ALLERGENS IN PERFUMES:GAS CHROMATOGRAPHY^MASS SPECTROMETRYS. C. Rastogi, National EnvironmentalResearch Institute, Rokilde, Denmark

Copyright^ 2000 Academic Press

Perfumes (fragrance substances) are used in the for-mulation of consumer products to provide pleasure tothe user and/or to mask malodours of some otheringredients in the products. Perfumes are also used inaromatherapy. A typical perfume may be composedof 10}300 substances selected from a battery of over3000 synthetic and natural fragrance materials. It hasbeen shown that approximately 2% of the generalpopulation is allergic to perfumes. Furthermore, per-fumes have also been shown to be one of the majorcause of allergic contact dermatitis from the use ofcosmetics and toiletries. Besides cosmetics, the use ofmany other consumer products such as perfumedlaundry detergents and dishwashers have also beenimplicated as the cause of perfume allergy in contacteczema patients.

Perfume allergy in contact eczema patients isdiagnosed by patch-testing with a fragrance mix con-taining 1% each of geraniol, eugenol, isoeugenol,cinnamic alcohol, cinnamic aldehyde, �-amylcin-namic aldehyde, hydroxycitronellal and an extractfrom oakmoss } oakmoss absolute. However, only50}80% of perfume allergy cases are diagnosed bythis test. For the management of allergy, it is impor-tant to identify the fragrance allergen responsible forcontact eczema in a patient, as this makes it possiblefor the patient to avoid the use of products containingthe sensitizing allergen(s). To establish the identity ofthe fragrance substance responsible for perfume al-lergy in a contact eczema patient, it is recommendedthat the product(s) used by a patient should be ana-lysed for the contents of fragrance allergens followedby patch-testing the patient with the relevant fra-grance allergens present in the product.

Gas chromatography}mass spectrometry (GC-MS)is frequently used for the analysis of fragrance sub-stances in essential oils. This approach is used for theidentiRcation and semiquantitative determination ofthe fragrance substances of interest in essential oils.In 1995, GC-MS was used for the identiRcationand quantiRcation of 10 selected fragrance substancesincluding the seven chemically deRned substancesof fragrance mix in perfumes, eau de toilette,

deodorants, creams, lotions, shampoos and other per-fumed consumer products which may containboth natural as well as synthetic fragrance materials.The method was later modiRed slightly so that quant-itative analysis of many more fragrance substances inperfumes or in perfumed products could be per-formed. This method, described in the presentarticle, has been applied to the analysis of perfumesin various consumer products. To demonstrate thepotential of the method for perfume analysis,example of analysis of fragrance substances in a de-odorant and in an eau de toilette are presented here.Sample preparation methods for the GC analysis offragrances in various types of consumer products isalso described. The quantitative data on fragrancesubstances in various consumer products are reportedin the publications described in the Further Readingsection.

Target Fragrance Substances

The analytical method has been developed for thequantiRcation of 21 fragrance substances which inrelatively high concentrations are commonly used inthe composition of perfumes, or which are estab-lished contact allergens:

1 geraniol: CAS registration number 106-24-1;2 eugenol: 97-53-0;3 isoeugenol: 97-54-1;4 linalool: 78-70-6;5 linalyl acetate: 115-95-7;6 citronellol: 106-22-9;7 cinnamic alcohol: 104-54-1;8 cinnamic aldehyde: 104-55-2;9 hydroxycitronellal: 107-75-5;

10 �-amylcinnamic aldehyde: 122-40-7;11 �-hexylcinnamic aldehyde: 101-86-0;12 �-isomethylionone: 127-51-5;13 coumarin: 91-64-5;14 piperonal: 120-50-7;15 benzyl alcohol: 100-51-6;16 benzyl acetate: 140-11-4;17 benzyl benzoate: 121-51-4;18 benzyl salicylate: 118-51-8;19 Lilial�: 80-54-6;20 Lyral�: 31906-04-4;21 Hedione�: 24851-98-7.

1974 III / ALLERGENS IN PERFUMES: GAS CHROMATOGRAPHY^MASS SPECTROMETRY

Approximately 1.0% (w/v) solutions of all of thesubstances in ethanol served as stock solutions. Thestock solutions were stored in closed vials at 43C andwere used within 1 month.

Sample Preparation

Perfumes, Eau de Toilette, Aftershave andDeodorant Sprays

These products were approximately diluted inethanol so that the concentrations of target fragrancesubstances were 40.1%. Depending on the concen-trations of the target fragrance substances ina sample, it may be necessary to analyse several dilu-tions of the sample.

Shampoos, Creams, Lotions, Lipsticks,Face Powders and Deodorant Sticks

Perfumes from 1 g sample were extracted in 10 mlmethanol at 603C (to facilitate the extraction) fol-lowed by removal of matrix components by silica gelcolumn chromatography. The extract was loaded ona 7�1.8 cm silica gel column, and the fragrancefraction was eluted with methanol. The perfume ex-tract was stored at 43C and analysed within 24 h.

Soap Bar and Laundry Detergents

Perfumes from 1 g sample dissolved in 50 ml waterwere extracted in 10 ml ethyl acetate employingliquid}liquid extraction. The perfume extract in ethylacetate was centrifuged to remove any solid or aque-ous contamination. The perfume extract was storedat 43C and analysed within 24 h.

Dishwashing liquid

The method used for the extraction of perfumes fromdishwashing liquids was the same as for shampoo.

GC-MS Analysis

MS Conditions

Electron impact ionization at 70 eV was used, scann-ing m/z 29}250 in 0.6 min.

Results

The method described here has been applied to thedetermination of 21 target fragrance substances inconsumer products. The chromatographic separationof these 21 compounds employing GC is shown inFigure 1. Day-to-day variation of retention times ofthe fragrance substances is (0.5%. The detectionlimits of all of the target substances are 41 p.p.m.,

the calibration curves for all of the target substancesare linear (coefRcient of correlation '0.995) inthe tested concentration range 10}2000 p.p.m.,the relative standard deviations of the determina-tion of all of the substances are (11%. The recov-ery of all of the target substances from the spikedsamples is 82}116%, and day-to-day variations ofquantitative analysis for all of the substances arewithin 5%.

The reconstructed ion chromatogram obtained byGC-MS analysis of fragrance substances in a deodor-ant (undiluted) is shown in Figure 2. The fragrancesubstances in the product were identiRed by compar-ing the retention times of the GC peaks with those ofthe reference materials, as well as by comparing thespectra of the GC peaks with the reference spectra ofstandard compounds in the mass spectrum library.Followed by GC-MS identiRcation, quantiRcation oftarget fragrance substances in the sample is carriedout with external standards.

Most consumer products contain many morefragrance substances other than the target com-pounds. The identiRcation of these substances wasonly performed by comparing the mass spectrum ofa GC peak with the mass spectra of reference com-pounds in the MS library. In this case, both thespectrum Rt and spectrum purity of match of theunknown spectrum with those of library spectra were'900. An example of identiRcation of fragrancesubstances in an eau de toilette is shown in Fig-ure 3A}E, where the results are divided in six win-dows for the clarity of peak identiRcation. ConRrma-tion of the identiRcation of these substances and theirquantiRcation were performed where a referencematerial was available.

In some cases it is not possible to identify all thepeaks because of the absence of mass spectra of thecompounds in the mass spectral library.

Discussion

For the analysis of perfumes on a routine basis,GC-MS identiRcation of the fragrance substancesfollowed by quantiRcation employing GC-Same ion-ization detection (FID) was found to be a more suit-able approach. The main reason for this is that the useof GC-FID allows relatively rapid production of vali-dated data. Thus, several relevant analysis recom-mended by quality assurance/quality control(QA/QC) protocol for a set of samples can be easilyperformed by GC-FID. FulRlling the requirements ofQA/QC protocol for the analysis by GC-MS is time-consuming, because it requires tuning and calibrationof the MS at regular intervals and frequent cleaningof the ion source. The detection limits of the target

III / ALLERGENS IN PERFUMES: GAS CHROMATOGRAPHY^MASS SPECTROMETRY 1975

Figure 1 GC-MS analysis of a mixture containing 83}117 p.p.m. of the 21 target fragrance substances. 50 m�0.32 mm, 1.2 �m filmthickness Chrompack fused silica capillary columns coated with CP-Sil-5CB, were used. 1 �L split injection; helium carrier gas flow30 ml min�1, column-head pressure 20 psi; injector temperature 3003C; column temperature program: 40}1403C in 4 min, thereafter53C min�1 to 2803C, 5 min at 2803C. 2 �L injection volume was used when the content of perfume in a sample was relatively low.

1976 III / ALLERGENS IN PERFUMES: GAS CHROMATOGRAPHY^MASS SPECTROMETRY

Figure 2 GC-MS analysis of the target fragrance in an undiluted deodorant. The following were present among the target fragrancesubstances: 102 p.p.m. benzyl alcohol, 1028 p.p.m. linalool, 141 p.p.m. citronellol, 136 p.p.m. geraniol, 614 p.p.m. linalyl acetate,205 p.p.m. hydroxycitronellal, 183 p.p.m. cinnamic alcohol, 408 p.p.m. eugenol, 1051 p.p.m. coumarin, 7 p.p.m. isoeugenol, 319 p.p.m.�-isomethylionone, 291 p.p.m. Lilial�, 199 p.p.m. Hedion�, 68 p.p.m. �-amylcinnamic aldehyde, 101 p.p.m. benzyl benzoate and112 p.p.m. benzyl salicylate. Quantification of Hedion� was performed by the analysis of 1 : 10 dilution of the sample, where nointerference by the sesquiterpene alcohol present in the sample was observed.

III / ALLERGENS IN PERFUMES: GAS CHROMATOGRAPHY^MASS SPECTROMETRY 1977

Figure 3 GC-MS analysis of an eau de toilette, diluted 1 : 10 in ethanol. The reconstituted ion chromatogram is divided in six windows(A}F) for the clarity of the compounds identified in the sample. Peaks with no name could not be identified.

1978 III / ALLERGENS IN PERFUMES: GAS CHROMATOGRAPHY^MASS SPECTROMETRY

Figure 3 Continued

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Figure 3 Continued

1980 III / ALLERGENS IN PERFUMES: GAS CHROMATOGRAPHY^MASS SPECTROMETRY

substances by GC-FID, however, are 2}5 p.p.m. So,unless the quantiRcation was required at 1 p.p.m.level, GC-FID was chosen for the determination offragrance substances after prior identiRcation byGC-MS.

Most of the fragrance substances in use, includ-ing the target fragrance substances, have a molecu-lar weight (250 Da. Therefore, the MS scan wasperformed only up to m/z 250. Occasionally, forexample in the identiRcation of musk ketone, it isnecessary to scan masses up to 300.

Not all the fragrance ingredients in all tested prod-ucts could be identiRed or quantiRed, in some casesdue to interferences. Occasionally the GC peak ofa relatively high amount of dipropylene glycol presentin a sample overlapped the peak by benzyl alcohol;a C11-alkyne interfered with the analysis of Lilial�;high amounts of triethyl citrate and/or a sesquiter-pene alcohol (C15H26O) interfered with the analysis ofHedione� and relatively high amounts of Hedione�interfered with the analysis of �-amylcinnamic al-dehyde. An unidentiRed compound was found to in-terfere with the analysis is benzyl salicylate. In mostcases these problems could be solved by analysingdiluted samples.

By using GC-MS, identiRcation of 226 substancesin deodorants has recently been reported. A struc-ture}activity relationship (SAR) analysis of contactallergens revealed that 84 of the identiRed com-pounds possess at least one structural alert (chemicalgroup) having sensitizing potential, and 70 belong to,or are susceptible to metabolize into, the chemicalgroups having sensitizing properties: aldehydes,ketones and �,�-unsaturated aldehydes, ketones oresters. The combination of GC-MS and SAR analysiscould be helpful in the selection of substances forsupplementary investigations regarding sensitizingproperties.

Analysis of as many fragrance ingredients aspossible in a perfumed product is of great import-ance for clinicians to establish the identity ofcontact allergens in each case. This informationis also important for clinical research to investi-gate cross-reactions of fragrance allergens. Thequantitative data on the fragrance ingredients inconsumer products make a basis for exposure as-sessment that is a help for establishing thresholdconcentrations of fragrances for the elicitation ofcontact allergy.

Conclusions

Chemical analysis of perfumes and perfumed prod-ucts is of great importance for the diagnosis andmanagement of perfume allergy. The GC-MS/GC-

FID method described here for the analysis of fra-grance substances in consumer products has provedto be valuable to identify allergens in patients withcontact eczema from the use of perfumes and per-fumed products. Using GC-MS in combination withSAR it has been possible to identify several fragrancesubstances in perfumes which possess sensitizing po-tential.

See also: II/Chromatography: Gas: Column Techno-logy; Derivatization; Detectors: General (Flame IonizationDetectors and Thermal Conductivity Detectors); Detectors:Mass Spectrometry; Detectors: Selective; HeadspaceGas Chromatography; Theory of Gas Chromatography.III/Flavours: Gas Chromatography: Sulphur Com-pounds: Gas Chromatography.

Further Reading

Calkin RR and Jellinek JS (1994) Perfumery Practice andPrinciples. New York: Wiley.

De Groot AC and Frosch P (1997) Adverse reactions tofragrance. A clinical review. Contact Dermatitis 36:57}86.

Frosch PJ, Pliz B, Andersen KE et al. (1995) Patch testingwith fragrances: results of a multicenter study of theEuropean Environmental and Contact Dermatitis Re-search Group with 48 frequently used constituents ofperfumes. Contact Dermatitis 33: 333}342.

Frosch PJ, Johansen JD and White IR (eds) (1998) Fragran-ces: BeneTcial and Adverse Effects. Berlin: Springer-Verlag.

Johansen JD, Rastogi SC and MenneH T (1996) Contactallergy to popular perfumes; assessed by patch test, usetest and chemical analysis. British Journal of Dermatol-ogy 135: 419}422.

Larsen W, Nakayama H, Lindberg M et al. (1996) Fra-grance contact dermatitis: a worldwide multicenterinvestigation (part I). American Journal of Contact Der-matitis 7: 77}83.

Pybus DH and Sell CS (eds) (1999) The Chem-istry of Fragrances. Cambridge: Royal Society ofChemistry.

Rastogi SC (1995) Analysis of fragrances in cosmetics bygas chromatography-mass spectrometry. Journal ofHigh Resolution Chromatography 18: 653}658.

Rastogi SC, Johansen JD and MenneH T (1996) Naturalingredients based cosmetics: content of selected fra-grance sensitizers. Contact Dermatitis 34: 423}426.

Rastogi SC, Johansen JD, Frosch P et al. (1998) Deodor-ants on the European market: quantitative chem-ical analysis of 21 fragrances. Contact Dermatitis 38:29}35.

Rastogi SC, Leppoitevin JP, Johansen JD et al. (1998)Fragrances and other materials in deodorants: search forpotentially sensitizing molecules using combined GC-MS and structure activity relationship (SAR) analysis.Contact Dermatitis 39: 293}303.

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