122 JOURNAL OF COSMETIC SCIENCE to the simultaneous assay of lactic acid, glycolic acid, and salicylic acid (and their salts) in a single capillary GC analysis. EXPERIMENTAL INSTRUMENTS AND CONDITIONS Analyses were performed on an Agilent/Hewlett-Packard Model 6890 gas chromato- graph system that included a flame ionization detector, a model 7673 autosampler, and Chemstation software (Agilent Technologies, Palo Alto, CA). The column was a 30-m x 0.32-ram i.d. HP-5 fused silica capillary column coated with 5% diphenyl-95% dimethylsiloxane copolymer (crosslinked) at 0.25-pm film thickness (Agilent Technolo- gies #19091J-413). The column was installed in a split/splitless injection port held at 300øC and connected to a flame ionization detector held at 310øC the carrier gas was helium held at 12-psi head pressure with a split ratio of about 25:1. The GC oven temperature was held at 90øC for two minutes, then programmed at a rate of 10.0øC/ rain to reach a temperature of 200øC, after which the column was cleaned out by increasing the rate to 30øC/rain to reach 280øC, where it was then held constant for five minutes. With these conditions, the retention times for silylated lactic acid, silylated glycolic acid, and silylated salicylic acid were approximately 4.1 minutes, 4.3 minutes, and 10.5 minutes, respectively. A Hamilton Microlab dispenser was used to accurately dispense acid-DMF reagent for sample preparations (Hamilton Company, Reno, NV). Ultrasonic/vortex mixers and disposable 20-ml glass scintillation vials with Polyseal cone caps were used for sample preparation (Fisher Scientific, Pittsburgh, PA). REAGENTS AND SOLUTIONS ACS reagent grade DMF (N,N-dimethylformamide) and HC1 (37% hydrochloric acid) were obtained from Fisher Scientific, Pittsburgh, PA. Acid-DMF reagent was prepared by adding 2.5 ml of HC1 to 500 ml of DMF and mixing. BSTFA reagent (bis- trimethylsilyltrifluoroacetamide containing 1% trimethylchlorosilane) was obtained from Regis Technologies, Morton Grove, IL. Sodium L-lactate, glycolic acid, and sali- cylic acid, all over 99% purity for use as analytical standards, were obtained from Aldrich Chemical Co., Milwaukee, WI. To prepare the mixed standard solution, about 0.15 g each of sodium L-lactate, glycolic acid, and salicylic acid standards was accurately weighed (+0.0001 g) into a 200-ml volumetric flask, then dissolved and diluted to volume with acid-DMF. Each day of use, a 250-pl portion was transferred to an auto- sampler vial where it was mixed with 500 pl of BSTFA reagent. ASSAY PROCEDURE AND CALCULATION A well-mixed sample (0.23-0.27 g) was weighed (+0.0001 g) into a 20-ml vial, and 9.75 ml of acid-DMF reagent was dispensed from the Microlab dispenser. The vial was capped, and ultrasonic or vortex mixing was used to dissolve the sample and force any salts into their acid form. After allowing undissolved solids to settle, 250 pl of super- natant was transferred to an autosampler vial and mixed with 500 pl of BSTFA reagent. Two microliters was then injected into the GC column and compared to 2-pl injections

DETERMINATION OF HYDROXY ACIDS 123 of the silylated standard mixture each was injected three times. Routine external stan- dard calculations were used to determine percent for each hydroxy acid. RESULTS AND DISCUSSION METHOD DEVELOPMENT OH H 0%C / OH , CH3--C --C I NOH --CNoH H H lactic acid glycolic acid salicylic acid In our laboratory, there was a need for a simple, straightforward procedure to determine alpha and beta hydroxy acid levels in personal care products. The Cosmetic Ingredient Review Safety Assessment of alpha hydroxy acids and their salts (June 6, 1997) contained a brief overview of previously published analytical methods, but none were straightfor- ward, directly applicable to consumer products, or amenable to routine laboratory in- strumentation. After reviewing published methods, we saw the opportunity to develop a new method that would require less operator time, be amenable to automated analysis, and provide reliable quantitation. Our laboratory was already experienced in the routine assay of salicylic acid using HPLC with a UV detector since lactic acid and glycolic and their salts do not possess a chromophore (an aromatic ring) as does salicylic acid/sodium salicylate, their UV absorptivities are low, and analysis by HPLC would require using a nonspecific lower wavelength or a conductivity detector. HPLC investigations in our laboratory showed that HPLC would not be adequate to simultaneously assay for the three hydroxy acids of interest for reliable automated analysis. These hydroxy acids and corresponding salts could not be assayed by GC directly, due to the polarity of the acid form or the lack of volatility of the neutralized form. However, if the salts could be transformed into their acid form, then all the original hydroxy acids and newly acidified salts could then be derivatized into their trimethylsilyl esters using BSTFA reagent, enabling GC analysis. To accomplish this, our previously invented reagent of acid-DMF (developed for the assay of sodium lauroyl sarcosinate) was utilized for one-step acidi- ficadon/dissolution, after which trimethylsilyl derivatization and capillary GC analysis could be performed. Previous experimentation had demonstrated that salts of carboxylic acids needed to be converted to the acid form for derivatization to occur the acid-DMF reagent would convert all salts of the hydroxy acids into the acid forms. This technology was tried on individual standards and mixtures of sodium lactate, glycolic acid, and salicylic acid, and a GC column and temperature program was chosen to produce proper chromatographic resolution. This technique worked so well that early plans to develop test methodology only for lactic and glycolic acids (and assay the salicylic acid separately by HPLC) were discarded so that this new technology could be used to determine all the analytes sz'm•/tameo•s/y in one sample preparation and GC run. Typical capillary GC