Development of a 2D-UPLC-MS/MS Assay for Therapeutic Monitoring for Patients with APRT Deficiency Unnur Arna Thorsteinsdottir (Presenter) University of Iceland Presenter Bio: I hold a master’s degree in cell biology from the University of Copenhagen (graduated in 2015). I’m currently a PhD student at the Faculty of Pharmaceutical Sciences at the University of Iceland (2016-current). In my PhD project I’m developing a clinical diagnostic method for a rare kidney stone disease using UPLC-MS/MS.

Authors: Unnur A. Thorsteinsdottir (1,2), Finnur F. Eiriksson (1,2), Hrafnhildur L. Runolfsdottir (1), Thorsteinn Hjortur Bjarnason (1,2), Vidar O Edvardsson (3), Runolfur Palsson (1,3), Margret Thorsteinsdottir (1,2)
(1)University of Iceland, Reykjavik, Iceland; (2) ArcticMass, Reykjavik, Iceland; (3) Landspitali – The National University Hospital of Iceland, Reykjavik, Iceland

Adenine phosphoribosyltransferase (APRT) deficiency results in excessive urinary excretion of poorly soluble 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol and febuxostat effectively reduces DHA excretion and prevents urinary stone formation. However, a reliable method for therapeutic monitoring of patients with APRT deficiency is lacking. A 2D-UPLC-MS/MS assay for simultaneous quantification of the purines DHA, adenine, adenosine, inosine, hypoxanthine and xanthine and the pharmacological agents allopurinol, its active metabolite oxypurinol, and febuxostat, in human plasma samples will be developed and optimized utilizing design of experiments (DoE). To our knowledge, this is the first report of an absolute quantification of DHA in human plasma.

Introduction

Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder characterized by excessive production and renal excretion of a poorly soluble purine metabolite, 2,8-dihydroxyadenine (DHA), which leads to kidney stone formation and chronic kidney disease (CKD). Treatment with the xanthine oxidoreductase (XOR) inhibitors allopurinol and febuxostat reduces urinary DHA excretion and decreases or even prevents stone formation and deposition of crystals in the renal parenchyma. The current practice is to monitor the effectiveness of pharmacotherapy by urine microscopy, where the absence of urinary DHA crystals has been considered indicative of adequate therapy. While this approach is simple and inexpensive, it lacks specificity and is operator dependent, rendering it unsatisfactory as the sole means for therapeutic monitoring. Therefore, a more reliable method for therapeutic monitoring of patients with APRT deficiency is needed. The aim of this study is to develop and optimize a two-dimensional ultra performance liquid chromatography-tandem mass spectrometry (2D-UPLC-MS/MS) plasma assay for simultaneous quantification of DHA, the drugs allopurinol, it's active metabolite, oxypurinol, and febuxostat, as well as the purines adenine, adenosine, inosine, hypoxanthine and xanthine, all of which are involved in the adenine metabolic pathway and, therefore, may be affected by treatment with XOR inhibitors. The assay allows for a simultaneous quantification of the medications used to treat APRT deficiency and biomarkers indicative of the disease activity. The results will provide important information for the monitoring of pharmacotherapy.

A design of experiments (DoE) will be used for optimization of the method, where the goal is to find the optimum conditions with as few experiments as possible. By using the chemometric approach, DoE, only a fraction of experiments is used that would have been required by changing one separate factor at a time (COST) approach. In addition, possible interaction effects between experimental factors can easily be detected.

Methods

The 2D-UPLC-MS/MS method for quantification of DHA, adenine, adenosine, inosine, hypoxanthine, xanthine, and the pharmaceutical agents allopurinol, oxypurinol, and febuxostat in human plasma will be optimized utilizing DoE. Experimental screening of the variables will be performed by D-optimal design to reveal significant factors influencing the retention time, peak height and peak area for all compounds. Significant variables will be optimized with central composite face design (CCF) and related to sensitivity and retention time utilizing partial least square (PLS) regression. The chemometric software MODDE 12 (Sartorius Stedim Data Analytics AB, Umeå, Sweden) will be used for the design of experiments and multivariate data analysis. A sample preparation method has been developed for all the compounds in human plasma using a protein precipitation plate (ISOLUTE® PLD+ Protein and Phospholipid Removal Plate, Biotage, Uppsala, Sweden) with 1% formic acid (FAC) in methanol as a crash solvent. Following protein precipitation, samples are evaporated under a gentle stream of nitrogen and redissolved in 10 mM ammonium hydroxide prior to analysis. Plasma samples from untreated APRT deficient patients and those on treatment with either allopurinol (300 or 400 mg/day) or febuxostat (80 mg/day) will be analyzed with the optimized 2D-UPLC-MS/MS assay.

Results, Conclusions & Discussion

Preliminary data show successful plasma sample cleanup with protein precipitation and that improved recovery of DHA was achieved by evaporating the samples and redissolving them in 10 mM ammonium hydroxide prior to analysis. Significant difference was observed in the plasma DHA concentration between untreated patients and those receiving allopurinol and febuxostat therapy. The DHA concentration in plasma samples from 3 APRT deficient patients without treatment was 459, 741 and 456 ng/mL, respectively, and 61, 27 and 130 ng/ml, on pharmacologic treatment, respectively. Moreover, DHA was not detected in plasma samples from healthy controls. The data suggest that the 2D-UPLC-MS/MS assay can be used for monitoring pharmacotherapy compliance and for therapeutic monitoring among patients with APRT deficiency, and thereby introducing an important step forward in their care.

In order to improve the chromatographic separation between all compounds, a multiple heart-cutting 2D-UPLC-MS/MS platform was utilized. The 2D-UPLC allows for different chromatographic conditions during the analysis compared to 1D-UPLC. This resulted in improved chromatographic separation between adenine, adenosine, allopurinol, hypoxanthine and inosine, as well as xanthine and oxypurinol, while maintaining the original chromatographic conditions for DHA. Furthermore, we will attempt to shorten the retention time for febuxostat, which has much higher retention time than the other compounds. Moreover, selecting which part of the effluent is directed to the detector will result in less ion suppression and improve the robustness of the method.

The 2D-UPLC-MS/MS quantification assay for DHA, allopurinol, oxypurinol, febuxostat, adenine, adenosine, inosine, hypoxanthine and xanthine in plasma will be validated according to the Food and Drug Administration (FDA) Guidelines for Bioanalytical Method Validation and Clinical and Laboratory Standards Institute Guideline (CLSI - C62-A). Furthermore, the plasma concentration of 2,8-DHA will be compared with the long-term clinical outcome of patients in order to establish a level of DHA that must be achieved in order to to prevent kidney stone episodes and progression of CKD in APRT deficient patients.