A reference measurement procedure for amino acids in blood using isotope dilution ultra-performance liquid chromatography-tandem mass spectrometry

•Improvement of measurement sensitivity and ionization stability with 5-sulfosalicylic acid.•The excellent measurement commutability in various blood matrices such as serum, plasma and whole blood as a reference measurement procedure.•Assessment of the quantification accuracy of the proposed method using certified reference material.•Determination of five amino acids in serum with strict estimation of measurement uncertainty using proposed method.

We described a reference measurement procedure for amino acid (AA) quantification in blood samples based on deproteinization with 5-sulfosalicylic acid (SSA) and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry (LC-MS) method. The serum was deproteinized with 15% v/v SSA and the supernatant was injected directly into the LC-MS system without further processing. Compared with the use of other precipitants and water as a control, five model AAs-valine, isoleucine, leucine, tyrosine, and phenylalanine-in the SSA-treated samples showed ionization enhancement as well as stable background signals without significant ion suppression effects. Five analytes were clearly separated within 3 min using gradient elution and ion-pair chromatography of water and acetonitrile containing 0.1% v/v trifluoroacetic acid. The limit of detection range of this method was 2-52 fmol, and the RSDs of accuracy and precision from intra- and inter-day assays were within 2.7%. The method was applied to various blood samples including serum, whole blood and plasma, with no reasonable measurement bias revealed. The quantification accuracy of this method was then assessed using commercially available plasma certified reference material (CRM) for AA, and the results agreed well within certified values. We finally applied this method to the determination of candidate serum CRM. The optimized protocol was found to be suitable for the accurate quantification of five AAs in serum, and may satisfactorily serve as a primary method for AA measurement in various blood matrices.