A Validated HPLC Method for the Monitoring of Thiopurine Metabolites in Whole Blood in Paediatric Patients with Inflammatory Bowel Disease

Abstract

Therapeutic drug monitoring (TDM) of major metabolites of thiopurine drugs is a widely used tool for assessing treatment efficacy and toxicity in patients with inflammatory bowel disease (IBD). We report the laboratory and clinical validation of a simple and reliable high performance liquid chromatography (HPLC) method for the measurement of 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) on paediatric patients with IBD. The aim of this paper is to develop and validate a method for the measurement of 6-TGN and 6-MMP applicable to routine practice and to evaluate the usefulness of the TDM of thiopurine drugs in children with IBD attending our Gastroenterology Unit. The HPLC method was validated following international guidelines starting from red blood cells (RBC) and whole blood (WB). A comparison between RBC and WB was assessed. The usefulness of TDM was then evaluated using the new method from WB in 47 paediatric patients with IBD treated with thiopurine drugs. WB and RBC resulted in interchangeable matrices. The majority of patients had the metabolite levels inside the therapeutic ranges. A moderate correlation was found between 6-MMP concentration and the dose of thiopurines. A higher percentage of non responders was found among patients with lower levels of 6-TGN. Toxicity was found in eight patients and was evaluated in respect to the metabolite concentration. The described HPLC method is applicable to routine practice and it is suitable for its use in multicentric studies. Our results of TDM on paediatric IBD patients can contribute to clarify its role in their therapeutic management.

Thiopurine monitoring in children with inflammatory bowel disease: a systematic review

“…Fifteen studies fulfilled the eligibility criteria . ‘Potentially’ eligible studies which were assessed and subsequently excluded are shown in http://onlinelibrary.wiley.com/doi/10.1111/bcp.12365/suppinfo.…”

“… and Cangemi et al . did not reach any conclusions due to very few reported cases. Therefore, a weak association between WBC and thiopurine metabolite concentrations was described in three studies ( n = 126 patients), inconclusive results were reported by four studies ( n = 120 patients) due to very few or no reported cases, and a possible inverse correlation between 6TGN and WBC was suggested by three studies ( n = 263 patients).…”

“… reported variable 6TGN concentrations in patients in remission, Cangemi et al . showed a trend of lower 6TGN values in non‐responders. However the association between a specific 6TGN target concentration and clinical remission was overall reported to be weak.…”

Association Between 6-Thioguanine Nucleotides Levels and Clinical Remission in Inflammatory Disease

“…[7][8][9] The reasons for discrepancies have not been found to date and remain to be explored. Hence, whether therapeutic drug monitoring of thiopurines should be used in clinical practice is still a matter of debate.…”

Cytosine derivatized diethylenetriaminepentaacetic acid (dtpa) and Eu(III) complexes for selective recognition of 6-Thioguanine

“…Currently, various kinds of analytical methods including high-performance liquid chromatogrphy, electrochemical method, phosphorimetry, localized surface plasmon resonance, fluorimetry, and SERS [9][10][11][12][13][14][15] have been reported for the determination and quantification of 6-TG. In fact, these analytic methods were thought to be rapid and sensitive, but they also show their limitations and demerits such as high cost, complicate separation process, tedious pretreatment steps and sophisticated instrumentation.…”