Developing LC-MS/MS methods to quantify rivaroxabanin human plasma and urine: Application to therapeutic drug monitoring.

Rivaroxaban is an oral anticoagulant directly inhibiting the activity of Factor Xa, which is widely used for the prophylaxis of thromboembolic disorders. Therapeutic drug monitoring (TDM) is required during therapy for individual dosage adjustment. This study aimed at developing a liquid chromatography/tandem mass spectrometry method that was suitable for rivaroxaban TDM in human plasma and urine and exploring the feasibility of urine drug monitoring in medical care. A 3 min run time of the LC-MS/MS methods was established by employing an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column using gradient elution of 10 mmol/L ammonium acetate containing 0.1% formic acid-0.1% formic acid acetonitrile as a mobile phase at a flow rate of 0.4 ml/min with calibration ranges of 0.5-400 and 10-10,000 ng/ml for human plasma and urine, respectively. Rivaroxaban was detected on a triple quadrupole tandem mass spectrometer with an electrospray ionization source in positive ion mode. The methods showed good linearity within the calibration range. The precision and accuracy, matrix effect, extraction recovery and stability in both human matrices were all validated and meet the international guideline requirements. These validated methods were successfully applied to support the TDM of an aged patient receiving rivaroxaban for therapy.

In Silico Modeling and Simulation to Guide Bioequivalence Testing for Oral Drugs in a Virtual Population.

Model-informed drug discovery and development (MID3) shows great advantages in facilitating drug development. A physiologically based pharmacokinetic model is one of the powerful computational approaches of MID3, and the emerging field of virtual bioequivalence is well recognized to be the future of the physiologically based pharmacokinetic model. Based on the translational link between in vitro, in silico, and in vivo, virtual bioequivalence study can evaluate the similarity and potential difference of pharmacokinetic and clinical performance between test and reference formulations. With the aid of virtual bioequivalence study, the pivotal information of clinical trials can be provided to streamline the development for both new and generic drugs. However, a regulatory framework of virtual bioequivalence study has not reached its full maturity. Therefore, this article aims to present an overview of the current status of bioequivalence study, identify the framework of virtual bioequivalence studies for oral drugs, and also discuss the future opportunities of virtual bioequivalence in supporting the waiver and optimization of in vivo clinical trials.

Accelerating Development of Benziamidazole-Class Proton Pump Inhibitors: A Mechanism-Based PK/PD Model to Optimize Study Design with Ilaprazole as a Case Drug.

Proton pump inhibitors (PPIs) are the mainstay for treatment of acid-related diseases. This study developed a mechanism-based pharmacokinetic (PK) and pharmacodynamics (PD) model with ilaprazole as case drug, so as to support and accelerate the development of novel PPIs. The model was established and verified using the PK and PD data from 26 subjects receiving 5 to 30 mg of ilaprazole and 22 subjects receiving the loading dose of ilaprazole 20 mg followed by 10 mg once daily for 2 days. The nonlinear mixed-effects modeling approach was performed for the PK/PD model. A two-compartment model with linear elimination and covariates (body weight and gender) described the observed data well. The relationship between plasma concentrations of ilaprazole and gastric acid pH was well quantified with individual variability, in which the synthesis and degradation of H+/K+-ATPase, the food effect, the circular rhythms of gastric acid secretion, and the irreversible inhibition of H+/K+-ATPase by ilaprazole were integrated. This PK/PD model well predicted the PK and PD profile of ilaprazole in healthy subjects and patients with duodenal ulcers receiving wide range dose regimens. The mechanism-based PK/PD model provided a potential strategy to accelerate the development of novel PPIs by waiving the unnecessary clinical trials.

Development of a rapid and sensitive UPLC-MS/MS assay for simultaneous quantitation of Vorolanib and its metabolite in human plasma and application to a pharmacokinetics study.

Vorolanib is an oral tyrosine kinase inhibitor that targets vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). A sensitive and specific LC-MS/MS assay was developed and fully validated for simultaneous quantification of vorolanib and its main metabolite X297 in human plasma. The two analytes were extracted from K2-EDTA plasma samples by protein precipitation (PP) with acetonitrile, and chromatographically separated on a C18 reverse-phase column using a gradient elution. A SCIEX 5500 QTRAP® mass spectrometer system was operated in multiple-reaction monitoring mode (MRM) and all components were detected using positive electrospray ionization (ESI). The results successfully demonstrated that the method had satisfactory linearity, sensitivity, and selectivity in the concentration ranges of vorolanib (1.00-1000 ng/mL) and X297 (0.500-500 ng/mL). In this study, two concentration related peaks in the vorolanib and X297 detection channels were observed, which were speculated to be isomers of vorolanib and X297. In order to standardize the sample pretreatment process, the effect of lamp light and pH on the isomer reconversion was evaluated. The results indicated, that the exposure of samples to lamp light during the handling procedures, did not cause the conversion of the isomers. For the first time a robust and specific ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the high-throughput quantification of vorolanib and X297 in human plasma was established and validated following bioanalytical validation guidelines. The proposed method was successfully applied to clinical trials evaluating the pharmacokinetics of vorolanib tablets in Chinese advanced solid tumor patients.

Simultaneous determination of indapamide, perindopril and its active metabolite perindoprilat in human plasma using UPLC-MS/MS method.

Lots of studies showed the combination therapy of perindopril, indapamide and amlodipine could increase BP lowering efficacy and the benefits of high-risk patients. To evaluate potential pharmacokinetic interaction, a simultaneous UPLC-MS/MS quantification method of perindopril, perindoprilat and indapamide in human plasma was developed and validated. The plasma samples were prepared by solid phase extraction, and then separated on an X-terra MS C18 (2.1 mm × 150 mm, 3.5 µm) with isocratic elution. The ion transitions at m/z 369.165 â†’ 172.000 (perindopril), m/z 341.146 â†’ 170.112 (perindoprilat), m/z 366.010 â†’ 132.100 (indapamide), m/z 389.120 â†’ 206.200 (S10211-1, IS1) and m/z 394.080 â†’ 160.200 (S1641, IS2) were monitored under the positive ion mode of electrospray ionization with multiple reaction monitoring. This method exhibited great sensitivity, linearity, accuracy, and precision for the determination of perindopril, perindoprilat and indapamide over the range of 0.250-50.0 ng/mL. The average extraction recovery of perindopril, perindoprilat and indapamide samples at low, medium, and high concentration levels were between 85.9% and 93.6%, respectively. The stability of analytes over different storage and processing conditions in the whole study was also validated. The method is fast, accurate, sensitive and reproducible, which is suitable for the detection of the concentration of perindopril, perindoprilat and indapamide in human plasma.

Development of an LC-MS/MS method for quantifying ASK120067, a novel mutant-selective inhibitor of the epidermal growth factor receptor (EGFR) as well as its main metabolite in human plasma and its application in a pharmacokinetic study.

ASK120067, an oral irreversible tyrosine kinase inhibitor (TKI) targeting the epidermal growth factor receptor (EGFR), is formulated for the management of patients with non-small cell lung cancer (NSCLC) who harbor T790M resistant and EGFR active mutations. Two rapid and high-throughput methods based on liquid chromatography-tandem mass spectrometry to detect ASK120067 and its primary metabolite CCB4580030 in human plasma were developed and applied in the clinical trials. A protein precipitation method using acetonitrile coupled with a gradient elution separation in a BEH C18 column (1.7 µm, 2.1 × 50 mm) was used to process plasma and separation analytes. The chromatographic separation was performed on the mobile phase of 5 mM ammonium acetate in water with 0.1% formic acid (A) and acetonitrile (B), and the flow rate was 0.4 mL/min. The multiple reaction monitoring (MRM) mode was selected to monitor the precursor-to-product ion transitions of m/z 546.2 â†’ m/z 431.2 for ASK120067 and m/z 532.1 â†’ m/z 420.2 for CCB4580030 at the positive ionization mode. The precision and accuracy of the two methods for ASK1200067 and CCB4580030 were within acceptable range for the linear range in 5.00-5000 ng/mL and 0.500-500 ng/mL, respectively. Further stabilities for the two analytes and internal standard were also investigated covered the entire experimental process beginning from harvesting whole blood to plasma extraction and analysis. ASK120067 was then administered without issue onto a dose-escalation, the first-in-human Phase I clinical trial in Chinese NSCLC patients to determine the pharmacokinetics of oral ASK120067 administration.

Development of an LC-MS/MS method for quantifying two main metabolites of abivertinib in human plasma.

Abivertinib represents a highly selective irreversible epidermal growth factor receptor tyrosine kinase inhibitor. Two major metabolites of abivertinib, M7 and MII-6, were detected in human plasma, which are recommended to be monitored for safety reasons in clinical trial. A high-throughput quantification method utilizing liquid chromatography-tandem mass spectrometry was designed and verified to quantify abivertinib's primary metabolites in human plasma. Solid-phase extraction was used to process the plasma, and then the analytes underwent a gradient elution separation in an Aquity UPLC BEH C18 column (1.7 µm, 2.1 × 50 mm) with mobile phase A (10 mm ammonium acetate containing 0.1% formic acid) and mobile phase B (methanol-acetonitrile, 2:8, v/v, with 0.1% formic acid). Ion transitions of M7 (m/z 490.2 → 405.1) and MII-6 (m/z 476.2 → 391.1) were monitored under multiple reaction monitoring mode and electrospray ionization in positive ion mode. This simultaneous determination method was found to have acceptable precision, accuracy and linearity in the 0.5-500 ng/mL range for M7 and the 0.5-500 ng/mL range for MII-6, accompanied by a mild matrix effect but high recovery. Further stability assessments indicated that both analytes remained stable throughout the entire experimental process from harvesting whole blood to plasma extraction and analysis.