Flow-Modulation Comprehensive 2D GC/MS for the Determination of Allergens in Fragrances

By: Flavio A. Franchina, Chromaleont; Mariarosa Maimone, University of Messina; Professor Luigi Mondello, Analytical Chemistry/Food Chemistry Laboratories, University of Messina, Italy, Reporter US, Volume 33.4

Introduction
Perfumes are complex mixtures comprising essential oils, aroma compounds, fixatives, and solvents. Contact dermatitis, an allergic response, may develop following skin contact with a sufficient amount of these substances in susceptible individuals. Rashes are most common on the face, under the arms, and on the hands, areas most often in direct contact with perfumes and other cosmetics. While per se a latent condition without visible signs or symptoms, the sensitivity can persist lifelong. The condition can be severe and generalized, with a significant impairment to quality of life and potential consequences for ability to work.

In 1999, the European Commission’s Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP) identified a set of 26 fragrance allergens with a well-recognized potential to cause contact allergy, for which information should be provided to consumers about their presence in cosmetic products[1]. Such a list has shown to be important in the clinical management of patients who are allergic to one or more of these 26 fragrance chemicals. In fact, these compounds must be reported in the ingredient list of cosmetic products sold in Europe so they can be avoided by patients with known allergic reactions. The aforementioned allergens are regulated by the European Directive 2003/15/EC fixing a maximum residue limit for “leave-on” and “rinse-off” products of 0.001% (10 ppm) and 0.01% (100 ppm), respectively[2].

In 2012, the revision of the SCCNFP Opinion (SCCS/1459/11, June 2012) on the clinical and experimental data published in 1999, confirmed the previously regulated allergens and stressed attention should be paid to additional fragrance substances which have been shown to be sensitizers in humans. Consequently, an updated list of allergens was reported, with a systematic and critical review of the scientific literature to identify them, both as single chemicals and as natural extracts. Eighty-two compounds were finally classified as contact allergens in humans, of which 54 were single chemicals and 28 were natural extracts[3].

The Scientific Committee on Consumer Safety (SCCS) examined available elicitation dose-response data to decide whether safe thresholds can be established for the fragrance allergens. The few studies available indicated that a general level of exposure of up to 0.8 ìg/cm² (0.01% in cosmetic products) may be tolerated by most consumers. The SCCS opinion is that levels of exposure below this would be sufficient to prevent elicitation for the majority of allergic individuals, unless there is experimental or clinical substance-specific data allowing the derivation of individual thresholds. With regards to the natural extracts, it was not possible to provide a safe threshold as no specific investigations exist and the model providing the general threshold (<0.01%) was based on individual chemicals. However, the SCCS considers that the maximum use concentration applied to the identified chemicals, both if added as chemicals or as an identified constituent of a natural ingredient, will reduce the risk of sensitization and elicitation from natural extracts.

The aim of the present research was the development of a flow-modulation comprehensive 2D GC/MS (FM GCxGC/MS) method for the determination of the recently indicated 54 allergens in fragrances. The detector was a rapid-scanning single quadrupole MS (qMS).

Experimental
Fifty-three allergen standards with purity higher than 95% were used. A standard for santolol (mixture of α- and β-santalol) was not commercially available, thus it was isolated from sandalwood oil using a preparative MDGC system developed in our laboratory. Methanol (purity >99 %) was used to prepare the calibration solutions. Two fragrances were purchased in a local store. Calibration solutions and both samples were analyzed using the conditions listed in Figure 1. Mass spectral matching was carried out by using the FFNSC database[4].

Conditions for Figure 1
column: (1D) SLB®-5ms, 20 m x 0.18 mm I.D., 0.18 µm (28564-U)
column: (2D) SLB-35ms, 5 m × 0.25 mm I.D., 0.25 µm; cut from a 30 m × 0.25 mm I.D., 0.25 µm column (29804-U)
oven: (1D) 45 °C, 3 °C/min to 230 °C
oven: (2D) 50 °C, 3 °C/min to 230 °C
oven: (flow modulator) stainless steel accumulation loop (20 cm x 0.71 mm O.D. x 0.52 mm I.D.), modulation period 5.4 sec (accumulation period 4.9 sec, injection period 0.5 sec)
inj. temp.: 310 °C
detector: qMS, 200 °C, m/z = 40-360
MSD interface: 250 °C
carrier gas: (1D) helium, 0.46 mL/min
carrier gas: (2D) helium, 7 mL/min
injection: 1 µL, 10:1 split
liner: 3.4 mm I.D., split/splitless type, wool packed straight FocusLiner™ design (2877601-U)
sample: perfume

Figure 1. GCxGC-qMS Chromatograms of Perfume Samples 1 and 2

Perfume Sample 1


Perfume Sample 2


Results and Conclusion
The 54 standard allergens, plus two internal standards (50 ppm), were used for calibration purposes (5, 10, 20, 50, and 100 ppm). Quantification was performed by using extracted ion chromatograms (Table 1). There are more than 54 allergens reported in Table 1 because some are considered to be mixtures (e.g. α- and β-santalol), and are here listed as single compounds. Method linearity was satisfactory, being in the range 0.9950-0.9988 (coefficients of determination). Method limits of quantification (LOQ) were all lower than 1 ppm, which is well below the European Commission’s regulated limit. LOQ values were extrapolated by considering a signal-to-noise (s/n) value of 10.

Two samples (1 and 2) of perfume were subjected to analysis. The quantification values expressed in ppm are reported in Table 1. Altogether 14 allergens were quantified in each sample (n = 2). The full-scan GCxGC-qMS chromatograms of sample 1 and 2 are shown in Figure 1. As can be seen, the SLB-5ms/SLB-35ms combination produced a satisfactory distribution of the analytes across the 2D space. Both columns are characterized by high thermal stability as can be seen from the absence of column bleed in the chromatogram. Moreover, retention time precision was excellent over a prolonged period on time (45 days of applications), confirming the stability of the 5ms and 35ms phases in each dimension.

The proposed method appears to be a reliable and sensitive approach for the determination of allergens in perfumes following the SCCNFP Opinion (SCCS/1459/11, June 2012).

Table 1. List of Allergens and Internal Standards, Linearity, Quantification Ions, and Concentrations in Two Perfume Samples
No. Compound R2 Quantifier Ion Sample
1 (ppm)
Sample
2 (ppm)
Cat. No.
1 α-Pinene 0.9969 93 76.8 27.4 80605
2 Benzaldehyde 0.9972 77 09143
3 ß-Pinene 0.9968 69 597.1 375.3 80607
4 Limonene 0.9981 93 353.6 322.4 62118,
62128
5 Benzyl Alcohol 0.9973 108 47509-U
6 Salicylaldehyde 0.9979 122 03273
7 Terpinolene 0.9960 93 4.9 5.3 86485
8 Linalool 0.9974 93 264.1 247.1 61706,
51782
9 Camphor 0.9968 95 23.3 21300
10 Menthol 0.9970 81 05174
I.S. Benzene, 2-Bromoethenyl   152 157449
11 Methyl 2-Octynoate 0.9983 95 68982
12 Methyl Salicylate 0.9980 120 76631
13 α-Terpineol 0.9972 93 40 26.7 83073
14 ß-Terpineol 0.9971 93
15 γ-Terpineol 0.9971 93
16 Citronellol 0.9952 69 303461,
303488,
W230901
17 Linalyl Acetate 0.9977 93 81 168.4 49599
18 Neral (Citral component) 0.9976 69 43318
19 Carvone 0.9988 82 22060,
22070
20 Geraniol 0.9966 69 32.1 48798
21 Geranial
(Citral component)
0.9958 69 268.2 43318
22 Farnesol 0.9958 69 43348
23 Cinnamal
(Cinnamaldehyde)
0.9958 131 4.4 W228613
24 Hydroxycitronellal 0.9964 59 36.8 54.8 66010
25 Anise Alcohol (Anisyl alcohol) 0.9965 138 W209902,
W209910
26 trans-Anethol 0.9951 148 10368
27 Dimethylbenzyl Carbinyl Acetate 0.9958 132 W363200
28 Amyl Cinnamyl Alcohol 0.9984 133 W206504
29 Eugenol 0.9978 164 50.5 36.1 35995
30 δ-Damascone 0.9972 69 55137
31 Rose Ketone-4
(Damascenone)
0.9965 69 W342017
32 α-Damascone 0.9969 69 59574
33 Vanillin 0.9950 152 259 30304
34 cis-ß-Damascone 0.9969 177 W324300
35 Trimethylbenzene Propanol (Majantol) 0.9970 106 69791
36 Ebanol 0.9976 121 44064
37 Isoeugenol 0.9982 164 34038
38 Coumarin 0.9981 118 27.9 72609
39 α-Isomethyl Ionone 0.9979 135 5.6 3.7 W271410
40 Butylphenyl
Methylpropional
0.9983 189 95338
41 Amyl Salicylate 0.9983 120 44041
42 6-Methylcoumarin 0.9952 160 69391
43 Propylidene Phthalide 0.9978 159 75012
44 ß-Caryophyllene oxide 0.9979 93 91034
45 Amyl Cinnamal
(α-Amylcinnamaldehyde)
0.9979 129 64397
46 Lyral 0.9982 136 55862
47 Tetramethyl
Acetyloctahydronaphthalenes
0.9986 109 551.1 480.4
48 Cinnamyl Alcohol 0.9974 92 93066
49 Hexyl Cinnamal
(α-Hexylcinnamaldehyde)
0.9979 129 09178
50 Benzyl Benzoate 0.9978 105 68183,
55177
51 Acetyl Cedrene 0.9981 43 18017
52 Hexamethylindanopyran 0.9983 243 W520608
53 Benzyl Salicylate 0.9977 91 51031
54 Hexadecanolactone 0.9982 69 14643
I.S. 4,4’-Dibromobiphenyl   152 442398
55 Benzyl Cinnamate 0.9955 192 69139
56 Sclareol 0.9981 95 49944
57 ß-Santalol 0.9950 94 75831
58 α-Santalol 0.9954 94

 

Featured Products
Description Cat. No.
Capillary GC Columns
SLB-5ms, 20 m × 0.18 mm I.D., 0.18 µm 28564-U
SLB-35ms, 30 m × 0.25 mm I.D., 0.25 µm (a 5 m portion was used in this study) 29804-U
Standards
See Table 1 for part numbers  

References

  1. European Commission, Health and Food Safety “SCCS final opinion on Fragrance allergens in cosmetic products now available.” http://ec.europa.eu/ dgs/health_consumer/dyna/enews/enews.cfm?al_id=1283 (accessed June 2, 2015).
  2. Directive 2003/15/EC of the European Parliament and of the Council of the European Union 27 February 2003 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:066: 0026:0035:en:PDF (accessed June 2, 2015).
  3. European Union Scientific Committee on Consumer Safety (SCCS) Opinion on fragrance allergens in cosmetic products. http://ec.europa.eu/health/ scientific_committees/consumer_safety/docs/sccs_o_102.pdf (accessed June 2, 2015).
  4. Mondello, Luigi. Flavors and Fragrances of Natural and Synthetic Compounds 2, 2nd Edition. John Wiley & Sons, Inc.: Hoboken, NJ, 2011; ISBN 978-1-118-14583-8.

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