September 2008

A simple, accurate and sensitive HPLC method was developed for measuring total and unbound mycophenolic acid (MPA) in human plasma. Total MPA was extracted by protein precipitation and ultrafiltration was used to assess unbound MPA concentrations. The supernatant (20 µL) or ultrafiltrate (100 µL) was injected onto a C18 HPLC column with a mobile phase of 0.05 m sodium phosphate buffer (pH 2.31)-acetonitrile (55:45, v/v for total MPA; 50:50 for unbound MPA) with UV detection at 254 nm. The extraction recovery was over 93% and reproducible. The assay was linear over the concentration range of 0.07-50 mg/L for total MPA and 4-1500 µg/L for unbound MPA. Intra- and inter-day assay reproducibility was less than 10%. Detection limits were 0.04 mg/L and 2 µg/L for total and unbound MPA, respectively. The assay utility was established in samples collected from five paediatric bone marrow transplant recipients who were receiving intravenous doses of mycophenolate mofetil. In these patients MPA concentrations ranged from 0.07 to 7.83 mg/L and unbound drug concentrations ranged from 2.1 to 107.5 µg/L. This method can be effectively applied to MPA pharmacokinetics in paediatric patients. Copyright © 2008 John Wiley & Sons, Ltd.

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The flavonoid content of several methanolic extract fractions of Navel orange peel (flavedo and albedo of Citrus sinensis) cultivated in Crete (Greece) was first analysed phytochemically and then assessed for its antioxidant activity in vitro. The chemical structures of the constituents fractionated were originally determined by comparing their retention times and the obtained UV spectral data with the available bibliographic data and further verified by detailed LC-DAD-MS (ESI+) analysis. The main flavonoid groups found within the fractions examined were polymethoxylated flavones, O-glycosylated flavones, C-glycosylated flavones, O-glycosylated flavonols, O-glycosylated flavanones and phenolic acids along with their ester derivatives. In addition, the quantitative HPLC analysis confirmed that hesperidin is the major flavonoid glycoside found in the orange peel. Interestingly enough, its quantity at 48 mg/g of dry peel permits the commercial use of orange peel as a source for the production of hesperidin. The antioxidant activity of the orange peel methanolic extract fractions was evaluated by applying two complementary methodologies, DPPH[bull] assay and the Co(II)/EDTA-induced luminol chemiluminescence approach. Overall, the results have shown that orange peel methanolic extracts possess moderate antioxidant activity as compared with the activity seen in tests where the corresponding aglycones, diosmetin and hesperetin were assessed in different ratios. Copyright © 2008 John Wiley & Sons, Ltd.

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Two methods are presented for the determination of ‘respectively’ the plasma protein unbound and total concentration of acyclovir in horse plasma and body fluids: first, a liquid-liquid extraction was performed on plasma, combined with HPLC-fluorescence detection for the total plasma concentration; second a more sensitive method using high-performance liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) was described for plasma and for body fluids analysis. To obtain the unbound concentration of acyclovir in plasma, a simple deproteinization step using a Microcon® filter was performed. Ganciclovir was used as an internal standard. Analysis was carried out on an Inertsil 5 ODS-3 column for the HPLC-fluorescence method. For the LC-HESI-MS/MS method a PLRP-S column was used. The limit of quantification (LOQ) for the total concentration was set at 50 and 2 ng mL-1 for the HPLC-fluorescence method and the LC-HESI-MS/MS method, respectively. The limit of quantification for the unbound concentration was set at 5 ng mL-1 and at 2 ng mL-1 for body fluids. The methods were successfully used to perform pharmacokinetic and clinical studies in horses after intravenous and oral dosage of acyclovir and its prodrug valacyclovir. Copyright © 2008 John Wiley & Sons, Ltd.

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A sensitive, selective and reliable method has been developed and validated for the determination of halofantrine and its metabolite desbutylhalofantrine in rat plasma using 9,10-diphenylanthracene as an internal standard. The method is based on peroxyoxalate chemiluminescence detection of hydrogen peroxide produced from fused aromatic rings in the structures of halofantrine and desbutylhalofantrine upon UV irradiation. Using spiked rat plasma, good linear relationships were obtained for both halofantrine and desbutylhalofantrine between peak height ratios (vs internal standard) and their corresponding concentrations over a range of 0.01-0.8 µg/mL with correlation coefficients of at least 0.997. The detection limits at signal-to-noise ratio of 3 using 0.2 mL of rat plasma were 1.5 and 1.4 ng/mL for halofantrine and desbutylhalofantrine, respectively. Relative standard deviations (n = 3) intra- and inter-day were between 0.5 and 5.4% for all the studied concentrations. Using this method with simple sample treatment, halofantrine and desbutylhalofantrine in rat plasma could be precisely determined without interference from endogenous substances. The method was successfully applied to the measurement of the time courses of plasma halofantrine concentration after oral administration of the drug (7 mg/kg) to rats. Copyright © 2008 John Wiley & Sons, Ltd.

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A selective, rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for assay of donepezil in human plasma using escitalopram as an internal standard. Chromatographic separation was achieved on a Betabasic-C8, 5 µm, 100 × 4.6 mm column using methanol:water:formic acid (90:9.97:0.03, v/v/v) as mobile phase. Detection of donepezil and internal standard was achieved by ESI MS/MS in positive ion mode using 380.20/91.10 and 325.13/262.00 transitions, respectively. The linearity over the concentration range of 0.15-50 ng/mL for donepezil was obtained and the lower limit of quantification was 0.15 ng/mL. For each level of quality control samples, inter-day and intra-day precisions (RSD) were [le]8.92 and 10.35% and accuracy (%RE) were [le]7.33% and 9.33%, respectively. The recovery was more than 88.50% for both donepezil and internal standard by solid-phase extraction, eliminating evaporation and reconstitution steps. Copyright © 2008 John Wiley & Sons, Ltd.

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A novel assay for the determination of l-asparaginase activity in human plasma is described that is based on the HPLC quantitation of l-aspartic acid produced during enzyme incubation. Methods for monitoring l-asparagine depletion are also described. Chromatography of l-aspartic acid, l-asparagine and l-homoserine (the internal standard) involved derivatization with o-pthaldialdehyde, then separation from other amino acids on a Phenomenex Luna C18 column using a 1 mL/min flow rate and a mobile phase consisting of di-potassium hydrogen orthophosphate propionate buffer, pH 6, with 10% methanol and 10% acetonitrile. Fluoresence detection was at excitation/emission wavelengths of 357/455 nm. Under these conditions l-aspartic acid, l-asparagine and l-homoserine had retention times of 3.5, 9.8 and 17.7 min, respectively. The l-asparaginase assay was linear from 0.1 to 10 U/mL activity and interday precision and accuracy were less than 13%. The limit of quantitation was approximately 0.03 U/mL. The assay utility was established in 12 children who received E. coli l-asparaginase as treatment for acute lymphoblastic leukaemia. Copyright © 2008 John Wiley & Sons, Ltd.

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New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform. Copyright © 2008 John Wiley & Sons, Ltd.

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