DETERMINATION OF 13 COMPONENTS IN OXIDATIVE HAIR DYES 323 SAMPLE PREPARATION About a 1.0 g sample was weighed and placed in a 25 ml calibrated fl ask, then 1 ml 1% sodium dithionite and 15 ml 50% (v/v) ethanol were added. The mixture was homoge- nized adequately by stirring before 15 min of the ultrasonic extraction process. Then the solution was diluted to volume with 50% (v/v) ethanol. About 1 ml aliquot of this solu- tion was fi ltered through a 0.45 μm membrane fi lter and ready for HPLC analysis. VALIDATION To evaluate the accuracy and precision of the method, standard recovery test was perfor med on real sample A (containing 536.6 mg/kg resorcinol) and sample B (con- taining 3528 mg/kg m-aminophenol, 6618 mg/kg p-aminophenol, and 8727 mg/kg p-phenylenediamine), with four addition levels of 1 time, 2 times, 10 times, and 20 or 100 times the LOQ (limit of quantifi cation) of target compounds. The method was also validated by fi ve different laboratories with real sample determination and standard recovery test. Figure 1. Chromatogram of dye intermediates at (a) 280 nm and (b) 331 nm. 1. Hydroquinone 2. resor- cinol 3. p-aminophenol 4. phenol 5. m-aminophenol 6. 4-methylaminophenol 7. o-phenylenediamine 8. p-phenylenediamine 9. 2,5-diaminotoluene 10. 1,5-naphthalenediol 11. 3,4-diaminetoluene 12. N,N-diethyl-p-phenylenediamine 13. 2,6-diaminopyridine.

JOURNAL OF COSMETIC SCIENCE 324 RESULTS AND DISCUSSION PRETREATMENT CONDITIONS To allow for both alcohol-soluble and water-soluble dye intermediates, 50% ethanol solu- tion was chosen as the extractant. The impact of sodium sulfi te and sodium dithionite as reductant on the extraction of easily oxidized dye intermediates was studied. Results showed that the recoveries of hydroquinone and 1,5-naphthalenediol were doubled by adding 1 ml 1% sodium dithionite compared to that by adding sodium sulfi te however, no difference was found for all the other dye inter- mediates studied. Therefore, sodium dithionite was chosen as the reductant. HPLC CONDITIONS The dye intermediates studied possess amino groups with two pKa values in the range of 4.2–6.5 and 1.7–3.0, and/or hydroxy groups with the fi rst and the second pKa values in Figure 2. Chromatogram of hair dye A at 280 nm. 1. Resorcinol. Figure 3. Chromatogram of hair dye B at 280 nm. 1. p-Aminophenol 2. m-aminophenol 3. p-phenylenediamine.