Daxesh P. Patel

Determination of (S)-(+)- and (R)-(-)-ibuprofen enantiomers in human plasma after chiral precolumn derivatization by reversed-phase LC–ESI-MS/MS

BACKGROUND A selective, sensitive and high-throughput LC-ESI-MS/MS method has been developed and validated for the chromatographic separation and quantitation of (S)-(+)-ibuprofen and (R)-(-)-ibuprofen after derivatization with (S)-(-)-1-(1-napthyl)ethylamine using 1-hydroxybenzotriazole as the activator of the carboxylic acid group and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as the coupling reagent in human plasma. RESULTS Both the analytes were chromatographically separated with a resolution factor of 1.27 on a Kinetex PFP (50 × 4.6 mm, 2.6 µm) analytical column. The method was validated over the concentration range of 0.10-32.0 µg/ml for both the enantiomers. The magnitude of matrix effect was assessed by post-column analyte infusion and also by precision (%CV) values for the calculated slopes of calibration curves. The mean extraction recovery was >91% for both the enantiomers. CONCLUSION The method was successfully applied to a bioequivalence study in 34 healthy human subjects. The assay reproducibility was confirmed by reanalysis of 130 subject samples.

Pharmacophore-based virtual screening of catechol-o-methyltransferase (COMT) inhibitors to combat Alzheimer’s disease

Alzheimer’s disease (AD) is one of the most significant neurodegenerative disorders and its symptoms mostly appear in aged people. Catechol-o-methyltransferase (COMT) is one of the known target enzymes responsible for AD. With the use of 23 known inhibitors of COMT, a query has been generated and validated by screening against the database of 1500 decoys to obtain the GH score and enrichment value. The crucial features of the known inhibitors were evaluated by the online ZINC Pharmer to identify new leads from a ZINC database. Five hundred hits were retrieved from ZINC Pharmer and by ADMET (absorption, distribution, metabolism, excretion, and toxicity) filtering by using FAF-Drug-3 and 36 molecules were considered for molecular docking. From the COMT inhibitors, opicapone, fenoldopam, and quercetin were selected, while ZINC63625100_413 ZINC39411941_412, ZINC63234426_254, ZINC63637968_451, and ZINC64019452_303 were chosen for the molecular dynamics simulation analysis having high binding affinity and structural recognition. This study identified the potential COMT inhibitors through pharmacophore-based inhibitor screening leading to a more complete understanding of molecular-level interactions.

Metabolic profiling by gas chromatography-mass spectrometry of energy metabolism in high-fat diet-fed obese mice

A novel, selective and sensitive single-ion monitoring (SIM) gas chromatography-mass spectrometry (GCMS) method was developed and validated for the determination of energy metabolites related to glycolysis, the tricarboxylic acid (TCA) cycle, glutaminolysis, and fatty acid β-oxidation. This assay used N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) containing 1% tert-butyldimethylchlorosilane (TBDMCS) as derivatizing reagent and was highly reproducible, sensitive, specific and robust. The assay was used to analyze liver tissue and serum from C57BL/6N obese mice fed a high-fat diet (HFD) and C57BL/6N mice fed normal chow for 8 weeks. HFD-fed mice serum displayed statistically significantly reduced concentrations of pyruvate, citrate, succinate, fumarate, and 2-oxoglutarate, with an elevated concentration of pantothenic acid. In liver tissue, HFD-fed mice exhibited depressed levels of glycolysis end-products pyruvate and lactate, glutamate, and the TCA cycle intermediates citrate, succinate, fumarate, malate, and oxaloacetate. Pantothenate levels were 3-fold elevated accompanied by a modest increased gene expression of Scl5a6 that encodes the pantothenate transporter SLC5A6. Since both glucose and fatty acids inhibit coenzyme A synthesis from pantothenate, it was concluded that these data were consistent with downregulated fatty acid β-oxidation, glutaminolysis, glycolysis, and TCA cycle activity, due to impaired anaplerosis. The novel SIM GCMS assay provided new insights into metabolic effects of HFD in mice.

Simultaneous analysis of glucocorticosteroid fluticasone propionate and its metabolite fluticasone propionate 17β-carboxylic acid in human plasma by UPLC–MS/MS at sub pg/mL level

&NA; A highly sensitive and rapid ultra performance liquid chromatography‐tandem mass spectrometry method has been developed for the simultaneous determination of fluticasone propionate (FP) and its major metabolite, fluticasone propionate‐17beta‐carboxylic acid (FP 17&bgr;‐CA) in human plasma. The analytes and their deuterated internal standards, FP‐d3 and FP 17&bgr;‐CA‐d3 were extracted from 500 &mgr;L plasma samples by solid phase extraction on Oasis MAX cartridges. The chromatographic analysis was performed on ACQUITY UPLC BEH C18 (50 mm × 2.1 mm, 1.7 &mgr;m) column using methanol‐acetonitrile (50:50, v/v) and 2.0 mM ammonium trifluroacetate (ATFA) (85:15, v/v) as the mobile phase. Following separation of the analytes, protonated precursor → product ion transitions (FP: m/z 501.1 → 293.2, FP17&bgr;‐CA: m/z 453.3 → 293.2, FP‐d3: m/z 504.2 → 293.2, FP 17&bgr;‐CA‐d3: m/z 456.3 → 293.2) were monitored on FP 17&bgr;‐CA a triple quadrupole mass spectrometer, operating in multiple reaction monitoring (MRM) and positive ionization mode. The calibration curves were established in the range of 0.5–100 pg/mL with a correlation coefficient (r2) ≥ 0.9992 for both the analytes. The intra‐batch and inter‐batch accuracy and precision varied from 95.5‐103.4% and 0.74‐5.06% across quality controls for both the analytes. The mean assay recoveries for FP and FP 17&bgr;‐CA were 84.2% and 93.5% respectively. The validated method was successfully applied to support a bioequivalence study of 200 &mgr;g FP, administered using nasal spray formulation in 18 healthy Indian subjects. Reproducibility of the method was assessed by reanalysis of 98 incurred study samples. Graphical abstract Figure. No caption available. HighlightsSub pg/mL level estimation of fluticasone propionate(FP) and FP 17&bgr;‐carboxylic acid.Simultaneous extraction of analytes from human plasma using SPE on MAX cartridge.Almost complete absence of FP 17&bgr;‐carboxylic acid in plasma samples of subjects.Bioequivalence study with 200 &mgr;g FP after nasal administration in healthy subjects.Method reproducibility is established by reanalysis of 98 incurred samples.

Metabolic alterations in triptolide-induced acute hepatotoxicity

Triptolide, a major active constitute of Tripterygium wilfordii Hook. F, is prescribed for the treatment of autoimmune diseases in China. One of its most severe adverse effects observed in the clinical use is hepatotoxicity, but the mechanism is still unknown. Therefore, the present study applied an LC/MS-based metabolomic analysis to characterize the metabolomic changes in serum and liver induced by triptolide in mice. Mice were administered triptolide by gavage to establish the acute liver injury model, and serum biochemical and liver histological analyses were applied to assess the degree of toxicity. Multivariate data analyses were performed to investigate the metabolic alterations. Potential metabolites were identified using variable importance in the projection values and Students t-test. A total of 30 metabolites were observed that were significantly changed by triptolide treatment and the abundance of 29 metabolites was correlated with the severity of toxicity. Pathway analysis indicated that the mechanism of triptolide-induced hepatotoxicity was related to alterations in multiple metabolic pathways, including glutathione metabolism, tricarboxylic acid cycle, purine metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism, pantothenate and CoA biosynthesis, pyrimidine metabolism and amino acid metabolism. The current study provides new mechanistic insights into the metabolic alterations that lead to triptolide-induced hepatotoxicity.

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.