Abstract Background Biochemical determinations of urinary amino acid excretion patterns may be required to diagnose and manage amino acid transport disorders. A simplified process for testing home collected urine specimens would benefit this patient community. We describe the validation of a volumetric absorptive microsampling (VAMS) strategy for the analysis of creatinine and several amino acids (N=22) in dried urine. Methods 30 µL of urine were volumetrically sampled and dried on two VAMS tips from a Mitra device (Neoteryx). An ultra-sonication based solvent extraction procedure was optimized to remove the creatinine and amino acids from the dried urine. Each were respectively quantified in a biochemical genetics laboratory using established clinical assays on a CobasPro (Roche) and MassTrak ultra-performance liquid chromatography (UPLC) amino acid analysis (AAA) solution (Waters). Performance of the VAMS strategy was determined through an assessment of accuracy, precision and stability. The effects of ground transit (time, relative humidity (% RH), and temperature) on dried microsampled urine was also examined. The devised VAMS strategy was applied to urine collected from a patients with lysinuric protein intolerance (LPI, OMIM # 222700), cystinuria (OMIM # 220100), and phenylketonuria (PKU, OMIM # 261600). Results Creatinine measured from microsampled dried urine had an average recovery of 104 ± 15% relative to neat urine. Accurate urine creatinine results were obtained from dried microsamples of archived College of American Pathology (CAP) and ERNDiM proficiency testing material. A 60:40 (v/v) water:methanol pH 11 extraction solvent provided the highest average amino acid recovery. The accuracy of the creatinine corrected amino acid results derived from the VAMS tips relative to neat urine was 107 ± 13%. The average inter-day precision (CV%) of the amino acids and creatinine was 12.4% and 7.7%, respectively. The average 10 day storage stability at 5.6°C/89.3%RH, 20.6°C/50.7%RH, and 39.8°C/18.7%RH were 111%, 107%, and 110% for the amino acids and 95%, 96% and 98%, for creatinine, respectively. Dried urine specimens were shipped from different locations in Massachusetts, Rhode Island, and New Hampshire to our laboratory for testing. The maximum transit duration, temperature, and %RH experienced were 6 days 22 hours 25 minutes, 47°C and 71.1%RH, respectively. Amino acids and creatinine were stable in the microsampled dried urine under all shipping conditions, with average recoveries of 104 ± 9% and 102 ± 6% relative to neat urine. Appropriate marked elevations in arginine, cystine, lysine, and ornithine were respectively observed in the urine of LPI and cystinuria patients and were consistent with the biochemical diagnosis. Marked elevation in phenylalanine was also identified in the dried urine from a PKU patient. Conclusion Creatinine corrected urine amino acid results from the microsampled dried urine testing strategy were accurate relative to traditional analyses of neat urine specimens. Testing dried urine from home collected specimens may be appropriate for patients with amino acid transport disorders.
Ptolemy et al. (Wed,) studied this question.