Urinary Metabolite Diagnostic and Prognostic Liquid Biopsy Biomarkers of Lung Cancer in Non-smokers and Tobacco Smokers.

PURPOSE: Non-smokers account for 10-13% of all lung cancer cases in the United States. Etiology is attributed to multiple risk factors including exposure to secondhand smoking, asbestos, environmental pollution, and radon, but these exposures are not within the current eligibility criteria for early lung cancer screening by low-dose computed tomography (LDCT). EXPERIMENTAL DESIGN: Urine samples were collected from two independent cohorts comprising 846 participants (exploratory cohort) and 505 participants (validation cohort). The cancer urinary biomarkers, creatine riboside (CR) and N-acetylneuraminic acid (NANA) were analyzed and quantified using liquid chromatography-mass spectrometry to determine if non-smoker cases can be distinguished from sex and age-matched controls in comparison to tobacco smoker cases and controls, potentially leading to more precise eligibility criteria for LDCT screening. RESULTS: Urinary levels of CR and NANA were significantly higher and comparable in non-smokers and tobacco smoker cases as compared to population controls in both cohorts. Receiver Operating Characteristics (ROC) analysis for combined CR and NANA levels in non-smokers of the exploratory cohort resulted in better predictive performance with the area under the curve (AUC) of 0.94, whereas the validation cohort non-smokers had an AUC of 0.80. Kaplan-Meier survival curves showed that high levels of CR and NANA were associated with increased cancer-specific death in non-smokers as well as tobacco smoker cases in both cohorts. CONCLUSIONS: Measuring CR and NANA in urine liquid biopsies could identify non-smokers at high risk for lung cancer as candidates for LDCT screening and warrant prospective studies of these biomarkers.

Intestinal peroxisome proliferator-activated receptor α-fatty acid-binding protein 1 axis modulates nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor α (PPARα) regulates fatty acid transport and catabolism in liver. However, the role of intestinal PPARα in lipid homeostasis is largely unknown. Here, intestinal PPARα was examined for its modulation of obesity and NASH. APPROACH AND RESULTS: Intestinal PPARα was activated and fatty acid-binding protein 1 (FABP1) up-regulated in humans with obesity and high-fat diet (HFD)-fed mice as revealed by using human intestine specimens or HFD/high-fat, high-cholesterol, and high-fructose diet (HFCFD)-fed C57BL/6N mice and PPARA -humanized, peroxisome proliferator response element-luciferase mice. Intestine-specific Ppara or Fabp1 disruption in mice fed a HFD or HFCFD decreased obesity-associated metabolic disorders and NASH. Molecular analyses by luciferase reporter assays and chromatin immunoprecipitation assays in combination with fatty acid uptake assays in primary intestinal organoids revealed that intestinal PPARα induced the expression of FABP1 that in turn mediated the effects of intestinal PPARα in modulating fatty acid uptake. The PPARα antagonist GW6471 improved obesity and NASH, dependent on intestinal PPARα or FABP1. Double-knockout ( Ppara/Fabp1ΔIE ) mice demonstrated that intestinal Ppara disruption failed to further decrease obesity and NASH in the absence of intestinal FABP1. Translationally, GW6471 reduced human PPARA-driven intestinal fatty acid uptake and improved obesity-related metabolic dysfunctions in PPARA -humanized, but not Ppara -null, mice. CONCLUSIONS: Intestinal PPARα signaling promotes NASH progression through regulating dietary fatty acid uptake through modulation of FABP1, which provides a compelling therapeutic target for NASH treatment.

Mutant Idh2 Cooperates with a NUP98-HOXD13 Fusion to Induce Early Immature Thymocyte Precursor ALL.

Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequently observed in a wide variety of hematologic malignancies, including myeloid and T-cell leukemias. In this study, we generated Idh2R140Q transgenic mice to examine the role of the Idh2R140Q mutation in leukemia. No leukemia developed in Idh2R140Q transgenic mice, suggesting a need for additional genetic events for leukemia development. Because myeloid cells from NUP98-HOXD13 fusion (NHD13) transgenic mice frequently acquire somatic Idh mutations when they transform to acute myeloid leukemia, we generated Idh2R140Q/NHD13 double transgenic mice. Idh2R140Q/NHD13 transgenic mice developed an immature T-cell leukemia with an immunophenotype similar to double-negative 1 (DN1) or DN2 thymocytes. Idh2R140Q/NHD13 leukemic cells were enriched for an early thymic precursor transcriptional signature, and the gene expression profile for Idh2R140Q/NHD13 DN1/DN2 T-ALL closely matched that of human early/immature T-cell precursor (EITP) acute lymphoblastic leukemia (ALL). Moreover, recurrent mutations found in patients with EITP ALL, including KRAS, PTPN11, JAK3, SH2B3, and EZH2 were also found in Idh2R140Q/NHD13 DN1/DN2 T-ALL. In vitro treatment of Idh2R140Q/NHD13 thymocytes with enasidenib, a selective inhibitor of mutant IDH2, led to a marked decrease in leukemic cell proliferation. These findings demonstrate that Idh2R140Q/NHD13 mice can serve as a useful in vivo model for the study of early/immature thymocyte precursor acute lymphoblastic leukemia development and therapy. SIGNIFICANCE: T-cell leukemia induced in Idh2R140Q/NUP98-HOXD13 mice is immunophenotypically, transcriptionally, and genetically similar to human EITP ALL, providing a model for studying disease development and treatment.

Improved detection and precise relative quantification of the urinary cancer metabolite biomarkers - Creatine riboside, creatinine riboside, creatine and creatinine by UPLC-ESI-MS/MS: Application to the NCI-Maryland cohort population controls and lung cancer cases.

Creatine riboside (CR) is a novel metabolite of cancer metabolism. It is a urinary diagnostic biomarker of lung and liver cancer risk and prognosis. The level of CR is highly positive correlated in tumor and urine indicating that it is derived from human lung and liver cancers. A precise and sensitive ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for simultaneous quantification of the noninvasive biomarker CR, along with creatinine riboside (CNR), and their precursors creatine and creatinine, utilizing the labeled internal standard creatine riboside-13C,15N2 (CR-13C,15N2). Chromatography was carried out on a hydrophilic interaction chromatography column under a gradient mobile phase condition. MRM transitions were monitored for CR (264.1 > 132.1, m/z), CNR (246.1 > 113.9, m/z), creatine (132.0 > 72.0, m/z), creatinine (114.0 > 85.8, m/z) and CR-13C,15N2 (267.1 > 134.9, m/z) with a 11.0 min run time in the positive mode ionization. The calibration plot of the method was linear over the concentration range of 4.50-10,000 nM. Method validation was performed according to regulatory guidelines established for sensitivity, selectivity, calibration curve, stability at different storage conditions, reinjection reproducibility, ruggedness with acceptable accuracy, and precision. This assay was applied for the quantification of CR along with CNR, creatine and creatinine in a subset of urine and serum samples from the National Cancer Institute - Maryland (NCI-MD) cohort population controls and lung cancer cases. It can be standardized and used in multiple laboratories for cancer diagnosis and determining the efficacy of cancer therapy and monitoring cancer recurrence.

A multiparametric organ toxicity predictor for drug discovery.

The assessment of major organ toxicities through in silico predictive models plays a crucial role in drug discovery. Computational tools can predict chemical toxicities using the knowledge gained from experimental studies which drastically reduces the attrition rate of compounds during drug discovery and developmental stages. The purpose of in silico predictions for drug leads and anticipating toxicological endpoints of absorption, distribution, metabolism, excretion and toxicity, clinical adverse impacts and metabolism of pharmaceutically active substances has gained widespread acceptance in academia and pharmaceutical industries. With unrestricted accessibility to powerful biomarkers, researchers have an opportunity to contemplate the most accurate predictive scores to evaluate drug's adverse impact on various organs.A multiparametric model involving physico-chemical properties, quantitative structure-activity relationship predictions and docking score was found to be a more reliable predictor for estimating chemical toxicities with potential to reflect atomic-level insights. These in silico models provide informed decisions to carry out in vitro and in vivo studies and subsequently confirms the molecules clues deciphering the cytotoxicity, pharmacokinetics, and pharmacodynamics and organ toxicity properties of compounds. Even though the drugs withdrawn by USFDA at later phases of drug discovery which should have passed all the state-of-the-art experimental approaches and currently acceptable toxicity filters, there is a dire need to interconnect all these molecular key properties to enhance our knowledge and guide in the identification of leads to drug optimization phases. Current computational tools can predict ADMET and organ toxicities based on pharmacophore fingerprint, toxicophores and advanced machine-learning techniques.

Withaferin A Improves Nonalcoholic Steatohepatitis in Mice.

Nonalcoholic steatohepatitis (NASH) is the progressive stage of nonalcoholic fatty liver disease that highly increases the risk of cirrhosis and liver cancer, and there are few therapeutic options available in the clinic. Withaferin A (WA), extracted from the ayurvedic medicine Withania somnifera, has a wide range of pharmacological activities; however, little is known about its effects on NASH. To explore the role of WA in treating NASH, two well defined NASH models were used, the methionine-choline-deficient diet and the 40 kcal% high-fat diet (HFD). In both NASH models, WA treatment or control vehicle was administered to evaluate its hepatoprotective effects. As assessed by biochemical and histologic analyses, WA prevented and therapeutically improved liver injury in both models, as revealed by lower serum aminotransaminases, hepatic steatosis, liver inflammation, and fibrosis. In the HFD-induced NASH model, both elevated serum ceramides and increased hepatic oxidative stress were decreased in the WA-treated group compared with the control vehicle-treated group. To further explore whether WA has an anti-NASH effect independent of its known action in leptin signaling associated with obesity, leptin signaling-deficient ob/ob mice maintained on an HFD were used to induce NASH. WA therapeutically reduced NASH in HFD-treated leptin-deficient ob/ob mice, thus demonstrating a leptin-independent hepatoprotective effect. This study revealed that WA treatment could be an option for NASH treatment.

Extrahepatic PPARα modulates fatty acid oxidation and attenuates fasting-induced hepatosteatosis in mice.

PPARα (PPARA), expressed in most oxidative tissues, is a major regulator of lipid homeostasis; hepatic PPARA plays a critical role during the adaptive fasting response by promoting FA oxidation (FAO). To clarify whether extrahepatic PPARA activity can protect against lipid overload when hepatic PPARA is impaired, lipid accumulation was compared in WT (Ppara+/+), total body Ppara-null (Ppara-/-), and hepatocyte-specific Ppara-null (PparaΔHep) mice that were fasted for 24 h. Histologic staining indicated reduced lipid accumulation in PparaΔHep versus Ppara-/- mice, and biochemical analyses revealed diminished medium- and long-chain FA accumulation in PparaΔHep mouse livers. Hepatic PPARA target genes were suppressed in both mouse models. Serum FFAs increased in all genotypes after fasting but were highest in Ppara-/- mice. In PparaΔHep mice, FAO genes were increased in brown adipose tissue, heart, and muscle, and total lipase activity was elevated in the muscle and heart, suggesting increased lipid utilization. Thus, extrahepatic PPARA activity reduces systemic lipid load when hepatic lipid metabolism is impaired by elevating FAO and lipase activity in other tissues and, as a result, protects against fasting-induced hepatosteatosis. This has important clinical implications in disease states with impaired hepatic PPARA function, such as nonalcoholic steatohepatitis and nonalcoholic fatty liver disease.

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 Student's 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.

Ultra-performance liquid chromatography-tandem mass spectrometry assay for determination of plasma nomegestrol acetate and estradiol in healthy postmenopausal women.

A highly sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry method is described for the simultaneous determination of nomegestrol acetate (NOMAC), a highly selective progestogen, and estradiol (E2), a natural estrogen in human plasma. NOMAC was obtained from plasma by solid-phase extraction, while E2 was first separated by liquid-liquid extraction with methyl tert-butyl ether followed by derivatization with dansyl chloride. Deuterated internal standards, NOMAC-d5 and E2-d4 were used for better control of extraction conditions and ionization efficiency. The assay recovery of the analytes was within 90-99%. The analytes were separated on UPLC BEH C18 (50 × 2.1 mm, 1.7 µm) column using a mobile phase comprising of acetonitrile and 3.0 mm ammonium trifluoroacetate in water (80:20, v/v) with a resolution factor (Rs ) of 3.21. The calibration curves were linear from 0.01 to 10.0 ng/mL for NOMAC and from 1.00 to 1000 pg/mL for E2, respectively. The intra- and inter-batch precision was ≤5.8% and the accuracy of quality control samples ranged from 96.7 to 103.4% for both analytes. The practical applicability of the method is demonstrated by analyzing samples from 18 healthy postmenopausal women after oral administration of 2.5 mg nomegestrol acetate and 1.5 mg estradiol film-coated tablets under fasting.

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.

Simultaneous determination of etonogestrel and ethinyl estradiol in human plasma by UPLC-MS/MS and its pharmacokinetic study.

A selective, sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of etonogestrel (ENG) and ethinyl estradiol (EE) in human plasma. The analytes and their deuterated internal standards, ENG-d7 and EE-d4, were extracted from plasma samples by solid-phase extraction on HyperSep™ Retain PEP cartridges. The chromatographic analysis was performed on an Acquity UPLC HSS Cyano column, 100 Å (50 × 2.1 mm, 1.8 µm), column using gradient mobile phase, acetonitrile and 2.0 mm ammonium trifluoroacetate at 0-1.7 min (65:35, v/v) and 1.8-2.7 min (95:5, v/v) with 0.250 mL/min flow rate. Analytes and IS protonated precursor → product ion transitions (ENG, m/z 325.2 → 257.2; EE, m/z 530.2 → 171.2; ENG-d7, m/z 332.2 → 263.2; EE-d4, m/z 534.2 → 171.2) were monitored on a Triple Quadrupole Mass spectrometer (TQMS), operating in multiple reaction monitoring and positive ionization mode. The calibration curves were established at 10.00-2500 pg/mL for ENG and 1.500-150.0 pg/mL for EE with a correlation coefficient (r2 ) ≥0.9996 for both. The validated method was successfully applied to support a bioequivalence study of 0.15 mg ENG and EE 0.03 mg tablet formulation, administered in 24 healthy Indian females. Method reliability was assessed by reanalysis of 94 incurred study samples.

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 ß-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 ß-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 17ß-carboxylic acid in human plasma by UPLC-MS/MS at sub pg/mL level.

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 17ß-CA) in human plasma. The analytes and their deuterated internal standards, FP-d3 and FP 17ß-CA-d3 were extracted from 500µL plasma samples by solid phase extraction on Oasis MAX cartridges. The chromatographic analysis was performed on ACQUITY UPLC BEH C18 (50mm×2.1mm, 1.7µm) column using methanol-acetonitrile (50:50, v/v) and 2.0mM 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, FP17ß-CA: m/z 453.3→293.2, FP-d3: m/z 504.2→293.2, FP 17ß-CA-d3: m/z 456.3→293.2) were monitored on FP 17ß-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 0.5-100pg/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 17ß-CA were 84.2% and 93.5% respectively. The validated method was successfully applied to support a bioequivalence study of 200µg FP, administered using nasal spray formulation in 18 healthy Indian subjects. Reproducibility of the method was assessed by reanalysis of 98 incurred study samples.

A novel, sensitive and selective method of UPLC/MS-MS for rapid simultaneous determination of midodrine and its active metabolite desglymidodrine in human plasma: Application to support bioequivalence study in healthy human volunteers.

A specific, rapid, sensitive and selective ultra-performance liquid chromatography - tandem mass spectrometry has been developed for the simultaneous determination of midodrine and desglymidodrine in human plasma. The analytes and its deuterated analogs were quantitatively extracted from 100µL of human plasma by solid phase extraction technique. Separation of analytes was achieved on the Waters Acquity UPLC BEH C18 (50×2.1mm, 1.7µm) column using acetonitrile-4.0mM ammonium formate, pH 2.5(90:10, v/v) as mobile phase. The protonated analytes were quantified by selected reaction monitoring in the positive ionization mode by triple quadrupole mass spectrometer. The calibration plots were linear over the concentration range of 0.050-50.0ng/mL. The intra-batch and inter-batch precision (%CV) across quality control levels was <4.0 and the% mean relative recovery was ≥96%. Various other parameters like stability in different conditions; matrix effect and reproducibility of the method were performed in accordance with the guidelines specified by the USFDA for bioanalytical method development and validation. The developed method was successfully administered to the pharmacokinetics study of 5 mg midodrine tablet in 12 healthy subjects. Reproducibility of assay was proved by reanalysis of 48 incurred samples.

Simultaneous quantification of atenolol and chlorthalidone in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry.

A simple, sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of atenolol, a ß-adrenergic receptor-blocker and chlorthalidone, a monosulfonamyl diuretic in human plasma, using atenolol-d7 and chlorthalidone-d4 as the internal standards (ISs). Following solid-phase extraction on Phenomenex Strata-X cartridges using 100 µL human plasma sample, the analytes and ISs were separated on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) column using a mobile phase consisting of 0.1% formic acid-acetonitrile (25:75, v/v). A tandem mass spectrometer equipped with electrospray ionization was used as a detector in the positive ionization mode for both analytes. The linear concentration range was established as 0.50-500 ng/mL for atenolol and 0.25-150 ng/mL for chlorthalidone. Extraction recoveries were within 95-103% and ion suppression/enhancement, expressed as IS-normalized matrix factors, ranged from 0.95 to 1.06 for both the analytes. Intra-batch and inter-batch precision (CV) and accuracy values were 2.37-5.91 and 96.1-103.2%, respectively. Stability of analytes in plasma was evaluated under different conditions, such as bench-top, freeze-thaw, dry and wet extract and long-term. The developed method was superior to the existing methods for the simultaneous determination of atenolol and chlorthalidone in human plasma with respect to the sensitivity, chromatographic analysis time and plasma volume for processing. Further, it was successfully applied to support a bioequivalence study of 50 mg atenolol + 12.5 mg chlorthalidone in 28 healthy Indian subjects.

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.

Application of a UPLC-MS/MS method for the analysis of alosetron in human plasma to support a bioequivalence study in healthy males and females.

A simple, rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the determination of alosetron (ALO) in human plasma. The assay method involved solid-phase extraction of ALO and ALO 13C-d3 as internal standard (IS) on a LichroSep DVB-HL (30 mg, 1 cm(3) ) cartridge. The chromatography was performed on an Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile and 2.0 mm ammonium formate, pH 3.0 adjusted with 0.1% formic acid (80:20, v/v) as the mobile phase in an isocratic mode. For quantitative analysis, the multiple reaction monitoring transitions studied were m/z 295.1/201.0 for ALO and m/z 299.1/205.1 for IS in the positive ionization mode. The method was validated over a concentration range of 0.01-10.0 ng/mL for ALO. Post-column infusion experiment showed no positive or negative peaks in the elution range of the analyte and IS after injection of extracted blank plasma. The extent of ion-suppression/enhancement, expressed as IS-normalized matrix factor, varied from 0.96 to 1.04. The assay recovery was within 97-103% for ALO and IS. The method was successfully applied to support a bioequivalence study of 1.0 mg alosetron tablets in 28 healthy Indian male and female subjects.

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

A simple, rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/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 mm×2.1 mm, 1.7 µm) column. The method was established over a concentration range of 0.5-1000 ng/mL for cilostazol and 0.5-500 ng/mL for 3,4-dehydro cilostazol. Intra- and inter-batch precision (% CV) and accuracy for the analytes were found within 0.93-1.88 and 98.8-101.7% for cilostazol and 0.91-2.79 and 98.0-102.7% for the metabolite respectively. The assay recovery was within 95-97% 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.

Parallel achiral-chiral determination of oxybutynin, N-desethyl oxybutynin and their enantiomers in human plasma by LC-MS/MS to support a bioequivalence trial.

A parallel achiral and chiral determination of oxybutynin, its pharmacologically active metabolite N-desethyl oxybutynin and their enantiomers in human plasma is described using LC-MS/MS. Both the methods were developed and validated using deuterated analogues as internal standards. Achiral analysis of racemic oxybutynin and N-desethyl oxybutynin was carried out on Phenomenex Gemini C18 (150mm×4.6mm, 5µm) column under isocratic conditions using acetonitrile-5.0mM ammonium acetate, pH 4.0 (90:10, v/v) as the mobile phase. Separation of (S)- and (R)-enantiomers of the analytes was performed on Phenomenex Lux Amylose-2 (150mm×4.6mm, 3µm) chiral column using a mixture of solvent A [acetonitrile:10mM ammonium bicarbonate, 80:20 (v/v)] and solvent B [2-propanol:methanol, 50:50 (v/v)] in 20:80 (v/v) ratio as the mobile phase. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate-diethyl ether-n-hexane solvent mixture. A linear range was established from 0.025 to 10.0ng/mL and 0.25 to 100ng/mL for the enantiomers of oxybutynin and N-desethyl oxybutynin respectively. The extraction recovery varied from 96.0 to 105.1%, while the IS-normalized matrix factors ranged from 0.96 to 1.07 for all the enantiomers. The validated method was applied for a pilot bioequivalence study with 5mg oxybutynin tablet formulation in 8 healthy subjects. The pharmacokinetic profiles showed that the plasma concentration of (R)-oxybutynin was lower than that of (S)-oxybutynin, while a reverse trend was observed for the enantiomers of N-desethyl oxybutynin. The reproducibility in the measurement of study data was demonstrated by reanalysis of 20 incurred samples.

Development of a sensitive and rapid method for quantitation of (S)-(-)- and (R)-(+)-metoprolol in human plasma by chiral LC-ESI-MS/MS.

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250 mm×4.6 mm, 5 µm) column. Solid phase extraction of (S)-(-)- and (R)-(+)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 µL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1% (v/v) diethyl amine in acetonitrile (50:50, v/v) as the mobile phase within 7.0 min. The precursor→product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500-500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.