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. 2022 Nov 24;12(12):1173.
doi: 10.3390/metabo12121173.

A Validated HPLC-Diode Array Detection Method for Therapeutic Drug Monitoring of Thiopurines in Pediatric Patients: From Bench to Bedside

Martina Franzin  1 , Debora Curci  1 , Marianna Lucafò  1 , Matteo Bramuzzo  1 , Marco Rabusin  1 , Antonella Fabretto  1 , Riccardo Addobbati  1 , Gabriele Stocco  1   2 , Giuliana Decorti  1   2
Affiliations

A Validated HPLC-Diode Array Detection Method for Therapeutic Drug Monitoring of Thiopurines in Pediatric Patients: From Bench to Bedside

Martina Franzin et al. Metabolites. .

Abstract

Thiopurine drugs are part of the therapeutic armamentarium for pediatric patients suffering from inflammatory bowel disease (IBD) and acute lymphoblastic leukemia (ALL). The therapeutic drug monitoring of these drugs, consisting of measurements of the thiopurine metabolites thioguanine nucleotides (TGN) and methylmercaptopurine nucleotides (MMPN) are used to optimize the effectiveness of treatment and prevent adverse effects. In this context, we developed and validated a high-performance liquid chromatography-diode array detection (HPLC-DAD) method for the simultaneous quantification of thiopurine metabolites according to the most recent International Council for Harmonisation (ICH) guidelines. The calibration curves were built in the clinically relevant range of concentrations for TGN of 300-12,000 nM and for MMPN of 3000-60,000 nM. The limit of detection and the lower limit of quantification were 100 and 300 nM for TGN and 900 and 3000 nM for MMPN, respectively. The percentage of inter-day accuracy and precision (CV%) varied between 85 and 104% and 1.6 and 13.8%. Stability was demonstrated for both of the metabolites for at least 50 days at -20 °C. The proposed HPLC-DAD method showed an appropriate selectivity, specificity, linearity, accuracy, precision and good applicability to samples from patients with IBD and ALL undergoing thiopurine treatment.

Keywords: pediatric patients; therapeutic drug monitoring; thiopurines.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Thiopurine pathways. Compounds: MMP: methylmercaptopurine, TIMP: thioinosine monophosphate, TIDP: thioinosine diphosphate, TITP: thioinosine triphosphate, TXMP: thioxanthosine monophosphate, TGMP: thioguanosine monophosphate, TGDP: thioguanosine diphosphate, TGTP: thioguanosine triphosphate. Enzymes: GST: glutathione S-transferase, GMPS: guanosine monophosphate synthetase, HPRT: hypoxanthine-guanine phosphoribosyltransferase, IMPDH: inosine monophosphate dehydrogenase, ITPA: inosine triphosphate pyrophosphatase, NUDT15: nudix type 15—nucleoside diphosphate-linked moiety X-type motif 15, XO: xanthine oxidase, TPMT: thiopurine S-methyltransferase, RAC1: Ras-related C3 botulinum toxin substrate 1.
Figure 2
Figure 2
Chromatogram showing the retention times in minutes of the analytes TGN and MMPN (green and blue lines) and of IS (purple line). * indicates the retention times.
Figure 3
Figure 3
Chromatogram showing blank sample after the injection of CAL with the highest concentration. * indicates the retention times.
Figure 4
Figure 4
Chromatogram derived from the injection of a real sample containing TGN and MMPN (517.94 and 12,255.06 pmol/8 × 108 RBC, respectively). * indicates the retention times.
See this image and copyright information in PMC

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