Sanjeev Giri

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues

A highly sensitive and specific LC-MS/MS method was developed for simultaneous estimation of acetyl co-enzyme A (ACoA) and malonyl co-enzyme A (MCoA) in surrogate matrix using n-propionyl co-enzyme A as an internal standard (IS). LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. Simple acidification followed by dilution using an assay buffer process was used to extract ACoA, MCoA and IS from surrogate matrix and tissue samples. The total run time was 3 min and the elution of both analytes (ACoA, MCoA) and IS occurred at 1.28 min; this was achieved with a mobile phase consisting of 5 mM ammonium formate (pH 7.5)-acetonitrile (30:70, v/v) delivered at a flow rate of 1 mL/min on a monolithic RP-18e column. A linear response function was established for the range of concentrations 1.09-2187 and 1.09-2193 ng/mL for ACoA and MCoA, respectively. The intra- and inter-day precision values for ACoA and MCoA met the acceptance as per FDA guidelines. ACoA and MCoA were stable in a battery of stability studies viz. bench-top, auto-sampler and long-term. The developed assay was used to quantitate ACoA and MCoA levels in various tissues of rat.

Development and validation of a highly sensitive LC-MS/MS-ESI method for the determination of nobiletin in rat plasma: application to a pharmacokinetic study.

A highly sensitive, rapid assay method has been developed and validated for the estimation of nobiletin in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves extraction of nobiletin and citalopram (internal standard, IS) from rat plasma with liquid-liquid extraction. Chromatographic separation was achieved using an isocratic mobile phase (0.2% formic acid-acetonitrile, 20:80, v/v) at a flow rate of 0.6 mL/min on an Atlantis dC₁₈ column (maintained at 40 ± 1 °C) with a total run time of 2.0 min. The MS/MS ion transitions monitored were 403.2 → 373.0 for nobiletin and 325.2 → 109.0 for IS. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range extended from 0.05 to 51.98 ng/mL. The intra- and inter-day precisions were in the range of 1.96-14.3 and 6.21-12.1, respectively.

LC-MS/MS-ESI method for simultaneous quantitation of metformin and repaglinidie in rat plasma and its application to pharmacokinetic study in rats

A highly sensitive and specific LC-MS/MS-ESI method has been developed for simultaneous quantification of metformin (MFN) and repaglinide (RGN) in rat plasma (50 μL) using phenacetin as an internal standard (IS). Simple protein precipitation was used to extract MFN and RGN from rat plasma. The chromatographic resolution of MFN, RGN and IS was achieved with a mobile phase consisting of 0.2% formic acid in water-acetonitrile (1:1, v/v) with a time program flow gradient on a Chromolith RP-18e column. The total chromatographic run time was 3.5 min and the elution of MFN, RGN and IS occurred at 1.64, 2.21 and 2.15 min, respectively. A linear response function was established for the range of concentrations 0.855-394 and 0.021-21.7 ng/mL for MFN and RGN, respectively. The intra- and inter-day precision values for MFN and RGN met the acceptance as per FDA guidelines. MFN and RGN were stable in battery of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in rats.

A highly sensitive LC-MS/MS method for the determination of S-citalopram in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive, rapid assay method has been developed and validated for the estimation of S-citalopram (S-CPM) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves a simple liquid-liquid extraction of S-CPM and phenacetin (internal standard, IS) from rat plasma with t-butyl methyl ether. Chromatographic separation was operated with 0.2% formic acid:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Symmetry Shield RP(18) column with a total run time of 3.0 min. The MS/MS ion transitions monitored were 325.26 → 109.10 for S-CPM and 180.10 → 110.10 for IS. Method validation and pre-clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.5 ng/mL and the linearity was observed from 0.5 to 5000 ng/mL. The intra- and inter-day precisions were in the range of 1.14-5.56 and 0.25-12.3%, respectively. This novel method has been applied to a pharmacokinetic study and to estimate brain-to-plasma ratio of S-CPM in rats.

A highly sensitive LC‐MS/MS method for the determination of S‐raclopride in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and rapid assay method has been developed and validated for the estimation of S-(-)-raclopride (S-RCP) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive ion mode. The assay procedure involves a simple liquid-liquid extraction technique for extraction of S-RCP and phenacetin (internal standard, IS) from rat plasma. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (80:20, v/v) at a flow rate of 0.30 mL/min on a Phenomenex Prodigy C(18) column with a total run time of 4.5 min. The MS/MS ion transitions monitored were 347.2 → 112.1 for S-RCP and 180.1 → 110.1 for IS. Method validation and pre-clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range was extended from 0.05 to 152 ng/mL in rat plasma. The intra-day and inter-day precisions were 0.23-10.5 and 3.74-7.29%, respectively. This novel method was applied to a pharmacokinetic study of S-RCP in rats.

Highly sensitive LC-MS/MS-ESI method for simultaneous quantitation of albendazole and ricobendazole in rat plasma and its application to a rat pharmacokinetic study

A highly sensitive and specific LC-MS/MS-ESI method was developed for simultaneous quantification of albenadazole (ABZ) and ricobendazole (RBZ) in rat plasma (50 μL) using phenacetin as an internal standard (IS). Simple protein precipitation was used to extract ABZ and RBZ from rat plasma. The chromatographic resolution of ABZ, RBZ and IS was achieved with a mobile phase consisting of 5 m m ammonium acetate (pH 6) and acetonitrile (20:80, v/v) at a flow rate of 1 mL/min on a Chromolith RP-18e column. The total chromatographic run time was 3.5 min and the elution of ABZ, RBZ and IS occurred at 1.66, 1.50 and 1.59 min, respectively. A linear response function was established for the ranges of concentrations 2.01-2007 and 6.02-6020 ng/mL for ABZ and RBZ, respectively. The intra- and inter-day precision values for ABZ and RBZ met the acceptance as per FDA guidelines. ABZ and RBZ were stable in battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in rats.

LC–MS/MS-ESI method for simultaneous quantitation of three endocannabinoids and its application to rat pharmacokinetic studies

An LC-MS/MS-ESI method has been validated for simultaneous estimation of the three endocannabinoids; N-arachidonoylethanolamine (AEA), N-oleoylethanolamine (OEA) and palmitoylethanolamide (PEA), in surrogate matrix using AEA-d (4) as an internal standard with highest sensitivity over the existing methods. Simple precipitation was used to extract analytes and these were subsequently analyzed on a monolithic column. Linear response function was established over the concentration range 12.3 to 1225 pg/ml for AEA (r > 0.994); 0.70 to 641 ng/ml for OEA (r > 0.999) and 0.54 to 321 ng/ml (r > 0.998) for PEA. The intra- and inter-day precision values met the acceptance to criteria as per US FDA guidelines. Analytes were found to be stable in the battery of stability studies. The method was applied to quantify endogenous levels of analytes in rat plasma.

Development and validation of a highly sensitive LC-MS/MS-ESI method for the determination of bicalutamide in mouse plasma: application to a pharmacokinetic study: Bicalutamide quantitation by LC-MS/MS in mouse plasma

A highly sensitive, rapid assay method has been developed and validated for the estimation of bicalutamide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative-ion mode. The assay procedure involves extraction of bicalutamide and tolbutamide (internal standard, IS) from mouse plasma with a simple protein precipitation method. Chromatographic separation was achieved using an isocratic mobile phase (0.2% formic acid:acetonitrile, 35:65, v/v) at a flow rate of 0.5 mL/min on an Atlantis dC₁₈ column (maintained at 40 ± 1°C) with a total run time of 3.0 min. The MS/MS ion transitions monitored were m/z 428.9 → 254.7 for bicalutamide and m/z 269.0 → 169.6 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 1.04 ng/mL and the linearity range extended from 1.04 to 1877 ng/mL. The intra- and inter-day precisions were in the ranges of 0.49-4.68 and 2.62-4.15, respectively.

Abstract A36: CA-170, an oral small molecule PD-L1 and VISTA immune checkpoint antagonist, promotes T cell immune activation and inhibits tumor growth in pre-clinical models of cancer

The clinical success of antibody-mediated immune checkpoint blockade therapies has transformed the cancer therapy paradigm by demonstrating that durable antitumor immune responses and long-term remissions may be achieved in a subset of patients across a diverse range of cancers. However, the majority of patients fail to respond to antibody therapies targeting single immune checkpoint pathways and antibodies exhibit a long in vivo half-life (>15-20 days with >70% target occupancy for months) which may contribute to the emergence of immune-related adverse events. Additionally, antibody therapies must be administered by intravenous infusion in a hospital or clinic which places an additional burden on patients who may have mobility challenges. Thus, there is a significant opportunity for a novel immune checkpoint therapy that can address the shortcomings associated with the current antibody therapies. CA-170 is a small molecule, orally bioavailable antagonist of the PD-L1, PD-L2 and VISTA/PD-1H immune checkpoint pathways which is currently undergoing Phase I clinical testing. In preclinical safety studies conducted in rodents and non-human primates, orally administered CA-170 shows no signs of toxicity when dosed up to 1000 mg/kg for 28 consecutive days. CA-170 exhibits an oral bioavailability of approximately 40% and Citation Format: Adam S. Lazorchak, Troy Patterson, Yueyun Ding, Pottayil G. Sasikumar, Naremaddepalli S. Sudarshan, Nagaraj M. Gowda, Raghuveer K. Ramachandra, Dodheri S. Samiulla, Sanjeev Giri, Rajesh Eswarappa, Murali Ramachandra, David Tuck, Timothy Wyant. CA-170, an oral small molecule PD-L1 and VISTA immune checkpoint antagonist, promotes T cell immune activation and inhibits tumor growth in pre-clinical models of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A36.

Highly sensitive method for the determination of adenosine by LC-MS/MS-ESI: method validation and scope of application to a pharmacokinetic/pharmacodynamic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of adenosine in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electro-spray ionization in the positive-ion mode. The assay procedure involves extraction of adenosine and phenacetin (internal standard, IS) from rat plasma with a simple protein precipitation extraction process. The method was validated using rat plasma with extinguished adenosine endogenous levels. Chromatographic separation was achieved using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.50 mL/min on an Atlantis dC(18) column with a total run time of 4.0 min. The MS/MS ion transitions monitored were 268 → 136 for adenosine and 180 → 110 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.48 ng/mL and the linearity range extended from 0.48 to 1210 ng/mL. The intra- and inter-day precisions were in the ranges 2.32-12.7 and 4.01-9.40%, respectively.