Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples

Abstract An HPLC method has been developed and validated for the determination of spironolactone, 7a-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 µl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30–1000 ng/ml for spironolactone and 25–1000 ng/ml for 7a-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7a-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7a-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 mu L), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes. (c) 2006 Elsevier B.V. All rights reserved.

The development and validation of liquid chromatography method for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma

This article describes the development of SPE and HPLC methods for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma. Several extraction and HPLC methods have been described previously for the determination of each of these analytes in plasma separately. The simultaneous determination of these analytes is important for the routine monitoring of diabetic patients who take combination medications and for studying the pharmacokinetics of the combined dosage forms. In addition this developed method can serve as a standard method for the plasma determination of these analytes therefore saving time, effort and money. The recoveries of the developed methods were found to be between 76.3% and 101.9%. The limits of quantification were between 5 and 22.5 ng/ml. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error %) was always less than 12%. Stability analysis showed that all analytes are stable for at least 3 months when stored at -70degreesC. (C) 2004 Elsevier B.V. All rights reserved.

Facile Syntheses of Three Major Metabolites of Thioridazine

NMR studies were conducted with the aim of determining the diastereoisomeric ratio of a commercially supplied sample of mesoridazine (MES) and to compare the results with a freshly synthesised sample of MES. The results indicated that the commercially supplied MES consisted almost entirely of one diastereoisomeric pair, which was in agreement with previous findings reported by Eap et al. (J Chromatogr 669:271-279, 1995). The synthesised sample of MES was analysed by NMR in two stages: 1) as the initial product isolated as the free base from the direct synthesis, and 2) as the free base isolated from the crystallised besylate salt of the synthetic product. The NMR results show that the initial synthetic product consisted of two equal pairs of diastereoisomers. The diastereoisomeric pairs were further separated by the addition of the chiral shift reagent (R)-(-)-N-(3,5 dinitrobenzoyl)-alpha-benzylamine to reveal equal quantities of all four enantiomers, clearly observed at the methyl sulfoxide proton peak of the NMR scan. The sample obtained from the crystallisation of MES besylate, however, indicated a significant difference, with a diastereoisomeric ratio of 75:25. The results suggest that MES besylate undergoes preferential crystallisation of one pair of diastereoisomers, with the other pair remaining in solution. (C) 2004 Wiley-Liss, Inc.

Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45 min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95 pmol/8 x 101 RBCs and 2, 5, 2, 3, 20 ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200 mu l of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients. (C) 2008 Elsevier B.V. All rights reserved.

Population pharmacokinetic and pharmacogenetic analysis of 6-mercaptopurine in paediatric patients with acute lymphoblastic leukaemia

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT center dot The cytotoxic effects of 6-mercaptopurine (6-MP) were found to be due to drug-derived intracellular metabolites (mainly 6-thioguanine nucleotides and to some extent 6-methylmercaptopurine nucleotides) rather than the drug itself. center dot Current empirical dosing methods for oral 6-MP result in highly variable drug and metabolite concentrations and hence variability in treatment outcome. WHAT THIS STUDY ADDS center dot The first population pharmacokinetic model has been developed for 6-MP active metabolites in paediatric patients with acute lymphoblastic leukaemia and the potential demographic and genetically controlled factors that could lead to interpatient pharmacokinetic variability among this population have been assessed. center dot The model shows a large reduction in interindividual variability of pharmacokinetic parameters when body surface area and thiopurine methyltransferase polymorphism are incorporated into the model as covariates. center dot The developed model offers a more rational dosing approach for 6-MP than the traditional empirical method (based on body surface area) through combining it with pharmacogenetically guided dosing based on thiopurine methyltransferase genotype. To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and examine the effects of various genetic polymorphisms on the disposition of these metabolites. Data were collected prospectively from 19 paediatric patients with ALL (n = 75 samples, 150 concentrations) who received 6-MP maintenance chemotherapy (titrated to a target dose of 75 mg m(-2) day(-1)). All patients were genotyped for polymorphisms in three enzymes involved in 6-MP metabolism. Population pharmacokinetic analysis was performed with the nonlinear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance for the active metabolites. The developed model revealed considerable interindividual variability (IIV) in the clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a significant part of 6-TGNs clearance IIV when incorporated in the model (IIV reduced from 69.9 to 29.3%). The most influential covariate examined, however, was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest reduction in the model's objective function (P