Katja Heinig

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.

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.

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.

Sensitive determination of a pharmaceutical compound and its metabolites in human plasma by ultra-high performance liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction.

This paper describes the determination of a drug candidate and two metabolites in human plasma by column-switching LC-MS/MS after protein precipitation. Starting from a standard method with a quantitation limit of 0.5 ng/mL, a highly sensitive assay was developed, employing UHPLC separation and detection on an API 5000 mass spectrometer. The injected plasma equivalent was increased from 6 to 20 μL; conventional column trapping for compound enrichment and removal of matrix constituents was combined with high-pressure analytical separation using small particle columns to improve resolution and signal-to-noise ratio. Quantitation limits were thus lowered to between 5 and 20 pg/mL, offering the possibility to provide bioanalytical support for microdosing studies in humans. Excellent assay quality and robustness were achieved by both methods.

Determination of Ganciclovir and its prodrug Valganciclovir by hydrophilic interaction liquid chromatography–tandem mass spectrometry

This manuscript describes the determination of Ganciclovir (GCV), active component of the antiviral drug Valcyte®, and its ester prodrug Valganciclovir (VGC) in human and rat plasma, using liquid chromatography coupled to tandem mass spectrometry. Protein precipitation with acetonitrile was followed by hydrophilic interaction liquid chromatography on a silica column with 4 min run time. After electrospray ionization, the compounds were detected in positive ion selected reaction monitoring (SRM) mode. The lower limits of quantification (LLOQ) were 16 ng/mL for GCV and 4 ng/mL for VGC in human and rat plasma. Inter-day and intra-day precisions and inaccuracies were below 15% and between 85 and 115%, respectively. Five-fold deuterated GCV and VGC were used as internal standards and compensated for any matrix effect. The method was successfully applied to samples from a rat pharmacokinetic study. The feasibility of blood analysis as dried blood spots (DBS) was investigated.

Dalcetrapib pharmacokinetics and metabolism in the cynomolgus monkey

The thioester dalcetrapib is undergoing Phase III clinical evaluation for the prevention and regression of atherosclerosis and the prevention of cardiovascular events through targeting cholesteryl ester transfer protein and increasing high-density lipoprotein cholesterol levels. Dalcetrapib undergoes rapid hydrolysis to generate the pharmacologically active form (dalcetrapib-thiol), which undergoes extensive metabolism via glucuronic acid conjugation, methylation, and hydroxylation, predominately forming the pharmacologically inactive S-methyl (dalcetrapib-S-Me) and S-glucuronide (dalcetrapib-S-Glu) metabolites. The purpose of this study was to characterize the absorption and disposition of dalcetrapib-thiol and its primary metabolites in cynomolgus monkeys following first pass through the intestines and liver using an in vivo dual portal and peripheral vein cannulation. Results showed the high influence of glucuronidation of dalcetrapib-thiol on the first-pass effect. Following passage through the primate intestinal wall, area under the plasma concentration–time curve indicated a marked loss (by ~85%) of active compound and formation of dalcetrapib-S-Glu and dalcetrapib-S-Me. Based on time to maximum drug concentrations (Tmax) values in the portal vein, metabolism of dalcetrapib-thiol to dalcetrapib-S-Glu appears to occur almost instantly (median Tmax 6.0 and 5.5u2009h, respectively), whereas methylation to dalcetrapib-S-Me occurs much more slowly (median Tmax, 24u2009h). A relatively modest impact on systemic exposure followed hepatic first pass, with a further decrease in dalcetrapib-thiol exposure of 58% (AUC), a 3-fold reduction in exposure levels of dalcetrapib-S-Me and near-complete decrease in exposure of dalcetrapib-S-Glu. Passage of drug-related material through the intestinal wall and the liver results in an overall decrease of exposure to dalcetrapib-thiol of >90%.

An improved LC‐MS/MS method for the determination of taspoglutide in plasma and urine using orthogonal HILIC–RP column switching, ultra‐performance LC separation and ‘wrong‐way‐round’ electrospray ionization

The synthetic peptide drug taspoglutide, developed for treatment of diabetes, must be quantified at low pg/mL levels in biological samples. This manuscript describes the improvement of a previous method, featuring orthogonal hydrophilic interaction to reversed-phase chromatography column switching and tandem mass spectrometric detection. Signal-to-noise ratio was enhanced and isobaric interferences were reduced by ultra-performance separation using a basic mobile phase in wrong-way-round ionization mode and monitoring a selective fragment ion. Tedious solid-phase extraction cleanup was abandoned in favor of simple protein precipitation. Urine required the addition of surfactants to prevent adsorptive drug loss. Dissociation of complexes with possibly formed anti-drug antibodies was achieved with formic acid. Lower limits of quantitation (LLOQ) were 4 pg/mL in human plasma and 10 pg/mL in urine using a 250 μL sample, and an LLOQ of 50 pg/mL was obtained in animal plasma using 50 μL. Precision, accuracy and incurred samples reproducibility fulfilled regulatory requirements. Simultaneous determination of unlabeled and stable isotope labeled taspoglutide, interesting for clearance studies in which both compounds are co-administered, was realized using a structural analog as internal standard. The described method offered excellent sensitivity with low sample consumption, reasonable throughput, moderate costs and high robustness for routine analysis.

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.

Role of the Intestinal Peptide Transporter PEPT1 in Oseltamivir Absorption: In Vitro and In Vivo Studies

It was reported that oseltamivir (Tamiflu) absorption was mediated by human peptide transporter (hPEPT) 1. Understanding the exact mechanism(s) of absorption is important in the context of drug-drug and diet-drug interactions. Hence, we investigated the mechanism governing the intestinal absorption of oseltamivir and its active metabolite (oseltamivir carboxylate) in wild-type [Chinese hamster ovary (CHO)-K1] and hPEPT1-transfected cells (CHO-PEPT1), in pharmacokinetic studies in juvenile and adult rats, and in healthy volunteers. In vitro cell culture studies showed that the intracellular accumulation of oseltamivir and its carboxylate into CHO-PEPT1 and CHO-K1 was always similar under a variety of experimental conditions, demonstrating that these compounds are not substrates of hPEPT1. Furthermore, neither oseltamivir nor its active metabolite was capable of inhibiting Gly-Sar uptake in CHO-PEPT1 cells. In vivo pharmacokinetic studies in juvenile and adult rats showed that the disposition of oseltamivir and oseltamivir carboxylate, after oral administration of oseltamivir, was sensitive to the feed status but insensitive to the presence of milk and Gly-Sar. Moreover, oseltamivir and oseltamivir carboxylate exhibited significantly higher exposure in rats under fasted conditions than under fed conditions. In humans, oral dosing after a high-fat meal resulted in a statistically significant but moderate lower exposure than after an overnight fasting. This change has no clinical implications. Taken together, the results do not implicate either rat Pept1 or hPEPT1 in the oral absorption of oseltamivir.

Small Molecule Specific Run Acceptance, Specific Assay Operation, and Chromatographic Run Quality Assessment: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Teams

Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.