Simultaneous determination of alpha and beta hydroxy

Transcription

1 J. Cosmet. Sci., 53, (March/April 2002) Simultaneous determination of alpha and beta hydroxy acids in personal care products by capillary Das chromatodraphy K. MOLEVER, Research and Development Department, The Dial Corporation, N. Scottsdale Road, Scottsdale, AZ Accepted for publication December 31, Synopsis A simple and rapid procedure is described for the isolation, silylation, and simultaneous capillary gas chromatographic quantitation of alpha hydroxy acids and beta hydroxy acids in various personal care products. The sample is dissolved in acidified N,N-dimethylformamide to simultaneously acidify/extract the hydroxy acids; a portion is then trimethylsilyl derivatized with BSTFA and quantified by capillary gas chromatography (GC) using flame ionization detection. INTRODUCTION Alpha hydroxy acids such as lactic acid and glycolic acid (and beta hydroxy acids such as salicylic acid) and their salts are commonly added to personal care products for their skin enhancement properties. However, the analysis of hydroxy acids has not been straightforward in finished product matrices except for the high-performance liquid chromatography (HPLC) assay of hydroxy acids containing a UV-absorbing chromophore, such as the beta hydroxy acid salicylic acid. Although other analytical methods for hydroxy acids in consumer products including gas and liquid chromatography have been reported in the literature (1-6), this gas chromatographic (GC) method combines specificity, quantitation, and easy sample preparation for the simultaneous analysis of these hydroxy acids in commercial personal care products. Advances in capillary gas chromatography have greatly enhanced capabilities for resolving complex mixtures; frequently, the resolving capacity of capillary columns can eliminate the need for extensive sample preparations or cleanups. In our analytical laboratory we routinely analyze consumer products for ingredients such as glycols, sorbitol, fatty acids, and similar ingredients, using adaptations of our previously published procedure for determining glycerin in soap bars (7); we also published a further adaptation, employing one-step acidification/dissolution using acidified N,N-dimethylformamide followed by silyl derivatization and capillary GC analysis to quantitate sodium lauroyl sarcosinate personal care products (8). This technology has been further adapted here 121

2 122 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 chromatograph 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 Technologies #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 (bistrimethylsilyltrifluoroacetamide containing 1% trimethylchlorosilane) was obtained from Regis Technologies, Morton Grove, IL. Sodium L-lactate, glycolic acid, and salicylic 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 ( 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 autosampler vial where it was mixed with 500 pl of BSTFA reagent. ASSAY PROCEDURE AND CALCULATION A well-mixed sample ( g) was weighed ( 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 supernatant 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

3 DETERMINATION OF HYDROXY ACIDS 123 of the silylated standard mixture; each was injected three times. Routine external standard calculations were used to determine percent for each hydroxy acid. RESULTS AND DISCUSSION METHOD DEVELOPMENT CH3--C --C OH H 0%C / OH, 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 straightforward, directly applicable to consumer products, or amenable to routine laboratory instrumentation. 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 acidificadon/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

4 124 JOURNAL OF COSMETIC SCIENCE GLYCOLIC ACID-TMS 400 LACTIC ACID-TMS ' 300 J SALICYLIC ACID-TMS 20O A i i I 1 o 112 ' ' mi pa 160 _ GLYCOLIC ACID-TMS 80 - LACTIC ACID-TM, J SALICYLIC ACID-TMS 60_ ' / 40- m. ß o- '... B i ' mir Figure 1. Typical capillary gas chromatograms of: (A) silylated standard hydroxy acid mix and (B) silylated hydroxy acid-containing personal care product. UNEARITY ' Concentration 400 Glycolic acid Lactic acid Salicylic acid Figure 2. Linearity.

5 DETERMINATION OF HYDROXY ACIDS 125

6 126 JOURNAL OF COSMETIC SCIENCE Table II Assay of Commercially Available Products % Total lactic % Total glycolic % Total acid acid salicylic acid Product 1: Alpha hydroxy lotion 4.1 Product 2: Refining toner Product 3: Alpha hydroxy lotion 3.7 Product 4: Alpha hydroxy lotion 1.7 Product 5: Hydroxy cream 3.6 Product 6: AHA facial treatment 5.5 Product 7: Lotion Product 8: AHA body lotion 5.1 Product 9: AHA hand cream 5.8 Product 10: AHA hand lotion 4.1 Product 11: Eye cream 0.48 Product 12: Facial masque O.O curves for derivatized hydroxy acid standard mix and a commercially available personal care product are shown in Figure 1. ACCURACY AND PRECISION Linearity studies demonstrated excellent linearity in the working range expected in commercial personal care products, detailed in Figure 2. Methodology validation studies were conducted in which placebo personal care products were spiked with varying levels of sodium L-lactate, glycolic acid, and salicylic acid, and analyzed using this method. Average recoveries of lactic acid, glycolic acid, and salicylic acid were 99.74%, 99.16%, and 99.40%, respectively, with an average standard deviation of 2.2, and are detailed in Table I. Results obtained from analyzing commercially available personal care products are detailed in Table II. REFERENCES (1) D. de Villiers, E. Wurster, T. Bergh, and K. Narsai, Stability-indicating HPLC assay of the alpha hydroxy acids lactic acid and glycolic acid in nonionic creams, Pharmazie, 53, (1998). (2) S. Scalia, R. Callegari, and S. Villani, Determination of glycolic acid in cosmetic products by solidphas extraction and reversed-phase ion-pair high-performance liquid chromatography,j. Ch,vmatogr., 795, (1998). (3) K. Nakamura, Y. Morikawa, and I. Matsumoto, Studies on high-speed liquid chromatography in cosmetic analysis, Bunseki Kagakz, 29, (1980). (4) J. D. Blake, M. M. Clarke, and G. N. Richards, Determination of organic acids in sugar cane process juice by high-performance liquid chromatography, J. Chromatogr., 398, (1987). (5) S.H. Ashoor and J. Welty, Determination of organic acids in foods by high-performance liquid chromatography, J. Chromatogr., 287, (1984). (6) P.S. Nassos, J. E. Schade, A.D. King, and A. E. Stafford, Comparison of HPLC and GC methods for measuring lactic acid in ground beef, J. Food Sci., 49, (1984). (7) K. Molever, Quantitative determination of glycerin in soap by capillary gas chromatography, J. Am. Oil Chem. Soc., 64, (1987). (8) K. Molever, Quantitative determination of sodium lauroyl sarcosinate by gas chromatography,j. Am. Oil Chem. Soc., 70, (1992).