nan M.B.Kekare

Determination of free and liposomal amphotericin B in human plasma by liquid chromatography-mass spectroscopy with solid phase extraction and protein precipitation techniques.

Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC-MS-MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25-15.0 microg/ml) and liposomal Amphotericin B (1.0-100.0 microg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.

Simultaneous HPTLC Determination of Clotrimazole and Tinidazole in a Pharmaceutical Formulation

An HPTLC method for simultaneous determination of clotrimazole and tinidazole in a pharmaceutical formulation has been developed and validated. The analytes were separated on silica gel 60F254 HPTLC plates with toluene-ethyl acetate-methanol-glacial acetic acid, 6.0 + 3.0 + 1.0 + 0.3 (v/v), as mobile phase, after chamber saturation for 10 min. The development distance was 8 cm. The plate was then dried in air and scanned and quantified at λ = 254 nm. Response to clotrimazole and tinidazole was a linear function of concentration in the ranges 120 to 320 μg mL−1 and 300 to 800 μg mL−1, respectively. The limits of detection for clotrimazole and tinidazole were 20 and 60 μg mL−1, respectively; the respective limits of quantification were 120 and 200 μg mL−1. The method enables accurate, precise, and rapid simultaneous analysis of clotrimazole and tinidazole.

Thin-Layer Chromatographic Determination of Alpha-Amyrin in the Bark of Mallotus philippensis Lamk

A simple, precise, and accurate high-performance thin-layer chro-matographic method has been established for determination of alpha-amyrin in a methanol extract of powdered bark from Mallotus philippensis. The amount of alpha-amyrin in the powder was 0.2 mg per 2 g dry weight. Separation was performed on silica gel 60 F254 TLC plates with dichloromethane-toluene, 9.5:0.5 (v/v), as mobile phase. Detection and quantification were performed by den-sitometry at 586 nm. The response to alpha-amyrin was a linear function of concentration over the range 10 to 40 μg mL-1. The sensitivity, accuracy, precision, and reproducibility of the TLC system were evaluated quantitatively.