Bioanalytical method for the simultaneous determination of d- and l-serine in human plasma by LC/MS/MS

d-Serine is an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors. Plasma concentrations of d-serine and the ratio of d-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the d- and l-isomers of serine in human plasma. Since d- and l-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. d- and l-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. d-Serine (m/z 106.1 > 60.0), l-serine (m/z 106.1 > 60.1) and dl-serine-d₃ (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of d- and l-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01–10 μg/mL (d-serine) and 0.1–100 μg/mL (l-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of d- and l-serine in human plasma were 0.124–0.199 and 7.97–13.1 μg/mL, respectively.     

Development of a high-throughput method for the determination of ethosuximide in human plasma by liquid chromatography mass spectrometry

A simple, rapid, sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the quantification of ethosuximide in human plasma is described. Analyte was chromatographed on a Hypersil Gold C₁₈ column (100 mm × 2.1 mm, i.d., 1.9 μm) with isocratic elution at a flow rate of 0.250 mL/min and pravastatin was used as the internal standard. The assay involves a simple solid-phase extraction procedure of 0.25 mL human plasma and the analysis was performed on a triple-quadrupole tandem mass spectrometer by MRM mode via electrospray ionization (ESI). The method was linear in the concentration range of 0.25–60.0 μg/mL. The lower limit of quantification (LLOQ) was 0.25 μg/mL. The within- and between-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 95.1% and 94.4% for ethosuximide and pravastatin, respectively. The analysis time for each sample was 1.8 min. The method was highly reproducible and gave peaks with excellent chromatography properties.     

An LC-MS/MS method for determination of forsythiaside in rat plasma and application to a pharmacokinetic study

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of forsythiaside in rat plasma using epicatechin as internal standard. The analytes were extracted by solid-phase extraction and chromatographied on a C₁₈ column eluted with a gradient mobile phase of acetonitrile and water both containing 0.2% formic acid. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 623 → 161 and m/z 289 → 109 for forsythiaside and epicatechin, respectively. The assay was linear over the concentration ranges of 2.0–50.0 and 50.0–5000.0 ng/mL with limits of detection and quantification of 0.2 and 1.0 ng/mL, respectively. The precision was <10.8% and the accuracy was >91.9%, and extraction recovery ranged from 81.3% to 85.0%. This method was successfully applied to a pharmacokinetic study of forsythiaside in rats after intravenous (20 mg/kg) and oral (100 mg/kg) administration, and the result showed that the compound was poorly absorbed with an absolute bioavailability being approximately 0.5%.     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.     

Determination of casopitant and its three major metabolites in dog and rat plasma by positive ion liquid chromatography/tandem mass spectrometry

A sensitive, selective and quantitative method for the simultaneous determination of casopitant, a potent and selective antagonist of the human Neurokinin 1 (NK-1) receptor, and its three major metabolites M12, M13 and M31 was developed and validated in dog and rat plasma. Acetonitrile containing stable labeled internal standards for the four analytes was used to precipitate proteins in plasma. Chromatographic separation was obtained using a reversed phase column with multiple reaction monitoring turboionspray positive ion detection. The lower and upper limits of quantification for casopitant and its metabolites were 15 and 15,000 ng/mL, using a 50 μL of dog or rat plasma aliquot, respectively. The inter-day precision (relative standard deviation) and accuracy (relative error) in dog plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 4.1% to 10.0% and −10.8% to 8.7%, respectively, for casopitant and its 3 major metabolites. The intra-day precision (relative standard deviation) and accuracy (relative error) in rat plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 3.9% to 6.6% and −9.6% to 8.3%, respectively, for casopitant and its three metabolites. All analytes were found to be stable in analytical solutions for at least 43 days at 4 °C, in dog and rat plasma at room temperature for at least 24 h, at the storage temperature of −20 °C for at least 6 months, and following the action of three freeze–thaw cycles from −20 °C to room temperature. All analytes were also found to be stable in processed extracts at 4 °C for at least 72 h. This assay proved to be accurate, precise, fast and was used to support long-term toxicology studies in dog and rat.     

Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis

The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.     

Development and validation of a liquid chromatography/mass spectrometry method for pharmacokinetic studies of OZ78, a fasciocidal drug candidate

Fascioliasis is a zoonotic disease of considerable public health and great veterinary significance and new drugs are needed. OZ78 is a promising fasciocidal drug candidate. In order to support the development of OZ78, including pharmacokinetic (PK) studies an accurate, precise, and selective liquid chromatography/mass spectrometry (LC/MS) method for OZ78 was developed for sheep plasma and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. Protein precipitation was used for sample clean up. Separation was performed through a Phenomenex C8(2) analytical column (50.0 mm × 2.0 mm, 5 μm) with a mobile phase of acetonitrile (buffer B) and 5 mM ammonium formate (buffer A) at a flow-rate of 0.3 mL/min and a gradient from 20% to 95% acetonitrile. The mass spectrometer was operated under selected ion monitoring, and orifice voltage set to −4.1 kV and ion spray temperature to 400 °C. Nitrogen was used as a nebulizer, curtain, and collision gas. OZ78 was monitored at 321.4 m/z (deprotonated parent compound, M-). The validated linear dynamic range was between 156.25 ng/mL and 5 μg/mL and the achieved correlation coefficient (r²) was greater than 0.99. The validation results demonstrated that the developed LC/MS method is precise, accurate, and selective for the determination of OZ78 in sheep plasma. The method was successfully applied to the evaluation of the PK profile of OZ78 in sheep.     

Development of high performance liquid chromatography/electrospray ionization mass spectrometry for assay of ginkgolic acid (15:1) in rat plasma and its application to pharmacokinetics study

A highly sensitive HPLC–ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50 μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C₁₈ column (2.1 × 100 mm, 3 μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8–1000 ng/mL with the square regression coefficient (r²) of 0.996. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n = 6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10 mg/kg. GA (15:1) pharmacokinetic parameters C_max, T_max, t_1/2, AUC_0–12h are 1552.9 ± 241.0 ng/mL, 0.9 ± 0.7 h, 5.5 ± 2.6 h, 3356.0 ± 795.3 ng h/mL, respectively.