Validation of high-performance liquid chromatography-tandem mass spectrometry assays quantifying omacetaxine mepesuccinate and its 4'­des-methyl and cephalotaxine metabolites in human plasma and urine.

Omacetaxine mepesuccinate (hereafter called omacetaxine) is a modified cephalotaxine and is registered (Synribo(®)) for the treatment of adult patients with chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs). To evaluate the pharmacokinetics of omacetaxine, sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for the quantification of omacetaxine and its inactive 4'-des-methyl (4'-DMHHT) and cephalotaxine metabolites in human plasma and urine were developed and validated. Since omacetaxine is mainly metabolised by esterases, the plasma samples were immediately stabilised after collection with an esterase inhibitor and stored at a nominal temperature of -80°C. Urine samples were stored at -80°C immediately after collection. Protein precipitation was applied as the sample pretreatment method for the plasma samples, and urine samples were processed using solid-phase extraction (SPE). For both assays, the dried and reconstituted extracts were injected on a XBridge BEH Phenyl column for analysis of all analytes. Gradient elution was applied with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionised using a turbospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer. The validated plasma assay quantifies all analytes in the concentration range of 0.1-100ng/mL and the urine assay in the range of 0.1-50ng/mL. At all concentrations, the accuracies were within ±15% of the nominal concentrations and precisions were ≤15%. The developed methods have successfully been applied in a human mass balance study of omacetaxine.

Development and validation of LC-MS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine.

A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay is described for the quantification of the anti-cancer agent bendamustine and its phase I metabolites γ-hydroxy-bendamustine (M3) and N-des-methylbendamustine (M4) and for its product of two-fold hydrolysis, dihydroxy-bendamustine (HP2), in human plasma and urine. Like most alkylating nitrogen mustards, bendamustine is prone to chemical hydrolysis in aqueous solution. To minimize degradation of bendamustine, urine samples were stabilized by a 100-fold dilution with human plasma and then processed identically to plasma samples. Sample aliquots of 200 µL were mixed with an internal standard solution and acidified before separation of the analytes from the biomatrix with solid phase extraction. Dried and reconstituted extracts were injected on a Synergi Hydro RP column for the analysis of bendamustine, M3 and M4 or a Synergi Polar RP column for the analysis of HP2. Gradient elution was applied using 5mM ammonium formate with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionized using an electrospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer. The quantifiable range for bendamustine, M3 and M4 was 0.5-500 ng/mL in plasma and 0.5-50 µg/mL in urine, and that for HP2 was 1-500 ng/mL in plasma and 0.1-50 µg/mL in urine. The assays were accurate and precise, with inter-assay and intra-assay accuracies within ± 20% of nominal and CV values below 20% at the lower limit of quantification and within ± 15% of nominal and below 15% at the other concentration levels tested. These methods were successfully applied to evaluate the pharmacokinetic profile of bendamustine and its metabolites in cancer patients treated with bendamustine.

Determination of the D- and L-enantiomers of modafinil in human plasma utilizing liquid-liquid extraction and high-performance liquid chromatography.

Modafinil, DL-2-[(diphenylmethyl)sulfinyl]acetamide (Provigil), which is chiral at its sulfur atom, is a novel wake-promoting agent currently being developed as the racemate in the United States by Cephalon, Inc. In order to characterize the pharmacokinetic properties of each enantiomer, a stereospecific high-performance liquid chromatography (HPLC) method has been developed for simultaneous determination of D- and L-modafinil in human plasma. The analytes are extracted from plasma into a mixture of hexane-methylene chloride-triethylamine (55:45:2, v/v/v) and then resolved on an EM Separations ChiraDex beta-cyclodextrin column at 12 degrees C using an isocratic mobile phase of 0.020 M, pH 3.0 phosphate buffer-acetonitrile (84:14, v/v). D- and L-modafinil, and the internal standard, 3,3-diphenylpropylamine, are monitored by UV detection at 225 nm. The two major circulating metabolites, modafinil acid and modafinil sulfone, have been shown not to interfere with the assay. Using 0.200 ml of plasma for extraction, the quantifiable range of the assay is 0.100 to 15.0 microg/ml for each enantiomer. The utility of the assay for the characterization of D- and L-modafinil pharmacokinetics in humans after single and multiple oral doses of racemic modafinil has been demonstrated.