Improved HPLC methodology for monitoring thiopurine metabolites in patients on thiopurine therapy

Clinical Biochemistry
Cristiana StefanZulfikarali Verjee

Abstract

Standardization of thiopurine metabolite testing is currently lacking. This paper presents in-depth methodological analysis and optimization of two currently available HPLC procedures (Lennard-Singleton [J. Chromatogr. 583 (1992) 83] and Dervieux-Boulieu [Clin. Chem. 44 (1998) 551]) to improve precision, turn-around time, ruggedness, and cost effectiveness. Reversed-phase chromatography with UV detection was performed on a Waters HPLC system. The two protocols were improved with regards to internal standardization (IS), chromatographic conditions, as well as reagent preparation, storage, and use. 6-Thioguanine nucleotides (6-TGNs) were analyzed by our optimized techniques in samples from patients on thiopurine therapy (n = 24) and the results were compared. 6-Mercaptopurine (6-MP) was an ideal internal standard in either procedure. Isocratic elution with 5% acetonitrile (ACN) in 20 mmol/l phosphate buffer pH 2.5 allowed for minimal background interference in both protocols. 6-Thioguanine, 6-mercaptopurine, and 6-methylmercaptopurine (6-MMP) eluted at around 4, 5, and 6 min, respectively. Dithiothreitol (DTT) was critical only during the acid hydrolysis step. Less mercury-containing waste was generated in the Lennard-Singleton p...Continue Reading

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Citations

Aug 22, 2006·Alimentary Pharmacology & Therapeutics·L J J DerijksD W Hommes
Jul 23, 2016·Journal of Microbiological Methods·Ramya MovvaIulia Oancea
Mar 8, 2018·Clinical Pharmacokinetics·Luc J J DerijksAdriaan A van Bodegraven
May 7, 2011·American Journal of Health-system Pharmacy : AJHP : Official Journal of the American Society of Health-System Pharmacists·Hamidreza Montazeri AliabadiAfsaneh Lavasanifar
Nov 30, 2005·The Pharmacogenomics Journal·A WoelderinkE Rodriguez-Cerezo
Nov 30, 2006·The Annals of Pharmacotherapy·Myla E MorettiGideon Koren

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