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BACKGROUNDAdvances in technology and the earlier release of newborns from hospitals have pressed the demand for accurate calibration and improved interlaboratory performance for newborn screening tests. As a first step toward standardization of newborn screening aminoacidopathy tests, we have produced six-pool sets of multianalyte dried-blood-spot amino acid reference materials (AARMs) containing predetermined quantities of five amino acids. We describe here the production of the AARMs, validation of their amino acid contents, and characterization of their homogeneity and their stability in storage.METHODSTo each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0-200 mg/L), Leu (0-200 mg/L), Met (0-125 mg/L), Tyr (0-125 mg/L), and Val (0-125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs.RESULTSThe absolute mean biases across the analytic range for five amino acids were 2.8-9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P >0.05). Regression slopes (0 +/- 0.01) for amino acid concentrations vs storage times and their P values (>0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 degrees C, or -20 degrees C during the intervals tested.CONCLUSIONThe validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.

Validation of Accuracy-based Amino Acid Reference Materials in Dried-Blood Spots by Tandem Mass Spectrometry for Newborn Screening Assays