Unnur Arna Thorsteinsdottir (Presenter)
University of Iceland
Bio: I received a M.Sc in Biology from the University of Copenhagen in 2015 and I am currently a PhD student at the Faculty of Pharmaceutical Sciences, University of Iceland, starting in 2016. The objective of my PhD research is to develop a quantitative UPLC-QqQ-MS/MS assay for clinical diagnosis and pharmacotherapy monitoring of patients with APRT deficiency. The project is a collaboration between the University of Iceland and the National University Hospital of Iceland. The use of mass spectrometry for clinical diagnosis in Iceland has gradually been increasing the past several years and I'm very interested in participating in the implementation of mass spectrometry in the clinical laboratory in Iceland.
Authorship: Unnur A. Thorsteinsdottir (1,2), Finnur F. Eiriksson (1,2), Hrafnhildur L. Runolfsdottir (1), 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
| Short Abstract 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, which is metabolized to the active metabolite oxypurinol, effectively reduces DHA excretion and prevents urinary stone formation. However, a reliable method for therapeutic monitoring of patients with APRT deficiency is lacking. An UPLC-QqQ-MS/MS assay for quantitative measurement of oxypurinol in patient’s plasma samples was successfully developed and optimized utilizing design of experiments. APRT enzyme assay was used to confirm diagnosis of patients with APRT deficiency. |
Long Abstract
Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder that is characterized by excessive production of 2,8-dihydroxyadenine (DHA). This is due to a dysfunctional APRT enzyme, which normally catalyzes the synthesis of adenosine monophosphate (AMP) from adenine and phosphoribosylpyrophosphate (PRPP). The poorly soluble DHA is excreted in the urine and leads to kidney stone formation and chronic kidney disease (CKD). Treatment with the xanthine dehydrogenase (XDH) inhibitor allopurinol reduces DHA excretion and prevents urinary stone formation. allopurinol is metabolized to the active metabolite oxypurinol, which has a plasma half-life of about 18 to 30 h in patients with normal renal function and up to a week in patients with severely impaired renal function. 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, which renders it unsatisfactory as the sole means for therapeutic drug monitoring. Therefore, a reliable method for diagnosis as well as therapeutic monitoring of patients with APRT deficiency is needed in order to facilitate the institution of timely pharmacotherapy to prevent kidney stone episodes and progressive CKD. The aim of this study was to develop and optimize an UPLC-QqQ-MS/MS assay for quantitative measurement of oxypurinol in human plasma. A design of experiments (DoE) was utilized for optimization of the method, where the goal was to find the optimum conditions with as few experiments as possible. APRT deficiency in patients was confirmed by an APRT enzyme assay.
Methods
The UPLC-QqQ-MS/MS quantification method for oxypurinol in human plasma was optimized utilizing DoE. Experimental screening of the variables was performed in negative ionization mode by D-optimal design to reveal both quantitative and qualitative significant factors influencing the retention time, peak height and peak area for oxypurinol. Significant variables were optimized with central composite face design (CCF) and related to sensitivity and retention time utilizing partial least square (PLS) regression. Furthermore, xanthine, a structural analogue of oxypurinol was included in the study, to ensure adequate separation between the two compounds, since xanthine plasma concentration can increase during allopurinol therapy. The chemometric software MODDE 11 (MKS Data Analytics Solutions) was used for the design of experiments. Following optimization of the quantification method, a sample preparation method was developed for oxypurinol in human plasma. Protein precipitation with a 96 well PPT plate (Sirocco, Waters) was used with methanol (MeOH) and 0.1% formic acid (FAC) as a precipitation solvent. The samples were diluted with 2 mM ammonium acetate prior to analysis of samples from patients receiving allopurinol therapy by the optimized UPLC-QqQ-MS/MS assay. A calibration curve was prepared by spiking oxypurinol standard in different concentrations into plasma from healthy volunteers. The APRT enzyme activity in erythrocyte lysates from patients, heterozygotes and controls was estimated by measuring the formation of AMP in erythrocytes. Erythrocytes were isolated from whole blood and incubated with adenine and PRPP. UPLC-QqQ-MS/MS assay in positive ionization mode was developed and AMP formation during incubation was measured and used to estimate the APRT enzyme activity.
Results
Significant changes were observed in the formation of AMP between APRT deficient patients, heterozygotes and controls. These results confirm that APRT enzyme activity is abolished in APRT deficient patients and reduced in heterozygotes.
An UPLC-QqQ-MS/MS assay in negative ionization mode for quantitative measurement of oxypurinol was successfully developed and optimized with short analysis time. By using the chemometric approach, design of experiments (DoE), only a fraction of experiments was used that would have been required by changing one separate factor at a time (COST) approach and possible interaction effects between experimental factors could easily be detected. A sufficient separation between oxypurinol and xanthine was obtained and the sensitivity of the assay for quantification of oxypurinol in plasma was furthermore improved. Plasma sample cleanup was successful with PTT extraction plate and the recovery was increased by diluting the samples with 2 mM ammonium acetate prior to analysis. The assay was used to measure the plasma concentration of oxypurinol in patients with APRT deficiency. Significant difference was observed in the concentration of oxypurinol in plasma between patients receiving allopurinol therapy and those who were untreated. The data suggest that the UPLC-QqQ-MS/MS assay will greatly facilitate therapeutic drug monitoring among patients with APRT deficiency and will be an important step forward in their care.
The UPLC-QqQ-MS/MS quantification assay for oxypurinol in plasma will be validated according to the Food and Drug Administration (FDA) Guidelines for Bioanalytical Method Validation and Clinical and Laboratory Standards Institute (CLSI) Guidelines. The validated UPLC-QqQ-MS/MS method will be used for measuring the concentration of oxypurinol in plasma samples from patients with APRT deficiency on allopurinol treatment. The next step will be to compare the concentration of DHA in both urine and plasma to the oxypurinol plasma concentration, in order to investigate if there is a correlation between oxypurinol concentration and DHA generation.
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