A selective and accurate liquid chromatography-tandem mass spectrometry method for the quantitation of the novel 5-HT4 receptor partial agonist SUVN-D4010 (Usmarapride) in human plasma and urine.

Liquid chromatography and the tandem mass spectrometry method to quantitate SUVN-D4010 (Usmarapride) in human plasma and urine have been developed and fully validated in compliance with regulatory guidelines. The sample preparation technique is simple and rapid consisting of acetonitrile precipitation followed by dilution of supernatant with a compatible solvent. Chromatographic separation was achieved on an X-Bridge C18 (2.1×50 mm, 3.5 µm) column using 0.1% v/v ammonium hydroxide and acetonitrile as mobile phase components, delivered at a flow rate of 0.75 mL min-1. Electrospray Ionization technique in positive mode was used for mass spectrometric detection. Selective reaction monitoring (SRM) transitions of m/z 384.2 → 352.1 for SUVN-D4010 and m/z 388.2 → 356.1 for SUVN-D4010-d4 were used for quantitation. Calibration curves for SUVN-D4010 were linear across the concentration range of 0.3-300 ng mL-1 in human plasma and 5.00-5000 ng mL-1 in human urine. The method generated results with acceptable accuracy (± 9.0%), precision (%CV, ≤8.7), and mean extraction recovery (≥93.4%) with negligible matrix effect in both plasma and urine. SUVN-D4010 was found to be stable in human plasma and urine at the defined storage conditions. The validated method was successfully applied to quantitate SUVN-D4010 in human plasma and urine from a clinical first-in-human study conducted to evaluate its safety, tolerability, and pharmacokinetics in healthy adults.

Evaluation of monoamine oxidase A and B type enzyme occupancy using non-radiolabelled tracers in rat brain.

Monoamine oxidase (MAO) enzymes, type A and B metabolise the amine neurotransmitters of the body. Selective inhibition of either enzyme is an approach for treating neurodegenerative and stress-induced disorders, and inhibition of an enzyme is proportional to the binding of the MAO inhibitor. Conventionally, the binding of test compounds to enzymes is assessed by radiolabelled ligands in ex vivo and in vivo occupancy assays. Regulatory restrictions and turnaround time are the limitations of the methods that use radiolabelled ligands. But the use of non-radiolabelled tracers and sensitive mass spectrometry (LC-MS/MS) based assays accelerated the determination of target occupancy in pre-clinical species. A report on use of non-radiolabelled ligand in in vivo MAO occupancy assay is not available. The objectives of the present study were to optimise non-radiolabelled harmine and deprenyl as selective tracers in MAO-A and MAO-B occupancy assays and evaluate MAO occupancy of test compounds in rat brain. Tracer optimisation resulted in a detectable, stable, and low ratio (<3.0) of tracer concentrations between any two brain tissues. In occupancy assay, tracer was intravenously administered (10 µg/kg, harmine or 60 µg/kg, L-deprenyl) after the treatment with test compound (clorgyline or tranylcypromine or pargyline or phenelzine or thioperamide). Specific brain tissues were isolated at a defined interval and tracer concentrations were quantified using LC-MS/MS method. Pre-treatment with MAO inhibitors resulted in a decrease (maximum, 80-85%) in harmine or an increase (maximum, 85-300%) in L-deprenyl concentrations. But we considered the change in tracer concentration, relative to the vehicle and positive control groups to calculate MAO occupancy. The observed selectivity and ratio of occupancies (ED50) of test compound towards MAO-A and MAO-B are comparable with the results from in vitro radiolabelled ligand-based inhibition assay. The results demonstrated the application of these non-radiolabelled tracers as suitable pre-clinical tools to determine MAO occupancy.

LC-MS/MS method for quantification of 3,4-dihydroxyphenylglycol, a norepinephrine metabolite in plasma and brain regions.

Aim: To evaluate suitability of the LC-MS/MS method to quantify 3,4-dihydroxyphenylglycol (DHPG) that is used as a biomarker for monoamine oxidase (MAO) inhibition. Methods: DHPG was extracted using alumina basic cartridges and quantified on a triple quadrupole mass spectrometer using negative electrospray ionization, without the use of derivatization reagents. Results: Modulation of DHPG levels was observed following administration of selective and nonselective MAO inhibitors and results were in correlation with historical MAO inhibition potential of compounds. Conclusion: The proposed method is sensitive enough to measure plasma DHPG levels and DHPG can be used as a biomarker to assess MAO inhibition potential of new therapeutic agents.

Development and validation of sensitive LC-MS/MS method for the quantification of SUVN-502 and its metabolite and its application for first in human pharmacokinetic study.

A sensitive and rapid LC-MS/MS method was developed and validated for the quantification of SUVN-502 and M1 of SUVN-502, a 5-HT6 receptor antagonist for the treatment of dementia associated with Alzheimer's disease. Following solid-phase extraction, SUVN-502 and M1 of SUVN-502 and IS were eluted with 10mM ammonium acetate (pH 4.0) and acetonitrile using a rapid gradient program on reverse phase column. Multiple reaction monitoring mode was used to monitor the respective transitions of m/z 478.2→377.7 for SUVN-502 and m/z 464.1→377.7 for M1 of SUVN-502. The assay exhibited a linear dynamic range of 10-10000pg/mL for SUVN-502 and 20-20000pg/mL for M1 of SUVN-502 in human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The within batch accuracy and precision were within acceptable limits. All the other validation parameters were within the acceptable limits. The validated method was applied to analyze human plasma samples obtained from a human pharmacokinetic study consisting single and multiple ascending doses.

LC-MS/MS method for the determination of pitolisant: application to rat pharmacokinetic and brain penetration studies.

A simple and sensitive LC-MS/MS method was developed and validated for the quantitation of pitolisant, an H3 receptor antagonist/inverse agonist. Acetonitrile protein precipitation technique was used to prepare rat blood and brain tissue homogenate samples by using aripiprazole as internal standard (IS). Chromatographic separation was performed by using Xbridge column (2.1 × 50 mm, 3.5 µm) with a gradient elution program. The mobile phase consists of ammonium formate (10 mm) with 0.2% formic acid and acetonitrile. Multiple reaction monitoring mode was used in positive polarity with a transition of m/z 296.3 → 98.2 for the pitolisant and m/z 448.2 → 285.3 for the IS. The calibration curves were linear in the range of 0.1-100 ng/mL in both the blood and brain homogenate samples. This method was applied to quantify samples obtained from the pharmacokinetic and brain penetration studies in male wistar rats. Mean maximum concentration, area under the curve from zero to infinity and half-life of the pitolisant were found to be 3.4 ± 1.7 ng/mL, 5 ± 4 ng h/mL and 1.9 ± 0.3 h, respectively, after a 3 mg/kg oral dose. The mean calculated concentrations in the brain were found to be 38, 60 and 52 ng/g at 0.5, 1 and 2 h, respectively.

LC-MS/MS method for the quantification of almotriptan in dialysates: application to rat brain and blood microdialysis study.

A sensitive LC-MS/MS method was developed and validated for the quantification of almotriptan in rat brain and blood dialysates. Almotriptan is a 5HT1B/1D receptor agonist used for the treatment of migraine pain. Method consists of rapid gradient elution program with 10mM ammonium formate (pH 3) and acetonitrile on a Xbridge column. The MRM transitions monitored were m/z 336.2-58.1 for almotriptan and m/z 448.2-285.3 for the IS. The assay was linear in the range of 0.1-20 ng/ml, with acceptable precision and accuracy along with adequate sensitivity. The between batch accuracy was in the range of 99.0-104.3% with precision in between 0.6% and 5.8%. Microdialysis is an important sampling technique, with the capability of capturing the concentrations of various analytes in different bio fluids, at a single time point. This method was applied to quantify brain and blood dialysate samples obtained from a microdialysis study of rats treated with almotriptan (10mg/kg, p.o.). In vivo recovery experiments were performed to correct the dialysate concentrations into extracellular concentrations. Mean peak dialysate concentrations of almotriptan were found to be 152 ± 78 and 7.4 ± 1.0 ng/ml in blood and prefrontal cortex, respectively. The brain penetration of almotriptan is characterized by the AUCbrain/AUCblood found to be 0.07 ± 0.05. The results revealed the importance of measuring the unbound almotriptan concentrations in the brain over the blood for understanding its PK/PD relationship.