• Application Note

Fast Analysis of Cosmetic Allergens Using UltraPerformance Convergence Chromatography (UPC2) with MS Detection

Fast Analysis of Cosmetic Allergens Using UltraPerformance Convergence Chromatography (UPC2) with MS Detection

  • Jane Cooper
  • Michael D. Jones
  • Stephane Dubant
  • Waters Corporation

Abstract

In the EU Cosmetic Regulations (1223/2009),  there are ‘currently’ 26 fragrance ingredients, 24 volatile chemicals, and two natural extracts (oak moss and tree moss), that are considered more likely to cause reactions in susceptible people. These 26 fragrance ingredients must be indicated in the list of ingredients of the final product, if the concentration exceeds 0.001% (10 mg/kg) in leave-on products, e.g. moisturizers, or 0.01% (100 mg/kg) in rinse-off products, e.g shampoos. Listing the regulated allergens on products can help identify the cause of an allergic reaction and also aids people to make informed choices about what they buy, particularly if they have a diagnosed allergy to a specific fragrance ingredient.

Current analytical methods used for the analysis of cosmetic allergens include Gas Chromatography Mass Spectrometry (GC-MS), Headspace-GC-MS, GC-GC/MS, Liquid Chromatography-UV (LC-UV), and LC-MS, which all have run times of approximately 30 to 40 minutes.

There are many challenges that need to be addressed for any method used for allergen analysis. For example, the resolution achieved between analyte, isomer, and matrix components all need to be optimized, and the sensitivity of the method should be at least 1 ppm (greater preferred).

Convergence Chromatography (CC) is a separation technique that uses carbon dioxide as the primary mobile phase, with the option if required to use an additional co-solvent such as acetonitrile or methanol to give similar selectivity as normal phase LC.

Benefits

This application note will consider how hyphenating UltraPerformance Convergence Chromatography (UPC2) with MS detection can be used to achieve specificity, selectivity, and sensitivity for the analysis of fragrance allergens in perfume, cosmetics, and personal care products in a fast 7-minute run. UPC2  is an ideal alternative to both HPLC and GC analysis, providing:

  • Ability to run LC and GC amenable compounds in a single analysis.
  • Greater selectivity and specificity compared to either HPLC or GC analysis alone. 
  • more than six times faster analysis times than existing HPLC and GC methods
  • 95% less solvent usage than existing HPLC methods.

Introduction

Fragrances are complex combinations of natural and/or man-made substances that are added to many consumer products to give them a distinctive smell, impart a pleasant odor, or mask the inherent smell of some ingredients, but ultimately to enhance the experience of the product user. Fragrances create important olfactory benefits that are ubiquitous, tangible, and valued. Fragrances can be used to communicate complex ideas such as creating mood, signaling cleanliness, freshness, softness, alleviating stress, creating well-being, or to trigger allure and attraction. 

In most types of cosmetics and skin care products, including perfumes, shampoos, conditioners, moisturizers, facial cosmetics, and deodorants, there are more than 5000 different fragrances present. Many people suffer from allergies, which are caused by an abnormal reaction of the body to a previously encountered allergen that can be introduced in a number of ways such as by inhalation, ingestion, injection, or skin contact. Allergies are often manifested by itchy eyes, a runny nose, wheezing, skin rashes (including dermatitis1), or diarrhea. 

In the EU Cosmetic Regulations (1223/2009),2 there are ‘currently’ 26 fragrance ingredients, 24 volatile chemicals, and two natural extracts (oak moss and tree moss), that are considered more likely to cause reactions in susceptible people. These 26 fragrance ingredients must be indicated in the list of ingredients of the final product, if the concentration exceeds 0.001% (10 mg/kg) in leave-on products, e.g. moisturizers, or 0.01% (100 mg/kg) in rinse-off products, e.g shampoos. Listing the regulated allergens on products can help identify the  cause of an allergic reaction and also aids people to make informed choices  about what they buy, particularly if they have a diagnosed allergy to a specific fragrance ingredient. 

Current analytical methods used for the analysis of cosmetic allergens include Gas Chromatography Mass Spectrometry3-5 (GC-MS), Headspace-GC-MS,6 GC-GC/MS, Liquid Chromatography-UV ( LC-UV),7 and LC-MS,8 which all have run times of approximately 30 to 40 minutes. 

The current 24 regulated volatile cosmetic allergens contain compounds from different classes and different polarities (phenols, cyclic hydrocarbons, alcohols, carbonyl compounds, esters, and lactones). Many are small molecules with similar structures that often produce non-specific fragment ions for mass spectrometric detection.

There are many challenges that need to be addressed for any method used for allergen analysis. For example, the resolution achieved between analyte, isomer, and matrix components all need to be optimized, and the sensitivity of the method should be at least 1 ppm (greater preferred). 

Convergence Chromatography (CC) is a separation technique that uses carbon dioxide as the primary mobile phase, with the option if required to use an additional co-solvent such as acetonitrile or methanol to give similar selectivity as normal phase LC.

This application note will consider how hyphenating Waters®UltraPerformance Convergence Chromatography (UPC2) with MS detection can be used to achieve specificity, selectivity, and sensitivity for the analysis of fragrance allergens in perfume, cosmetics, and personal care products in a fast 7-minute run. 

Experimental

Sample preparation

Cosmetic and personal care sample analysis

  • 0.2 g sample was added to 2.5 mL water and 2.5 mL (methanol + 20 mM ammonium formate).
  • Mixture vortexed for 2 min (1600 rpm).
  • Mixture further extracted in an ultrasonic bath for 30 min.
  • Approximately 1 mL of extract centrifuged for 5 min (10,000 rpm).
  • Centrifuged extract transferred to LC vials ready for analysis.

Perfume

100 μL sample + 900 μl (methanol + 20 mM ammonium hydrogen carbonate).

 

Gradient

Time (min)

Flow rate(mL/min)

%A

%B

Curve

1.0

Initial

1.5

99.5

0.5

2.0

4.5

1.5

85.4

14.6

6.0

3.0

4.6

1.5

80.0

20.0

6.0

4.0

5.0

1.5

80.0

20.0

6.0

5.0

5.05

1.5

99.5

0.5

6.0

6.0

7.0

1.5

99.5

0.5

6.0

Table 1. ACQUITY UPC2 mobile phase gradient.

The MS conditions were optimized for the analysis of 24 currently regulated cosmetic allergens. Six additional compounds were also analyzed, considering cosmetic allergens that could potentially be added during future regulation changes, and two compounds that are potential carcinogens (methyl eugenol and 4-allyl anisole). CAS numbers, empirical formulas, and structures are detailed in Table 2 and Table 3 respectively. The established MRM method (Table 4) utilizes fast polarity switching available on the Xevo TQD, which enables the analysis of positive and negative allergens within the same analytical analysis.

Table 2. Cosmetic allergens considered, as regulated under current EU Cosmetic Regulations 1223/2009,2 associated CAS numbers, empirical formulas, and structures.
Table 3. Additional compounds considered, associated CAS numbers, empirical formulas, and structures.
Table 4. Expected retention times, ionization mode, cone voltages, MRM transitions, and associated collision energy values for 24 regulated cosmetic allergens and six additional compounds.

Instrument control, data acquisition, and results processing

MassLynx Software was used to control the ACQUITY UPC2 and the Xevo TQD, and also for data acquisition. Data quantitation was achieved using the TargetLynx Application Manager.

Results and Discussion

The analysis of the 24 regulated and 6 additional compounds was achieved using the Xevo TQD in MRM mode with APCI ionization (+/-), coupled  to an ACQUITY UPC2 System.

Optimum MRM and UPC2 conditions were  developed with the elution of all compounds within a 7-minute run. 

Mixed calibration standards, 0.25 to 25 ppm,  were prepared and analyzed. An example calibration curve generated for cinamyl alcohol, shown in  Figure 1, with an r2 value of 0.9999. The MRM chromatograms for each compound are shown in Figure 2.

The developed 7-minute UPC2 method, is more than six times faster than existing HPLC and GC methods, with an excess of 95% less solvent usage than existing HPLC methods. 

Figure 1. TargetLynx Quantify results browser showing the calibration curve for cinnamyl alcohol.
Figure 2. MRM chromatograms for 24 regulated cosmetic allergens and six additional compounds in 10 ppm calibration standards (1 ppm for chloratranol and atranol).

Shampoo and perfume analysis

The MRM mass detection method (Table 4) was used after appropriate sample preparation for the analysis of the 24 regulated and four additional compounds in shampoo and perfume samples.

Perfume samples were fortified at 10 mg/kg (0.001%) with 24 cosmetic allergens, and four additional compounds. They were then prepared for analysis as detailed in the Experimental section. Example MRM chromatograms achieved for fortified perfume are shown in Figure 3.

Figure 3. MRM chromatograms for 24 cosmetic allergens and four additional compounds in perfume, fortified at 10 mg/kg (0.001%). 

Shampoo samples were fortified at 100 mg/kg (0.01%) with 24 cosmetic allergens and 4 additional compounds, then prepared for analysis as detailed in the Experimental section. Example MRM chromatograms achieved for fortified shampoo are shown in Figure 4.

Figure 4. MRM chromatograms for 24 cosmetic allergens and 4 additional compounds in shampoo fortified at 100 mg/kg (0.01%). 

Various cosmetic allergens compounds are isomeric, for example Farnesol where potentially four isomeric forms can be produced (Figure 5).  For the example of farnesol, normally trans,trans-farnesol is the major isomer, with trans,cis-farnesol and cis,trans-farnesol being the minor forms, leaving cis,cis-farnesol which is rarely seen. This is demonstrated by the MRM chromatograms (Figure 6) for  farnesol in a shampoo sample fortified at 10 mg/Kg (one tenth of the regulated limit of 0.01%), and the nearest equivalent standard (0.5 ppm), which illustrated several isomeric farnesol peaks. For comparison, a blank shampoo sample MRM chromatogram for farnesol  is also shown in Figure 6.

Additional benefits of using ACQUITY UPC2 coupled to the Xevo TQD over previous methodology include improved selectivity and sensitivity for the analysis of cosmetic allergens. The established method achieves resolution between analytes, isomers, and matrix. Additionally, the attained sensitivity is four times less than required (0.25 ppm).

Figure 5. Four isomers of farnesol. 
Figure 6. MRM chromatograms for shampoo fortified at 10 mg/Kg (one-tenth of the regulated limit of 0.01%), the nearest equivalent standard (10 mg/Kg), and a blank shampoo sample. 

Conclusion

  • Separation by UPC2  is an ideal alternative to both HPLC and GC analysis.
  • Ability to run LC and GC amenable compounds in a single analysis.
  • Fast 7-minute analysis of the 24 regulated cosmetic allergens, 4 non-regulated cosmetic allergens, and 2 potential carcinogenic compounds containing:
    • different classes of compounds;
    • different polarities.
  • UPC2  with MS detection offers an orthogonal technique, which enables greater selectivity and specificity compared to either HPLC or GC analysis alone.
  • The developed 7-minute UPC2  method is more than six times faster than existing HPLC and GC methods, with 95% less solvent usage than existing HPLC methods.

Acknowledgements

Celine Roy (ERINI, France), Beatrice Grimaud and Isabelle Dubrulle (Yves Rocher, France) for guidance and advice during the development of this application note.

References

  1. Larson W, Nakayama H, Fischer T, et al. Fragrance contact dermatitis: a worldwide multicenter investigation (Part II). Contact Dermatitis. 44(6): 344–346, June 2001.
  2. 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:2009:342:0059:0209:en:PDF
  3. Debonneville C, Chaintreau A. Quantitation of suspected allergens in fragrances: evaluation of comprehensive GCconventional MS. J Chromatogr A. 1027: 109–15, 2004.
  4. Rastogi S C. Analysis of Fragrances in Cosmetics by Gas Chromatography-Mass Spectrometry. J High Resolution Chrom. 18: 653–658, 1995.
  5. López-Nogueroles M, Chisvert A, Salvador A. Determination of atranol and chloratranol in perfumes using simultaneous derivatization and dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry. Analytica Chimica Acta. 826: 28–34, 2014.
  6. Desmedt B, Canfyn M, Pype M, et al. HS-GC-MS method for the analysis of fragrance allergens in complex cosmetic matrices. Talanta. 131: 444–451, 2015.
  7. Villa C, Gambaro R, Mariani E, Dorato S. High-performance liquid chromatographic method for the simultaneous determination of 24 fragrance allergens to study scented products. J Pharmaceutical and Biomedical Analysis. 44: 755–762, 2007.
  8. Rudback J, Islam N, Nilsson U, et al. A sensitive method for determination of allergenic fragrance terpene hydroperoxides using liquid chromatography coupled with tandem mass spectrometry. J Sep Sci. 36:1370–1378, 2013.

720005553, January 2017

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