Porous graphitic carbon based chromatography hyphenated with mass spectrometry: A new strategy for profiling thiopurine nucleotides in patients with inflammatory bowel diseases

Abstract

Thiopurine (TP) treatment is discontinued in up to 30% of patients suffering from inflammatory bowel diseases (IBD) due to various adverse effects. Therapeutic drug monitoring of biologically active TP metabolites, i.e. thiopurine nucleotides (TPN), can help to optimize the efficacy and safety of the TP treatment. In our work, a novel strategy for TPN profiling, based on a porous graphitic carbon (PGC) chromatography, was developed. The validated PGC-MS method was compared with ion-exchange LC-MS, a currently leading analytical approach established for the determination of TPN. The innovative approach enabled an enhancement of several key performance parameters demanded in a clinical routine use, namely (i) selectivity (time- and mass-recognition of all 12 TPN in one run), (ii) sensitivity (2-5-fold increase in intensities of the analytical signals), (iii) sample throughput (25% shorter analysis time). Application of the novel TPN profiling strategy to a pilot clinical study (12 patients) revealed significantly higher levels of 6-methylthioguanine 5'-diphosphate (MeTGDP) in non-responsive IDB patients treated with azathioprine. Some other TPN are very close to the critical level (p = 0.05) and they will need larger groups of IBD patients to confirm definitively their relevance. In conclusion, the developed PGC-MS method represents a significant improvement to currently available methods for detailed profiling of TPN and its use in bigger clinical studies should lead to a better understanding of the relationship between TPN profiles and therapeutic outcome.

Keywords: Azathioprine; Inflammatory bowel diseases; Mass spectrometry; Porous graphitic carbon based chromatography; Therapeutic drug monitoring; Thiopurine nucleotides.