A liquid chromatography-tandem mass spectrometry method for the determination of apixaban in human plasma and its application to pharmacokinetics studies in the Indian population.

Apixaban is a novel oral anticoagulant intended to treat and prevent blood clots and to prevent strokes in patients with nonvalvular atrial fibrillation. The development and validation of a fast, selective, accurate, and precise method using high-performance liquid chromatography tandem mass spectrometry is described for the estimation of apixaban in human plasma, with apixaban 13CD3 as an internal standard (IS). Using a reverse phase Gemini C18 column (50 mm × 4.6 mm, 3 µm) and a mixture of acetonitrile (2 mM) and ammonium formate buffer (50 : 50 v/v) as the mobile phase, chromatographic separation was achieved following extraction via a solid-phase extraction process. To track multiple reaction monitoring transitions set at 460/443 (m/z) and 464/447 (m/z) for apixaban and apixaban 13CD3, respectively, liquid chromatography coupled with triple quadrupole mass spectrometry was employed. A concentration linearity range between 1.01 and 280.00 ng mL-1 was validated with regression ≥0.99, and the method was successfully applied to apixaban pharmacokinetics analysis. At a flow rate of 1.0 mL min-1, the run time was around 1.8 min, which is short. With an extraction recovery of >73% for both apixaban and apixaban 13CD3, the method was sensitive, with a limit of quantitation of 1.01 ng mL-1. The inter-day/between-run precision ranged from 1.21% to 3.21%, while the accuracy ranged from 96.5% to 102%. For pharmacokinetics analysis, the validated method was applied. The percentage difference between findings from samples that were reanalyzed and samples that were initially analyzed was within ±20%. With high-quality assay specificity and accuracy in relation to apixaban analysis in human plasma under the experimental conditions used, the method provided is accurate.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of free and total dabigatran in human plasma and its application to a pharmacokinetic study.

A high-performance liquid chromatography-tandem mass spectrometric method for the determination of free and total dabigatran in human plasma has been developed and validated using a stable labeled internal standard (IS) as dabigatran D4. The extraction of analyte and IS was accomplished by the solid-phase extraction technique. Chromatographic separations were achieved using Peerless basic C8 (150 × 4.6) mm, 5 µ column eluted at a flow rate of 1 mL/min with mobile phase Acetonitrile: 5 mM ammonium formate: Methanol and 0.2% formic acid (30:20:50, v/v/v). The run time of the method was about 2.5 min with elution times of dabigatran and dabigatran D4 at around 1.2 min. The multiple reaction monitoring transitions (Q1/Q3) were set at 472/289, 172 (m/z) for dabigatran, and 476/293 (m/z) for dabigatran D4. The calibration curves were linear (r2 ≥0.99) over the range of 1.04-406.49 ng/mL. The presented method was successfully employed in the analysis of pharmacokinetic studies with the added advantage of demonstrating the effect of co-administration of dabigatran with the proton pump inhibitor pantoprazole on bioavailability and pharmacokinetic characteristics. Re-analysis of incurred sample resulted in >98% compliance indicating good assay precision of target analytes.

Liquid chromatography-tandem mass spectrometry method for determination of rivaroxaban in human plasma and its application to a pharmacokinetic study.

A high-performance liquid chromatography tandem mass spectrometric method for the determination of Rivaroxaban in human plasma has been developed and validated using Rivaroxaban D4 as an internal standard. The extraction of analyte and internal standard was accomplished by solid phase extraction technique. The method has been validated over a concentration range of 5.96-801 ng/mL. Chromatographic separations were achieved using Gemini C18, 150 mm × 4.6 mm, 5 µm, column eluted at flow rate of 1.5 mL/min with mobile phase (acetonitrile: ammonium acetate buffer (80:20 v/v)). The overall run time of method was about 1.8 min with elution times of Rivaroxaban and its internal standard Rivaroxaban D4 at around 1.18 min. The multiple reaction monitoring transitions were set at 436/145 (m/z) and 440/145 (m/z) for Rivaroxaban and Rivaroxaban D4, respectively. The calibration curves were linear (r2 ≥ 0.99) over the range of 5.96-801 ng/mL with lower limit of quantitation validated at 5.96 ng/mL. Extraction recoveries were >88% for both rivaroxaban and its stable labeled internal standard rivaroxaban D4. The inter-day/between run precisions were ranged from 1.08% to 3.75%, while accuracy ranged from 96.3% to 102%. The presented method was used in pharmacokinetic study in healthy volunteers. Results of incurred sample reanalysis were within the acceptance range of ±20% of original value, for 98.3% of samples reanalyzed. This indicated good assay precision of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for the determination of the pharmacokinetic parameters was demonstrated.

Development of simultaneous determination of empagliflozin and metformin in human plasma using liquid chromatography-mass spectrometry and application to pharmacokinetics.

A rapid and sensitive liquid chromatography-mass spectrometry method was developed, optimized, and validated for simultaneous quantification of empagliflozin and metformin in human plasma using empagliflozin D4and metformin D6 as an internal standard. Analytes and internal standard were extracted from plasma by optimized solid-phase extraction technique using Strata X polymeric reverse phase (30 mg-1cc) solid-phase extraction cartridges. The prepared samples were chromatographed on Orosil C18 column (150 × 4.6 mm, 3 µ). Separation was done by pumping isocratic mobile phase consisting of methanol and 10 mM ammonium trifluoroacetate (90:10, v/v) in positive ion mode at a flow rate of 0.8 mL/min. The API 3200 liquid chromatography-mass spectrometry system having turbo ion spray as an ion source coupled with Shimadzu Prominence ultrafast liquid chromatography system was operated under the selected reaction monitoring mode. Turbo ion spray ionization was used for mass transition of m/z 468.070/355.100 and m/z 130.072/71.200 for empagliflozin and metformin, respectively. A method was successfully validated for concentration range of 10.09-5013.46 ng/mL for both the analytes and according to the United States Food and Drugs Administration guidelines. The linearity was found to be in the range of 10.09-403.46 ng/mL for empagliflozin and 25.44-5013.46 ng/mL for metformin. The limit of quantification was found to be 10.09 ng/mL for empagliflozin and 25.44 ng/mL for metformin. Intra- and inter-day/between batch precision determination for empagliflozin and metformin, expressed as coefficient of variation were within the acceptance limits and ranged below 13.16%. A short run time of 3.3 min allows analysis of more than 400 plasma samples per day. The developed method was successfully applied to fasting pharmacokinetic study in healthy human volunteers. Results of incurred sample re-analysis were within the acceptance range of ±20% of original value, for 97.2% of samples reanalyzed for empagliflozin and 100% of samples reanalyzed for metformin.

High- performance liquid chromatography method applied to investigate mechanism, kinetics, isotherm, and thermodynamics of bile acid adsorption onto bile acid sequestrants.

The purpose of the study was to develop and validate a high-performance liquid chromatography (HPLC) method which can be further applied to understand the mechanism, kinetics, isotherm, and thermodynamics of bile acid adsorption onto bile acid sequestrants. To investigate these properties a HPLC method was developed using peerless C-8 (150 x 4.6 mm, 5 µm) column with a detection wavelength of 200 nm and run time of about 12.5 min. Bile salts glycocholic (GC), glycochenodeoxycholic (GCDC), and taurodeoxycholic acid (TDC), were used and colesevelam hydrochloride was employed as the bile acid sequestrant. The calibration range was found linear from 10 to 6500 mgL-1 for GC and GCDC and 4to 2400 mg L-1 for TDC. The precision was less than 8.8% and accuracy was found well within the range of 85 to 115%. On treating the data with various established models, it was known that, the adsorption kinetics followed the pseudo second order equation indicating chemisorption mechanism. Equilibrium isotherms revealed that the linear form of Langmuir model was the best fit. The separation factor (RL) calculated revealed that the reaction is favorable and reversible. The positive value of heat of sorption (B) calculated from Temkin model indicated towards the exothermic nature of adsorption. The adsorption energy (E) calculated from Dubinin-Kaganer-Radushkevich model was found to be greater than 8 KJmol-1 conforming chemisorption mechanism. The Gibbs free energy calculated established the affinity of bile salts as TDC > GCDC > GC.

A novel analytical approach towards in-vitro bile acid binding studies to Colesevelam Hydrochloride tablets: An ultra-high performance liquid chromatography tandem mass spectrometric method.

Colesevelam hydrochloride is a bile acid sequestrant used as a low density lipoprotein (LDL) reducing agent in hyperlipidemia with an additional advantage to improve glycemic control in type 2 diabetes patients. The objective of the study was to develop and validate a liquid chromatography tandem mass spectroscopic method for the simultaneous in-vitro estimation of bile acid salts of Glycocholic acid (GC), Glycochenodeoxycholic acid (GCDC) and Taurodeoxycholic acid (TDC) and its application in performing in-vitro binding study with Colesevelam Hydrochloride tablets. The method was developed using C-18 (50 x 4.6 mm, 3 µm) column with detection on negative ion mode and acquisition time of 3.5 min. The calibration range was linear from 0.0002 mM to 0.0065 mM for GC, 0.0002 mM to 0.0065 mM for GCDC and 0.0001 mM to 0.0021 mM for TDC. The precision was less than 3.0% and accuracy was found well within the range of 85 to 115%. The validated method was further applied to conduct in-vitro equilibrium binding study. The data was subjected to Langmuir isotherm and affinity constant (k1) and capacity constant (k2) were calculated.