Simultaneous analysis of allopurinol and oxypurinol using a validated liquid chromatography-tandem mass spectrometry method in human plasma.

The present study describes a simple, reliable and reproducible liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the simultaneous determination of allopurinol and its active metabolite, oxypurinol in human plasma for a pharmacokinetic/bioequivalence study. After protein precipitation (PPT) of 100 µL plasma sample with 1.0% formic acid in acetonitrile, the recovery of the analytes and allopurinol-d2 as an internal standard ranged from 85.36% to 91.20%. The analytes were separated on Hypersil Gold (150 mm×4.6 mm, 5 µm) column using 0.1% formic acid-acetonitrile (98:2, v/v) as the mobile phase. Quantification was done using electrospray ionization in the positive mode. The calibration concentration range was established from 60.0 to 6000 ng/mL for allopurinol and 80.0-8000 ng/mL for oxypurinol. Matrix effect in human plasma, expressed as IS-normalized matrix factors ranged from 1.003 to 1.030 for both the analytes. The developed method was found suitable for a clinical study with 300 mg allopurinol tablet formulation in healthy subjects.

An improved LC-MS/MS method for the quantification of alverine and para hydroxy alverine in human plasma for a bioequivalence study☆.

A highly sensitive and selective high performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of alverine (ALV) and its active metabolite, para hydroxy alverine (PHA), in human plasma. For sample preparation, solid phase extraction of analytes was performed on Phenomenex Strata-X cartridges using alverine-d5 as the internal standard. The analytes were separated on Symmetry Shield RP18 (150 mm×3.9 mm, 5 µm) column with a mobile phase consisting of acetonitrile and 10 mM ammonium formate (65:35, v/v). Detection and quantitation was done by electrospray ionization mass spectrometry in the positive mode using multiple reaction monitoring. The assay method was fully validated over the concentration range of 15.0-15,000 pg/mL for ALV and 30.0-15,000 pg/mL for PHA. The intra-day and inter-day accuracy and precision (% CV) ranged from 94.00% to 96.00% and 0.48% to 4.15% for both the analytes. The mean recovery obtained for ALV and PHA was 80.59% and 81.26%, respectively. Matrix effect, expressed as IS-normalized matrix factor ranged from 0.982 to 1.009 for both the analytes. The application of the method was demonstrated for the specific analysis of ALV and PHA for a bioequivalence study in 52 healthy subjects using 120 mg ALV capsules. The assay reproducibility was also verified by reanalysis of 175 incurred subject samples.

Application of an LC-MS/MS method for reliable determination of amodiaquine, N-desethylamodiaquine, artesunate and dihydroartemisinin in human plasma for a bioequivalence study in healthy Indian subjects.

A sensitive and high throughput bioanalytical method has been developed for reliable determination of amodiaquine (AQ), N-desethylamodiaquine (DEAQ), artesunate (AS) and dihydroartemisinin (DHA) in human plasma by LC-MS/MS. The method employs a solid phase extraction procedure without an evaporation step and with optimum use of organic solvents to circumvent degradation of artemisinin derivatives. The analytes and their deuterated internal standards (ISs) were analyzed on Hypersil Gold (100 mm × 4.6mm, 5 µm) column using acetonitrile and 2.0mM ammonium formate (pH 2.50) in 80:20 (v/v) ratio as the mobile phase. A triple quadrupole mass spectrometer equipped with an electrospray ionization interface was used to detect and quantify the analytes. The method was established over the concentration range of 0.250-30.0 ng/mL, 1.50-180 ng/mL, 2.00-600 ng/mL and 5.00-1400 ng/mL for AQ, DEAQ, AS and DHA respectively using 250 µL human plasma. The intra-day and inter-day accuracy and precision (% CV) across quality controls varied from 93.3-105.0% and 1.7-8.3 respectively for all the analytes. The stability was assessed in whole blood as well as in plasma samples under different conditions. All four analytes were stable in whole blood up to 2h on melting ice. The long term stability in plasma was ascertained up to 90 days. IS-normalized matrix factors ranged from 0.988-1.023 for all the analytes. The method was successfully applied to a bioequivalence study using 50mg artesunate and 135 mg amodiaquine fixed dose formulation in 14 healthy subjects.

Liquid chromatography--tandem mass spectrometry method for simultaneous determination of albendazole and albendazole sulfoxide in human plasma for bioequivalence studies.

An improved high performance liquid chromatography--tandem mass spectrometry (LC-MS/MS) method has been developed for sensitive and rapid determination of albendazole (ABZ) and its active metabolite, albendazole sulfoxide (ABZSO), in the positive ionization mode. The method utilized solid phase extraction (SPE) for sample preparation of the analytes and their deuterated internal standards (ISs) from 100 µL human plasma. The chromatography was carried out on Hypurity C18 column using acetonitrile-2.0 mM ammonium acetate, pH 5.0 (80:20, v/v) as the mobile phase. The assay exhibited a linear response over the concentration range of 0.200-50.0 ng/mL for ABZ and 3.00-600 ng/mL for ABZSO. The recoveries of the analytes and ISs ranged from 86.03%-89.66% and 89.85%-98.94%, respectively. Matrix effect, expressed as IS-normalized matrix factors, ranged from 0.985 to 1.042 for the both analytes. The method was successfully applied for two separate studies in healthy subjects using single dose of 400 mg conventional tablets and 400 mg chewable ABZ tablets, respectively.