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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.

Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The objective of the current study was to develop a sensitive, fast and high-throughput HPLC-ESI-MS/MS method to measure etoricoxib levels in human plasma using a one-step methanol protein precipitation technique. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive mode and multiple reaction monitoring (MRM) were used for data collection. The quantitative MRM transition ions were m/z 359.15 > 279.10 and m/z 363.10 > 282.10 for etoricoxib and IS. The linear range was from 10.00 to 4000.39 ng/mL and the validation parameters were within the acceptance limits of the European Medicine Agency (EMA) and Food and Drug Analysis (FDA) guidelines. The present method was sensitive (10.00 ng/mL with S/N > 40), simple, selective (K prime > 2), and fast (short run time of 2 min), with negligible matrix effect and consistent recovery, suitable for high throughput analysis. The method was used to quantitate etoricoxib plasma concentrations in a bioequivalence study of two 120 mg etoricoxib formulations. Incurred sample reanalysis results further supported that the method was robust and reproducible.

Objective: The study aimed to develop a rapid, simple and sensitive LC/ESI-MS/MS method to measure prazosin concentration in human plasma and apply bedside sampling in bioequivalence study of two prazosin tablets to resolve the adverse effect of orthostatic hypotension., Significance: The LC/ESI-MS/MS prazosin method was highly sensitive and selective. Bedside sampling reduced the orthostatic hypotension incidence and subject dropout rate., Methods: After sample preparation, prazosin and terazosin (IS) were detected on mass spectrometer operating in multiple reaction monitoring mode using positive ionization. Mobile phase flow rate was set at 0.40 mL/min with sample run time of 1.75 min. The bioanalytical method was validated as per EMEA and FDA guidelines. Bedside sampling was performed in bioequivalence study for the first 4 h after dosing. The three primary pharmacokinetic parameters, C max , AUC 0- t and AUC 0-∞ and 90% confidence interval were determined., Results: The small injection volume of 1 μL minimized instrumentation contamination and prolonged the analytical column lifespan. Linearity was obtained between 0.5 and 30.0 ng/mL, with coefficient of determination, r 2 ≥ 0.99. The mean extraction recovery of prazosin and IS was >92%, with precision value (CV, %) ≤ 10.3%. Only two orthostatic hypotension adverse events were reported. The two prazosin formulations were found to be bioequivalent., Conclusion: The LC/ESI-MS/MS method has shown robustness and reliability exemplified by the incurred sample re-analysis result. Bedside sampling should be proposed for bioequivalence or pharmacokinetic studies of drugs demonstrating adverse event of orthostatic hypotension.

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-C 18 (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).

A simple, rapid, sensitive, and reproducible LC-MS/MS method was developed for simultaneous quantification of flavoxate and 3-methyl-flavone-8-carboxylic (MFCA) in human plasma, using diphenhydramine HCl as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7 μm) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1 mmID, 2.7 μm). The mobile phase consisted of 0.1 % v/v formic acid and acetonitrile (30:70, v/v) run at a flow rate of 0.40 mL/min. The standard calibration curve was linear over the concentration range of 2.00 - 2,000.31 ng/mL and 240.00 - 24,000.04 ng/mL for flavoxate and MFCA. For flavoxate and MFCA, the within-run precision was 0.81-6.67 % and 1.68-4.37 %, while accuracy was 100.21-108.25 % and 103.99-110.28 %. The between-run precision was 2.01-9.14 % and 2.31-11.11 %, and accuracy was 96.09-103.33 % and 102.37-109.52 %. The extended run precision was 7.78-11.04 % and 2.22-3.33 %, while accuracy was 100.72-101.88 % and 102.34-105.60 %. Flavoxate and MFCA in plasma were stable 4 h at bench top (short term), 24 h in autosampler and instrumentation room (post-preparative), after 7 freeze-thaw cycles, and 89 days in the freezer. Both analytes and IS stock solutions were stable for 31 days when kept at room temperature (25 ± 4 °C) and refrigerated (2-8 °C). The validated method was successfully applied to a bioequivalence study of two flavoxate formulations involving 24 healthy volunteers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: The aim of the study was to develop a simple, highthroughput and sensitive LC-MS/MS method and apply to a bioequivalence study of montelukast, a light sensitive drug., Method: The effects of organic modifiers in mobile phase, protein precipitation agent to plasma sample ratio, and light on montelukast stability in unprocessed and processed human plasma, were evaluated. Validation was conducted in accordance with European Medicines Agency Guideline on bioanalytical method validation., Results: No interference peak was observed when acetonitrile was used as an organic modifier. Acetonitrile to plasma ratio of 4:1 produced clean plasma sample. Approximately 3 % of cis isomer was detected in unprocessed plasma samples while 21 % of cis isomer was detected in processed plasma samples after exposing to fluorescent light for 24h. The standard calibration curve was linear over 3.00-1200.00 ng/mL. All method validation parameters were within the acceptance criteria., Conclusion: The validated method was successfully applied to a bioequivalence study of two montelukast formulations involving 24 healthy Malaysian volunteers. The light stability of a light sensitive drug in unprocessed and processed human plasma samples should be studied prior to pharmacokinetic/bioequivalence studies. Measures could then be taken to protect the analyte in human plasma from light degradation.

A simple, fast and sensitive LC-MS/MS method was developed to quantify terazosin in human plasma. The mobile phase consisted of acetonitrile-0.1% (v/v) formic acid (70:30, v/v). Prazosin was used as internal standard (IS). As deproteinization agent, acetonitrile produced a clean sample. A higher response intensity with more symmetrical peak was obtained using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7 μm) compared with Kinetex XB-C18 (100 × 2.1 mm, 2.6 µm) column. The response of terazosin and IS were approximately two times in citrate phosphate dextrose (CPD) plasma compared with dipotassium ethylenediaminetetraacetic acid (K 2 EDTA) plasma. Plasma calibration curve was linear from 1.0 to 100.0 ng/mL, with coefficient of determination r 2  ≥ 0.99. The within-run and between-run precision values (CV, %) were <5.2% and <7.8%, while accuracy values were 102.8-112.7% and 103.4-112.2%. The extended run accuracy was 98.6-102.8% and precision (CV, %) 4.3-10.4%. The recovery of analyte was >98% and IS >94%. Terazosin in plasma kept at benchtop was stable for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h, for 7 freeze-thaw cycles and in freezer for 140 days. Terazosin and IS stock standard solutions were stable for 140 days at room temperature and in the chiller. The high throughput method was successfully utilized to measure 935 samples in a bioequivalence study of terazosin., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A simple, rapid, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine sitagliptin in human plasma. Diphenhydramine HCl was used as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1mmID, 2.7 µm). The mobile phase consisted of 0.1% v/v formic acid and methanol (45:55, v/v) run at a flow rate of 0.45 mL/min at 30 °C. Methanol produced relatively cleaner plasma sample as deproteinization agent. Polytetrafluoroethylene membrane was preferred over nylon membrane as the former produced clear plasma samples. The standard calibration curve was linear over the concentration range of 5-500.03 ng/mL. The within-run precision was 0.53-7.12% and accuracy 87.09-105.05%. The between-run precision was 4.74-11.68% and accuracy 95.02-97.36%. The extended run precision was 3.60-6.88% and accuracy 93.18-95.82%. The recovery of analyte and IS was consistent. Sitagliptin in plasma was stable at benchtop (short term) for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h (post-preparative), after 7 freeze-thaw cycles (-20 ± 10 °C), and 62 days in the freezer (-20 ± 10 °C). Both sitagliptin (analyte) and IS stock solutions were stable for 62 days when kept at room temperature (25 ± 4 °C) and in chiller (2-8 °C). The validated method was successfully applied to a bioequivalence study of two sitagliptin formulations involving 26 healthy Malaysian volunteers., (Copyright © 2020 Elsevier B.V. All rights reserved.)