The introduction of small-molecule tyrosine kinase VEGFR2 (vascular endothelial growth factor receptor) inhibitors has added another dimension in the treatment of several oncology indications as they offer a unique mechanism. The VEGFR2 inhibitors have demonstrated superior benefits in treating certain types of cancer, such as renal cell carcinoma and hepatocellular carcinoma, as a monotherapy option. Many of the approved VEGFR2 inhibitors have also shown promise when used in combination with other anticancer agents. There are numerous bioanalytical methods published for the analysis of VEGFR2 inhibitors in preclinical and clinical samples. This review covers VEGFR2 inhibitors such as sunitinib, sorafenib, pazopanib and JI-101. In addition to providing a comprehensive review of the available methods for the above-mentioned VRGFR2 inhibitors, it also provides information on assays that can simultaneously measure multiple tyrosine kinase inhibitors, including VEGFR2 molecules. Based on the review, the published methodologies using LC/MS-MS or HPLC-UV are adequate for the quantification of the VEGFR2 inhibitors and can easily be established in a modern day bioanalytical laboratory. The availability of a plethora of assays for multiple tyrosine kinase inhibitors makes it easy to analyze a panel of compounds to support either therapeutic drug monitoring and/or clinical pharmacokinetics.

1. An 'open access' generic high-performance liquid chromatography method was developed for different combination sets each containing specific cytochrome P450 probe substrate and the corresponding metabolite. Method development, optimization and validation were carried out with the following combinations: phenacetin + paracetamol + internal standard (IS, celecoxib), bufuralol + hydroxybufuralol + IS, testosterone + 6beta-hydroxytestosterone + IS, chlorzoxazone + 6-hydroxychlorzoxazone + IS, coumarin + 7-hydroxycoumarin + IS, tolbutamide + hydroxytolbutamide + IS, and diazepam + desmethyldiazepam + IS. 2. The assay procedure involved a simple one-step liquid/liquid extraction followed by reverse phase chromatography (Inertsil ODS 3V column) employing a ternary gradient system and the eluate was monitored by a photodiode array/fluorescence detector. The standard curve for each compound, in the concentration range 0.1-10 microg ml(-1), in various sets was linear (r(2)>0.99) and absolute recoveries of all analytes were >90%. The lower limit of quantification was 0.1 microg ml(-1). The intraday precision and accuracy in the measurements of quality control were <15% relative standard deviation and <15% deviation from nominal values, respectively. 3. Each combination set was tested with individual chemical inhibitors (furafylline, quinidine, ketoconazole, disulfiram, diethyldithiocarbamate, sulphaphenazole and tranylcypromine) and all analytes were well resolved. Overall, the assay is simple, uses conventional instrumentation and provides a scope to analyse all cytochrome P450 combination sets continuously. The application of the method in the cytochrome P450 liability screen of novel compounds is also presented.

Bendamustine is an alkylating agent administered as 1 h intravenous infusion in the clinic for the treatment of malignant haematological cancers. The aim of the study was to evaluate the pharmacokinetics of bendamustine and its key cytochrome P 450 (CYP) 1A2 mediated γ-hydroxybendamustine (M3) metabolite after 30- and 60-min intravenous infusion of bendamustine in rats. 2 groups were assigned to receive bendamustine either as 30- or 60-min infusion and doses were normalized to 15 mg/kg for the sake of statistical evaluation. Serial pharmacokinetic samples were collected and were analysed for the circulatory levels of bendamustine and its M3 metabolite. Standard pharmacokinetic parameters were generated for bendamustine and its M3 metabolite. Regardless of the intravenous regimens, Cmax coincided with end of infusion for both bendamustine and its M3 metabolite. Immediately after stoppage of infusion, a rapid decline in the plasma levels occurred for both bendamustine and M3 metabolite. The Cmax and AUC0-∞ parameters for bendamustine after 60-min infusion were 1.90 and 1.34-fold higher; while CL was lower by 1.32-fold as compared to the 30-min infusion. In contrast, the Cmax and AUC0-∞ after 30-min infusion for the M3 metabolite was 2.15- and 2.78-fold greater; while CL was 2.32-fold lower when compared to the 60-min infusion. However, T1/2 and Vz values were similar between the 2 intravenous treatments for bendamustine or the M3 metabolite. The data unequivocally confirmed the existence of differential pharmacokinetics of bendamustine and its M3 metabolite as the function of the duration of intravenous infusion.

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A valproic acid is primarily being used in the treatment of epilepsy is a histone deacetylase inhibitor and it is under investigation for treatment of HIV and various cancer indications. A specific, sensitive and fast bioanalytical LC-MS/MS method was developed with furosemide as an internal standard (IS) and thoroughly validated for the quantitation of valproic acid using turbo ion spray in negative ion mode. The analyte and IS was extracted using protein precipitation. The chromatographic separation of analytes from extracted matrix was achieved using a Chromolith RP 18e (2.0×50 mm) column with a gradient mobile phase comprising of acetonitrile and purified water with acetic acid. The elution of both peaks was achieved within 5.2 min, with retention times of 2.55 min and 1.67 min for valproic acid and IS, respectively. Quantitation of valproic acid was achieved by the pseudo SRM transition pairs ( 142.8→ 142.8), and SRM transition pair ( 328.8 → 204.6) for internal standard.The calibration standards of valproic acid showed linear over a range from 50 to 40 000 ng/mL, with a lower limit of quantitation of 50 ng/mL with accuracy of 3.74% and precision of 5.06%. The bias for inter- and intra-batch assays was 1.24-6.14% and 3.85-11.84%, respectively; while the corresponding precision was 2.56-16.37% and 1.29-11.34%, respectively. The developed method was used to monitor valproic acid levels in clinical samples. Because of higher sensitivity, this method can be used for therapeutic drug monitoring in pediatric subjects.