Volumetric absorptive microsampling (VAMS) for the quantification of ten kinase inhibitors and determination of their in vitro VAMS-to-plasma ratio.

Personalized dosing of kinase inhibitors (KI) might be beneficial in oral anti-cancer therapy to overcome individual pharmacokinetic variability. Volumetric absorptive microsampling (VAMS) has emerged as an attractive alternative compared to conventional invasive sampling methods enabling remote and frequent specimen collection. Therefore, an LC-MS/MS VAMS method was developed and validated to monitor drug exposure of ten KI from 20 µL dried capillary blood. The assay includes the KI cabozantinib, dabrafenib, nilotinib, and osimertinib with a calibration range of 6-1500 ng/mL and afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib and trametinib within a range of 2-500 ng/mL. Using acetonitrile containing isotope labelled internal standards (IS) as solid-liquid extraction solvent, analytes and IS were detected by multiple reaction monitoring (MRM) after electro-spray ionization (ESI) in positive ionization mode after chromatographic separation using a phenyl-hexyl column. The method was validated according to the FDA and EMA guidelines for bioanalytical method validation and in accordance with the guideline of the International Association for Therapeutic Drug Monitoring and Clinical Toxicology for dried blood spot-based methods. The calibration model was linear and reproducible for all KI (R2> 0.994). Furthermore, the validation demonstrated that the VAMS method is accurate, precise, and sensitive. The method fulfilled the acceptance criteria for matrix effects, recovery, carry over, selectivity as well as for the haematocrit effect and all substances proved to be stable in dried condition for at least six weeks at room temperature. In vitro experiments using spiked venous blood were conducted to establish a VAMS-to-plasma conversion factor for each analyte for comparison of VAMS and plasma concentrations. The method was successfully used in a real-life setting demonstrating its applicability in clinical routine. VAMS concentrations of afatinib, cabozantinib, dabrafenib, nilotinib, ruxolitinib and trametinib were assessed in capillary blood samples collected from either trained healthcare professionals or patients at home.

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

PURPOSE: Knowledge on Ruxolitinib exposure in patients with graft versus host disease (GvHD) is scarce. The purpose of this prospective study was to analyze Ruxolitinib concentrations of GvHD patients and to investigate effects of CYP3A4 and CYP2C9 inhibitors and other covariates as well as concentration-dependent effects. METHODS: 262 blood samples of 29 patients with acute or chronic GvHD who were administered Ruxolitinib during clinical routine were analyzed. A population pharmacokinetic model obtained from myelofibrosis patients was adapted to our population and was used to identify relevant pharmacokinetic properties and covariates on drug exposure. Relationships between Ruxolitinib exposure and adverse events were assessed. RESULTS: Median of individual mean trough serum concentrations was 39.9 ng/mL at 10 mg twice daily (IQR 27.1 ng/mL, range 5.6-99.8 ng/mL). Applying a population pharmacokinetic model revealed that concentrations in our cohort were significantly higher compared to myelofibrosis patients receiving the same daily dose (p < 0.001). Increased Ruxolitinib exposure was caused by a significant reduction in Ruxolitinib clearance by approximately 50%. Additional comedication with at least one strong CYP3A4 or CYP2C9 inhibitor led to a further reduction by 15% (p < 0.05). No other covariate affected pharmacokinetics significantly. Mean trough concentrations of patients requiring dose reduction related to adverse events were significantly elevated (p < 0.05). CONCLUSION: Ruxolitinib exposure is increased in GvHD patients in comparison to myelofibrosis patients due to reduced clearance and comedication with CYP3A4 or CYP2C9 inhibitors. Elevated Ruxolitinib trough concentrations might be a surrogate for toxicity.

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.

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. Graphical abstract.

Quantification of letermovir in human serum using high-performance liquid chromatography with diode array detection.

A simple, rapid, cost-effective and sensitive high-performance liquid chromatography method with diode array detection was developed and validated for the quantification of letermovir, a compound approved for prophylaxis of cytomegalovirus infection and disease in adult recipients of an allogeneic hematopoietic stem cell transplant. Sorafenib was used as internal standard. Samples were pre-treated by liquid-liquid extraction with tert-butyl methylether. Separation was achieved on a XTerra® RP18 column (150 × 2.1 mm, 5 µm) at 30 °C using gradient elution with a mobile phase of 20 mM ammonium bicarbonate pH 7.9 (mobile phase A) and acetonitrile:20 mM ammonium bicarbonate (9:1 v/v) (mobile phase B). Samples were eluted at a flow rate of 0.3 mL/min throughout the 20-min run. UV wavelength mode was used, letermovir and sorafenib were monitored at 260 nm. The calibration curve was linear in a concentration range of 25-5000 ng/mL with correlation coefficients ≥ 0.99. Intra-day and inter-day accuracy expressed as relative error were -11.4-20% and -7.96-10.62%, respectively. Precision expressed as coefficient of variation was 1.44-3.15% (intra-day) and 1.17-1.93% (inter-day). The method was successfully applied for analysis of 128 letermovir levels demonstrating its usefulness for letermovir monitoring in routine clinical practice.