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The analysis of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-mMP) in biological samples is not straight forward and requires pre-treatment of samples. There are no validated published methods for the analysis of azathioprine/6-mercaptopurine (AZA/6-MP) metabolites in dried blood spot (DBS) samples that study the correlation with red blood cells (RBC) concentrations. DBS was prepared by applying fifteen microliters of blood [spiked with analytes or samples obtained from patients] to a Guthrie card which was then dried at room temperature overnight. The sample treatment procedure used protein precipitation followed by a hydrolysis step in which, 6-mMP was converted into 4-amino-5-(methylthio)carbonyl imidazole (AMTCI) then analytes were transferred to a solid phase extraction cartridge. The extracted sample was chromatographed using a reversed phase system (C18) column preceded by a guard column of matching chemistry. The method gave a linear calibration over the range 0.5-15 μmol/L and 3.75-175 μmol/L for 6-TG and 6-mMP, respectively. The method has been applied successfully to the determination of 6-TG and 6-mMP concentrations in DBS finger-prick samples from 27 paediatric patients with IBD who were receiving (AZA/6-MP). Patient 6-TG and 6-mMP RBC concentrations, calculated from whole blood finger prick DBS samples and those measured in RBCs derived from matched venous samples (analyzed using conventional HPLC-UV technique) showed good agreement using the Bland-Altman test. This is the first published method for determining 6-TG and 6-mMP in DBS that studies their correlation with RBCs concentrations. It is applicable to a range of clinical studies such as adherence and pharmacokinetic studies involving AZA/6-MP., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 45min 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, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/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 (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.