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<p>Thiopurines are widely used in the management of inflammatory bowel disease (IBD), particularly in resource-limited settings such as India. Therapeutic drug monitoring is conventionally based on total 6-thioguanine nucleotide (6-TGN) concentrations; however, their correlation with clinical response remains inconsistent. Recent evidence highlights the importance of individual phosphorylated metabolites, especially thioguanosine triphosphate (TGTP), as key mediators of thiopurine efficacy. We developed and validated a sensitive ultra-high-pressure liquid chromatography (UPLC) method for the simultaneous quantification of thioguanosine monophosphate (TGMP), diphosphate (TGDP), and triphosphate (TGTP) in red blood cells. Method validation was performed according to the bio-analytical guidelines, assessing linearity, precision, accuracy, sensitivity, specificity and recovery. The phosphorylated metabolite levels were assessed in 30 IBD patients on thiopurine therapy and compared with routinely measured total 6-TGN concentrations. The method demonstrated excellent selectivity and linearity (R<sup>2</sup> &gt; 0.99) over ranges of 0.25–250&#xa0;pmol/3.2 × 10<sup>8</sup> RBCs for TGMP and 0.48–500&#xa0;pmol/3.2 × 10<sup>8</sup> RBCs for TGDP and TGTP, with lower limits of quantification of 0.125&#xa0;pmol/3.2 × 10<sup>8</sup> RBCs and 0.24&#xa0;pmol/3.2 × 10<sup>8</sup> RBCs, respectively. TGTP was the predominant phosphorylated metabolite in patient samples. The sum of phosphorylated metabolites correlated with total 6-TGN levels while providing additional differentiation between active and inactive metabolites. To conclude this UPLC-based method enables reliable quantification of individual phosphorylated thiopurine metabolites and offers an improved approach for therapeutic drug monitoring beyond total 6-TGN measurement in IBD patients.</p>

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Development and Validation of an Ultra-high Performance Liquid Chromatography Method for Simultaneous Quantification of Phosphorylated Thiopurine Metabolites in Patients with Inflammatory Bowel Disease

  • 期刊论文

  • 发表日期：

    2026年4月13日

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  • Khushboo G. Upadhyay,
  • Mihika B. Dave,
  • Devendra C. Desai,
  • Prasad R. Naik,
  • Sarita D. Pol,
  • Rohan V. Lokhande,
  • Tester F. Ashavaid,
  • Alpa J. Dherai

摘要

Thiopurines are widely used in the management of inflammatory bowel disease (IBD), particularly in resource-limited settings such as India. Therapeutic drug monitoring is conventionally based on total 6-thioguanine nucleotide (6-TGN) concentrations; however, their correlation with clinical response remains inconsistent. Recent evidence highlights the importance of individual phosphorylated metabolites, especially thioguanosine triphosphate (TGTP), as key mediators of thiopurine efficacy. We developed and validated a sensitive ultra-high-pressure liquid chromatography (UPLC) method for the simultaneous quantification of thioguanosine monophosphate (TGMP), diphosphate (TGDP), and triphosphate (TGTP) in red blood cells. Method validation was performed according to the bio-analytical guidelines, assessing linearity, precision, accuracy, sensitivity, specificity and recovery. The phosphorylated metabolite levels were assessed in 30 IBD patients on thiopurine therapy and compared with routinely measured total 6-TGN concentrations. The method demonstrated excellent selectivity and linearity (R2 > 0.99) over ranges of 0.25–250 pmol/3.2 × 108 RBCs for TGMP and 0.48–500 pmol/3.2 × 108 RBCs for TGDP and TGTP, with lower limits of quantification of 0.125 pmol/3.2 × 108 RBCs and 0.24 pmol/3.2 × 108 RBCs, respectively. TGTP was the predominant phosphorylated metabolite in patient samples. The sum of phosphorylated metabolites correlated with total 6-TGN levels while providing additional differentiation between active and inactive metabolites. To conclude this UPLC-based method enables reliable quantification of individual phosphorylated thiopurine metabolites and offers an improved approach for therapeutic drug monitoring beyond total 6-TGN measurement in IBD patients.