A targeted reversed-phase gradient UPLC-MS/MS assay has been developed for the quantification /monitoring of 66 amino acids and amino-containing compounds in human plasma and serum using precolumn derivatization with 6-aminoquinolyl-N hydroxysuccinimidyl carbamate (AccQTag Ultra). Derivatization of the target amines required minimal sample preparation and resulted in analytes with excellent chromatographic and mass spectrometric detection properties. The resulting method, which requires only 10 μL of sample, provides the reproducible and robust separation of 66 analytes in 7.5 min, including baseline resolution of isomers such as leucine and isoleucine. The assay has been validated for the quantification of 33 amino compounds (predominantly amino acids) over a concentration range from 2 to 20 and 800 μM. Intra- and interday accuracy of between 0.05 and 15.6 and 0.78−13.7% and precision between 0.91 and 16.9% and 2.12−15.9% were obtained. A further 33 biogenic amines can be monitored in samples for relative changes in concentration rather than quantification. Application of the assay to samples derived from healthy controls and patients suffering from acetaminophen (APAP, paracetamol)-induced acute liver failure (ALF) showed significant differences in the amounts of aromatic and branched chain amino acids between the groups as well as a number of other analytes, including the novel observation of increased concentrations of sarcosine in ALF patients. The properties of the developed assay, including short analysis time, make it suitable for high-throughput targeted UPLC-ESI-MS/MS metabonomic analysis in clinical and epidemiological environments.
|UPLC-MS of Multiple Amines in induced Liver Failure_25JAN17||1434.2kB|