Simultaneous determination of tramadol and paracetamol in human plasma using LC-MS/MS and application in bioequivalence study of -fixed-dose combination.

The study aimed to develop a sensitive and high-throughput liquid chromatography coupled with tandem mass spectrometry method to quantify concentrations of tramadol and paracetamol simultaneously in human plasma. Sample preparation involved single-step protein precipitation using methanol and two deuterated internal standards, tramadol D6 and paracetamol D4. Agilent Poroshell 120 EC-C18 (100 × 2.1 mm, 2.1 µm) analytical column was employed to achieve chromatographic separation. Detection was in positive ion multiple reaction monitoring mode. A tailing factor (Tf) of <1.2, separation factor (K prime) of >1.5 from the column dead time and signal-to-noise (S/N) ratio >10, were obtained for analytes and internal standards. The standard curve was linear over the concentration range of 2.5-500.00 ng/mL for tramadol and 0.025-20.00 µg/mL for paracetamol. A small injection volume of 1 µL, low flow rate of 440 µL/min and short analysis time of 3.5 min reduced the solvent consumption, analysis cost and system contamination. The results of method validation parameters fulfilled the acceptance criteria of bioanalytical guidelines. The method was successfully applied to a bioequivalence study of fixed-dose combination products of tramadol and paracetamol in Malaysian healthy subjects.

Sample preparation and quantification of polar drug, allopurinol, in human plasma using LCMSMS.

A fast, selective and reproducible LC-MS/MS method with simple sample preparation was developed and validated for a polar compound, allopurinol in human plasma, using acyclovir as internal standard (IS). Chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 (100 × 2.1 mmID, 2.7 µm) analytical column. The mobile phase was comprised of 0.1%v/v formic acid-methanol (95:05; v/v), at a flow rate of 0.45 mL/min. The effect of different protein precipitation agents used in sample preparation such as methanol, acetonitrile, a mixture of acetonitrile-methanol and a mixture of acetonitrile-acetone were evaluated to optimize the extraction efficiency of allopurinol and IS. The use of acetone-acetonitrile (50:50, v/v) as protein precipitating agent shortened the sample preparation time and improved the recovery of allopurinol to above 93%. The IS-normalised matrix factors at two concentration levels were 1.0, with CV of 5.1% and 4.2%. Allopurinol in plasma was stable at benchtop for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h, in freezer after 7 freeze-thaw cycles and in freezer for 140 days. Allopurinol stock standard solutions were stable for 140 days at room temperature and in the chiller. The short sample run time of the validated bioanalytical method allowed high throughput analysis of plasma samples in pharmacokinetic study of an allopurinol formulation. The robustness and reproducibility of the bioanalytical method was reaffirmed through incurred sample reanalysis (ISR).