Validation of an LC-MS/MS assay for rapid and simultaneous quantification of 21 kinase inhibitors in human plasma and serum for therapeutic drug monitoring.

Kinase inhibitors have revolutionized cancer treatment in the past 25 years and currently form the cornerstone of many treatments. Due to the increasing evidence for therapeutic drug monitoring (TDM) of kinase inhibitors, the need is growing for new assays to rapidly evaluate kinase inhibitor plasma concentrations. In this study, we developed an LC-MS/MS assay for the rapid and simultaneous quantification of 21 kinase inhibitors. First, a literature search was conducted to ensure that the linear ranges of the analytes were in line with the reported therapeutic windows and/or TDM reference values. Subsequently, the assay was validated according to FDA and EMA guidelines for linearity, selectivity, carry-over, accuracy, precision, dilution integrity, matrix effect, recovery, and stability. The assay was fast, with a short run-time of 2 min per sample. Sample pre-treatment consisted of protein precipitation with methanol enriched with stable isotope-labeled internal standards (SIL-IS), and the mixture was vortexed and centrifuged before sample injection. Separation was achieved using a C18 column (3 µm,50 × 2.1 mm) with a gradient of two mobile phases (ammonium formate buffer pH 3.5 and acetonitrile). Analyte detection was conducted in positive ionization mode using selected reaction monitoring. The assay was accurate and precise in plasma as well as in serum. Extraction recovery ranged between 95.0% and 106.0%, and the matrix effect was 95.7%-105.2%. The stability of the analytes varied at room temperature and in refrigerated conditions. However, all drugs were found to be stable for 7 days in the autosampler. The clinical applicability of the analytical method (486 analyzed samples between 1 July 2022-1 July 2023) as well as external quality control testing results were evaluated. Taken together, the results demonstrate that the analytical method was validated and applicable for routine analyses in clinical practice.

A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects.

Aim: The aim of this study was to develop and validate a LC-MS/MS assay for tacrolimus, sirolimus, everolimus, cyclosporin A and mycophenolic acid using volumetric absorptive microsampling tips as a sampling device and to investigate the effect on the recoveries of the analyte concentration in combination with the hematocrit (HT), which included temsirolimus (a structural analog). Results: The maximum observed overall bias was 9.6% for the sirolimus LLOQ, while the maximum overall coefficient of variation was 8.3% for the everolimus LLOQ. All five immunosuppressants demonstrated to be stable in the volumetic absorbtive microsampling tips for at least 14 days at 25°C. Biases caused by HT effects were within 15% for all immunosuppressants between HT levels of 0.20 and 0.60 l/l, except for cyclosporin A, which was valid between 0.27 and 0.60 l/l. Reduced recoveries were observed at high analyte concentrations in combination with low HT values for sirolimus, everolimus and temsirolimus. Conclusion: A robust extraction and analysis method in volumetric absorptive microsampling tips was developed and fully validated. HT- and concentration-related recovery effects were observed but were within requirements of the purpose of the analytical method.

Quantification of co-trimoxazole in serum and plasma using MS/MS.

BACKGROUND: Co-trimoxazole is frequently used in the prophylaxis and treatment of Pneumocystis carinii pneumonia. High plasma concentrations of sulfamethoxazole or trimethoprim are correlated with toxicity. There is, however, a large variation in PK observed which can lead to underexposure or toxicity. RESULTS: We developed a novel LC-MS/MS method to analyze the components of co-trimoxazole, trimethoprim and sulfamethoxazole and its metabolite sulfamethoxazole-N-acetyl. This new method is expeditious due to its limited sample preprocessing and a relatively short run-time of only 3 min. CONCLUSION: This new method met the US FDA requirements on linearity, selectivity, precision, accuracy, matrix effects, recovery and stability and is suitable for routine analysis and future prospective studies.

Quantification of amikacin and kanamycin in serum using a simple and validated LC-MS/MS method.

BACKGROUND: Amikacin and kanamycin are frequently used in the treatment of multidrug-resistant TB. The current commercially available immunoassay is unable to analyze kanamycin and trough levels of amikacin. The objective was therefore to develop a LC-MS/MS method for the quantification of amikacin and kanamycin in human serum. MATERIALS & METHODS: Using apramycin as internal standard, selectivity, accuracy, precision, recovery, matrix effects and stability were evaluated. RESULTS: The presented LC-MS/MS method meets the recommendations of the US FDA with a low LLOQ of 250 ng/ml for amikacin and 100 ng/ml for kanamycin. No statistical significant difference was found between the LC-MS/MS method and the immunoassay of amikacin (Architect(®) assay, p = 0.501). CONCLUSION: The low LLOQ of amikacin and the ability to analyze kanamycin makes the LC-MS/MS method the preferred method for analyzing these aminoglycosides.