Development and validation of a sensitive liquid chromatography tandem mass spectrometry assay for the simultaneous determination of ten kinase inhibitors in human serum and plasma

Abstract

A liquid chromatography tandem mass spectrometry method for the analysis of ten kinase inhibitors (afatinib, axitinib, bosutinib, cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib) in human serum and plasma for the application in daily clinical routine has been developed and validated according to the US Food and Drug Administration and European Medicines Agency validation guidelines for bioanalytical methods. After protein precipitation of plasma samples with acetonitrile, chromatographic separation was performed at ambient temperature using a Waters XBridge® Phenyl 3.5 μm (2.1 × 50 mm) column. The mobile phases consisted of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A and methanol-water (9:1, v/v) with 10 mM ammonium bicarbonate as phase B. Gradient elution was applied at a flow rate of 400 μL/min. Analytes were detected and quantified using multiple reaction monitoring in electrospray ionization positive mode. Stable isotopically labeled compounds of each kinase inhibitor were used as internal standards. The acquisition time was 7.0 min per run. All analytes and internal standards eluted within 3.0 min. The calibration curves were linear over the range of 2–500 ng/mL for afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib, and trametinib, and 6–1500 ng/mL for cabozantinib, dabrafenib, nilotinib, and osimertinib (coefficients of correlation ≥ 0.99). Validation assays for accuracy and precision, matrix effect, recovery, carryover, and stability were appropriate according to regulatory agencies. The rapid and sensitive assay ensures high throughput and was successfully applied to monitor concentrations of kinase inhibitors in patients.

A rapid, simple and sensitive LC-MS/MS method for lenvatinib quantification in human plasma for therapeutic drug monitoring

“…The IS normalized matrix effect was 1.27±0.03 (SD) with a CV% ≤ 2.8% for QCL and 1.09±0.02 (SD) for QCH with a CV% ≤ 2.1. These data are slightly higher than previously published methods based on PP but with a much lower CV% [ 10 , 12 , 13 ]. The obtained CV% values were lower than the guidelines acceptance criteria (<15%) and so the matrix effect was considered negligible in affecting analyses results.…”

“…The percentage of LENVA recovery resulted high, ≥ 95.6% (range from 95.6 to 102, CV% ≤ 4.6), and reproducible over the concentrations ranges tested ( Table 3 ). These percentages are the highest (89.5%) among the published methods ( Table 1 ) based on the same sample treatment (PP) [ 7 , 9 , 12 , 13 ].…”

“…In the literature three other methods were reported for LENVA quantification with other 8, 9 or 2 kinase inhibitors, respectively [ 10 , 12 , 13 ]. These methods overcome the above-mentioned limitations: they required a small sample volume (50/100 μL), had a fast chromatographic run (between 3.5 and 7min) and a simple sample preparation (PP).…”

Ruxolitinib exposure in patients with acute and chronic graft versus host disease in routine clinical practice—a prospective single-center trial

“…Ruxolitinib concentrations were determined using a fully validated liquid chromatography-tandem mass spectrometry method (LC–MS/MS) [ 28 ]. In brief, after protein precipitation with acetonitrile separation was achieved on a Waters XBridge Phenyl 3.5 µm (2.1 × 50 mm) column using gradient elution (flow rate 400 μL/min).…”

An Easily Expandable Multi-Drug LC-MS Assay for the Simultaneous Quantification of 57 Oral Antitumor Drugs in Human Plasma

“…Currently, there is a lack of appropriate methods to determine plasma concentrations for the wide variety of clinically used oral anticancer drugs. Existing methods are limited to the quantification of only a few oral anticancer drugs simultaneously, with a maximum of 17 published [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. Each of these methods requires an individual analytical setup, which makes them suitable for a limited number of drugs only and therefore hampers the use in studies or clinical situations with a wide range of oral anticancer drugs.…”