Highlights of the 12th Japan Bioanalysis Forum Symposium.

Approximately 300 people associated with pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 12th Japan Bioanalysis Forum Symposium. The webinar was conducted from 9 to 11 March 2021. The theme of the symposium was 'for the next generation', and the event provided 'an opportunity for young researchers in bioanalysis (including students)' and 'an opportunity to discuss new frontiers of bioanalysis'. The speakers focused on hot topics of bioanalysis, including biomarker analysis, patient centric sampling, virtual clinical trials, gene therapy, cancer genome medicine and therapeutic middle molecules. The symposium presented a platform for the discussion of the prospects and challenges facing bioanalysts working in the field of pharmacokinetics. This report presents the key issues discussed.

The 10th Japan Bioanalysis Forum Symposium conference: a report.

The Japan Bioanalysis Forum Symposium was held on 12-14 February 2019 (Yokohama, Japan), in celebration of its 10th anniversary, and over 370 participants from pharmaceutical industries, contractors, academia and regulatory authorities from home and abroad came together in Yokohama. The 3-day symposium particularly aimed to foster collaboration with the scientists surrounding bioanalysts, according to the theme 'Open to the Public.' The symposium also included a broad range of pioneering programs, such as lectures by speakers from DMPK/metabolomics fields, discussions of future bioanalysis and poster presentations by publicly offered presenters as well as the regular ones we had organized. This report summarizes the major topics as a conference report.

CYP3A but not P-gp plays a relevant role in the in vivo intestinal and hepatic clearance of the delta-specific phosphoinositide-3 kinase inhibitor leniolisib.

This study investigated the effect of itraconazole, a strong dual inhibitor of cytochrome P450 (CYP) 3A4 and P-glycoprotein (P-gp), on the single dose pharmacokinetics of leniolisib. In order to differentiate the specific contribution of CYP3A from P-gp, the potential interaction with quinidine, a strong inhibitor of P-gp but not CYP3A, was studied as well. Using a fixed-sequence, 3-way crossover design, 20 healthy male subjects received single oral doses of 10 mg leniolisib during three phases separated by a washout: (1) leniolisib alone, (2) 200 mg itraconazole once daily for 9 days plus leniolisib on day 5, and (3) 300 mg quinidine administered 1 h before and 3 h after leniolisib. Itraconazole increased the leniolisib oral drug exposure (AUCinf ) by on average 2.1-fold, whereas the peak drug concentration (Cmax ) was less impacted (1.25-fold). The terminal elimination half-life (T1/2 ) of leniolisib was also increased by ~2-fold. Neither oral AUCinf nor Cmax or T1/2 was found to be altered by quinidine. These findings suggest that the interaction with itraconazole occurred mainly systemically through inhibition of CYP3A, and corroborate our in vitro findings that leniolisib is neither a sensitive CYP3A substrate nor a relevant in vivo substrate for intestinal or hepatic P-gp. Assuming itraconazole levels achieved complete inhibition of CYP3A, the fractional contribution of CYP3A to the overall disposition of leniolisib is estimated to be about 50%. The concomitant use of leniolisib with strong inhibitors of CYP3A as well as strong and moderate inducers of CYP3A is best avoided.

Pharmacokinetic Interaction Between Fingolimod and Carbamazepine in Healthy Subjects.

This open-label, single-sequence study in healthy subjects investigated the effects of steady-state carbamazepine on the pharmacokinetic (PK) profile of a single 2-mg dose of fingolimod. In period 1, a single oral dose of fingolimod 2 mg (day 1) was followed by PK and safety assessments up to 36 days. In period 2, carbamazepine was administered in flexible, up-titrated doses (600 mg twice daily maximum) for 49 days. Fingolimod was administered on day 35, followed by a study completion evaluation (day 71). The PK analysis included 23 of 26 of the enrolled subjects (88.5%). Coadministration of fingolimod at steady-state carbamazepine concentrations resulted in increased fingolimod CL/F by 67% through the induction of CYP3A4, a cytochrome with negligible involvement in fingolimod clearance in an uninduced state. Fingolimod Cmax was reduced by 18% and AUCinf by 40%, as was T1/2 (106 vs 163 hours). A similar trend was observed for fingolimod-P. Models linking fingolimod-P blood concentrations to lymphocyte count or annual relapse rate suggest that such a decrease would have a low impact on the treatment effect. However, in the absence of efficacy data of fingolimod at doses lower than the therapeutic dose, their coadministration should be used with caution.

Determination of Seminal Concentration of Fingolimod and Fingolimod-Phosphate in Multiple Sclerosis Patients Receiving Chronic Treatment With Fingolimod.

The safety profile of fingolimod 0.5 mg, approved therapy for relapsing multiple sclerosis, is well established in clinical and real-world studies. As fingolimod is teratogenic in rats, it was considered important to assess the concentrations of fingolimod and its active metabolite, fingolimod-phosphate, in the semen of male patients on treatment and the risk of harming a fetus in a pregnant partner. In this multicenter open-label study, 13 male patients receiving fingolimod for at least 6 months provided 1 semen and 1 blood sample for analyte concentration measurements. The steady-state seminal concentrations of fingolimod and fingolimod-phosphate were close to those simultaneously observed in blood. The amount of fingolimod-related material in 10 mL of ejaculate was estimated to be 47.5 ng. The estimated fingolimod and fingolimod-phosphate blood Cmax values in a woman having regular sexual intercourse with a male patient treated with fingolimod 0.5 mg were approximately 400 and 2400 times smaller than the estimated values in the embryo-fetal development study in rats at the no-observed-adverse-event level. Consequently, the risk of harming a fetus in a pregnant woman is considered extremely unlikely.

Pharmacokinetics and safety of indacaterol and glycopyrronium (IND/GLY) following repeated once daily inhalation from a fixed-dose combination in healthy Chinese subjects
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OBJECTIVE: Indacaterol/glycopyrronium (IND/GLY) is a once-daily fixed-dose combination of two long-acting bronchodilators: indacaterol 110 µg (long-acting ß2-adrenergic agonist, LABA) and glycopyrronium 50 µg (long-acting muscarinic antagonist, LAMA). This study assessed the pharmacokinetics of IND/GLY 110/50 µg following multiple once-daily inhaled administrations in healthy Chinese subjects. METHODS: This was a single-centre, open-label, multiple-dose study of inhaled IND/GLY delivered via the Breezhaler® device. Pharmacokinetic samples were collected on day 1 after first dose, on days 5, 7, 10, and 12 (predose (trough)) and on day 14 (steady state) after last dose for pharmacokinetic analysis using non-compartmental analysis. RESULTS: Both IND and GLY were absorbed rapidly after inhalation of IND/GLY (tmax: IND, 15 minutes; GLY, 5 minutes). Accumulation through systemic exposure of both IND and GLY from day 1 to day 14 was observed (mean accumulation ratio (Racc) of AUC0-24h (day 14/day 1): IND, 3.02; GLY 2.94; estimated accumulation ratio of Cmax: IND 1.56; GLY 1.33). Mean effective half-life (t1/2,acc) was 41.3 h and 40.0 h for IND and GLY, respectively. Pharmacokinetic steady states were reached after 12 and 10 days of daily dosing for IND and GLY, respectively. There was one mild adverse event (AE) not related to the study drug. No discontinuations due to treatment related AEs/SAEs (adverse event/serious adverse event) were reported. CONCLUSIONS: In healthy Chinese subjects, multiple once-daily inhaled doses of IND/GLY 110/50 µg were rapidly absorbed and were safe and well tolerated. The comparison of systemic exposure data following inhalation of IND/GLY 110/50 µg in Chinese vs. the non-Chinese populations did not indicate any clinically relevant differences across ethnicities.
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Lung Delivery of Indacaterol and Mometasone Furoate Following Inhalation of QMF149 in Healthy Volunteers.

This single-dose, 4-period crossover study evaluated the pharmacokinetics (PK) of the ß2 -agonist indacaterol maleate and the corticosteroid mometasone furoate (MF) after inhalation of a fixed-dose combination (QMF149, indacaterol maleate/MF, 500/400 µg) via the Twisthaler (TH) device with and without activated charcoal and postdose mouth rinsing in healthy volunteers. The PK of indacaterol maleate 300 µg inhaled via the Breezhaler (BRZ) device was also characterized. Relative bioavailability of indacaterol and MF for inhalation with versus without charcoal, based on AUClast, was 0.25 (90% confidence interval [CI], 0.18-0.35) and 0.70 (90%CI, 0.52-0.93), respectively. Thus, 25% and 70% of systemic exposure of indacaterol and MF, respectively was due to pulmonary absorption and 75% and 30%, respectively, was due to gastrointestinal absorption. Mouth rinsing reduced the systemic exposure of indacaterol by approximately 35% but had no relevant effect on the exposure of MF. Dose-normalized AUClast for indacaterol inhaled via the BRZ device was 2.3-fold higher than QMF149 via the TH device. All treatments had a good safety profile and were well tolerated. Data from this study and comparison with inhalation of indacaterol via the BRZ device suggest that the latter was more efficient than the TH device regarding lung delivery of indacaterol.

Pharmacokinetics of indacaterol and mometasone furoate delivered alone or in a free or fixed dose combination in healthy subjects.

PURPOSE: QMF149 is a fixed-dose combination of the long-acting ß2 agonist, indacaterol and the corticosteroid, mometasone furoate that is currently under development for treatment of patients with asthma and chronic obstructive pulmonary disease. We describe here a study designed to assess any pharmacokinetic (PK) and/or biopharmaceutical interaction between indacaterol and mometasone furoate when administered via the Breezhaler(®) device, either alone or in a free or fixed combination (QMF149) in healthy adult subjects. METHODS: In this randomized, open-label, four-way crossover study, subjects were randomized to receive indacaterol acetate 150 µg, mometasone furoate 320 µg, alone and as free combination of the individual components, or QMF149 (indacaterol acetate 150 µg/mometasone furoate 320 µg) once daily for 14 days in each period, followed by a 7-day washout between periods. PK profiles were characterized on Day 14 up to 168 h post-dose. RESULTS: Indacaterol AUC0-24h,ss and Cmax,ss after administration of QMF149 were 13% [ratio: 1.13; 90%CI: 1.09, 1.17] and 18% [ratio: 1.18; 90%CI: 1.12, 1.25] higher, respectively, than indacaterol monotherapy. Mometasone furoate AUC0-24h,ss and Cmax,ss after administration of QMF149 were 14% [ratio: 1.14; 90%CI: 1.09, 1.20] and 19% [ratio: 1.19; 90%CI: 1.13, 1.26], higher, respectively than mometasone furoate monotherapy. The majority (three of four comparisons between QMF149 and monotherapy) of the 90% confidence intervals of the between-treatment ratios for AUC0-24h,ss and Cmax,ss were within the 0.80 to 1.25 interval and therefore fulfilled bioequivalence criteria. The 90% confidence interval for Cmax,ss for MF for the QMF149 vs. monotherapy comparison was [1.13, 1.26]. Although no definitive data can be provided on the basis of the present study results, it is unlikely that the small observed differences in expsoure are clinically meaningful. Multiple inhaled doses of indacaterol and mometasone furoate, when administered alone, in free combination or as QMF149 were well tolerated. CONCLUSIONS: The QMF149 fixed dose combination treatment showed comparable systemic exposure to the free combination and monotherapy treatments in terms of AUC0-24h,ss and Cmax,ss for both indacaterol and mometasone furoate, indicating an absence of clinically relevant PK or biopharmaceutical interactions. These data support further development of QMF149 without dose adjustment.

Pharmacokinetics of Glycopyrronium Following Repeated Once-Daily Inhalation in Healthy Chinese Subjects.

BACKGROUND AND OBJECTIVES: Glycopyrronium is a once-daily long-acting muscarinic antagonist for the maintenance treatment of patients with chronic obstructive pulmonary disease. This study assessed the pharmacokinetics of inhaled glycopyrronium 50 µg once-daily for 14 days in healthy Chinese subjects. METHODS: In this open-label study, 12 Chinese healthy subjects (six males and six females; mean age 23.1 years [range 18-26 years]) were enrolled and completed the study. Glycopyrronium in plasma was determined using validated liquid chromatography-mass spectrometry method with a lower limit of quantification of 1.5 pg/mL. Plasma pharmacokinetic parameters were determined on Day 1 after first dose and on Day 14 (steady state) after last dose using non-compartmental analysis. Trough pharmacokinetic samples (Days 5, 7, 10 and 12) were collected. Safety was also assessed. RESULTS: Glycopyrronium was rapidly absorbed into the systemic circulation after inhalation and its plasma concentrations decreased rapidly thereafter. Median time to reach maximum concentration (T max) was reached within 5 min after inhalation on both Days 1 and 14. Accumulation in the systemic exposure to glycopyrronium was observed from the time of first dose administration on Day 1 up to Day 14 and the observed accumulation ratio (R acc) values of area under the plasma drug concentration-time curve [AUC] from time 0 to 24 h post-dose (AUC0-24h) and maximum plasma drug concentration (C max) (Day 14/Day 1) were 2.77 and 1.59, respectively. The elimination half-life (T 1/2) was not reported. Mean effective half-life (T 1/2,acc) was 37.7 h. Pharmacokinetic steady state was reached after 5 days of daily dosing. One subject experienced dry mouth; otherwise glycopyrronium was well tolerated. CONCLUSIONS: Comparison of systemic exposure to glycopyrronium in Chinese versus the non-Chinese population did not indicate clinically relevant ethnic differences. Multiple inhaled doses of glycopyrronium were safe and well tolerated.

Pharmacokinetics of QMF149 in Japanese versus Caucasian subjects: an open-label, randomized phase I study.

OBJECTIVES: This study aimed to evaluate influence of ethnic factors on the pharmacokinetics of orally inhaled QMF149, a novel combination of an approved longacting ß2-agonist, indacaterol (Onbrez® Breezhaler® for COPD), and an approved inhaled corticosteroid, mometasone furoate (MF), (Asmanex® Twisthaler® for asthma), following multiple dose administration of QMF149 (indacaterol acetate/MF) 150/80 µg and 150/320 µg via the Breezhaler® device in healthy Japanese and Caucasian subjects. METHODS: This was a single-center, openlabel, multiple-dose, two-period, complete crossover study that randomized healthy Japanese and, age and weight matched Caucasian subjects to QMF149 150/80 µg or 150/320 µg once daily (o.d.) for 14 days in each period. Pharmacokinetics (PK) were assessed up to 24 hours on days 1 and 14. RESULTS: 24 Japanese and 24 Caucasian healthy subjects were enrolled. Indacaterol and MF had similar PK profiles across both the doses and both ethnic groups. The maximum geometric mean ratios (90% confidence interval (CI)) for Japanese vs. Caucasian subjects for Cmax were 1.23 (1.11 - 1.38) and 1.24 (1.11 - 1.38) for indacaterol and MF, respectively. For AUC, the maximum ratios were 1.22 (1.09 - 1.36) and 1.30 (1.18 - 1.44) for indacaterol and MF, respectively. The mild trend towards higher exposure in Japanese subjects could be explained by the fact that the mean body weight was 14% higher for Caucasians compared to their Japanese counterparts. No serious adverse events or discontinuations related to study medication were reported. CONCLUSION: The study demonstrated increase of mean exposure parameters in Japanese subjects vs. Caucasian subjects, which ranged between 19 - 23% and 17 - 30%, for indacaterol and MF components, respectively. Multiple doses of both the QMF149 dose levels were safe and well-tolerated in all subjects. Body weight was considered a key contributory factor for the observed difference in exposure. These results suggest no dose adjustment for QMF149 is required in Asian populations.

Glycopyrronium does not affect QT interval in healthy subjects: a randomized, three- period, cross-over, placebo- and positive-controlled study.

OBJECTIVE: Glycopyrronium (NVA237), a once-daily long-acting muscarinic antagonist, has recently been approved for the treatment of patients with chronic obstructive pulmonary disease (COPD). This study evaluated the effect of glycopyrronium on the QT interval and other cardiac parameters in healthy subjects. METHODS: This randomized, partially blinded, single-dose, placebo- and positive- (moxifloxacin) controlled, three-way cross-over study investigated the effect of a single inhaled supra-therapeutic dose (8-fold clinical dose in COPD patients) of 400 µg glycopyrronium on the Fridericia-corrected QT interval (QTcF; primary objective), Bazettcorrected QT interval (QTcB), heart rate, blood pressure, pharmacokinetics (PK), safety, and tolerability. RESULTS: A total of 73 healthy male (n = 35) and female (n = 38) subjects, aged between 18 and 45 years, were randomized. Glycopyrronium did not cause significant QTcF prolongation compared to placebo. The largest time-matched mean difference to placebo was 2.97 ms at 5 minutes, with the upper limit of the two-sided 90% confidence interval (CI) being 4.80 ms, excluding a relevant QT effect as defined by the ICH E14 guideline. Glycopyrronium had a slight bradycardic effect with a mean change of -2.88 (90% CI: -3.78, -1.99) beats per minutes (bpm) and a maximum of -5.87 (90% CI: -7.82, -3.92) bpm at 5 hours post-inhalation. No clinically relevant effects were seen on QTcB, other electrocardiogram (ECG) intervals, or blood pressure. Maximum plasma concentration (Cmax) of glycopyrronium was achieved shortly after inhalation (median tmax = 7 minutes). All the treatments were well tolerated with no serious adverse events. CONCLUSION: A supra-therapeutic dose of glycopyrronium had a favorable cardiovascular safety profile with no clinically relevant effect on QT interval.

Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors.

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

Effect of dual bronchodilation with QVA149 on cardiac safety in healthy volunteers.

OBJECTIVES: QVA149 is a dual bronchodilator, containing a fixed-dose combination of the long-acting ß2-agonist indacaterol and long-acting muscarinic antagonist glycopyrronium, for the treatment of chronic obstructive pulmonary disease (COPD). Here we assess the potential of QVA149 (440/200 µg) at 4-fold the therapeutic dose for causing cardiac pharmacodynamic (PD) effects. METHODS: This double-blind, randomized study estimated the time-matched largest heart rate (HR) change and average HR change (over 24 hours) from baseline for QVA149 vs. placebo in healthy subjects. Similar analyses were done for QVA149 vs. indacaterol 600 µg, glycopyrronium 200 µg, and salmeterol 200 µg. The time-matched and average change from baseline in QT interval corrected for HR using Fridericia's formula (QTcF), effects on serum potassium and blood glucose, pharmacokinetic (PK) parameters, and safety were also assessed. RESULTS: Of 50 subjects randomized, 43 completed the study. QVA149, when compared with placebo, showed the time-matched largest mean increase and decrease in HR of 5.69 bpm and -2.51 bpm, respectively, and average HR change from baseline of 0.62 bpm. QVA149 showed no tachycardic potential compared with indacaterol and no relevant tachycardic effect compared with glycopyrronium. No consistent differences were seen in the time-matched largest mean change and average change from baseline in QTcF for QVA149 vs. other treatments. There were no relevant effects of QVA149 on serum potassium and blood glucose. There was no apparent PK/PD relationship between the observed exposures to indacaterol and glycopyrronium in QVA149 on HR and QTcF. There were no deaths or serious adverse events. CONCLUSION: Overall, short-term administration of QVA149 showed a good cardiovascular safety and tolerability profile in healthy subjects.

Recommendations from the Global Bioanalysis Consortium Team A8: documentation.

The A8 Global Harmonization Team focused on the documentation needed to support both small and large molecule bioanalysis. Current regulatory requirements were compiled and compared. The scope of the team's discussions included the validation report, the bioanalytical report, study plans, raw data, and bioanalytical summaries for the common technical document (CTD). A common high-level table of contents for method validation and sample analysis reports is proposed. Suggestions have been made as to how the CTD can be standardized to improve usability and review. Additional comments have been made on reports of failure investigations, study plans, and raw data documentation. The recommendation is that no prescriptive guidelines are required in these areas but should be led by good scientific practices subject to particular circumstances.

Systemic pharmacokinetics of indacaterol, an inhaled once-daily long-acting beta2-agonist, in different ethnic populations.

RATIONALE: To assess ethnic sensitivity of indacaterol systemic pharmacokinetics in Japanese vs. non-Japanese patients. METHODS: Analyses were in three parts: data from a single "all Asian" clinical study; and two on pooled data - one using a linear mixed effects (LME) model and the other a non-linear mixed effects (NLME) model. The NLME model analyzed pharmacokinetic data from nine indacaterol studies; the LME model analyzed peak (C(max)) and trough (C(min)) serum concentration using data from four of these studies. RESULTS: In the all-Asian study, indacaterol serum concentration-time pharmacokinetic profiles in Japanese patients (n = 102) were similar to those in the overall population (n = 229). In the LME model, C(max) (4,392 observations, 1,845 patients) and C(min) (4,664 observations, 1,796 patients) for Japanese patients (n = 94) were on average 25% and 18% higher, respectively, than non-Japanese patients. However, after adjusting for study differences, this apparent ethnicity effect was not significant (p = 0.25 and 0.39, respectively). In the NLME model (25,540 observations, 2,857 patients), there was no statistically significant effect of Japanese (n = 230) ethnicity on indacaterol serum pharmacokinetics. CONCLUSION: No ethnicity effect was observed on indacaterol systemic pharmacokinetic profile for Japanese patients when compared with the overall Asian patient population or with the Caucasian patient population.

Development and validation of a method for quantitative determination of valsartan in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of valsartan in human plasma was developed and validated. A 0.5 ml aliquot was extracted using solid-phase extraction in an Empore high performance extraction disk plate, universal resin 96-well format. The estimated calibration range of the method was 2-2000 ng/ml. The method was fully validated with intra-day mean accuracy and precision of 94.8-107% and 2.19-5.40% and inter-day mean accuracy and precision of 93.5-105% and 1.87-5.67%, respectively. No significant loss of valsartan in processed samples was confirmed in processed samples for up to 24 h at 10 degrees C. Sample dilution up to 50-fold with blank human plasma provided acceptable analyses. No interference peaks or matrix effects were observed. No effect of QC sample location results was observed in a 96-well plate. This LC-MS/MS technique was found to improve quantitative determination of valsartan allowing its pharmacokinetic evaluation with clinically relevant doses.

Simultaneous analytical method for the determination of TCH346 and its four metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

TCH346 (dibenzo[b,f]oxepin-10-ylmethyl-prop-2-ynylamine) is a novel propargylamine compound under investigation as a putative agent in the treatment of chronic neurodegenerative illnesses. To support clinical studies an analytical method was developed for TCH346 plus its three amine metabolites and a carboxylic acid metabolite in human plasma. Using a two-step liquid-liquid extraction, one under acidic and one under basic conditions, by pH-switching both the basic and acidic analytes were extracted from 0.5 mL of plasma. All these basic and acidic compounds could be analyzed simultaneously using gradient high-performance liquid chromatographic (HPLC) separation with positive/negative selected reaction monitoring mass spectrometry. As a result of the validation study, the analytical method was shown to be appropriate for the determination of TCH346 and its metabolites CGP70861, GP42120, CGP71090, and GP54840 in plasma for forthcoming clinical studies. The LLOQs were set to 2, 200, 20, 20, and 200 pg/mL for TCH346, CGP70861, GP42120, CGP71090, and GP54840, respectively, and the ULOQ for all analytes was 20 000 pg/mL. All analytes were stable in 50% MeOH at 4 degrees C for at least one year, in human plasma stored below -70 degrees C for at least 7 months, in human plasma below -18 degrees C for at least 6 months, in human plasma at room temperature for at least 1 day, and in the final extract solution at 4 degrees C for at least 3 days.