Simultaneous quantification of 2′,2′‐difluorodeoxycytidine and 2′,2′‐difluorodeoxyuridine nucleosides and nucleotides in white blood cells using porous graphitic carbon chromatography coupled with tandem mass spectrometry

A novel assay for the simultaneous quantification of the widely used anticancer agent 2′,2′‐difluorodeoxycytidine (gemcitabine; dFdC), its deaminated metabolite 2′,2′‐difluorodeoxyuridine (dFdU) and their mono‐, di‐ and triphosphates (dFdCMP, dFdCDP, dFdCTP, dFdUMP, dFdUDP and dFdUTP) in peripheral blood mononuclear cells (PBMCs) is described. Separation of all eight compounds was achieved within 15 min using a porous graphitic carbon column (Hypercarb) with a gradient from 0 to 25 mM ammonium bicarbonate in acetonitrile/water (15:85, v/v). Calibration ranges in PBMC lysate from 4.29 to 429, 29.0 to 2900, 31.4 to 3140 and 36.9 to 3690 nM for dFdC, dFdCMP, dFdCDP and dFdCTP and from 42.1 to 4210, 25.4 to 2540, 43.2 to 4320 and 52.7 to 5270 nM for dFdU, dFdUMP, dFdUDP and dFdUTP, respectively, were validated. Accuracies were within 82.3–119% at the lower limit of quantification (LLOQ) and the precisions were less than 20.0%. At the other tested levels accuracies were within 91.4–114% and precisions less than 14.9%. Mixtures of ¹³C,¹⁵N₂‐labeled dFdC and dFdU nucleotides were synthesized and used as internal standards. Whole blood samples showed extensive ongoing dFdC metabolism when stored at room temperature, but not on ice‐water, which made the addition of enzyme inhibitors unnecessary. Stock solutions and samples were stable under all analytically relevant conditions. The method was successfully applied to clinical samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Biodetermination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan, a metabolite of vintriptol, by high-performance liquid chromatography with fluorescence detection.

The determination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan (vintriptol acid, VtrpA), a metabolite of the investigational semi-synthetic vinca alkaloid vintriptol [N-(deacetyl-O-4-vinblastoyl-23)-L-ethyltryptophan, VtrpE], in plasma and urine samples is described. Sample pretreatment included liquid-liquid extraction of the buffered (pH 5.0) biological samples with chloroform-2-propanol (95:5, v/v). The analyses were performed by ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection. The assay was applied to the analysis of samples from cancer patients who had been treated with VtrpE in a phase I clinical study. VtrpA was found to be a principal metabolite of VtrpE with up to 1.2% of the administered dose excreted in the urine.     

Screw sense of polyisocyanides

From steric considerations as well as from an analysis of CD spectra a (P)-screw configuration could be attributed to the (?)-rotating molecules of poly(t-butyl isocyanide).     

A new,validated HPLC‐MS/MS method for the simultaneous determination of the anti‐cancer agent capecitabine and its metabolites: 5′‐deoxy‐5‐fluorocytidine, 5′‐deoxy‐5‐fluorouridine, 5‐fluorouracil and 5‐fluorodihydrouracil,in human plasma

A rapid and selective liquid chromatography/tandem mass spectrometric method was developed for the simultaneous determination of capecitabine and its metabolites 5′‐deoxy‐5‐fluorocytidine (5′‐DFCR), 5′‐deoxy‐5‐fluorouracil (5′‐DFUR), 5‐fluorouracil (5‐FU) and dihydro‐5‐fluorouracil (FUH₂) in human plasma. A 200 μL human plasma aliquot was spiked with a mixture of internal standards fludarabine and 5‐chlorouracil. A single‐step protein precipitation method was employed using 10% (v/v) trichloroacetic acid in water to separate analytes from bio‐matrices. Volumes of 20 μL of the supernatant were directly injected onto the HPLC system. Separation was achieved on a 30 × 2.1 mm Hypercarb (porous graphitic carbon) column using a gradient by mixing 10 mm ammonium acetate and acetonitrile–2‐propanol–tetrahydrofuran (1 : 3 : 2.25, v/v/v). The detection was performed using a Finnigan TSQ Quantum Ultra equipped with the electrospray ion source operated in positive and negative mode. The assay quantifies a range from 10 to 1000 ng/mL for capecitabine, from 10 to 5000 ng/mL for 5′‐DFCR and 5′‐DFUR, and from 50 to 5000 ng/mL for 5‐FU and FUH₂ using a plasma sample of 200 μL. Correlation coefficients (r²) of the calibration curves in human plasma were better than 0.99 for all compounds. At all concentration levels, deviations of measured concentrations from nominal concentration were between ?4.41 and 3.65% with CV values less than 12.0% for capecitabine, between ?7.00 and 6.59% with CV values less than 13.0 for 5′‐DFUR, between ?3.25 and 4.11% with CV values less than 9.34% for 5′‐DFCR, between ?5.54 and 5.91% with CV values less than 9.69% for 5‐FU and between ?4.26 and 6.86% with CV values less than 14.9% for FUH₂. The described method was successfully applied for the evaluation of the pharmacokinetic profile of capecitabine and its metabolites in plasma of treated cancer patients. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatographic assay for the protease inhibitor atazanavir in plasma

Atazanavir is the most recently introduced protease inhibitor for the suppression of the anti-human immunodeficiency virus. A sensitive and selective reversed-phase liquid chromatographic assay for this drug in human plasma has been developed and validated. Atazanavir was isolated from a 500 microL plasma sample using liquid-liquid extraction with dichloromethane. After evaporation and reconstitution of the extract the sample was analysed using liquid chromatography and ultraviolet detection at 280 nm. In the evaluated concentration range (44-4395 ng/mL atazanavir), intra-day precisions were < or =7% and inter-day precisions were < or =14%. Accuracies between 96 and 106% were found. The lower limit of quantification was 44 ng/mL with an intra-day precision of 7%, an inter-day precision of 14% and an accuracy of 87%. There was no interference from 32 tested potentially co-administrated drugs and metabolites. The usefulness of the assay was demonstrated for samples obtained from an HIV-infected patient treated with atazanavir.     

Quantitative analysis of gemcitabine triphosphate in human peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Gemcitabine triphosphate (dFdCTP) is a highly active metabolite of gemcitabine. It is formed intra-cellularly via the phosphorylation of gemcitabine by deoxycytidine kinase. The monitoring of dFdCTP in human peripheral blood mononuclear cells (PBMCs), in addition to plasma concentrations of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine, is considered very useful in determining pharmacokinetic-pharmacodynamic relationships.We describe a novel sensitive assay for the quantification of dFdCTP in human PBMCs. The method is based on weak anion-exchange liquid chromatography and detection with tandem mass spectrometry (LC-MS/MS). The assay has been validated from 1 ng/ml (lower limit of quantification, LLOQ) to 25 ng/ml (upper limit of quantification, ULOQ) using 180 microl aliquots of PBMC extracts containing approximately 0.648 mg protein or 3.8 x 10(6) lysed PBMCs. The LLOQ is equivalent to 94 fmol/10(6) cells (1 ng/ml = 0.18 ng/180 microl or 0.18 ng/0.648 mg protein = 0.047 ng/10(6) cells or 94 fmol/10(6) cells). This highly sensitive assay is capable of quantifying about 200-fold lower concentrations of dFdCTP in human PBMCs than currently available methods.     

Simultaneous quantification of fludarabine and cyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Fludarabine and cyclophosphamide are anticancer agents mainly used in the treatment of hematologic malignancies. We have developed and validated an assay using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry for the quantification of fludarabine in combination with cyclophosphamide in human heparin and human EDTA plasma. Sample pre-treatment consisted of a protein precipitation with cold acetonitrile (-20 degrees C) using 250 microL of plasma. Separation was performed on an Extend C18 column (150 x 2.1 mm i.d.; 5 microm) with a stepwise gradient using 1 mM ammonia solution and acetonitrile at a flow rate of 400 microL/min. The analytical run time was 12 min. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over a concentration range of 1 to 100 ng/mL for fludarabine and cyclophosphamide in human heparin and human EDTA plasma. The coefficients of variation were <13.9% for inter- and intra-day precisions. Mean accuracies were also within the designated limits (+/-15%). The analytes were stable in plasma, processed extracts and in stock solution under all relevant conditions.     

Validated method for the determination of platinum from a liposomal source (SPI-77) in human plasma using graphite furnace Zeeman atomic absorption spectrometry

A sensitive analytical method based on flameless atomic absorption spectrometry with Zeeman correction has been validated for the quantitative determination in human plasma of platinum originating from cisplatin in a liposomal source, SPI-77. The performance of the method was acceptable over a sample concentration range of 0. 125-1.25 micromol platinum/L and the lower limit of quantification was determined to be 1.25 micromol platinum/L in undiluted clinical samples. The performance data of the assay were investigated using both a calibration curve with carboplatin in plasma ultrafiltrate and diluted human plasma samples spiked with SPI-77. The recoveries, between-day and the within-day precisions of both methods of calibration were not significantly different allowing carboplatin ultrafiltrate calibration standards to be used to quantify platinum derived from SPI-77 in human plasma. Apparently, the liposomal formulation had no significant influence on the determination of platinum. The usefulness of the presented method was demonstrated in a phase I clinical and pharmacokinetic study. In addition, in vitro experiments were carried out to determine the distribution of SPI-77 in blood. The results indicated that platinum from SPI-77 mainly concentrates in plasma and that binding to and/or endocytosis in red blood cells is negligible.     

Determination of total platinum in plasma and plasma ultrafiltrate,from subjects dosed with the platinum-containing N-(2-hydroxypropyl)methacrylamide copolymer AP5280, by use of graphite-furnace Zeeman atomic-absorption spectrometry

AP5280 is an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer to which are linked tetrapeptide side chains containing bioactive platinum complexes at their C-terminal sides. We have developed and validated a rapid and sensitive analytical assay for the determination of total platinum concentrations in plasma, and free platinum of an AP5280 origin in plasma ultrafiltrate (PUF), of subjects dosed with AP5280. The total platinum levels were determined by use of graphite-furnace atomic-absorption spectrometry (GF-AAS) with Zeeman correction after appropriate dilution of the plasma sample with plasma-hydrochloric acid 0.2 mol L(-1) (1:5) as diluent. The limit of quantitation of this assay is 0.25 micromol L(-1) platinum in plasma. Linear calibration curves were obtained over the concentration range 0.25-5.0 micromol L(-1). Accuracy was between 87.7% and 104.2% and precision was 15.3% at the lowest concentration and less than 14% for all other levels tested. Accuracy and precision were thus in accordance with generally accepted criteria for analytical methods. Analysis of samples obtained from patients receiving AP5280 demonstrated the applicability of the described assay.Analysis of free platinum in PUF was performed by use of a previously validated and reported assay from our institute in which the same instrumental method is used.