Simultaneous determination of 14 underivatized amino acids using ion-pairing chromatography and isotope dilution...

We describe the simultaneous quantification of 14 underivatized amino acids (AAs) in various matrices using ion-pairing liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with isotope dilution mass spectrometry (IDMS) as a newly developed reference measurement procedure. The underivatized AAs were successfully separated on a reversed-phase column using mixed ion-pairing reagents of trifluoroacetic acid and heptafluorobutyric acid within 6 min through optimized gradient elution. The method was applied to different matrices, including buffer and plasma with appropriate sample treatments, followed by dilution with water to adjust the concentration range from 1 to 20 mg/kg, ensuring high measurement accuracy and precision. To facilitate exact-matched double IDMS, isotope-labeled AAs were added to the sample prior to treatment as internal standards. The optimized procedure was rigorously validated for linearity, sensitivity, accuracy, and precision, while its reliability was confirmed using commercially available certified reference materials for the AAs. Finally, the method was applied to certify AAs in candidate certified reference material batches. The optimized protocol proved to be suitable for the accurate quantification of 14 AAs in various matrices, and thus may serve as a reliable primary method for AA measurement across different applications.