j. Cosmet. sci., 54, 47-52 (January/February 2003) Determination of methylparaben in o/w emulsions by solid-phase extraction and hioh-performance liquid chromatooraphy NONGNUCH PONGCHAROENKIAT, ARUNEE WITTAYANUKULLUK, and STANLEY L. HEM, Department of Indratrial and Physical Pharmacy, Purdue University, W. Lafayette, IN 47907. Accepted for publication April 29, 2002. Synopsis A simple, fast, and accurate solid-phase extraction (SPE) using a 1-cc Oasis HLB cartridge for sample clean-up followed by an HPLC analysis for the assay of methylparaben (MP) in an o/w emulsion is described. One milliliter of methanol followed by 1 ml of 10% methanol in water was used to activate the cartridge sorbent. The sample was loaded into the cartridge and MP was then separated from oil-soluble excipients by washing the cartridge with 1 ml of 10% CH3CN in water. MP was finally eluted from the cartridge with mobile phase, acetonitrile and water (60:40), and quantified by HPLC analysis on a Nova-pak © C-18 column at 254 nm. INTRODUCTION Methylparaben (MP) is one of the most widely used preservatives in pharmaceutical, cosmetic, and personal care emulsions due to its broad antimicrobial spectrum and relatively low toxicity (1). The determination of MP in these products is required to assure its efficacy. One of the frequently employed assay methods is high-performance liquid chromatography (HPLC) (2-5). Due to the complicated matrices of emulsions and the relatively small concentration of MP present, direct injection of emulsions into the HPLC system may result in lower accuracy. A convenient alternative to direct injection of emulsions into the HPLC system is to dissolve the emulsion in a water-miscible solvent or mixture of solvents. Unfortunately, the wide range of hydrophilic and hy- drophobic components of a cosmetic or pharmaceutical emulsion frequently precludes this approach. Thus, extraction is frequently needed prior to HPLC analysis. Nongnuch Pongcharoenkiat's current address is Government Pharmaceutical Organization, Bangkok 10400, Thailand. Address all correspondence to Stanley L. Hem. 47

48 JOURNAL OF COSMETIC SCIENCE A classical procedure for extracting MP from complicated matrices is liquid-liquid extraction (LLE) (6). However, LLE is time-consuming and requires a large volume of organic solvent to isolate the analyte. On the other hand, solid-phase extraction (SPE) can be performed in a shorter period of time and generates a significantly smaller amount of waste. SPE is based on a principle similar to that of chromatography and may utilize normal- phase, reversed-phase, and ion-exchange adsorbents. Reversed-phase SPE, in which a nonpolar stationary phase, the sorbent, retains the nonpolar compounds including the analyte and allows the polar compounds to pass through, was used in this study. An eluent is then used to extract the retained analyte from the sorbent. Although SPE procedures have been proposed to extract MP, evaporation of the extract in order to remove the organic solvent was still necessary (7,8). The drying process in these procedures may reduce assay accuracy due to the degradation of the MP. The goal of this study was to develop a simple, fast, and accurate SPE technique using a small volume of organic solvent without a drying step in order to separate MP from other components of o/w emulsions. EXPERIMENTAL REAGENTS MP, butylparaben (BP), butylated hydroxytoluene (BHT) (Aldrich, Milwaukee, WI), Miglyol 810 (Huls Aktiengesellschaft, Hillside, NJ), olive oil (ICN Biomedical, Aurora, OH), glycerol (J. T. Baker Inc., Phillipsburg, NJ), methanol, and acetonitrile (HPLC grade, Mallinckrodt, Paris, KY) were obtained commercially. Egg phospholipid was a gift of Pharmacia & Upjohn (Clayton, NC). Doubly distilled water (ddH20) was used throughout the study. PREPARATION OF EMULSIONS A 20% w/w oil-in-water emulsion containing 0.1% MP was prepared according to the procedure reported in a previous study (9). The oil phase was composed of 0.1% w/w MP, 0.2% w/w BHT, and 10% w/w each of Miglyol 810 and olive oil. The aqueous phase consisted of 1.2% w/w egg phospholipid, 2.4% w/w glycerol, and ddH20. The oil phase was prepared by dissolving BHT and MP in a mixture of Miglyol 810 and olive oil using a sonicator (model 1200, Branson Ultrasonic Corporation, Danbury, CT). The aqueous phase was prepared by dispersing egg phospholipid in the mixture of glycerol and ddH20 by use of a constant-speed stirrer (Stedi-Speed, Fisher, Fairlawn, NJ) at 1000 rpm. A pre-emulsion was prepared by adding the oil phase to the aqueous phase and mixing at 1000 rpm for 30 min. Final emulsification was completed by passing the pre-emulsion through a microfluidizer (model 110Y, Microfluidics Corporation, New- ton, MA) at 20,000 psi five times. Nitrogen gas was bubbled continuously during the preparation of the emulsion. The precision and accuracy of the quantification of MP was tested by adding MP to the finished emulsion. Two aqueous stock solutions of MP containing 1.5 mg MP/ml or 2.0 mg MP/ml were prepared. Spiked emulsions were prepared by adding 0.10 ml of the 1.5