DETERMINATION OF 13 COMPONENTS IN OXIDATIVE HAIR DYES 325 water–acetonitrile system. When the pH value was below 3.0, all substances got good peak shape, but the retention times of some basic dyes (such as benzenediamines) were too short to reach the baseline separation. When the pH value was between 4.0 and 5.0, some peaks tailed off slightly, but when the pH value was above 5.0, peak overlapping and broadening were obvious. According to the formula: pH = pKa + log ([A−]/[A]), 99% of the compound will exist in one species when buffer pH value is above or below its pKa of two units. Since most dye intermediates contain amino groups, low pH values contribute to reducing the interac- tion between amino groups and Si-OH and enable sharp chromatographic peak. At the optimum buffer pH below 3.0, substances containing amino groups exist as cationic spe- cies, while substance without amino groups but containing hydroxyl groups exist as neu- tral species therefore, most substances obtained good chromatographic peak shape. To improve the separation of basic dyes below pH 3.0, sodium 1-octanesulfonate was added as ion-pair reagent to extend their retention times. Results showed that 13 kinds of dye intermediates achieved good separation within 25 min. The typical chromatograms of standards and two real samples (hair dyes A and B) are shown in Figures 1–3. Online UV absorption spectra (Figure 4) of dye intermediates by DAD (diode array de- tector) can be used for initial qualitative analysis and reducing false-positive results. The target compounds have char acteristic absorption bands in the range of 215–240 nm and 270–320 nm with the exception of 2,6-diaminopyridine, which has an absorption maxi- mum at 331 nm. LINEAR RANGE AND DETECTION LIMIT Quantitative determination was performed on 13 dye intermediates. The calibration curves for these compounds were linear within 2–500 μg/ml with 0.999 as a typical cor- relation coeffi cient, and the detection limits (S/N = 3) were in the range of 0.2–2 μg/ml (Table I). Table I The Linear Ranges and Detection Limits of 13 Dye Intermediates Determined by HPLC Compound Linear regression equation R2 Detection limit (μg/ml) Hydroquinone Y = 1.02e + 004 X − 3.90e + 003 1 0.2 Resorcinol Y = 8.18e + 003 X − 2.52e + 003 1 0.3 p-Aminophenol Y = 5.79e + 003 X − 1.44e + 003 0.9999 0.6 Phenol Y = 4.75e + 003 X − 2.81e + 003 0.9999 0.9 m-Aminophenol Y = 7.47 e + 003 X − 1.51 e + 004 0.9998 0.6 4-Methylaminophenol Y = 3.14e + 003 X − 4.85e + 003 0.9999 1 o-Phenylenediamine Y = 1.04e + 004 X − 1.46e + 004 1 0.4 p-Phenylenediamine Y = 3.70e + 003 X − 3.63e + 002 0.9999 0.8 2,5-Diaminotoluene Y = 2.35e + 003 X + 2.31e + 003 0.9999 1 1,5-Naphthalenediol Y = 1.39e + 004 X + 4.58e + 003 0.9999 0.4 3,4-Diaminetoluene Y = 7.03e + 003 X − 2.59e + 004 0.9998 1 N,N-Diethyl-p-phenylenediamine Y = 2.11e + 003 X − 1.15e + 003 0.9999 2 2,6-Diaminopyridine Y = 6.32e + 004 X + 6.99e + 004 0.9999 0.1

JOURNAL OF COSMETIC SCIENCE 326 Figure 4. Continued