Vikas V. Vaidya

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of tenofovir and emtricitabine in human plasma and its application to a bioequivalence study

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of Tenofovir (TEN) and Emtricitabine (EMT) in human plasma using Chromolith Speed Rod RP18. The mass transition ion-pair has been followed as m/z 288.10-->176.10 for TEN, m/z 248.20-->130.20 for EMT and m/z 230.10-->112.10 for Lamivudine (LAM). The method involves solid phase extraction from plasma, simple isocratic chromatographic conditions and mass spectrometric detection using an API 5000 instrument that enables detection at nanogram levels. Lamivudine was used as the internal standard. The proposed method has been validated with a linear range of 10-600 ng/ml for TEN and 25-2,500 ng/ml for EMT. The intrarun and interrun precision values are within 12.0% for TEN and 15.6% for EMT at their respective LOQ levels. The overall recoveries for TEN and EMT were 84.3% and 68.5%, respectively. Total elution time was as low as 2 min.

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 determination of hydrochlorothiazide, quinapril and quinaprilat in human plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous estimation of hydrochlorothiazide, quinapril and its metabolite quinaprilat in human plasma. After solid phase extraction (SPE), the analytes and IS were chromatographed on a hypurity C8 (100 mm x 2.1 mm i.d., 5 microm particle size) column using 2 microL injection volume with a run time of 2.8 min. An isocratic mobile phase consisting of 0.5% (v/v) formic acid:acetonitrile (25:75, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The proposed method was validated over the range of 5-500 ng/mL for hydrochlorothiazide method and 5-1500 ng/mL for quinapril and quinaprilat. Inter-batch and intra-batch precision (coefficient of variation - % CV) across five validation runs lower limit of quantitation (LLOQ), lower quality control (LQC), middle quality control (MQC), higher quality control (HQC) and upper limit of quantitation (ULOQ) was less than 15. The accuracy determined at these levels was within +/-13% in terms of relative percentage error.

Validated LC-MS/MS method for determination of Alverine and one of its hydroxy metabolites in human plasma along with its application to a bioequivalence study.

The present research work involves a first of its kind rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed and validated for simultaneous analysis of Alverine (ALV) and one of its hydroxy metabolites, para hydroxy Alverine (PHA) in human plasma. The analytes were extracted from the matrix using a simple solid-phase extraction procedure. Mebeverine was used as the internal standard for both analytes. A Kromasil C8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatography conditions and mass spectrometric detection in the positive ionization mode using an API 5000 MS/MS system. The proposed method has been validated with a linear range of 100-10,000 pg/mL for both ALV and PHA. The interrun and intrarun precision values are within 6.3%, 3.7% for ALV and 6.3%, 3.2% for PHA at LOQ levels. The intrarun accuracy in terms of % RE was within the range of -7.0% to -0.1% and -8.1% to -1.7% for ALV and PHA, respectively whereas the interrun accuracy was within the range of -5.1% to -0.5% for ALV and -8.6% to 0.4% for PHA, respectively. The overall recoveries for ALV and PHA were 83.5% and 86.2% respectively. Total elution time was about 4 min which allowed quantitation of more than 150 plasma samples per day. This validated method was used successfully for analysis of real samples from a bioequivalence study.

HPTLC method for determination of colchicine in a pharmaceutical formulation

An HPTLC method, using an internal standard, for analysis of colchicine in a pharmaceutical formulation, has been established and validated. The analyte and internal standard were separated on aluminum plates precoated with silica gel 60 F254; the mobile phase was ethyl acetate—acetonitrile-water-formic acid 8.0:1.0:0.5:0.5 (v/v). Quantification was by densitometric scanning at 358 nm. Response was a linear function of colchicine concentration in the range 5 to 35 μg mL−1. The limits of detection and quantification for colchicine were 1 and 5 μg mL−1, respectively. Average recovery of colchicine was 100.48%, which showed the method was free from interference from excipients present in the formulation. The established method enabled accurate, precise, and rapid analysis of colchicine in the pharmaceutical formulation.

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.

Quantification of puerarin in Pueraria tuberosa DC. by high-performance thin-layer chromatography

In Ayurveda, ‘Vidari’ is botanically equated to Pueraria tuberosa DC. of the Fabaceae family. Reported chemical constituents in Pueraria tuberosa DC. are puerarin [1] and tuberosin [2]. Puerarin has antihyperglycemic effect in streptozotocin-induced diabetic rats [3]. Pueraria tuberosa is a large, perennial climber with huge tuberous roots, used to treat many ailments. The pharmacological activity of the plant includes antihepatotoxic [4] and anti-implantation activity in rats [5]. A simple, rapid, economical, precise, and accurate HPTLC method has been established for determination of puerarin in Pueraria tuberosa DC.