Monitoring of thiopurine metabolites – A high-performance liquid chromatography method for clinical use

A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials. Ten patient samples were analysed to demonstrate clinical application and to establish pilot ranges for all analytes.The method measured thioguanosine mono-(TGMP), di-(TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine).LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8 × 108 RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 4 °C for 8 h after sampling.

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► A novel HPLC method for thiopurine monitoring was developed and validated.
► TGNs and meTINs were measured as intact nucleotides.
► The most sensitive method for thioguanosine monophosphate (TGMP).
► The method was sensitive, simple, cheap, robust and well characterised.
► The method was tested on 10 patient samples to demonstrate clinical application and a pilot range established.