Development and Validation of an LC-MS-MS Method for Determination of Simvastatin and Simvastatin Acid in Human Plasma: Application to a Pharmacokinetic Study.

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of simvastatin (SV) and simvastatin acid (SVA) in human plasma. To improve assay sensitivity and achieve simultaneous analysis, SVA monitored in (-)ESI (electrospray ionization) mode within the first 4.5 min and SV thereafter in (+)ESI mode. The separation of all compounds was achieved in about 6.2 min using a C18 reverse-phase fused-core(®) column (Ascentis(®) Express C18) and a mobile phase, which was composed of 2.00 ± 0.05 mM ammonium acetate buffer titrated to pH 3.8 with glacial acetic acid-acetonitrile (25:75, v/v), in isocratic mode at a flow rate of 0.500 mL/min. Additionally, a solid-phase extraction step was performed to reduce any ion-suppression and/or enhancement effects. The developed method was linear in the concentration range of 0.100-74.626 ng/mL for SV, and 0.100-48.971 ng/mL for SVA, with correlation coefficient greater than 0.99 for both analytes. The method has shown tremendous reproducibility, with intra- and inter-day precision <7.6%, and intra- and interday accuracy within ±10.9% of nominal values, for the both analytes. The method was successfully applied to characterize the pharmacokinetic profiles of SV and SVA following an oral administration of 40 mg SV tablet to healthy human volunteers.

Liquid chromatography tandem mass spectrometry method for the quantitation of mycophenolate mofetil in human plasma: Application to a bioequivalence study and metabolite identification.

We established a sensitive, selective, and rapid analytical method for the quantitation and pharmacokinetic investigation of mycophenolate mofetil in human plasma. To our knowledge, this is the first method that characterizes presence of mycophenolate mofetil glucuronide in clinical samples through tandem mass spectrometry detection and resolves mycophenolate mofetil from its glucuronide metabolite. Liquid chromatography coupled to tandem mass spectrometry detection in positive ion mode was selected to provide optimal selectivity and sensitivity. Due to the ionizable characteristics of the mycophenolate mofetil, a mixed-mode cation-exchange disposable extraction cartridge was prudently chosen. The chromatographic separation was achieved on Luna(®) C18(2) (100×4.60 mm) column using mobile phase consisting of a mixture of 1±0.05 mM ammonium formate in water, titrated to pH 3.1±0.1 with formic acid, and methanol (20:80, v/v), at a flow rate of 0.7 mL/min. The detection was led at m/z ratios of 434.4→ 114.2 and 438.4→ 118.3, for mycophenolate mofetil and mycophenolate mofetil-D4, respectively. The developed method was linear between 40.2-4986.0 pg/mL. All validation parameters were within the defined limits. The validated method was then successfully applied for the evaluation of bioequivalence parameters of mycophenolate mofetil after an oral administration of 500 mg mycophenolate mofetil tablet to healthy male Indian volunteers.

Simultaneous quantitation of atorvastatin and its two active metabolites in human plasma by liquid chromatography/(-) electrospray tandem mass spectrometry.

A sensitive, accurate and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the simultaneous quantitation of atorvastatin (AT) and its equipotent hydroxyl metabolites, 2-hydroxy atorvastatin (2-AT) and 4-hydroxy atorvastatin (4-AT), in human plasma. Electrospray ionization (ESI) interface in negative ion mode was selected to improve the selectivity and the sensitivity required for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce any ion-suppression and/or enhancement effects. The separation of all compounds was achieved in less than 6 min using a C18 reverse-phase fused-core® column and a mobile phase, composed of a mixture of 0.005% formic acid in water:acetonitrile:methanol (35:25:40, v/v/v), in isocratic mode at a flow rate of 0.6 mL/min. The method has lower limit of quantitation (LLOQ) of 0.050 ng/mL for all analytes. The method has shown tremendous reproducibility, with intra- and inter-day precision less than 6.6%, and intra- and inter-day accuracy within ±4.3% of nominal values, for all analytes, and has proved to be highly reliable for the analysis of clinical samples.

Simultaneous quantitation of atorvastatin and its two active metabolites in human plasma by liquid chromatography/(-) electrospray tandem mass spectrometry / 药物分析学报

A sensitive, accurate and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the simultaneous quantitation of atorvastatin (AT) and its equipotent hydroxyl metabolites, 2-hydroxy atorvastatin (2-AT) and 4-hydroxy atorvastatin (4-AT), in human plasma. Electrospray ionization (ESI) interface in negative ion mode was selected to improve the selectivity and the sensitivity required for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce any ion-suppression and/or enhancement effects. The separation of all compounds was achieved in less than 6 min using a C18 reverse-phase fused-cores column and a mobile phase, composed of a mixture of 0.005%formic acid in water:acetonitrile:methanol (35:25:40, v/v/v), in isocratic mode at a flow rate of 0.6 mL/min. The method has lower limit of quantitation (LLOQ) of 0.050 ng/mL for all analytes. The method has shown tremendous reproducibility, with intra-and inter-day precision less than 6.6%, and intra- and inter-day accuracy within 74.3% of nominal values, for all analytes, and has proved to be highly reliable for the analysis of clinical samples.

Liquid chromatography tandem mass spectrometry method for the estimation of lamotrigine in human plasma: Application to a pharmacokinetic study.

A reliable, selective and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of lamotrigine in human plasma using lamotrigine-13C3, d3 as an internal standard. Analyte and internal standard were extracted from human plasma by solid-phase extraction and detected in positive ion mode by tandem mass spectrometry with electrospray ionization (ESI) interface. Chromatographic separation was performed on a Chromolith® SpeedROD; RP-18e column (50-4.6 mm i.d.) using acetonitrile: 5±0.1 mM ammonium formate solution (90:10, v/v) as the mobile phase at a flow rate of 0.500 mL/min. The calibration curves were linear over the range of 5.02-1226.47 ng/mL with the lower limit of quantitation validated at 5.02 ng/mL. The analytes were found stable in human plasma through three freeze (-20 °C)-thaw (ice-cold water bath) cycles and under storage on bench-top in ice-cold water bath for at least 6.8 h, and also in the mobile phase at 10 °C for at least 57 h. The method has shown good reproducibility, as the intra- and inter-day precisions were within 3.0%, while the accuracies were within ±6.0% of nominal values. The validated LC-MS/MS method was applied for the evaluation of pharmacokinetic and bioequivalence parameters of lamotrigine after an oral administration of 50 mg lamotrigine tablet to thirty-two healthy adult male volunteers.

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: application to a pharmacokinetic study.

A rapid, selective and sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher 60 RP Select B column (125 mm x 4 mm i.d., 5 microm particle size) using acetonitrile:5+/-0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0-15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within +/-6.3% of nominal values. No matrix effect was observed in this method. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.

Simultaneous determination of propranolol and 4-hydroxy propranolol in human plasma by solid phase extraction and liquid chromatography/electrospray tandem mass spectrometry.

A very sensitive, reliable, reproducible and highly selective assay for the simultaneous determination of free and total (conjugated and unconjugated) propranolol and its equipotent hydroxyl metabolite, 4-hydroxy propranolol, in human plasma was developed and validated. The analytes were simultaneously extracted from 0.300 mL of human plasma using solid phase extraction and detected in positive ion mode by tandem mass spectrometry with a turbo ionspray interface. Deuterium-labeled propranolol and 4-hydroxy propranolol, propranolol-d7 and 4-hydroxy propranolol-d7, were used as internal standards. The method has a lower limit of quantitation (LOQ) of 0.20 ng/mL for both analytes with the limits of detection (LOD) 50 and 100 pg/mL for propranolol and 4-hydroxy propranolol, respectively, based on a signal-to-noise ratio of 5. The assay was linear over a range 0.20-135.00 ng/mL for free propranolol and 0.20-25.00 ng/mL for free 4-hydroxy propranolol and linear over range 1.00-500.00 ng/mL for total propranolol and 1.00-360.00 ng/mL for total 4-hydroxy propranolol, with coefficient of determination greater than 0.99 for both analytes. The extraction recoveries were >96 and >64% on an average for propranolol and 4-hydroxy propranolol, respectively. The analytes were found stable in human plasma through five freeze (-15 degrees C)-thaw (room temperature) cycles and under storage on bench-top for at least 6.5 h, and also in mobile phase at 10 degrees C for at least 48 h. The method has shown tremendous reproducibility, with intra- and inter-day precision <11.3% (RSD), and intra- and inter-day accuracy <11% of nominal values, for both analytes, and has proved to be highly reliable for the analysis of clinical samples.

Solid-phase synthesis of 2-aminoquinazolinone derivatives with two- and three-point diversity.

A versatile solid-phase method for the synthesis of various substituted 2-amino-4(3H)-quinazolinones with two- and three-point diversity is described. The synthesis commenced with the generation of polymer-bound S-methylisothiourea followed by N-acylation with different substituted o-nitrobenzoic acid. Finally, reduction of the nitro group triggered intramolecular cyclization via formation of guanidine to afford 2-amino-4(3H)-quinazolinone and its derivatives in high yields and purities.

New application of Pictet-Spengler reaction leading to the synthesis of an unusual seven-membered heterocyclic ring system.

A novel strategy for the Pictet-Spengler reaction is reported. Our strategy involves reaction of arylamines, linked to the N-1 of disubstituted imidazoles, with aldehydes in the presence of p-TsOH. The iminium ion generated in situ undergoes C-C bond formation with the C-5 of the imidazoles to furnish triazabenzoazulenes as a novel heterosystem. Our strategy differs from conventional Pictet-Spengler reaction since the latter utilizes only aliphatic amines in which the amine functionality is linked to a C instead of N of the activated aromatic moiety.