Validation of LC-MS/MS methods for quantitative analysis of kynurenine pathway metabolites in human plasma and cerebrospinal fluid.

Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

A sensitive LC-MS/MS method for simultaneous quantification of ulotaront and its N-desmethyl metabolite in human plasma and application to a clinical study.

Ulotaront (SEP-363856) is a novel non-D2-receptor-binding agent under development for the treatment of patients with schizophrenia. A highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with lower limit of quantitation of 0.0200 ng/mL (i.e. 20.0 pg/mL) was successfully developed and validated for the simultaneous quantitation of ulotaront and its N-desmethyl metabolite (M11A) in human plasma. Plasma samples were extracted by solid phase extraction with Oasis MCX 96-well plate, followed by a reversed phase LC separation coupled with MS/MS detection in positive mode (m/z 184.1 → 135.0 for ulotaront and 170.1 → 135.0 for M11A). Stable isotope-labeled compounds SEP-363856-d3 and M11A-d4 were used as internal standards (IS) for corresponding analytes. The validated calibration curve range was 0.0200-20.0 ng/mL for both analytes using a 0.200 mL plasma. Extraction recoveries were found to be 75.7% and 75.1% for ulotaront and IS1, and 82.7% and 83.9% for M11A and IS2, respectively. Frozen plasma samples were confirmed to be stable for up to 730 days at both -20 °C and -70 °C. The validated method has been successfully used to evaluate the pharmacokinetics (PK) of ulotaront and M11A in clinical studies. The application to the first-in-human PK study (single ascending dose) presented in this work demonstrated that ulotaront exhibited near dose proportionality for both Cmax (maximum concentration) and AUC (area under the curve) over the dose range from 5 to 125 mg. M11A was found as a minor metabolite with an exposure of about 2-3% of the parent compound.

Determination of carboplatin in human plasma using HybridSPE-precipitation along with liquid chromatography-tandem mass spectrometry.

The main purpose of this study was to develop and validate a rapid, specific, sensitive, and reliable LC-MS/MS-based bioanalytical method for the determination of carboplatin in human plasma. The optimal chromatographic behavior of carboplatin was achieved on a Biobasic SCX column (50 mm × 2.1 mm, 5 µm) using ion exchange chromatography. The total LC analysis time per injection was 2.6 min with a flow rate of 1.5 mL/min with a gradient elution. Optimization with regard to improving recovery and minimizing matrix effects using HybridSPE-precipitation (HybridSPE-PPT) has been evaluated under various extraction conditions. As a result, sample preparation via HybridSPE-PPT with 1% formic acid in acetonitrile in a 96-well format was applied for method validation and sample analysis and showed acceptable recovery of greater than 25% and negligible matrix effects. The method validation was conducted over the curve range of 2.00-2000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤4.8% relative standard deviation (RSD) and -13.2 to -3.6% relative errors (RE). The method was successfully applied to determine carboplatin in human plasma samples.