Received on March 15, 2005
Accepted on August 11, 2005

Drug Monitoring and Toxicology

Liquid Chromatography-Tandem Mass Spectrometry Analysis of Erythrocyte Thiopurine Nucleotides and Effect of Thiopurine Methyltransferase Gene Variants on these Metabolites in Patients Receiving Azathioprine/6-Mercaptopurine Therapy

¹ Prometheus Laboratories, San Diego, CA
² Department of Clinical Pharmacy, Claude Bernard University, Lyon, France
³ Division of Gastroenterology, Research Institute of the McGill University Health Sciences Center, Montreal, Quebec, Canada

^* To whom correspondence should be addressed. E-mail: tdervieux{at}prometheuslabs.com.

Background: Polymorphic thiopurine S-methyltransferase (TPMT) is a major determinant of thiopurine toxicity.

Methods: We extracted 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) from erythrocytes with perchloric acid and converted them to 6-thioguanine (6-TG) and a 6-methylmercaptopurine (6-MMP) derivative during a 60-min acid hydrolysis step. 8-Bromoadenine was the internal standard. The liquid chromatography system consisted of a C₁₈ column with an ammonium acetate-formic acid-acetonitrile buffer. Analytes were measured with positive ionization and multiple reaction monitoring mode. With PCR-restriction fragment length polymorphism analysis and TaqMan allelic discrimination, common TPMT alleles (*1, *2, *3A, *3B, *3C) were determined in 31 792 individuals. We used perchloric acid extraction, acid hydrolysis, and HPLC with ultraviolet detection to measure erythrocyte 6-TG and 6-MMP concentrations in 6189 patients with inflammatory bowel disease receiving azathioprine/6-mercaptopurine therapy.

Results: Intra- and interday imprecision were <10% at low and high analyte concentrations. The conversion of 6-TG and 6-MMP nucleoside mono-, di-, and triphosphates was complete after hydrolysis. Allelic frequency for TPMT variant alleles ranged from 0.0063% (*3B) to 3.61% (*3A). Compared with wild types, TPMT heterozygotes had an 8.3-fold higher risk for 6-TGNs >450 pmol/8 x 10⁸ erythrocytes (concentration associated with increased risk for leukopenia), but an 8.2-fold lower risk for 6-MMPNs >5700 pmol/8 x 10⁸ erythrocytes (concentration associated with increased risk for hepatotoxicity).

Conclusions: The liquid chromatography-tandem mass spectrometry method can be applied to the routine monitoring of thiopurine therapy. The association between TPMT genotype and metabolite concentrations illustrates the utility of pharmacogenetics in the management of patients undergoing treatment with thiopurines.