifying agent was readily identied as an alkali metal (sodium) soap. For the characterization of the lipophilic components, the n-hexane + chloroform extract was then
Rapid Communications in Mass Spectrometry, Vol. 11, 13291334 (1997)

submitted to GC/MS analysis (Fig. 2); several classes of compounds were identied: (a) perfume components, such as terpenes (peak 1 retention time (RT) 11.45 = -pinene) or sesquiterpenes (peaks 56 = -gurjunene RT 16.44; caryophyllene RT 16.58), the sesquiterpenyl alcohol farnesol (peak 11, RT 19.00), p-hydroxybenzoic acid (peak 13, RT 20.55); (b) the preservative mixture, mainly composed of phenoxyethanol (peak 2 = RT 12.32, scan number 744), and to a lesser extent of methylparaben and propylparaben (peaks 78; RT 17.40 and 18.07); (c) the antioxidant butylhydroxytoluene (BHT, peak 10, RT 18.45); (d) volatile silicon compounds (peaks 3, 4, 9, 12, 14, 18, whose mass spectra contain only a main peak at m/z 73, typical of a (CH3)3Si- moiety), probably belonging to the class of dimethylpolysiloxanes (foam-suppressing agents).

Figure 1. Negative-ion FAB mass spectrum of mascara (crude methanol extract).

Figure 2. GC/MS analysis of mascara (n-hexane + chloroform extract). 1997 by John Wiley & Sons, Ltd.

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The prole of the emulsifying mixture already identied preliminarily by FAB-MS, was conrmed by GC/ MS, from the peaks of myristic (peak 17, RT 23.13), palmitic (peak 22, RT 27.02), and stearic (peak 24, RT 29.48) acids. Peaks 15 [RT 22.00] and 16 [RT 22.49] show almost identical spectra, with ions at m/z 138, 121 (base peak), 92, 65, indicating esters of salicyclic acid with long-chain fatty alcohols, e.g. octyl salicylate or tridecyl salicylate (tentatively identied only: no library information); peak 19 [RT 24.37; ions at m/z 284, 129, 117 (base peak), 75 and 55] was not identied. The main fraction of the lipophilic phase is constituted by a series of homologous compounds (peaks 20, 21, 23, 2535; RT between 25 and 50 min) which show no molecular ions but almost identical fragmentation patterns, with ions at m/z 43, 57, 71, 85, 99, 113 and 127; the distribution of the relative intensities (m/z 57 base peaks; m/z 43 and 71 = 80 and 70%) are typical of saturated, unbranched hydrocarbon chains (data not shown). This indicates that this commercial mascara contains low cost mineral waxes, such as parafn, ceresin, ozokerite or petrolatum, all of which consist of high molecular weight hydrocarbons (derived from the higher petroleum fractions). In an attempt to obtain a deeper insight into the composition of this fraction, the n-hexane + chloroform extract was analysed in parallel by another combined technique, the GC/FTIR approach. Fig. 3 reports the chromatogram obtained by injecting 1 L samples of the extract: the pattern is qualitatively similar to that obtained by GC/MS, but only the 13 main peaks furnish diagnostic spectra. Peaks 1 (RT 5.45 min), 2 (RT 15.2 min) and 3 (RT 17.2) were unequivocally conrmed as phenoxyethanol (Fig. 4), palmitic acid (Fig. 5) and stearic acid (data not shown). The FTIR spectra of peaks 4-13 are typical of hydrocarbon derivatives, and give only the C-H stretchings at 2932/2865 cm1 and the CH3 and CH2 bendings at 1352 and 1464 cm1 (data not shown), but do not furnish

additional information to achieve denitive structural assignment. Because of the lower sensitivity of IR detection vs mass spectrometry (approximately 100 pg vs 100 ng), it was not possible to conrm the structure of peaks with retention times between those of phenoxyethanol (peak 1) and palmitic acid (peak 2), previously identied by GC/MS as essential oils, preservatives, antioxidants, which are present in the cosmetic product in small amounts. A rough estimate of the percentage composition of the product, obtained by determining rst the amount of volatile matter (105 C), then the amount of material extractable with the organic solvent and nally the inorganic residue, gives a satisfactory prole of mascara constituents in terms of recovery (analytical details in Table 1), where inorganic pigments, identied as Pigment Black 11 (Fe2O3) and Pigment Blue 29 (Na8Al6SiS2O24) account for 8.5%. Semipermanent hair dye The FAB-MS plus GC/MS approach was applied to the analysis of another widely used cosmetic product, a red semipermanent hair dye. Semipermanent hair dyes, so called because they will withstand ve to six shampoo

Figure 4. FTIR spectrum of peak 1.

Figure 3. GC/FTIR analysis of mascara (n-hexane + chloroform extract). 1997 by John Wiley & Sons, Ltd.

Figure 5. FTIR spectrum of peak 2. Rapid Communications in Mass Spectrometry, Vol. 11, 13291334 (1997)

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Table 1. Percentage composition semipermanent hair dye

Mascara

of

mascara

and

Involatile matter (residue 105C) = 42% (gravimetric) n-hexane extract = 32% (gravimetric) 12 (a) fatty acids = 8.5% (titration) (b) hydrocarbons, antioxidants, preservatives = 23.5% Inorganic pigments = 8.5% (a) HCI soluble Pigment Black 11 = 5.5% (b) HCI insoluble Pigment Blue 29 = 3% Aqueous residue (hydrolyzed cheratine)* = 1.5%
Semipermanent hair dye

Involatile matter (residue 105C) = 22% (gravimetric) n-hexane extract pH1 = 9% (a) fatty acids = 4.5% (titration) (b) ethoxylated fatty alcohols, antioxidants, etc. = 4.5% CHCl3 extracts (different pH values) (a) hair dyes <5% Cationic surfactants = 8% (titration) Aqueous residue ??? = 2%
* The
13

assigned to the three series the structures of polyethoxylated (n = 3-14) oleyl (A)/linoleyl(B)/ricinoleyl(C)-methylammonium chloride, a mixture of cationic tensides, widely used in hair products owing to their conditioning and softening properties. In the negative-ion FAB mass spectrum, the ions at m/z 143, 171, 199, 227, 255 and 283 (data not shown), which correspond to the carboxylate anions of caprylic (C8), capric (C10), lauric (C12), myristic (C14), palmitic (C16) and stearic (C18) acids, point to a mixture of fatty acids from a natural source (i.e. coconut). Next we examined by GC/MS the organic extracts of the hair dye formulation: in the more lipophilic fraction (n-hexane, Fig. 7) 30 compounds were identied (Table 2), among which were: perfume components (peaks 4, 11, 12, 18); dye solvents (peaks 1 and 2); antioxidants (peaks 9, 10); C8C18 fatty acids, already detected by FAB-MS, negative-ion mode (peaks 3, 6, 13, 16, 20, 22); fatty alcohols (peaks 8, 14) and the corresponding ethoxylated derivatives: C16En(n = 1 6; peaks 15, 21, 24, 26, 28, 30); C18En (n = 1 5; peaks 19, 23, 25, 27, 29).

only component reported on the label.

washings, contain dye molecules (generally low molecular weight, nonionic) formulated in a more complex matrix. To obtain a rapid screening of the main ionic species of the cosmetic base, the product was again directly analysed by FAB-MS (loading a few g of the product dissolved in methanol on the probe tip). In the positive-ion mode three series of ions A,B,C were clearly detectable (Fig. 6): A B C = m/z 414, 458, 502, 546, 590, 634, 678, 722, 766, 810, 854, 898; = m/z 412, 456, 500, 544, 588, 632, 676, 720, 764, 808, 852, 896; = m/z 430, 474, 518, 562, 606, 650, 694, 738, 782, 826, 870, 914;

indicative of three series of homologous compounds which differ by 44u (ethoxylated derivatives). On the basis of molecular weight and ion distribution, we

Figure 6. Positive-ion FAB mass spectrum of the semipermanent hair dye. Rapid Communications in Mass Spectrometry, Vol. 11, 13291334 (1997)

These compounds, which do not furnish molecular ions, were identied on the basis of their typical fragmentation patterns, previously described in detail.1 Compounds 7 and 17 (only tentatively identied because of the poor library match as ethyllaurate and ethylisostearate) are probably homologous compounds: they give almost identical fragmentation patterns (Table 2). The colouring mixture was selectively and quantitatively recovered in the chloroform extracts. The GC/MS prole of this fraction (Fig. 8) shows four main peaks, all of them identied as the components of the colouring matter on the basis of the molecular ions, the fragmentation patterns and the colour developed on TLC plates. Colour 1 (RT 21.24) was the only one identied by library match as 2-amino-5-nitrophenol (Fig. 9(a)), colour 2 (Fig. 9(b)) shows a molecular ion at m/z 182 and a fragmentation pattern governed by the loss of 31u (ion at m/z 151, base peak; M-CH2OH, typical of ethanolamine derivatives); the fragment ions at m/z 121 (151 - 30) and 105 (151 - 46) typical of a nitro

Figure 7. GC/MS analysis of the semipermanent hair dye (n-hexane extract). 1997 by John Wiley & Sons, Ltd.

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Table 2. Hair dye: mass spectral data of the components of the n-hexane extract
Peak RT (min) Scan number Ions (m/z) Assigned structure

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
a

6.48 7.12 8.42 9.00 11.50 13.00 13.30 14.54 15.06 15.30 15.48 16.06 16.48 18.60 19.30 20.06 20.50 21.54 22.60 23.12 23.70 26.00 26.50 27.50 30.00 30.80 33.10 34.00 36.80 38.00

206 218 262 271 347 391 401 449 454 466 476 485 505 560 582 606 616 660 680 698 712 782 798 826 902 927 995 1022 1106 1142

108 [M] ; 91; 79 (b.p.); 77; 51 + 122 [M] ; 91 (b.p.); 65 + 144 [M] ; 101; 85; 73; 60 (b.p.) + 154 [M] ; 136; 121; 93; 71; 59 (b.p.) + 214 [M] ; 125; 97; 83; 69; 54; 43 (b.p.) + 172 [M] ; 129; 115; 101; 87; 73; 60 (b.p.); 43 129; 115; 101; 88 (b.p.); 73 + 242 [M] ; 111; 97; 83; 69; 55 (b.p.) + 150 [M] ; 135 (b.p.); 115; 107; 91 + 220 [M] ; 205 (b.p.); 177; 145; 81; 57 + 206 [M] ; 191; 150; 135; 121 (b.p.); 107; 93 + 208 [M] ; 138; 120 (b.p.); 92; 65 + 200 [M] ; 171; 157; 143; 129; 115; 101; 85; 73 (b.p.); 60; 43 + 270 [M] ; 125; 111; 97; 83; 69; 55 (b.p.) Ref. 1 + 228 [M] ; 185; 129; 97; 73 (b.p.); 60; 43 129; 115; 101; 88 (b.p.); 73 + 228 [M] ; 91 (b.p.) Ref. 1 + 256 [M] ; 213; 129; 97; 73; 60; 43 (b.p.) Ref. 1 + 284 [M] ; 241; 185; 129; 97; 73; 43 (b.p.) Ref. 1 Ref. 1 Ref. 1 Ref. 1 Ref. 1 Ref. 1 Ref. 1 Ref. 1

Phenylmethanol Phenylethanol Caprylic acid Terpineol Myristyl alcohol (C14OH) Capric acid Not identied (ethyl-laurate?) Cetyl alcohol (C16OH) 2-t-butylphenol Butylated hydroxytoluene (BHT) -Methyl-ionone Amyl salicylate Lauric acid Stearyl alcohol (C18OH) C16H33-OCH2CH2OH (C16E1) Myristic acid Not identied (ethyl-isostearate?) Benzyl salicylate C18H37-OCH2CH2OH (C18E1) Palmitic acid C16E2 Stearic acid a C18E2 C16E3 C18E3 C16E4 C18E4 C16E5 C18E5 C16E6

E represents an ethoxylated derivative.

group, indicate the compound to be HC Yellow 2 (N-2-hydroxyethyl-o-nitroaniline). Colour 3 (orange) with molecular ion at m/z 198 and a fragmentation pattern with ions at m/z 167 (base peak), 137, 121 similar to that of HC-Yellow 2, was identied as O-(2-hydroxyethyl)-4-amino-3-nitrophenol (Fig. 10(a)); colour 4 (Fig. 10(b)) as HC Red 3, N-(2-hydroxyethyl)-2-nitro-p-phenylendiamine, with molecular ion at m/z 197, and fragments at m/z 166 (M - 31), 136 and 119, shifted by one mass unit with respect to those of colour 3. The other minor peaks were identied as due to N-methyl-pyrrolidone (RT 8.12), lauramide DEA (diethanolamide of lauric acid; RT

19.24), t-butyl-cymene (RT 12.48), and diisooctylphalate (RT 33.24), the rst two being dye solvents. The percentage composition of the product, established by gravimetric and titrimetric analyses, is shown in Table 1.

Figure 8. GC/MS analysis of the semipermanent hair dye (chloroform extracts). 1997 by John Wiley & Sons, Ltd.

Figure 9. EI mass spectra of (a) colour 1 and (b) colour 2. Rapid Communications in Mass Spectrometry, Vol. 11, 13291334 (1997)

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CONCLUSIONS The potentialities of mass spectrometry have hitherto been demonstrated in several elds of interest, such as pharmacology, biochemistry, toxicology, environmental pollution, clinical chemistry, etc. Surprisingly a eld still lacking a systematic investigation by mass spectroscopists is the cosmetic one, in which analysis is still carried out by conventional/traditional methods which are time-consuming, sometimes unreliable and which do not furnish an unequivocal characterization of the analyte. This last becomes urgent because of the increasing body of unwanted reactions (irritation is the

most common side effect to cosmetics) experienced by the consumer. Several studies carried out in USA,5,6 Sweden,7 Spain,8 France,9 The Netherlands10,11 indicate that 3 to 8% of adverse skin reactions are caused by hair colorants and up to 23% by eye make-up products. The approach proposed here seems adequate to satisfy the requirements outlined in the objectives, since it gives, at least from a qualitative point of view, a fairly complete picture of the functional and base ingredients of these formulations, containing more than 30/40 analytes each. Above all it is rapid and gives unequivocal structural information, this last being of outstanding importance when the identication of the causative allergen(s) is urgent for proper medical treatment. The percentage composition achieved for both the cosmetics shows the method to offer a successful approach to the problems. REFERENCES
1. U. Vettori, S. Issa, R. Maffei Facino and M. Carini, Biomed. Environ. Mass Spectrom. 17, 193 (1988). 2. R. Maffei Facino, M. Carini, P. Minghetti, G. Moneti, E. Arlandini and S. Melis, Biomed. Environ. Mass Spectrom. 18, 673 (1989). 3. R. Maffei Facino, M. Carini, S. Sala, P. Minghetti and P. Traldi, Biomed. Environ. Mass Spectrom. 19, 493 (1990). 4. R. Maffei Facino, M. Carini, G. Depta, P. Bernardi and B. Casetta, J. Am. Oil Chem. Soc. 72, 1 (1995). 5. W. F. Schorr, Cutis 14, 844 (1974). 6. R. M. Adams and H. I. Maibach, J. Am. Acad. Derm. 13, 1062 (1985). 7. E. Skog, Contact Dermatitis 6, 449 (1980). 8. C. Romaguera, J. M. G. Camarasa, A. Alomar and F. Grimalt, Contact Dermatitis 9, 167 (1983). 9. Z. Ngangu, M. Samsoen and J. Foussereaus, Dermatosen 31, 126 (1983). 10. A. C. De Groot, Contact Dermatitis 17, 26 (1987). 11. A. C. De Groot, D. P. Bruynzeel, J. D. Bos, H. L. van der Meeren, T. van Joost, B. A. Jagtman and J. W. Weyland, Arch. Dermatol. 124, 1525 (1988). 12. D. C. Abbott, Analyst 87, 286 (1962). 13. H. K. Biswas and B. M. Mandal, Anal. Chem. 44, 1636 (1972).

Figure 10. EI mass spectra of (a) colour 3 and (b) colour 4.

Rapid Communications in Mass Spectrometry, Vol. 11, 13291334 (1997)

1997 by John Wiley & Sons, Ltd.