Quantitative UPLC–MS/MS assay of urinary 2,8-dihydroxyadenine for diagnosis and management of adenine phosphoribosyltransferase deficiency

Abstract

Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary disorder that leads to excessive urinary excretion of 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol or febuxostat reduces DHA production and attenuates the renal manifestations. Assessment of DHA crystalluria by urine microscopy is used for therapeutic monitoring, but lacks sensitivity. We report a high-throughput assay based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) for quantification of urinary DHA. The UPLC–MS/MS assay was optimized by a chemometric approach for absolute quantification of DHA, utilizing isotopically labeled DHA as an internal standard. Experimental screening was conducted with D-optimal design and optimization of the DHA response was performed with central composite face design and related to the peak area of DHA using partial least square regression. Acceptable precision and accuracy of the DHA concentration were obtained over a calibration range of 100 to 5000 ng/mL on three different days. The intra- and inter-day accuracy and precision coefficients of variation were well within ±15% for quality control samples analyzed in replicates of six at three concentration levels. Absolute quantification of DHA in urine samples from patients with APRT deficiency was achieved wihtin 6.5 min. Measurement of DHA in 24 h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10 mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563 ng/mL and 24 h excretion of 816, 1327 and 1649 mg, respectively. A rapid and robust UPLC–MS/MS assay for absolute quantification of DHA in urine was successfully developed. We believe this method will greatly facilitate diagnosis and management of patients with APRT deficiency.

Adenine Phosphoribosyltransferase Deficiency Due to Novel Mutation

“…A newer technique of identifying DHA crystals using high performance liquid chromatography–linked assay and liquid chromatography-electrospray assay tandem mass spectrometry can be used, but none of these have been established for use in clinical practice. Recently an ultra-performance liquid chromatography-tandem mass spectrometry has been developed for absolute quantification of urinary DHA, using isotopically labeled DHA as an internal standard 8 . The gold standard technique for diagnosing APRT deficiency is measuring APRT enzyme activity, and detection of bi-allelic pathogenic mutations can also confirm the diagnosis.…”

Long-term renal outcomes of APRT deficiency presenting in childhood

“…However, detection of crystalluria may not be reliable enough for monitoring of drug treatment. Our group has recently developed a urinary DHA assay using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which has the potential to greatly facilitate both clinical diagnosis and therapeutic monitoring of pharmacotherapy of patients with APRT deficiency [16]. As the current study is based on observational registry data only, we did not have the opportunity to include the novel urinary DHA assay in this work.…”

APRT deficiency: the need for early diagnosis

“…However, it is not a reliable method for surveillance of therapeutic effect of treatment with XOR inhibitors. A high-throughput ultra-performance liquid chromatography–electrospray tandem mass spectrometry assay for measurement of 2,8-DHA levels in urine samples was published by Thorsteinsdottir et al  and shows promise for monitoring of pharmacotherapy in the future 25. In fact, a recent pilot study comparing the effect of allopurinol 400 mg/day and febuxostat 80 mg/day in the treatment of APRT deficiency used this method to monitor the levels of urinary excretion of 2,8-DHA and found febuxostat to be more efficacious, pending confirmation in larger studies 26.…”