title: 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 5000ng/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 6.5min. Measurement of DHA in 24h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563ng/mL and 24h excretion of 816, 1327 and 1649mg, 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.
authors: University of Iceland, Reykjavik, Iceland; ArcticMass, Reykjavik, Iceland. Electronic address: margreth@hi.is.
authors: Margret
authors: M
authors: Thorsteinsdottir
authors: University of Iceland, Reykjavik, Iceland; ArcticMass, Reykjavik, Iceland. Electronic address: u.thorsteinsdottir@gmail.com.
authors: Unnur A
authors: UA
authors: Thorsteinsdottir
authors: University of Iceland, Reykjavik, Iceland; ArcticMass, Reykjavik, Iceland. Electronic address: finnur@arcticmass.is.
authors: Finnur F
authors: FF
authors: Eiriksson
authors: University of Iceland, Reykjavik, Iceland. Electronic address: hrafnhr@lsh.is.
authors: Hrafnhildur L
authors: HL
authors: Runolfsdottir
authors: Childreńs Medical Center, Landspitali - The National University Hospital of Iceland, Reykjavik, Iceland. Electronic address: ingeragu@lsh.is.
authors: Inger M Sch
authors: IM
authors: Agustsdottir
authors: Department of Clinical Biochemistry, Landspitali - The National University Hospital of Iceland, Reykjavik, Iceland. Electronic address: steinodd@lsh.is.
authors: Steinunn
authors: S
authors: Oddsdottir
authors: ArcticMass, Reykjavik, Iceland; Center for Biomedicine, European Academy of Bolzano/Bozen, Bolzano, Italy. Electronic address: Baldur.Sigurdsson@eurac.edu.
authors: Baldur B
authors: BB
authors: Sigurdsson
authors: University of Iceland, Reykjavik, Iceland. Electronic address: hordurkari@gmail.com.
authors: Hordur K
authors: HK
authors: Hardarson
authors: University of Iceland, Reykjavik, Iceland. Electronic address: nrk2@hi.is.
authors: Nilesh R
authors: NR
authors: Kamble
authors: University of Iceland, Reykjavik, Iceland. Electronic address: snorrisi@hi.is.
authors: Snorri Th
authors: ST
authors: Sigurdsson
authors: University of Iceland, Reykjavik, Iceland; Childreńs Medical Center, Landspitali - The National University Hospital of Iceland, Reykjavik, Iceland. Electronic address: vidare@lsh.is.
authors: Vidar O
authors: VO
authors: Edvardsson
authors: University of Iceland, Reykjavik, Iceland; Division of Nephrology, Landspitali - The National University Hospital of Iceland, Reykjavik, Iceland. Electronic address: runolfur@lsh.is.
authors: Runolfur
authors: R
authors: Palsson
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000652:urine; D000228:Adenine Phosphoribosyltransferase / Q000172:deficiency* / Q000652:urine; D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D008661:Metabolism, Inborn Errors / Q000175:diagnosis / Q000652:urine*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D016482:Urinalysis / Q000379:methods; D052878:Urolithiasis / Q000175:diagnosis / Q000652:urine*
chemical_list: C002993:2,8-dihydroxyadenine; D000228:Adenine Phosphoribosyltransferase; D000225:Adenine
keywords: APRT deficiency; Chemometrics; Clinical mass spectrometry; D-optimal design; Therapeutic drug monitoring