Background/Objectives: This study aimed to develop a fully validated HPLC-MS/MS method for quantifying total and unbound lenalidomide concentrations in human plasma. Methods: Unbound concentrations were measured using plasma ultrafiltrate prepared with Amicon^® Centrifugal Filters. Lenalidomide and lenalidomide-d5 (internal standard) were extracted from 50 μL of human plasma using liquid–liquid extraction. Chromatography was conducted with a Halo^® C18 column using 0.1% formic acid and methanol (20:80, v/v) as the mobile phase. The mass spectrometer was operated in a positive ion mode with an electrospray ionization interface and multiple reaction monitoring modes. Results: Calibration curves were linear over the range of 5 to 1000 ng/mL (r² > 0.996) for both the total and unbound lenalidomide. For total lenalidomide concentrations, between-run precision (coefficients of variation) and accuracy were 1.70–7.65% and 94.45–101.10%, respectively. For unbound concentrations, inter-day precision and accuracy were 1.98–10.55% and 93.95–98.48%, respectively. Conclusions: We developed a highly reproducible, sensitive, and efficient bioanalytical method using a smaller volume of plasma sample (50 μL) with a relatively short run time (2.5 min). The proposed analytical method was successfully applied to measure total and unbound lenalidomide concentrations at various time points in multiple myeloma patients with renal impairment.

This study developed a simple, rapid, reproducible, and analytical method using liquid chromatography and electrospray ionization (ESI) with tandem mass spectrometry (LC-MS/MS) to simultaneously quantify pentoxifylline (PTX), its pharmacological active metabolites, lisofylline (PTX-M1) and 1-(3-carboxypropyl)-3,7-dimethylxanthine (PTX-M5), and donepezil (DNP) in rat plasma, using PTX-d6 and DNP-d7 as the internal standards. The LC-MS/MS procedure was performed at the ESI interface, operating in positive ionization and multiple reaction monitoring (MRM) modes; the monitoring of transitions comprised m/z 279.3 > 181.1 for PTX, m/z 281.1 > 263.1 > 160.90 for PTX-M1, m/z 267.1 > 249.0 > 220.9 for PTX-M5, m/z 380.3 > 90.9 for DNP, m/z 285.3 > 187.1 for PTX-d6 (IS1), and m/z 387.3 > 98.3 for DNP-d7 (IS2). After plasma protein precipitation (PP) with methanol, chromatographic separation was performed with an Imtakt Cadenza^® CD-C18 (100 × 3 mm, 3 µm) column, using an isocratic mobile phase consisting of 0.1% formic acid in water and methanol (20:80, v/v) at a flow rate of 0.2 mL/min. The retention times of DNP, PTX-M5, PTX, and PTX-M1 were 2.24, 2.50, 2.68, and 2.72 min, respectively, with a total run time of 5 min. This method was validated over a linear concentration range of 5–8000, 10–5000, 20–15,000, and 2–500 ng mL⁻¹ for PTX, PTX-M1, PTX-M5, and DNP, respectively, with a high correlation coefficient (r² ≥ 0.99). The established method was fully validated in terms of selectivity, the lower limit of quantitation, precision, accuracy, recovery, matrix effect, stability, and dilution integrity according to the regulatory guidelines from the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety. The validated method was successfully applied to a pharmacokinetic study on the concurrent administration of DNP and PTX in rats.

A highly accurate, precise, and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for ketotifen (KTF) estimation from Beagle dog plasma was developed and validated, with ketotifen-d3 (KTF-d3) as the internal standard (IS). KTF and IS were detected on an API 4000 mass spectrometer in multiple reaction monitoring (MRM) mode in electrospray ionization (ESI) positive ionization mode. The transitions were monitored at m/z 310.2 → 96.0 for KTF and m/z 313.2 → 99.1 for IS. KTF and IS were extracted from plasma using liquid-liquid extraction with methyl tertiary-butyl ether and then analyzed for 3 min with extracted samples (7 µL) into the LC–MS/MS system. Analytes were separated on a Luna^® Hilic column (50 × 2.0 mm i.d., 3 μm) using the Nexera X2 HPLC. The mobile phase A consisted of 10 mmol/L ammonium formate (pH 3.0), while mobile phase B consisted of 0.05% formic acid in acetonitrile. The ratio of mobile phase was 5:95 (v/v) at a flow rate of 0.2 mL/min. The method has been thoroughly validated in accordance with the bioanalytical method validation guidelines established by the Ministry of Food and Drug Safety in Korea and the U.S. Food and Drug Administration, addressing selectivity, lower limit of quantification, linearity, carryover, precision, accuracy, recovery, matrix effect, and stability. The developed LC–MS/MS method was effectively utilized for the bioequivalence assessment of ketotifen in Beagle dog plasma following the oral administration of ketotifen syrup.

Sitagliptin is an orally bioavailable selective DPP4 inhibitor that reduces blood glucose levels without significant increases in hypoglycemia. The aim of this study was to design and validate an innovative, rapid, and highly sensitive LC–MS/MS assay for the precise measurement of sitagliptin concentrations in human plasma. This analytical method, utilizing sitagliptin-d4 as the internal standard, is performed using only 100 μL of plasma and a liquid–liquid extraction procedure based on methyl tert-butyl ether (MTBE). Chromatographic separation is expertly achieved with a Kinetex^® C18 column under isocratic elution, employing a perfect 1:1 blend of 5 mM ammonium acetate (with 0.04% formic acid) and acetonitrile, and maintaining an efficient flow rate of 0.2 mL/min. Detection occurs in positive ionization mode through multiple reaction monitoring, precisely targeting transitions of m/z 408.2 → 193.0 for sitagliptin and 412.2 → 239.1 for the IS. The total runtime of this assay is under 2 min. Comprehensive validation in line with MFDS and FDA criteria demonstrates outstanding linearity (5–1000 ng/mL, r² > 0.998), alongside impressive levels of accuracy, precision, recovery and sample stability. Due to its minimal sample requirement and high-throughput capability, the validated approach is highly appropriate for pharmacokinetic and bioequivalence assessments involving sitagliptin.

Background/Objectives: Selinexor is a selective nuclear-export inhibitor approved for hematologic malignancies, characterized by extensive plasma protein binding (>95%). However, a validated analytical method to accurately measure the clinically relevant unbound fraction of selinexor in human plasma has not yet been established. This study aimed to develop a fully validated bioanalytical assay for simultaneous quantification of total and unbound selinexor concentrations in human plasma. Methods: We established and fully validated an analytical method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) capable of quantifying total and unbound selinexor concentrations in human plasma. Unbound selinexor was separated using ultrafiltration, and selinexor was efficiently extracted from 50 μL of plasma by liquid–liquid extraction. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase (0.1% formic acid:methanol, 12:88 v/v) with a relatively short runtime of 2.5 min. Results: Calibration curves showed excellent linearity over a range of 5–2000 ng/mL for total selinexor (r² ≥ 0.998) and 0.05–20 ng/mL for unbound selinexor (r² ≥ 0.995). The precision (%CV ≤ 10.35%) and accuracy (92.5–104.3%) for both analytes met the regulatory criteria. This method successfully quantified selinexor in plasma samples from renally impaired patients with multiple myeloma, demonstrating potential inter-individual differences in unbound drug concentrations. Conclusions: This validated bioanalytical assay enables precise clinical pharmacokinetic assessments in a short runtime using a small plasma volume and, thus, assists in individualized dosing of selinexor, particularly for renally impaired patients with altered protein binding.

Background/Objectives: Colistin is a complex polymyxin antibiotic with a narrow therapeutic window and significant interindividual pharmacokinetic variability, necessitating precise concentration monitoring. Current analytical methods often utilize colistin mixtures or require large sample volumes, potentially limiting the precision and resolution of individual component quantification. This study aimed to develop a sensitive and component-specific bioanalytical assay for the simultaneous quantification of colistin A and colistin B in human plasma. Methods: A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed using pure, component-specific reference standards to ensure rigorous independent quantification of each component. Analytes were efficiently extracted from a small volume of plasma (50 µL) using solid-phase extraction. Chromatographic separation was achieved on a C18 column with a total runtime of 4 min, and detection was performed using negative-ion multiple reaction monitoring (MRM). Results: Calibration curves showed excellent linearity over a range of 0.1–20 µg/mL for both colistin A and B (R² > 0.99). The precision (%CV ≤ 8.8%) and accuracy (86.4–105.7%) for both components met the predefined regulatory criteria. This method was clinically validated using 60 plasma samples from 15 patients, demonstrating its applicability for capturing individual concentration–time profiles within the clinically relevant range (0.323–19.579 µg/mL for colistin A and 0.065–6.132 µg/mL for colistin B). Conclusions: This validated bioanalytical assay enables precise clinical pharmacokinetic assessments in a high-throughput workflow using a small plasma volume. Therefore, it serves as a practical tool for therapeutic drug monitoring (TDM)-guided dose optimization and further clinical investigations of colistin therapy.