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Aim: Monitoring the internal standard (IS) response is common practice in bioanalysis by LC-MS/MS. IS response variation may raise questions on assay quality and should trigger investigations into the root cause. Results: In two case studies with IS variability, re-analysis of diluted samples and spiking predose study samples revealed no effect of IS variability on results. The D 17 -labeled IS in a third case proved not to be suitable during method development and was replaced by a differently labeled IS. Conclusion: Determining the exact root cause for varying IS response is not always feasible; however, assay accuracy and reliability of results should be demonstrated. In some cases, assay re-development is needed to solve the problem.

Aim: Alectinib (Alecensa ® ) is an anaplastic lymphoma kinase inhibitor for the treatment of anaplastic lymphoma kinase positive non-small-cell lung cancer, and M4 is its major pharmacologically active metabolite. To characterize the pharmacokinetics and excretion of alectinib and M4 in human urine, a bioanalytical method was required., Results: An LC-MS/MS method using supported liquid extraction was developed for the determination of alectinib and M4 in human urine over the concentration range 0.5-500 ng/ml. Accuracy ranged from 92.0 to 112.2% and precision (CV) was below 9.6%., Conclusion: The method was successfully employed to determine alectinib and M4 concentrations in urine samples from a clinical mass balance study. Addition of the surfactant Tween-20 to urine prevented nonspecific binding of the analytes.

Background: Alectinib is a novel anaplastic lymphoma kinase (ALK) inhibitor for treatment of patients with ALK-positive non-small-cell lung cancer who have progressed on or are intolerant to crizotinib. To support clinical development, concentrations of alectinib and metabolite M4 were determined in plasma from patients and healthy subjects., Methods: LC-MS/MS methods were developed and validated in two different laboratories: Chugai used separate assays for alectinib and M4 in a pivotal Phase I/II study while Roche established a simultaneous assay for both analytes for another pivotal study and all other studies., Conclusion: Cross-validation assessment revealed a bias between the two bioanalytical laboratories, which was confirmed with the clinical PK data between both pivotal studies using the different bioanalytical methods.

Background: During development of biologics, safety and efficacy assessments are often hampered by immune responses to the treatment. To assess active exposure of a drug peptide in a toxicology study, we developed an ex vivo potency assay which complemented the total drug quantification assay., Methodology: Compound activity was assessed in samples of treated monkeys by cell-based cAMP measurements. For each animal, activity was compared with its predose sample to which the compound has been added at the postdose concentration as determined by a total LC-MS/MS assay., Conclusion: We were able to show that despite a high total test compound level, activity was reduced tremendously in antidrug-antibody-positive monkeys. Therefore, the applied ex vivo potency assay supplements drug quantification methods to determine active exposures.

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.

An acylated peptide with MW ~4.5 kDa was measured in samples from pharmacokinetic, toxicology and clinical studies using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lower limits of quantitation of 2 ng/mL and 50 pg/mL were achieved for animal and human plasma, respectively. Repeated drug administration may lead to anti-drug antibodies (ADA) which can inactivate the drug by formation of drug-ADA complexes. Hence, the LC-MS/MS assay incorporated cleavage of potential drug-ADA complexes to quantify the total plasma concentration. To obtain information on active drug levels, an assay that measures the free concentration or alternatively the ADA-unbound concentration would be needed. Ultrafiltration experiments through 100 kD cutoff membranes to remove Ig-bound peptide were not successful due to nonspecific binding. Extraction of Ig-bound drug using Protein A or G (bacterial cell wall proteins with high affinity to the Fc region of IgG) was suitable to distinguish between ADA-bound drug and [free+protein bound (not ADA-bound)] drug and correlated with findings from ELISA ADA measurement., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012. Published by Elsevier B.V.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: A thin-layer chromatography-mass spectrometry interface was previously reported to extract compounds from dried blood spots online. Here, we integrated this interface into a column-switching LC-MS/MS system for online solid-phase extraction and addressed internal standard addition., Results: A drug was analyzed in dried blood spots with a lower limit of quantitation of 50 pg/ml. The internal standard was added via an autosampler to the extraction solvent. The method featured online aqueous dilution of the dried blood spots extract with subsequent trapping and gradient analytical separation. Good precision, accuracy and recovery were obtained., Conclusion: The thin-layer chromatography-mass spectrometry interface provided high reproducibility and good extraction efficiency. Although manually operated, it significantly reduced sample preparation efforts. The combination with online solid-phase extraction allowed enrichment of larger sample/extract volumes and efficient clean-up.

A highly sensitive liquid chromatographic method with online solid-phase extraction (SPE) and tandem mass spectrometric detection was developed for the quantification of the synthetic peptide drug taspoglutide in human and animal plasma. Sample preparation consisted of simple protein precipitation or automated off-line SPE using mixed mode cation exchange material, optionally preceded by cleavage of potential antidrug antibody complexes. Excellent selectivity was obtained by a novel orthogonal column switching approach, employing hydrophilic interaction liquid chromatography (HILIC) for online SPE followed by chromatographic separation on a 300 A pore size C18 column. The use of a stable isotope labeled drug (C-terminal arginine containing six (13)C and four (15)N atoms) as internal standard improved the quality and ruggedness of the method. The lower limits of quantitation (LLOQ) in human and animal plasma were 10.0 and 50.0 pg/mL, respectively, extracting only 250 or 100 microL sample aliquots. Precisions and accuracies were below 15% CV and between 89% and 114%, respectively. The method was successfully employed to analyze samples from pharmacokinetic studies.

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.

Abstract: 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 50mm C18 analytical column, followed by electrospray ionization and detection in positive ion selected reaction monitoring (SRM) mode. The total run time was 2.5min. The lower limit of quantification (LLOQ) was 10pg/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. [Copyright &y& Elsevier]