J. Cosmet. Sci., 63, 321–331 (September/October 2012) 321 Simultaneous determination of 13 components in oxidative hair dyes by high-performance chromatography using an ion-pair reagent YING LAI, HONGHUI WANG, QINGMU DONG, HEXIU CHEN, RUI LIN, and YANPING CAI, Technology Center of Xiamen Entry-Exit Inspection and Quarantine Bureau, Xiamen 361026, People’s Republic of China. Accepted for publication March 12, 2012. Synopsis A reliable high-performance liquid chromatography (HPLC) method was developed for the simultaneous deter- mination of 13 dye intermediates, including benzenediamines, aminophenols, benzenediols, naphthalenediol, and diaminopyridine, in oxidative hair dyes. Samples were extracted with 50% ethanol by adding sodium dithionite to prevent oxidation. The infl uences of buffer type, buffer pH, ion-pair reagent, and elution gradient were studied. A C18 column with aqueous compatibility and acetonitrile–citric acid mobile phase system (pH 2.6) with sodium 1-octanesulfonate as ion-pair reagent were selected for the separation of target com- pounds. Detection was performed by a diode array detector, (DAD) and two different wavelengths (280 and 331 nm) were used for quantifi cation. Results showed that 13 dye intermediates got good separation within 25 min. The detection limits of these compounds were in the range of 0.2–2 mg/l. The calibration curves were linear within 2–500 mg/l with 0.999 as a typical correlation coeffi cient. The recoveries of target compounds in hair dyes ranged from 81.7% to 102.0% with four addition levels. The method described was validated by fi ve different laboratories and successfully applied to the analysis of commercial oxidative hair dyes. INTRODUCTION Oxidative hair dye, the most widely used permanent dye, usually contains a mixture of aromatic compounds including benzenediamines, aminophenols, benzenediols, naphtha- lenediol, and diaminopyridine. Such substances are of great health concern due to their mutagenic or even carcinogenic activity (1,2). The use of these compounds in hair dyes is regulated by legislation in many countries. Therefore, a simple and rapid method for the determination of these dye intermediates in hair dyes is of utmost importance. Several methods have been developed for the analysis of dye intermediates in hair dyes, including thin layer chromatography (TLC) (3,4), high-performance liquid chromatography (HPLC) (5–11), high performance liquid chromatography-mass Address all correspondence to Ying Lai at laiy@xmciq.gov.cn.

JOURNAL OF COSMETIC SCIENCE 322 spectrometry(HPLC-MS) (12), gas chromatography (GC) (13), gas chromatography-mass spectrometry (GC-MS) (14–16), capillary electrophoresis (CE) (17), and micellar electroki- netic chromatography (MEKC) (18,19). Usually, GC and CE have better separation ability than HPLC. However, GC has some restrictions for the determination of hydrophilic sub- stances in hair dyes due to their high polarity and low volatility, and CE is not suitable for quantitative determination because of low reproducibility. Therefore, only HPLC is the most convenient method for the determination of hair dye intermediates, which has high polarity, low volatility, and thermostability. Several studies contribute to improving the amount of oxidative hair dyes analyzed in a single HPLC run by optimizing the separation parameters (7–10) or prolonging the analysis time (11). Up to now, the best result achieved (11) is the separation of 22 oxidative hair dyes in 50 min. Usually, a HPLC runs less than 30 min and allows the simultaneous determination of two to eight oxidative hair dyes (7–10). Therefore, the aim of this work was to develop a reliable HPLC method to analyze the most commonly used hair dye intermediates, such as benzenediamines, aminophenols, benzenediols, naphthalenediol, and diaminopyridine, with less time. EXPERIMENTAL CHEMICALS AND REAGENTS Hydroquinone (99.5%), resorcinol (99.5%), p-aminophenol (99.0%), and phenol (99.5%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) o-phenylenediamine (99.5%), 1,5-naphthalenediol (99%), and 3,4-diaminetoluene (99.8%) from ChemService (West Chester, PA) N,N-diethyl-p-phenylenediamine sulfate (97%) from IL (South San Francisco, CA) m-aminophenol (99%), 4-methylaminophenol sulfate (99%), p-phenylenediamine (99%), 2,5-diaminotoluene sulfate (99%), and 2,6-diaminopyridine (98%) from ACROS (Geel, Belgium) 1-octanesulfonic acid sodium salt monohydrate (97%) from J&KCHEMICA (Beijing, China). Water (Milli-Q Purifi cation System, Millipore (Billerica, MA)), acetonitrile, and ethanol were of HPLC grade. Sodium dithionite and sodium sulfi te were of AR grade. Standard stock solutions of 13 dye intermediates each at a concentration of 2500 μg/ml was prepared in 50% (v/v) ethanol solution containing 0.1% sodium sulfi te in brown bottles, and stored at −10°C. A calibration curve was prepared by injecting eight diluted solutions, obtained from the stock solution, in the concentration range of 2–500 μg/ml. APPARATUS HPLC system was composed of a Waters 2695 pump equipped with a liquid autosam- pler, and a Waters 2996 diode array detector. Analysis was performed at 30°C on a ZORBAX SB-Aq C18 column (5 μm, 250 × 4.6 mm I.D., Agilent (Santa Clara, CA)) with a same packing precolumn (5 × 4.6 mm I.D.) through a gradient elution. The fl ow rate was 1.0 ml/min. Eluent A was a buffer solution (pH 2.6) containing 10 mmol/l citric acid and 10 mmol/l sodium 1-octanesulfonate. Eluent B was acetonitrile. Time program: 0–5 min, B 12% 5.01–15 min, B 12–20% 15.01–24 min, B 12%. Injection volume: 5 μl. Detection was performed by scanning from 190 to 400 nm. Quantitation was performed at 331 nm for 2,6-diaminopyridine and at 280 nm for all the other dye intermediates.