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Development of an analysis method for quantification of colophonium components in cosmetic products

Appendix A: Literature study on the detection of the main components of unmodified colophonium in commodities - regarding contact allergy

1 Introduction

1.1 Analytes

The two main components of colophonium are the diterpenoids abietic acid (AbA), CAS no.. 514-10-3 and dehydroabietic acid (DeA), CAS no.. 1740-19-8 [1]. Consequently, these two acids are the most commonly examined analytes when studying the presence of unmodified colophonium in commercial products [2-11]. An additional large number of other terpene species, often oxidised, have also been identified in unmodified colophonium as such [8, 12-17]. Some important allergens are generated through auto-oxidation of AbA and DeA, while the amounts of original acids present in the sample decrease. Therefore, it is important to include one or more oxidised species in the analysis as well as AbA and DeA when determining the presence of colophonium in a sample [8, 12, 14-16]. To be suitable as a marker for unmodified colophonium, an oxidation product should be stable enough for the analysis and preferably also be one of the major constituents of the oxidation mixture as to be present in maximum amounts. 7-oxo-dehydroabietic acid (7-O-DeA), CAS no. 18684-55-4, was chosen for the purpose [8, 12, 14, 15].

1.2 Investigated samples

Commodities investigated for colophonium content include disposable diapers [2], sanitary pads [6], herbal oils and ointments [4], sulphate soaps [9], mascaras [11, 18], adhesives [3, 5, 7], various forms of cardboard and paper [3, 8, 10, 19, 20], floor polish [8] and cutting fluid, soldering flux, and paint products [3]. As the heating of rosin flux during soldering causes a colophonium-containing aerosol, such fumes have been analysed [21]. Dust [8], wood [9] and papermaking process water [22] have also been examined. Furthermore, to study the exposure to colophonium, DeA has been used as a biomarker in urine of factory workers performing soldering [23].

1.3 Sample preparation

Prior to analysis, the samples are prepared by extraction of the analytes into a suitable solvent. Depending on the investigated sample different solvents have been utilized: acetone [2, 7, 8, 10, 19], methanol [3, 12, 19], ethanol [14], acetonitrile [4, 5], dichloromethane [3, 8, 15], ethyl acetate [3], methylene chloride [21], diethyl ether [4, 9, 23] and methyl tert-butyl ether (MTBE) [22]. If necessary, the following sample clean-up may involve filtration [3, 8, 12], centrifugation [4] and/or solid-phase extraction (SPE) [4, 5].

1.4 Analytical methods, separation and detection

To determine the presence of colophonium in the samples, the sample components are often derivatised and separated by gas chromatography (GC) [2, 6, 8-10, 13, 21-23] or separated by high-performance liquid chromatography (HPLC) in their unmodified form [3-5, 8, 12, 13]. Detection of the separated components by GC has been performed by flame ionization [2, 8-10, 22] or mass spectrometry (MS) [21, 23]. For detection of the HPLC separated samples, ultraviolet [3-5, 8, 12, 13], fluorescence detection [4, 5] and MS [12, 13] are normally used. Analytes which are sensitive to the high temperatures in GC have been introduced into the mass spectrometer through a direct insertion probe [13, 14]. An alternative method for such analytes is matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), where a cellulose coated thin layer chromatography (TLC) plate serves as the sample probe [15].

2 Method description and evaluation

2.1 GC methods

2.1.1 GC-FID

Method [9]

  • Description: Heptadecanoic acid as internal standard. Extraction with diethyl ether. AbA and DeA derivatised to methyl esters by diazomethane.
    Advantages: Peak identification based on both retention time of known, pure analytes and mass spectra.
  • Disadvantages: Modification of analytes. Diazomethane is classified as carcinogenic. No oxidation products analysed. Not quantitative.
  • Used for: wood extractives, sulphate soaps, crude and distilled tall oil

Method [22]

  • Description: Heneicosanoic acid, betulinol, cholesteryl heptadecanoate and 1,3-dipalmitoyl-2-oleoyl glycerol as four internal standards. Extraction with MTBE. AbA and DeA silylated by bis(trimethylsilyl)-trifluoro-acetamide (BSTFA) and trimethylchlorosilane (TMCS).
  • Advantages: Quantitative. Many other components also analysed.
  • Disadvantages: Limit of detection unspecified. Modification of analytes. Silylated samples degrade and should be analyzed within 12 hours. No oxidation products analysed. Not optimised specifically for separating AbA and DeA. Method for peak identification unspecified.
  • Used for: papermaking process waters and effluents

Method [2, 8, 10]

  • Description: Methyl stearate as internal standard. Extraction with acetone. Analytes derivatised to methyl esters by diazomethane.
  • Advantages: Detection of AbA, DeA, and 7-O-DeA. Quantitative.
  • Disadvantages: Modification of analytes. Diazomethane is carcinogenic and explosive. Peak identification based on retention time of known, pure analytes; risk of interferences by and confusion with unknown peaks. Need to synthesise reference substance 7-O-DeA, since not commercially available. Detection limit unspecified.
  • Used for: paper and linoleum floor covering, diapers

2.1.2 GC-MS

Method [21]

  • Description: Heptadecanoic acid as internal standard. Extraction with methylene chloride. Detection of AbA and DeA; derivatised to methyl esters by potassium carbonate and methyl iodide.
  • Advantages: Peak identification based on both mass spectra and retention time of known, pure analytes. Quantitative.
  • Disadvantages: Modification of analytes. No oxidation products detected. Limit of detection unspecified.
  • Used for: soldering fumes

Method [23]

  • Description: Heptadecanoic acid as internal standard. Extraction with diethyl ether. DHA derivatised by dimethylformamide dimethylacetal. Limit of detection; 50nM DHA.
  • Advantages: Peak identification based on both mass spectra and retention time of known, pure analytes. Quantitative.
  • Disadvantages: No detection of AbA or oxidation products.
  • Used for: biomarker in urine

2.2 HPLC methods

2.2.1 HPLC-UV

Method [3]

  • Description: UV detection of AbA at 242nm and DeA at 265nm. Calibration with pure acids. Extraction with methanol, ethyl acetate or dichloromethane. Limits of detection; 10ppm AbA, 150ppm DeA.
  • Advantages: No modification of analytes. Additional peak identification by UV-spectra at peak apex. Quantitative.
  • Disadvantages: No oxidation product detected. Peak identification based on retention time of known, pure analytes; risk of interferences by and confusion with unknown peaks. DeA has low absorption at 267nm; at max absorption 215nm there is high matrix interference.
  • Used for: adhesives, cutting fluid, soldering flux, and paint products

Method [13]

  • Description: UV detection of 15-HPDA at 254nm. 15-HPDA thermally instable; for MS collect HPLC fraction; DIP.
  • Advantages: No modification of analytes. Detection of the oxidation product 15-hydroperoxydehydroabietic acid (15-HPDA). Additional peak identification by MS.
  • Disadvantages: AbA and DeA not detected. Not quantitative. Need to synthesise reference substance 15-HPDA, which is not commercially available and unstable. Peak identification based on retention time of known, pure analytes; risk of interferences by and confusion with unknown peaks.
  • Used for: unmodified colophonium as such

Method [8]

  • Description: Method according to [3]. Extraction with acetone or dichloromethane.
  • Advantages: No modification of analytes. Detection of the oxidation product 7-O-DeA, also AbA and DeA.
  • Disadvantages: Must synthesise reference substance 7-O-DeA, since it is not commercially available. Peak identification based on retention time of known, pure analytes; risk of interferences by and confusion with unknown peaks. DeA has low absorption at 267nm; at max absorption 215nm there is high matrix interference. Not quantitative.
  • Used for: Paper, floor polish, dust

Method [12]

  • Description: UV detection of AbA and DeA at 245nm. Extraction with methanol. Separation optimised to for isolation of max number of components.
  • Advantages: Detection of the oxidation product 7-O-DeA, also AbA and DeA and a number of additional components. No modification of analytes. Identification of peaks by MS.
  • Disadvantages: Peak identification based on retention time of known, pure analytes; risk of interferences by and confusion with unknown peaks. Need to synthesise reference substance 7-O-DeA, since it is not commercially available. DeA has low absorption at 245nm; at max absorption 215nm there is high matrix interference. Not quantitative.
  • Used for: unmodified colophonium as such

2.2.2 HPLC-UV and fluorescence detection in combination

Method [5]

  • Description: UV detection of AbA at 240nm and fluorescence detection of DeA excitation 225nm, emission 285nm. Calibration with pure acids. Extraction with acetonitrile. SPE prior to HPLC. Limits of detection; 1.25ng AbA, 0.5ng DeA.
  • Advantages: No modification of analytes. SPE limits matrix interferences and enhances detection specificity. Detection of DeA by fluorescence is more than 100 times more sensitive than UV. Both detectors on the same column. HPLC separation time only approx. 10 minutes. Quantitative.
  • Disadvantages: No oxidation products detected. Peak identification based on retention time of known, pure analytes; risk of interferences by and confusion with unknown peaks.
  • Used for: makeup adhesive

Method [4]

  • Description: UV detection of AbA at 200nm and fluorescence detection of DeA excitation 225nm, emission 285nm. Calibration with pure acids. Extraction with acetonitrile or diethyl ether. SPE prior to HPLC. Limit of detection: 0.4ppm or 1ng for AbA and DeA.
  • Advantages: No modification of analytes. SPE limits matrix interferences and enhances detection specificity. Detection of DeA by fluorescence is more than 100 times more sensitive than UV. Both detectors on the same column. HPLC separation time only approx. 10 minutes. Quantitative.
  • Disadvantages: No oxidation products detected. Peak identification based on retention time of known, pure analytes; risk of interferences by and confusion with unknown peaks.
  • Used for: herbal oils and ointments

2.2.3 HPLC-MS

Method [13]

  • Description: HPLC-UV fractionation (see above); fractions analysed by DIP-MS.
  • Advantages: No modification of analyte. Mass spectra of the oxidation product 15-HPDA, which is thermally instable.
  • Disadvantages: AbA and DeA not detected. Not quantitative. Need to synthesise reference substance 15-HPDA, since it is not commercially available.
  • Used for: unmodified colophonium as such

Method [12]

  • Description: HPLC-UV fractionation (see above); fractions analysed by chemical ionization-MS.
  • Advantages: Detection of the oxidation product 7-O-DeA, also AbA and DeA and a number of additional components. No modification of analytes.
  • Disadvantages: Not quantitative. For 7-O-DeA identification; IR and NMR.
  • Used for: unmodified colophonium as such

2.2.4 Direct insertion probe MS method

Method [14]

  • Description: Extraction with ethanol. Chemical ionization and electron impact ionization MS. Compound identification based on reference mass spectra.
  • Advantages: Detection of the oxidation product 7-O-DeA, also AbA and DeA and a number of additional components, including oxidation products. No modification of analytes.
  • Disadvantages: No separation; complex mass spectra. Not quantitative.
  • Used for: unmodified colophonium as such

2.2.5 MALDI-MS method

Method [15]

  • Description: Extraction with dichloromethane. TLC plate coated with cellulose as sample probe.
  • Advantages: Detection of 7-O-DeA and other oxidation products. No modification of analytes.
  • Disadvantages: No separation; complex mass spectra. Not quantitative. Requires mechanical modification of instrument sample probe.
  • Used for: unmodified colophonium as such

2.3 Conclusions

The content of AbA may vary between 30 – 50% for gum rosin and 35 – 40% for tall oil rosin. Assuming that the resin acid content is 90%, a detection limit of 10 microgram/g AbA thus corresponds to a detection limit of approximately 30 microgram/g (ppm) of colophonium. This could be considered acceptable compared to the reactivity in allergic patients [3]. A detection limit of 10 ppm AbA may therefore serve as a guide to determine the order of magnitude for the desired limit of detection in the chosen analytical method.

Suggested method I, existing
The GC-FID-method [2, 8, 10] includes the quantitative analysis of AbA, DeA and 7-O-DeA. Amounts down to 1 ppm AbA [10], 1 ppm DeA [2] and 2 ppm 7-O-DeA [2] have been presented, however the limit of detection is unspecified. Note that the necessary derivatisation of the analytes includes the use of hazardous chemicals.
To avoid peak interferences and confusion with unknown peaks, a complementary CG-MS analysis could be developed and performed when needed [12].

Suggested method II, method development
The HPLC-UV-fluorescence method [4, 5] is quantitative for AbA and DeA down to 0.5-1.25 ng, corresponding to about 0.4 ppm. No sample derivatisation of the analytes is needed. Matrix interferences are limited and the detection specificity is enhanced by SPE.
The oxidation product 7-O-DeA may be included in this method. This analyte has been investigated by HPLC-UV method [8], however not quantitatively.
As the HPLC-UV-fluorescence method is non-destructive, fractionation and further analysis is an option, e.g. directly by MS to avoid confusion with potential unknown peaks [12-14].

2.4 References

  1. Hausen, B.M., et al., Contact Allergy Due to Colophony.3. Sensitizing Potency of Resin Acids and Some Related Products. Contact Dermatitis, 1989. 20(1): p. 41-50.
  2. Karlberg, A.T. and K. Magnusson, Rosin components identified in diapers. Contact Dermatitis, 1996. 34(3): p. 176-8O.
  3. Ehrin, E. and A.T. Karlberg, Detection of rosin (colophony) components in technical products using an HPLC technique. Contact Dermatitis, 1990. 23(5): p. 359-66.
  4. Lee, B.L., et al., High-performance liquid chromatographic determination of dehydroabietic and abietic acids in traditional Chinese medications. Journal of Chromatography A, 1997. 763: p. 221-226.
  5. Lee, B.L., et al., High-Performance Liquid-Chromatographic Method for Determination of Dehydroabietic and Abietic Acids, the Skin Sensitizers in Bindi Adhesive. Journal of Chromatography A, 1994. 685(2): p. 263-269.
  6. Kanerva, L., et al., Colophonium in sanitary pads. Contact Dermatitis, 2001. 44(1): p. 59-60.
  7. Gafvert, E. and G. Farm, Rosin (colophony) and zinc oxide in adhesive bandages - An appropriate combination for rosin-sensitive patients? Contact Dermatitis, 1995. 33(6): p. 396-400.
  8. Karlberg, A.T., et al., Airborne contact dermatitis from unexpected exposure to rosin (colophony) - Rosin sources revealed with chemical analyses. Contact Dermatitis, 1996. 35(5): p. 272-278.
  9. Holmbom, B., Improved Gas Chromatographic Analysis of Fatty and Resin Acid Mixtures with Special Reference to Tall Oil. J Am Oil Chem Soc, 1977. 54: p. 289-293.
  10. Karlberg, A.T., E. Gafvert, and C. Liden, Environmentally Friendly Paper May Increase Risk of Hand Eczema in Rosin-Sensitive Persons. Journal of the American Academy of Dermatology, 1995. 33(3): p. 427-432.
  11. Karlberg, A.T., C. Liden, and E. Ehrin, Colophony in Mascara as a Cause of Eyelid Dermatitis - Chemical-Analyses and Patch Testing. Acta Dermato-Venereologica, 1991. 71(5): p. 445-447.
  12. Sadhra, S., C.N. Gray, and I.S. Foulds, High-performance liquid chromatography of unmodified rosin and its applications in contact dermatology. J Chrom B, 1997. 700: p. 101-110.
  13. Shao, L.P., et al., 15-Hydroperoxydehydroabietic Acid - a Contact Allergen in Colophony from Pinus Species. Phytochemistry, 1995. 38(4): p. 853-857.
  14. Scalarone, D., et al., Direct-temperature mass spectrometric detection of volatile terpenoids and natural terpenoid polymers in fresh and artificially aged resins. Journal of Mass Spectrometry, 2003. 38(6): p. 607-617.
  15. Scalarone, D., et al., MALDI-TOF mass spectrometry on cellulosic surfaces of fresh and photo-aged di- and triterpenoid varnish resins. J Mass Spectrom, 2005. 40(12): p. 1527-35.
  16. Hausen, B.M., et al., Contact Allergy Due to Colophony.9. Sensitization Studies with Further Products Isolated after Oxidative-Degradation of Resin Acids and Colophony. Contact Dermatitis, 1993. 29(5): p. 234-240.
  17. Karlberg, A.T. and E. Gafvert, Isolated colophony allergens as screening substances for contact allergy. Contact Dermatitis, 1996. 35(4): p. 201-207.
  18. Sainio, E.L., M.L. HenriksEckerman, and L. Kanerva, Colophony, formaldehyde and mercury in mascaras. Contact Dermatitis, 1996. 34(5): p. 364-365.
  19. Karlberg, A.T. and C. Liden, Colophony (Rosin) in Newspapers May Contribute to Hand Eczema. British Journal of Dermatology, 1992. 126(2): p. 161-165.
  20. Bergh, M., T. Menne, and A.T. Karlberg, Colophony in Paper-Based Surgical Clothing. Contact Dermatitis, 1994. 31(5): p. 332-333.
  21. Smith, P.A., et al., Detection of resin acid compounds in airborne particulate generated from rosin used as a soldering flux. American Industrial Hygiene Association Journal, 1997. 58(12): p. 868-875.
  22. Örså, F. and B. Holmbom, A convenient method for the detemination of wood extractives in papermaking process waters and effluents. Journal of Pulp and Paper Science, 1994. 20(12): p. 361-365.
  23. Jones, K., et al., Identification of a possible biomarker for colophony exposure. Occup Med (Lond), 2001. 51(8): p. 507-509.

 

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