Chem. Pharm. Bull. 55(2) 251—254 (2007)

Orlistat (Xenical) (Fig. 1), a highly lipophilic tetrahydro derivative of lipstatin, is an inhibitor of gastric and pancreatic lipases required for the hydrolysis of dietary fat (triacylglycerols) in the gastrointestinal tract into free fatty acids and monoacylglycerols. A major contributing factor to the development and maintenance of obesity is excessive intake of dietary triglycerides. Inhibition of pancreatic lipase and gastric lipase, leading to prevention of lipid absorption is a treatment of severe obesity. Orlistat acts locally in the intestine to block absorption of approximately one-third of all dietary fat. By effectively limiting dietary fat absorption, orlistat promotes weight loss, maintenance of lost weight, and prevention of weight regain in obese patients. Orlistat yields low, if not undetectable plasma concentrations when administered orally. Quantitative determination of orlistat in human plasma has been reported using LC/MS/MS or GC/MS/MS with a low detection limit. To our best knowledge, there is no pharmacopeial monographs and dissolution tests on orlistat. The dissolution profile has emerged as a valuable quality control tool to assess batch-tobatch release rate properties and to assure the availability of the drug. This study, describes the development of a fast, accurate and precise HPLC method with isocratic elution for determination of orlistat in pharmaceutical formulations and in dissolution media for drug quality control purposes. The developed method was validated and applied to determination of the content of orlistat in Xenical capsules and to the samples obtained from in vitro dissolution studies. The dissolution method was also developed and validated according to USP guidelines. Experimental Materials Orlistat was from Biocon (Bangalore, India, Lot No: B0520199) and obtained from Osveh Pharmaceutical Company, Tehran, Iran. Acetonitrile was HPLC grade and purchased from Merck (Darmstadt, Germany). Xenical tablets (Roche, Switzerland, Lot No: M1162C) were purchased from a local pharmacy. All other chemicals and solvents were of analytical grade and prepared from Merck. Deinonized distilled water were used for mobile phase preparation, solubility, assay and dissolution experiments. Instrumentation The HPLC system consisted of a 600 pump, 710 plus Autosampler and a variable 480 UV detector, all from Waters (Milford, MA, U.S.A.). The data processing system was a multi-channel Chrom and Spec software for chromatography, version 1.5x. Chromatographic Conditions Separation was achieved using a NovaPak C18 4 mm steel column (3.9 mm 150 mm, Waters, Milford, MA, U.S.A.). The isocratic mobile phase pumped at a flow rate of 1 ml/min consisted of orthophosphoric acid 0.1%–acetonitrile (10 : 90, v/v) prepared daily and degassed by passing through a 0.45 mm filter (Millipore, Milford, MA, U.S.A.) and sonication for 10 min. The injection volume was 25 m l and the wavelength for UV detection was 205 nm. All sepatations were performed at room temperature. Preparation of Standard Solutions Stock standard solution of orlistat was prepared by dissolving appropriate amount of the compound in methanol to give a final concentration of 1000 mg/ml. Standard solutins of orlistat (10, 20, 40, 80, 120, 160 mg/ml) were prepared by subsequent dilution. No decrease in responses was observed after 1 week of storage at 4 °C. Validation Seven series of standard calibration solutions in the range of 10—160 mg/ml were prepared and analyzed as described above. Calibration curves were constructed by plotting the measured peak area of orlistat versus concentrations of standard samples and statistical analysis was performed. To establish the within-day and between-day accuracy and precision of the method, three replicate of standard solutions at three different concentrations (10, 40, 160 mg/ml) were assayed on one day and three separate days. Specificity The specificity of the proposed method was performed by analyzing spiked samples with appropriate amount of drug and also samples of the excipients of capsule formulation. The solutions were prepared and analyzed as described before. Application of the Method The content of 20 capsules were combined and weighed. An amount of powder equivalent to about 120 mg of orlistat was accurately weighed, transferred to a 100 ml volumetric flask, made up to volume with methanol and placed in an ultrasonic bath for 15 min. After filtration through a 0.45 mm membrane filter the solution was diluted with methanol to obtain a concentration of about 120 mg/ml. The drug concentration of six parallels were determined by HPLC using the calibration curve. To test the content uniformity of orlistat capsules, the content of ten capsules were individually transferred to a 100 ml volumetric flask and methanol was added. The mixture was placed in an ultrasonic bath for 15 min. The solution was diluted ten times and injected to the HPLC system after filtration. HPLC Analysis of Orlistat and Its Application to Drug Quality Control Studies