Validation of an UHPLC–MS/MS Assay for the Quantification of Isoniazid and Four Major Metabolites in Human Plasma

A validated bioanalytical LC-MS/MS method for the simultaneous determination of molnupiravir, N-hydroxycytidine, and baricitinib in human plasma.

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.

Development and validation of an HPLC-MS/MS method for the quantification of Aficamten in human plasma and urine and application to a clinical pharmacokinetic study.

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.

Sustainable UHPLC-MS/MS assay for ultra-sensitive simultaneous quantification of amoxicillin and flucloxacillin with application to a bioequivalence study.

A Simple and Accurate LC–MS/MS Method for the Determination of Indobufen in Human Plasma and Its Clinical Application

As an antithrombotic agent, indobufen is widely used in China. However, the relationship between drug concentration and therapeutic efficacy in specific patient populations remains unclear. Therefore, it is crucial to establish a method for quantifying indobufen concentrations in human plasma to guide dosage recommendations. An electrospray ionization (ESI) power supply was utilized in conjunction with multireaction monitoring (MRM) in positive ion mode. Protein precipitation was employed to extract indobufen from the plasma sample. Separation was carried out using a ChromCORE‐C18 column with a gradient elution of the mobile phase, which was composed of 0.1% formic acid in water (Solvent A) and methanol (Solvent B) at a flow rate of 0.3 mL/min. The typical standard curve equation was Y = 0.763X + 0.0986 (r = 0.9993), demonstrating a good linear relationship within the range of 0.5–30 μg/mL. All methodological outcomes complied with regulatory guidelines. In patients with moderate renal insufficiency, it is advisable to reduce the dosage to 100 mg once daily. For patients with severe renal insufficiency, the dosage should be reduced to 1/3–1/4 of the standard dose. For patients undergoing continuous renal replacement therapy (CRRT), indobufen can be gradually eliminated, and most patients do not require dose adjustment.

Enhanced selectivity in alpelisib quantification in human plasma: a validated LC-MS/MS method with deuterated internal standard

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.

Development and Validation of a Sensitive UHPLC-MS/MS Analytical Method for Fulzerasib in Rat Plasma and its Application to Pharmacokinetics Studies.

Fulzerasib is a novel, potent, and irreversible covalent inhibitor targeting the KRAS G12C mutation, recently approved in China for advanced nonsmall cell lung cancer. This study aimed to develop and fully validate a rapid, specific and sensitive UHPLC-MS/MS method for the quantification of Fulzerasib in rat plasma. Sample preparation was streamlined using a simple protein precipitation technique with acetonitrile. Chromatographic separation was achieved within a 4.0-min run on a C18 column, with verapamil as the internal standard and detection via multiple reaction monitoring. The method was rigorously validated over a linear range of 5 to 5000 ng/mL (R2 > 0.9933), demonstrating excellent accuracy (102%-112.7%) and precision (≤ 10.3% for QCs). Stability was confirmed under various storage and processing conditions. The validated method was successfully applied in the first pharmacokinetic study of Fulzerasib in Sprague-Dawley rats following single intravenous (1 mg/kg) and oral (3 mg/kg) administration. Fulzerasib exhibited favorable pharmacokinetic properties, including low plasma clearance (5.02 mL/min/kg), moderate volume of distribution and notably high oral bioavailability of 38.6%. The established method is reliable, efficient and readily applicable, thereby providing a vital tool for accelerating the ongoing preclinical and translational research of Fulzerasib.