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 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.

Vitamin E-stabilised UHMWPE for orthopaedic implants:quantitative determination of vitamin E and characterisation of its transformation products

The fate of vitamin E and the formation and identification of its transformation products were investigated at different stages of the manufacturing process of commercially produced cross-linked (by γ-irradiation) UHMWPE stabilised with vitamin E (vitamin E infused-post irradiation) used for tibia-components (as articulating surfaces) in total knee arthroplasty (total knee replacement). Vitamin E (α-tocopherol) and its transformation products were extracted from microtomed Tibia films and the different products were separated, isolated, purified using high performance liquid chromatography (HPLC), and characterised by spectroscopic methods and LC-MS. The amount of vitamin E and that of the products formed in the different Tibia samples and in their extracts were also quantified using FTIR and HPLC analysis and calibration curves. Thorough analysis of the Tibia extracts has shown that a number of vitamin E transformation products were formed at different concentrations at two selected stages of the implant manufacturing process that is before and after sterilisation by γ-irradiation. The identified products were found to correspond mainly to different stereoisomeric forms of a small number of vitamin E transformation products. Most of the observed products were of dimeric and trimeric nature with their identity confirmed through a detailed study of their spectral and chromatographic characteristics. It was found that the products of vitamin E, prior to the sterilisation step but after the crosslinking and doping of vitamin E, were mainly the dihydroxydimers and trimers (Tibia samples at this stage are referred to as “Tibia-VEPE”). After sterilisation and completion of the manufacturing process, additional dimers of vitamin E were also formed (Tibia samples at this stage are referred to as ‘Tibia-VEPE-Sterile’), Furthermore, two tocopherol-derived aldehydes (aldehyde 5-formyl-γ-tocopherol and aldehyde 7-formyl-γ-tocopherol) were also formed but at very low concentrations especially in the Tibia-VEPE-Sterile samples. The question of whether vitamin E becomes chemically reacted (grafted) onto the polymer matrix during the manufacturing process of the Tibia is also addressed.