Risdiplam distributes and increases SMN protein in both the central nervous system and peripheral organs.

Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small-molecule SMN2 pre-mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.

Determination of dalcetrapib by liquid chromatography-tandem mass spectrometry.

The cholesteryl ester transfer protein modulator dalcetrapib is currently under development for the prevention of dyslipidemia and cardiovascular disease. Dalcetrapib, a thioester, is rapidly hydrolyzed in vivo to the corresponding thiophenol which in turn is further oxidized to the dimer and mixed disulfides (where the thiophenol binds to peptides, proteins and other endogenous thiols). These forms co-exist in an oxidation-reduction equilibrium via the thiol and cannot be stabilized without influencing the equilibrium, hence specific determination of individual components, i.e., in order to distinguish between the free thiol, the disulfide dimer and mixed disulfide adducts, was not pursued for routine analysis. The individual forms were quantified collectively as dalcetrapib-thiol (dal-thiol) after reduction under basic conditions with dithiothreitol to break disulfide bonds and derivatization with N-ethylmaleimide to stabilize the free thiol. The S-methyl and S-glucuronide metabolites were determined simultaneously with dal-thiol with no effect from the derivatization procedure. Column-switching liquid chromatography-tandem mass spectrometry provided a simple, fast and robust method for analysis of human and animal plasma and human urine samples. Addition of the surfactant Tween 80 to urine prevented adsorptive compound loss. The lower limits of quantitation (LLOQ) were 5 ng/mL for dal-thiol, and 5 ng/mL for the S-methyl and 50 ng/mL for the S-glucuronide metabolites. Using stable isotope-labeled internal standards, inter- and intra-assay precisions were each <15% (<20% at LLOQ) and accuracy was between 85 and 115%. Recovery was close to 100%, and no significant matrix effect was observed.

Determination of oseltamivir (Tamiflu®) and oseltamivir carboxylate in dried blood spots using offline or online extraction.

BACKGROUND: Using dried blood spots (DBS) for quantitation of the antiviral drug oseltamivir (Tamiflu(®)), an ester prodrug, and its active metabolite oseltamivir carboxylate could provide ethical and logistic benefits. Hence, its feasibility was investigated using a previously developed column-switching LC-MS/MS method. RESULTS: Sensitivity, precision and accuracy in DBS were comparable to standard plasma assays. Chemically treated cards provided enhanced ex vivo stability of the ester prodrug in rodent blood. Online extraction was realized using the manual TLC-MS interface or the fully automated Sample Card and Prep system. Rat pharmacokinetic study data showed good correlation between plasma, liquid blood and DBS. CONCLUSION: From a bioanalytical perspective, DBS is potentially suited for Tamiflu analysis in animals and humans. Automation of the process by online DBS extraction promises workload reduction and throughput increase.

Determination of mycophenolic acid and its phenyl glucuronide in human plasma, ultrafiltrate, blood, DBS and dried plasma spots.

BACKGROUND: Analysis of mycophenolic acid (MPA), the active form of the immunosuppressive drug mycophenolate mofetil, and its glucuronide metabolite MPAG is required for therapeutic monitoring and postmarketing clinical studies. Dried blood spots (DBS) and dried plasma spots (DPS) could be alternatives to conventional assays for small-volume sampling and easy shipment. RESULTS: A LC-MS/MS method with online SPE was established using stable isotope labeled analytes as internal standards. The quantitation limits were set at 0.1 and 1 µg/ml, for total MPA and MPAG, respectively, in plasma, blood, DBS and DPS, but 100-fold lower for free MPA in ultrafiltrate. Ahlstrom 226 or Whatman FTA(®) DMPK-B cards were well suited for DBS and DPS analyses. CONCLUSION: MPA and MPAG were analyzed in human plasma and blood either as liquid or dried on cards with similar assay quality. Care should be taken to avoid back-conversion of an instable acyl glucuronide metabolite to MPA.

Bioanalytics and pharmacokinetics of the nociceptin/orphanin FQ peptide receptor agonist RO0646198 in Wistar rats and Cynomolgus monkeys.

This manuscript describes the determination of the nociceptin/orphanin FQ peptide (NOP) receptor agonist RO0646198 in rat and Cynomolgus monkey plasma using liquid chromatography coupled to tandem mass spectrometry. The structural analogue RO0658791 served as internal standard. After protein precipitation with ethanol, clean up and enrichment was carried out by on-line solid-phase extraction on a 10mm C18 trapping column. Gradient separation was performed on a 50 mm C18 analytical column, followed by electrospray ionization and detection in positive ion selected reaction monitoring (SRM) mode. The total run time was 2.5 min. The lower limit of quantification (LLOQ) was 10 pg/mL. Precisions were below 19% and accuracies were between 86% and 118%. The recoveries were above 90%, and no significant matrix effect was observed. The method was successfully applied to low dose pharmacokinetic studies. The bioavailability after oral dosing was below 5% for rats and below 1% in Cynomolgus monkeys, limiting the application of RO0646198 in the clinic to parenteral routes.

Sensitive determination of oseltamivir and oseltamivir carboxylate in plasma, urine, cerebrospinal fluid and brain by liquid chromatography-tandem mass spectrometry.

This manuscript describes the determination of oseltamivir (OP) and oseltamivir carboxylate (OC) in rat plasma, cerebrospinal fluid (CSF) and brain and in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry. Threefold deuterated OP and OC served as internal standards. Protein precipitation with perchloric acid was followed by on-line solid-phase extraction and gradient separation on a reversed-phase column. After electrospray ionization, the compounds were detected in positive ion selected reaction monitoring (SRM) mode. Run time was 3.6 min. The lower limits of quantification (LLOQ) were 0.1 ng/mL in rat plasma and CSF, 0.5 ng/g in brain and 1 ng/mL in human plasma and urine. Inter-day and intra-day precisions and inaccuracies in rat matrices were below 10.2% and 13.9% (below 19.0% at LLOQ), respectively. Intra-assay precisions and inaccuracies in human matrices were below 11.7% and 8.9%, respectively. The recoveries were close to 100%, and no significant matrix effect was observed. The method was successfully applied to rat study samples.

Fast liquid chromatographic-tandem mass spectrometric (LC-MS-MS) determination of metformin in plasma samples.

Liquid chromatographic-tandem mass spectrometric (LC-MS-MS) methods for the determination of metformin in plasma from different species are presented. The first method employed a YMC cyano 2mm x 50 mm, 3 microm analytical column. For minimum sample preparation direct injection of samples after protein precipitation was performed. The polar column used with highly organic mobile phases provided a normal phase retention mechanism. The elution conditions were optimized to obtain reproducible peak areas and good peak shape. A step gradient from 100% acetonitrile to acetonitrile-water 80:20 (v/v) containing 10mM ammonium acetate and 1% acetic acid was applied, leading to a sample-to-sample cycle time of 2 min. In a second method, a column-switching LC-MS-MS assay for on-line trapping was developed. The analyte and internal standard were trapped on a YMC cyano 2 mm x 10 mm, 5 microm column using acetonitrile-methanol 95:5 (v/v). Elution was performed isocratically in back-flush mode on to the analytical column (YMC cyano 2 mm x 50 mm, 3 microm) using 10 mM ammonium acetate in acetonitrile-water 80:20 (v/v) with 1% formic acid. With this approach, the signal-to-noise ratio was improved and the run time could be shortened to 1 min. Calibration samples were prepared in the matrix to be assayed in the range of 10-10,000 ng/ml. Quality control (QC) samples were prepared at 40, 400 and 4000 ng/ml and interspersed with the unknown study samples in the assays. Deviations for precision and accuracy were less than 20% for the lower limit of quantification (LLOQ) and low QC sample and less than 15% for other calibrators and QCs.

Ultra-fast quantitative bioanalysis of a pharmaceutical compound using liquid chromatography-tandem mass spectrometry.

This paper describes the ultra-fast determination of a pharmaceutical compound using TurboIonSpray LC-MS-MS on an API 4000 mass spectrometer. Sample preparation consisted of plasma protein precipitation, centrifugation and dilution of the supernatant. The use of small analytical column dimensions (2.1 mm x 10 mm) and high eluent flow rates (up to 2.2 ml/min) in isocratic mode led to a retention time of 9s. A sample-to-sample cycle time of only 10s was achieved by coupling two autosamplers. Partial separation of the drug and its main metabolite could be obtained. The d5-labeled drug used as internal standard compensated for matrix suppression effects. The assay was linear in the concentration range 1-1000 ng/ml, using standards prepared in human plasma. Inter-assay accuracy and precision were 98.5 and 6.2%, respectively. Mean intra-assay accuracy and precision calculated from quality control (QC) samples in human, rat and dog plasma at 3, 30 and 800 ng/ml were 100.8 and 3.8%, respectively. The ultra-fast LC-MS-MS method was successfully cross-validated against a commonly used column-switching LC-MS-MS assay with 2.3 min run time by analyzing real study samples.

Application of column-switching liquid chromatography-tandem mass spectrometry for the determination of pharmaceutical compounds in tissue samples.

Information on plasma-tissue distribution which is important for drug development may be obtained by "in silico" prediction tools. To support the validation of computer models, drug concentrations in rat plasma and tissues (brain, liver, kidney, testes, spleen, gut, lung. heart, muscle, skin and fat) had to be determined. In our work, we established analytical assays for a variety of substances including nicardipine, nitrendipine, felodipine and benzodiazepines. Sample preparation had to be simple and method development as well as analytical run time short to allow a high sample throughput and to minimize resources. Column-switching HPLC after homogenization and protein precipitation served as an efficient, easy and rapid sample preparation method, followed by selective MS-MS detection. Optimization of the trapping procedure was performed in order to reduce the influence of endogenous interferences and to obtain good recovery. Chromatographic separation was necessary to increase the selectivity. The use of small analytical column dimensions (2.1 x 10 mm) was investigated to achieve higher sample throughput without compromising the assay quality. Mass spectrometric parameters, such as ionization modes (positive vs. negative) and ion source types (TurbolonSpray vs. APCI) were screened to find suitable conditions for sensitive analysis of the compounds. Matrix suppression effects were taken into consideration. Calibration samples were prepared in plasma only, whereas quality control samples were prepared in both plasma and tissues to save animals and time. Accuracy and precision were in the range of 84.4-119.1% and 1-16.5%, respectively. Limits of quantification were in the range of 0.5-2.5 ng/ml for plasma and 2-10 ng/ml for tissues. Run times as short as 2.2 min could be achieved.