THE DEVELOPMENT AND VALIDATION OF A HPLC-UV METHOD TO DETECT AND MEASURE CAFFEINE AND PHENOLPHTHALEIN, ADULTERANTS IN OVER-THE-COUNTER SLIMMING PHYTOTHERAPEUTIC FORMULATIONS

Abstract

Slimming products based on phytotherapeutic formulations are popular due to their affordability and the misconception that they are safe because they are „natural products‟. However, adulterants have been found to be added into these herbal formulations to improve the weight lost effect of the product. Consumption of these adulterated products may lead to health problems, and even death. We hypothesise that over-the-counter (OTC) slimming phythotherapeutic formulations sold in Malaysia are adulterated with synthetic and natural drugs. Caffeine and phenolphthalein are popular adulterants due to their respective properties as an anorexic (appetite suppressor) and laxative. The objective of this study is to develop and validate a simple, precise and specific high-performance liquid chromatography coupled with ultraviolet detector (HPLC-UV) analytical method for the simultaneous detection and measurement of caffeine and phenolphthalein in slimming phytotherapeutic formulations. The method was then applied to analyse eight OTC slimming phythotherapeutic formulations purchased from the Malaysian market. The method was developed using a HPLC connected to a variable-wavelength detector (VWD). Separation was achieved with a C18 reversed-phase column using gradient elution. Mobile phase used were solvent A (aqueous): 25 mM ammonium acetate buffer (pH 5), and solvent B (organic): methanol. Gradient elution was achieved by a ratio of time (minutes) over methanol (%): 0/10, 1/10, 3/40, 5/55, with a run time of 10 minutes. Flow rate was maintained at 1.1 mL/min and the effluents monitored at 254 nm wavelength. The retention time of caffeine and phenolphthalein were approximately 5.5 minutes and 8.8 minutes, respectively. The developed method was validated for linearity, precision, accuracy and selectivity according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines (Validation of Analytical Procedures: Text and Methodology, Q2(R1)). The calibration curve area versus concentration was found to be linear (coefficient of determination, R2 ≥ 0.999) in the range of 10–100 μg/mL for both caffeine and phenolphthalein. The limit of detection (LOD) which indicates the lowest concentration of caffeine and phenolphthalein that can be detected were 0.77 and 0.47 μg/mL, respectively. The limit of quantification (LOQ) that express the lowest concentration of caffeine and phenolphthalein which can be confidently quantified were 2.35 and 1.44 μg/mL, respectively. The method was found to be accurate for both compounds with percent recovery values of 98–102% for both caffeine and phenolphthalein. The method had good intra-day and inter-day precision with percent relative standard deviation (%RSD) values of less than 2%. The validated method was applied to analysis of eight OTC slimming phytotherapeutic formulations purchased from the Malaysian market. Phenolphthalein was detected and quantified in five of these slimming products, at levels ranging from 0.68–126.83 mg/g. Caffeine was detected in six of these slimming products but only quantified in two products (sample A, 3.39 ± 0.21 mg/g and sample B, 0.99 ± 0.01 mg/g) as most of the products claimed to be of plant origin and hence caffeine detected were likely from natural sources. In conclusion, a simple and fast HPLC-UV method to detect for caffeine and phenolphthalein as adulterants was successfully developed and validated. The method was applied to actual OTC slimming products, and proved the hypothesis that OTC slimming phythotherapeutic formulations sold in Malaysia are adulterated. As future work, the developed method can be used to analyse more variety of slimming products. The analytical technique is also suitable to be used in quality control laboratories for routine detection of phenolphthalein and caffeine in slimming products.