A-317 Development of a patient-focused volumetric absorptive microsampling strategy to assess urinary amino acid excretion
Authors: Ptolemy A , Pickett S , Garrett R , Guarino B D , Kellogg M , Peake R 
Published: October 2024

Abstract

Background
Biochemical determinations of urinary amino acid excretion patterns can diagnose specific inborn errors of metabolism (IEM). Clinical management of these genetic metabolic patients may require frequent urine amino acid tests. A simplified process for transporting home collected urine specimens would benefit the IEM community. Volumetric absorptive microsampling (VAMS) is an emergent alternative sampling technique for the collection of dried biological specimens. We describe the development and preliminary validation results of a VAMS strategy for the analysis of creatinine and amino acids (N=22) from microsampled dried urine.

Methods
Urine creatinine and amino acids were respectively measured in a clinical laboratory setting using established assays on a CobasPro (Roche) and a MassTrak ultra-performance liquid chromatography (UPLC) amino acid analysis (AAA) solution (Waters). Two 30 µL VAMS tips from a Mitra device (Neoteryx) were used to sample each random urine specimen. An ultrasonication-based solvent extraction procedure was optimized to extract the amino acids and creatinine from the VAMS tips. Solvent extracts were subsequently dried by centrifugal evaporation. Extract from one tip was re-dissolved in water and tested for creatinine using an enzymatic method. The other extract was re-dissolved in 0.02 N sodium hydroxide and tested according to the MassTrak AAA procedure, which deploys pre-column AccQTag derivatization with reversed-phase UPLC on a C18 column (2.1 × 150 mm; 1.7 µm) and UV detection at 260 nm. Accuracy, within-batch precision and short-term stability of the optimized VAMS protocol were determined.

Results
Extraction solvent with a 60:40 (v/v) water:methanol mixture provided the highest average amino acid recovery. This extraction solvent produced higher average amino acid recoveries at pH 11 (89±13%), than pH 3 (82±10%) or pH 7 (72±11%). Creatinine measured from the VAMS tips (N=44) had a consistent negative bias of 20% relative to neat urine (R=0.9905, slope=0.802, intercept=0.02). The creatinine corrected urine amino acid results from the Mitra device were corrected for this observed concentration bias. The accuracy of the amino acid results derived from the VAMS tip vs neat urine was 104 ± 14%. The average within-batch precision (CV%) of the amino acids was 9.8% (range=6.7% (cystine) to 17.6% (arginine)) and 6.2% for creatinine. The average 1 week storage stability at ambient temperature was 91% (range=73% (ornithine) to 107% (glycine)) for the amino acids and 107% for creatinine. The devised VAMS strategy was applied to the analysis of microsampled dried urine from a patient with cystinuria (OMIM no. 220100). Marked elevations in arginine, cystine, lysine and ornithine were respectively observed and are consistent with the biochemical diagnosis of the patient.

Conclusions
Creatinine corrected amino acid results from the devised VAMS strategy exhibit good precision and accuracy when compared to tradition analyses of neat urine. The potential use of microsampled dried urine for the diagnosis and monitoring of selected IEM has been demonstrated. The analytical and clinical performance of this VAMS protocol must be further validated, including the use of microsampled dried urine from home specimen collections.