ESTIMATION OF CELECOXIB IN HUMAN PLASMA BY RAPID AND SELECTIVE LC-MS/MS METHOD FOR A BIOEQUIVALENCE STUDY

Objective: A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for the determination of celecoxib (CXB) in negative ionization mode.Methods: Celecoxib and celecoxib-D7 (CXB-D7) as internal standard (IS) were extracted from 300 µl human plasma by solid-phase extraction using strata-X SPE cartridges. Chromatographic separation was achieved on ACE C8-300 (50 × 4.0 mm, 3.0 μm) column using methanol-1.0 mmol ammonium acetate solution in 80:20 (v/v) ratio. The protonated precursor to product ion transitions studied for CXB and CXB-D7 were m/z 380.0 → 315.9 and 387.0 → 323.0, respectively.Results: The limit of detection (LOD) and lower limit of quantitation of the method were 2.50 and 10.0 ng/ml respectively with a linear dynamic range of 10.0-4000 ng/ml for CXB. The intra-batch and inter-batch precision (% CV) and mean relative recovery across quality control levels is<7.2 % and 85.5 % respectively. Matrix effect in human plasma, expressed as IS-normalized matrix factor ranged from 0.99-1.03.Conclusion: The method was successfully applied in healthy subjects using a single dose of 400 mg celecoxib capsules under fasting and fed conditions. The reproducibility in the measurement of study data is demonstrated by incurred sample reanalysis

Determination of asenapine in presence of its inactive metabolites in human plasma by LC-MS/MS

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been described for the determination of asenapine (ASE) in presence of its inactive metabolites N-desmethyl asenapine (DMA) and asenapine-N-glucuronide (ASG). ASE, and ASE 13C-d3, used as internal standard (IS), were extracted from 300 µL human plasma by a simple and precise liquid-liquid extraction procedure using methyl tert-butyl ether. Baseline separation of ASE from its inactive metabolites was achieved on Chromolith Performance RP8e (100 mm × 4.6 mm) column using acetonitrile-5.0 mM ammonium acetate-10% formic acid (90:10:0.1, v/v/v) within 4.5 min. Quantitation of ASE was done on a triple quadrupole mass spectrometer equipped with electrospray ionization in the positive mode. The protonated precursor to product ion transitions monitored for ASE and ASE 13C-d3 were m/z 286.1 → 166.0 and m/z 290.0 → 166.1, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were 0.0025 ng/mL and 0.050 ng/mL respectively in a linear concentration range of 0.050–20.0 ng/mL for ASE. The intra-batch and inter-batch precision (% CV) and mean relative recovery across quality control levels were ≤ 5.8% and 87.3%, respectively. Matrix effect, evaluated as IS-normalized matrix factor, ranged from 1.03 to 1.05. The stability of ASE under different storage conditions was ascertained in presence of the metabolites. The developed method is much simpler, matrix free, rapid and economical compared to the existing methods. The method was successfully used for a bioequivalence study of asenapine in healthy Indian subjects for the first time. Keywords: Asenapine, Asenapine 13C-d3, Metabolites, LC-MS/MS, Bioequivalence study, Human plasm

Highly sensitive LC–MS/MS method to estimate doxepin and its metabolite nordoxepin in human plasma for a bioequivalence study

A selective, sensitive and rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay has been developed for the simultaneous determination of doxepin (Dox) and its pharmacologically active metabolite, nordoxepin (NDox) in human plasma. The analytes and their internal standards (IS) were extracted from 500 µL of human plasma by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved on Hypurity C8 column (100 mm × 4.6 mm, 5 µm) using a mixture of acetonitrile-methanol (95:5, v/v) and 2.0 mM ammonium formate in 93:7 (v/v) ratio. Detection was accomplished by tandem mass spectrometry in the positive ionization and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions studied for Dox, NDox, and their corresponding ISs, propranolol and desipramine, were m/z 280.1→107.0, 266.0 →107.0, 260.1→116.1 and 267.1→72.1, respectively. A linear dynamic range of 15.0–3900 pg/mL for Dox and 5.00–1300 pg/mL for NDox was established with mean correlation coefficient (r2) of 0.9991 and 0.9993, respectively. The extraction recovery ranged from 86.6%–90.4% and 88.0%–99.1% for Dox and NDox, respectively. The intra-batch and inter-batch precision (% CV) across quality control levels was ≤ 8.3% for both the analytes. Stability evaluated under different storage conditions showed no evidence of degradation and the % change in stability samples compared to nominal concentration ranged from 4.7% to 12.3%. The method was successfully applied to a bioequivalence study of 6 mg doxepin hydrochloride orally disintegrating tablet in 41 healthy Indian subjects under fasting and fed conditions. Keywords: Doxepin, Nordoxepin, LC–MS/MS, Liquid-liquid extraction, Human plasma, Bioequivalence stud

Determination of cilostazol and its active metabolite 3,4-dehydro cilostazol from small plasma volume by UPLCâMS/MS

A simple, rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLCâMS/MS) method has been developed for the simultaneous determination of cilostazol and its pharmacologically active metabolite 3,4-dehydro cilostazol in human plasma using deuterated analogs as internal standards (ISs). Plasma samples were prepared using solid phase extraction and chromatographic separation was performed on UPLC BEH C18 (50 mmÃ2.1 mm, 1.7 µm) column. The method was established over a concentration range of 0.5â1000 ng/mL for cilostazol and 0.5â500 ng/mL for 3,4-dehydro cilostazol. Intra- and inter-batch precision (% CV) and accuracy for the analytes were found within 0.93â1.88 and 98.8â101.7% for cilostazol and 0.91â2.79 and 98.0â102.7% for the metabolite respectively. The assay recovery was within 95â97% for both the analytes and internal standards. The method was successfully applied to support a bioequivalence study of 100 mg cilostazol in 30 healthy subjects. Keywords: Cilostazol, 3,4-dehydro cilostazol, UPLCâMS/MS, Sensitive, High throughpu

Determination of lercanidipine in human plasma by an improved UPLC–MS/MS method for a bioequivalence study

An improved and reliable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the determination of lercanidipine in human plasma. Plasma samples with lercanidipine-d3 as an internal standard (IS) were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 100 µL of human plasma. Chromatographic analysis was performed on UPLC BEH C18 (50 mm×2.1 mm, 1.7 µm) column under isocratic conditions. Linear calibration curves were obtained over a wide dynamic concentration range of 0.010–20.0 ng/mL. Matrix effect was assessed by post-column infusion, post-extraction spiking and standard-line slope methods. The mean extraction recovery was >94% for the analyte and IS. Inter-batch and intra-batch precision (% CV) across five quality controls was <5.8%. Bioequivalence study was performed with 36 healthy subjects after oral administration of 10 mg of lercanidipine and the assay reproducibility was evaluated by reanalysis of 133 incurred samples

Complete Genome Sequence of Cluster J Mycobacteriophage Superphikiman

Mycobacteriophage Superphikiman is a cluster J bacteriophage which was isolated from soil collected in Philadelphia, PA. Superphikiman has a 109,799-bp genome with 239 predicted genes, including 2 tRNA genes