title: Monitoring of thiopurine metabolites - a high-performance liquid chromatography method for clinical use.
abstract: 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.
authors.affiliation: Division of Drug Research, Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, SE-58185 Linköping, Sweden. svante.vikingsson@liu.se
authors.forename: Svante
authors.initials: S
authors.lastname: Vikingsson
authors.forename: Sven
authors.initials: S
authors.lastname: Almer
authors.forename: Curt
authors.initials: C
authors.lastname: Peterson
authors.forename: Björn
authors.initials: B
authors.lastname: Carlsson
authors.forename: Martin
authors.initials: M
authors.lastname: Josefsson
mesh_terms: D000328:Adult; D053000:Analytic Sample Preparation Methods; D000964:Antimetabolites, Antineoplastic / Q000097:blood / Q000493:pharmacokinetics* / Q000627:therapeutic use; D001379:Azathioprine / Q000097:blood / Q000493:pharmacokinetics / Q000627:therapeutic use; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D016903:Drug Monitoring / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008297:Male; D015122:Mercaptopurine / Q000097:blood / Q000493:pharmacokinetics / Q000627:therapeutic use; D019384:Nucleic Acid Synthesis Inhibitors / Q000097:blood / Q000493:pharmacokinetics* / Q000627:therapeutic use; D010865:Pilot Projects; D011196:Potassium Permanganate / Q000737:chemistry; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood / Q000188:drug therapy; D011687:Purines / Q000097:blood* / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013866:Thioguanine / Q000097:blood / Q000493:pharmacokinetics / Q000627:therapeutic use; D013873:Thionucleotides / Q000097:blood* / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D007202:Indicators and Reagents; D019384:Nucleic Acid Synthesis Inhibitors; D011687:Purines; D000644:Quaternary Ammonium Compounds; D013873:Thionucleotides; D011196:Potassium Permanganate; C009405:tetrabutylammonium; D015122:Mercaptopurine; D013866:Thioguanine; D001379:Azathioprine