Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study.

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.

Direct injection, column switching-liquid chromatographic technique for the estimation of rabeprazole in bioequivalence study.

A rapid, simple and sensitive high-performance liquid chromatography-ultra violet (HPLC-UV) method with column switching between sample pre-treatment column and analytical column was developed for the quantitation of rabeprazole in human plasma; on a Bio-Sample Analysis system (Co-sense for BA) from Shimadzu Corporation, Kyoto, Japan. Zaleplon was used as an internal standard. The method was validated as per USFDA guidelines for the concentration range of 20.0-1200.0 ng/mL and the correlation coefficient were found to be better than 0.999. Recovery of rabeprazole as well as the internal standard from human plasma was more than 90.0%. Rabeprazole was stable in human plasma for 4 months at -70 +/- 5 degrees C and for 20.0 h at ambient temperature. In the auto sampler, the drug was stable for 24.0 h at 4 degrees C. The method was specific as there were no interfering peaks in the human plasma eluting at the retention times of the rabeprazole and the internal standard. The frozen plasma samples containing rabeprazole were stable to three freeze thaw cycles. The bioanalytical method was rugged in terms of inter- and intra-day accuracy and precision. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of 20mg rabeprazole tablet of German Remedies Ltd. (A division of Cadila Healthcare Ltd.), India versus Pariet tablet of Eisai Ltd. & Janssen-Cilag Ltd., Japan in male human subjects.

Liquid chromatography--electrospray ionisation mass spectrometry method for the determination of escitalopram in human plasma and its application in bioequivalence study.

A novel liquid chromatographic--electrospray ionisation mass spectrometric (LC--ESI-MS) method has been developed for the determination of escitalopram, an antidepressant in human plasma using paroxetine as internal standard. The method involved liquid--liquid extraction of the analyte from human plasma with a mixture of diethyl ether and dichloromethane (70:30, v/v). The chromatographic separation was achieved within 7.0 min by using 2.0 mM ammonium acetate (pH 5.0)--acetonitrile (54:46, v/v) as mobile phase and a ODS YMCAQ 150 mm x 4.6 mm analytical column; the flow-rate was 1.0 ml/min. Ion signals m/z 325.0 and 330.0 for escitalopram and internal standard, were measured in the positive mode. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range of 1.0-200 ng/ml with a mean correlation coefficient more than 0.99. The absolute recovery was more than 75% for both escitalopram and internal standard. The method was simple, sensitive, precise, accurate and was successfully applied to the bioequivalence study of escitalopram in healthy, male, human subjects.

Validation of LC/MS electrospray ionisation method for the estimation of ursodiol in human plasma and its application in bioequivalence study.

A novel High Performance Liquid Chromatography-electrospray mass spectrometric method has been developed for the estimation of Ursodiol (Ursodeoxycholic acid)--a bile acid, in human plasma using Ornidazole as internal standard. The methodology involved solid phase extraction of the analyte from human plasma matrix. The chromatographic separation was achieved within seven minutes by an isocratic mobile phase containing 1.0 mM ammonium acetate and Acetonitrile (65:35, v/v), flowing through XTerra MS C18, 100 x 2.1, 3.5 microm analytical column, at a flow rate of 0.2 ml/min. Ion signals were measured in negative mode for Ursodiol and internal standard at m/z 391.3 and 278.1, respectively. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range 50.0 ng/ml to 3000.0 ng/ml with the mean correlation coefficient more than 0.99. The absolute recovery was more than 54.90% for Ursodiol and 76.51% for internal standard. Ursodiol was stable for sixty-nine days at -70 degrees C and for eight hours at ambient temperature. After extraction from plasma, the reconstituted samples of Ursodiol were stable in autosampler at 10 degrees C for forty-eight hours. Upon subjecting to three freeze thaw cycles, there was no change in the recovery of the analyte. The integrity of the plasma samples remained unaffected even upon four-fold dilution with drug free human plasma. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of Ursodiol in male human subjects.