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Optimization of UPLC-MS/MS assay for clinical diagnosis and therapeutic drug monitoring in patients with APRT deficiency by design of experiments

Unnur Arna Thorsteinsdóttir1, Hrafnhildur L. Runolfsdottir3, Vidar O. Edvardsson1,3, Runolfur Palsson1,3, Margrét Thorsteinsdóttir1,2

  1. University of Iceland, Reykjavik, Iceland
  2. ArcticMass, Reykjavik, Iceland
  3. Landspitali – The National University Hospital of Iceland, Reykjavik, Iceland

e-mail: margreth@hi.is

Design of experiments (DoE) is an efficient tool for development and optimization of UPLC-MS/MS bioanalytical method which involves many experimental factors that need to be simultaneously optimized to obtain maximum sensitivity with adequate resolution at minimum retention time. Adenine phosphoribosyltransferase deficiency (APRTd) is an inborn error of adenine metabolism, characterized by excessive urinary excretion of poorly soluble 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease (CKD) [1]. Treatment with the xanthine oxidoreductase (XOR) inhibitors allopurinol or febuxostat effectively reduces DHA excretion and prevents urinary stone formation and renal crystal deposition [2]. Currently, diagnosis and therapeutic drug monitoring (TDM) are performed by urine microscopy, which lacks specificity and is operator dependent. The aim of this study was to optimize a UPLC-MS/MS assay for clinical diagnosis and TDM of patients with APRTd utilizing DoE.

D-optimal design with several quantitative factors and multi-level qualitative factors was selected for experimental screening to reveal significant factors influencing the analysis of DHA, adenine, adenosine, 2-deoxyandenosine, inosine, 2-deoxyinosine and hypoxanthine in human urine by UPLC-MS/MS. Significant factors were studied via central composite face design and related to sensitivity, resolution and retention time utilizing PLS-regression. Urine samples from APRTd patients and healthy controls before and after treatment were analyzed with the optimized UPLC-MS/MS assay.

A sensitive UPLC-MS/MS assay for simultaneous quantification DHA and the main purine metabolites was successfully optimized utilizing DoE. There was a strong interaction effect between several variables, indicating that these variables cannot be independently controlled to obtain optimal conditions. DHA was detected in all urine samples from untreated APRTd patients but not in any specimens from healthy controls. Significant changes were observed in the urinary excretion of DHA and adenine with drug therapy and DHA excretion in APRTd patients decreased with conventional doses of both allopurinol and febuxostat. Today the UPLC-MS/MS assay is used for clinical diagnosis and TDM of patients with the rare kidney stone disorder APRTd.

This study demonstrates the utilization of DoE in ensuring that selected experiments contain maximum information and optimization is conducted efficiently. We believe the optimized clinical UPLC-MS/MS assay will greatly facilitate clinical diagnosis of patients with APRTd.

References

  1. Runolfsdottir H.L.; Palson R.; Augustdottir I.M.; Indridason O.S.; Edvardsson V.O. Kidney disease in adenine phosphoribosyltransferase deficiency. Am. J. Kidney Dis. 2015, 67, 431-438.
  2. Edvardsson V.O.; Runolfsdottir H.L.; Thorsteinsdottir U.A.; Augustdottir I.M.; Oddsdottir S.; Eiriksson F.; Goldfarb D.S.; Thorsteinsdottir M.; Palsson R. Comparison of the effect of allopurinol and febuxostat on urinary 2,8-dihydroxyadenine excretion in patients with Adenine phosphoribosyltransferase deficiency (APRTd): A clinical trial. Eur J Intern Med. 2018, 48, 75-79.