Short-Term Stabilities of 21 Amino Acids in Dried Blood Spots

To the Editor: Dried blood spot (DBS)1 sampling is used in disease diagnosis, epidemiological surveillance, and newborn screening, and enables sample collection far from analytical laboratories and shipment via mail services. Amino acids (AAs) measured in DBS are routinely used for the diagnosis of inborn errors of metabolism (1) and childhood malnutrition (2), but environmental stresses incurred during sampling, transportation, and storage can impact the short-term (3) and long-term (4) stabilities of some AAs. Here, we systematically investigated the stabilities of 21 routinely analyzed AAs in DBS under environmental conditions simulating a global health work flow. DBSs were prepared by precisely blotting 30 μL of whole blood onto cellulose-based Whatman 903, FTA DMPK-C, and cotton-based PerkinElmer 226 cards. The optimized sample preparation used 200 μL of 0.02% trifluoroacetic acid in water to completely resuspend the entire DBS spot, followed by ultrasonic extraction with 800 μL of methanol/acetonitrile (1:1, v/v) containing 0.02% trifluoroacetic acid. The AAs were dansylated (5) at pH 9.2 and analyzed by C18 reversed-phase liquid chromatography/positive electrospray ionization/multiple-reaction monitoring mass spectrometry, with 21 13C- or 2H-labeled internal standards, for precise measurements of their molar concentrations (CVs ≤ 7.9% and recoveries of 88.4%–112.8%). Four sets of stability-testing experiments were conducted, and the molar …