Jul 2026· Journal of Applied Pharmaceutical Research· Vol 14, pp. 247-256· 0 citations
TL;DR
A reliable, sensitive, and selective liquid chromatography-tandem mass spectrometry method suitable for high-throughput pharmacokinetic studies and therapeutic drug monitoring of ALB in human plasma is developed and validated.
Abstract
Background: To enhance efficacy and manage dose-limiting toxicities such as hyperglycemia, therapeutic drug monitoring of alpelisib (ALB), a phosphatidylinositol 3-kinase alpha (PI3Kα) inhibitor used in advanced breast cancer, is essential. This study aimed to develop and validate a selective, sensitive, and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of ALB in human plasma to support pharmacokinetic studies and therapeutic drug monitoring. Methodology: Alpelisib-D3 was used as a deuterated internal standard (IS). Analytes were extracted from 50 µL of plasma by liquid-liquid extraction using n-hexane and methyl tert-butyl ether (20:80, v/v). Chromatographic separation was performed on an X-Terra RP8 column using an isocratic mobile phase of 5 mM ammonium acetate buffer (pH 5.00) and acetonitrile (10:90, v/v) at 0.4 mL/min. Detection was carried out in positive electrospray ionization mode using multiple reaction monitoring of m/z 442.15→141.07 for ALB and m/z 445.36→144.05 for the IS. Validation included linearity, precision, accuracy, recovery, matrix effect, carryover, dilution integrity, and stability. Results and discussion: The method was linear over 50.0–10,000 ng/mL. Intra- and inter-day precision (CV%) was <4.2%, and accuracy ranged from 94.5% to 106.2%. Mean extraction recovery exceeded 90%, with no significant matrix effects. Carryover, stability, and dilution integrity also met acceptance criteria. Conclusion: A reliable, sensitive, and selective LC-MS/MS method was successfully developed and validated. The method is suitable for high-throughput pharmacokinetic studies and therapeutic drug monitoring of ALB in human plasma.
Introduction Ensartinib is a second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small cell lung cancer (NSCLC). However, its in vivo disposition and metabolic characteristics, particularly those of its major metabolite M465, remain incompletely understood. This study aimed to develop and validate a sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous quantification of ensartinib and M465 in rat plasma and to apply the method to a pharmacokinetic study in rats. Methods Crizotinib was used as the internal standard (IS). Plasma samples were processed by one-step protein precipitation with acetonitrile. Chromatographic separation was achieved on a Waters Acquity BEH C18 column using gradient elution with 0.1% formic acid in water and acetonitrile. The method was validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), precision, accuracy, recovery, matrix effect, stability, carry-over and dilution integrity. The validated method was subsequently applied to determine plasma concentrations of ensartinib and M465 after oral administration of ensartinib to rats. Results The method showed good linearity over the concentration ranges of 0.1–100 ng/mL for ensartinib and 0.05–10 ng/mL for M465, with LLOQs of 0.1 ng/mL and 0.05 ng/mL, respectively. The intra- and inter-day precision values ranged from 2.2% to 16.4%, and the accuracy ranged from −12.3% to 8.0%, meeting the acceptance criteria for bioanalytical method validation. Recovery, matrix effect, stability, carry-over and dilution integrity were all within acceptable limits. After oral administration of ensartinib to rats, the maximum plasma concentrations (Cmax) of ensartinib and M465 were approximately 64.7 ng/mL and 6.3 ng/mL, respectively. The AUC(0–t) values of ensartinib and M465 were 577.6 ng/mL*h and 32.5 ng/mL*h, respectively, indicating lower systemic exposure of M465 compared with the parent drug. Conclusion A sensitive, reliable, and rapid UPLC–MS/MS method was successfully developed and validated for the simultaneous determination of ensartinib and M465 in rat plasma. The method was successfully applied to a rat pharmacokinetic study and provides useful analytical support for further preclinical investigations of ensartinib and its major metabolite.
Unknown authors· Drug Design, Development and...· 0 citations
Bosutinib is a second-generation tyrosine kinase inhibitor (TKI) primarily used for the treatment of chronic myeloid leukemia (CML). The aim of this study was to develop and validate a rapid and accurate ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of bosutinib and its major metabolite M5. Chromatographic separation was performed on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using a gradient elution with acetonitrile and 0.1% formic acid as the mobile phase, and gefitinib was used as the internal standard (IS). Biological analysis showed that both bosutinib (2-400 ng/mL) and its metabolite M5 (1-60 ng/mL) exhibited good linearity in their respective concentration ranges, with correlation coefficients (r2) of 0.996 and 0.993, respectively. Both of them exhibited intra- and inter-day precision (RSD%) below 15% and accuracy (RE%) within ± 15%. Recoveries were all within the range of 85.3-98.0%, and matrix effects were all within the range of 91.6-101.6%. Ultimately, human liver microsomes (HLM) was used to study in vitro metabolic stability, while in vivo pharmacokinetics were evaluated in rats. In vivo pharmacokinetic studies confirmed that this method could be successfully used for the analysis of bosutinib after oral administration of 50 mg/kg in rats. In vitro results showed that bosutinib had the intrinsic clearance (CLint) value of 0.03 mL/min/mg and the half-life (t1/2) value of 79.94 min. This study provides a potential reference for the clinical investigation of bosutinib.
Peiqi Wang, Yan Chen, Jun Wu et al.· Chemico-Biological Interacti...· 0 citations
A sensitive and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of the anti-COVID-19 agents molnupiravir (MPV), its active metabolite N-hydroxycytidine (NHC), and baricitinib (BARI) in human plasma. As MPV is rapidly converted to NHC in vivo, whereas BARI is predominantly eliminated unchanged, NHC was generated in vitro by alkaline hydrolysis to enable accurate determination of the active metabolite. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column using methanol and 0.1% formic acid in water (90:10, v/v). Escitalopram was selected as the internal standard owing to its consistent extraction recovery, and satisfactory chromatographic performance. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate, yielding recoveries of 87.5-91.6% for MPV, 87.5-89.4% for NHC, and 87.9-94.9% for BARI. The method was validated according to FDA bioanalytical guidelines over concentration ranges of 20-500, 20-10.000, and 20-500 ng/mL for MPV, NHC, and BARI, respectively. Accuracy, precision, stability, recovery, carryover, and matrix effect met the acceptance criteria. Normalized matrix factors for MPV, NHC, and BARI ranged from 0.948-0.984, 0.900-0.902, and 0.912-1.014, respectively, with CV values below 1%, indicating negligible matrix interference. The proposed method provides a reliable analytical tool for future pharmacokinetic and therapeutic drug monitoring studies.
Fatma H. Ghazy, Ramzia I. El-Bagary, Samar H. Fahim et al.· Bioanalysis· 0 citations
Aficamten (CK-3773274) is a next-in-class selective cardiac myosin inhibitor now approved for use for chronic oral therapy in hypertrophic cardiomyopathy (HCM). The present study established an innovative, rapid, and highly sensitive HPLC-MS/MS approach for quantifying aficamten concentrations in human plasma and urine. Samples were prepared using protein precipitation with acetonitrile containing CK-3942912 (D5-labelled aficamten) as the internal standard (IS). Sample separation was achieved using a C18 analytical column, with a mobile phase comprising acetonitrile and ammonium acetate. A Xevo TQS tandem mass spectrometer performed the detection in electrospray ionization (ESI) positive mode. The following MRM ion transitions were monitored: m/z 338.2 → 213.2 for CK-3773274 and m/z 343.2 → 218.2 for IS. This method was validated with excellent selectivity and sensitivity. Linear calibration curves (1-500 ng/mL) demonstrated excellent accuracy (RE% ±15%) and precision (RSD% ≤15%) for both intra- and inter-run analyses. Furthermore, critical validation parameters including matrix effects, extraction recovery, carryover, and stability were also assessed. The validated method was subsequently utilized to quantify aficamten concentrations in a clinical pharmacokinetic study involving healthy Chinese adults.
Xiao-Fei Wu, Aijing Liu, Adrienne Griffith et al.· Journal of chromatography. B...· 0 citations
A robust and highly sensitive LC-MS/MS method was developed and validated for the quantification of diosgenin in mice plasma in accordance with ICH M10 guidelines. Chromatographic separation was achieved using Kinetex C8 column (50 x 2.1 mm, 5 µm) with 0.1% formic acid in distilled water and methanol as mobile phase at a flow rate of 0.6 mL/min, and the run time was 17 min. Detection was performed in positive ESI+ mode using MRM at m/z 415.10 → 271.20, employing sarsasapogenin as the internal standard. Protein precipitation yielded efficient extraction with minimal matrix interference. The method exhibited excellent linearity over 3.125-1000 ng/mL (r2 = 0.996) with an LLOQ of 3.125 ng/mL. The accuracy during within-day and between-day evaluations ranged from 91.7% to 110.99%, and precision (%CV) remained below 5.14%. Dilution integrity indicated that the analyte can be reliably quantified in biological samples up to 24,000 ng/mL. The stability studies confirmed that the analyte was stable under various conditions. Further, this validated method was applied for pharmacokinetic studies in female mice via oral (10mg/kg) and intramuscular (5mg/kg) administration. Overall, the method is reliable for trace-level quantification and demonstrated its applicability in pharmacokinetic, bioavailability, and formulation studies of diosgenin.
The first validated liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalytical method for the quantification of atirmociclib in mouse plasma is reported, validated in compliance with international council for harmonisation-M10 (ICH-M10) guidelines.
Ashwani Gaur, U. Patel, Ashutosh Gavali et al.· Journal of chromatography. B...· 0 citations
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