A novel simultaneous determination of native and isotopically-labeled plasma amino acids in pigs by gas chromatography mass spectrometry

The ultimate goal of the study was to develop a method that allows simultaneous quantification of both native and isotopically-labeled AA in plasma and other physiological fluids in pigs. The stable isotope infusion technique is commonly used in humans and animals to study various aspects of protein and AA metabolism in vivo (e.g. plasma AA kinetics). Simultaneous quantification of these bio-compounds in physiological fluids is difficult, because native and labeled AA elute at the same time during gas chromatography; and quantification is usually achieved using separate chromatographic runs. The latter can potentially increase the intra-assay variation, and the time required for analysis. Quantification of derivatized (Phenomenex EZ:faast™ free AA analysis Kit) native and labeled AA in standard and plasma samples was achieved using GC-MS (Agilent 6890 GC coupled with an Agilent 5973 mass selective detector). The method was developed employing selective ion monitoring (SIM) to identify and quantify multiple native and labeled AA simultaneously. The validation of the method was related to linearity, sensitivity, accuracy and repeatability. In the validated method linearity was achieved within concentration range 0.93 to 58.62 ng/µl for the labeled AA (R²=0.93), and 3.52 to 12.36 ng/µl for native AA (R²=0.92). The limits of detection for Lys, Met, Thr, Trp, Ile, Leu, Val, Phe, and Gln were, 0.03, 0.03, 0.02, 0.02, 0.03, 0.01, 0.02, 0.03, and 1.46 ng/µl, respectively. Coefficient of variation for the inter-day and the intra-day analysis, which is a precision and accuracy parameter, did not exceed 15 %. The practical application of this method was supported by determining the isotopic enrichments of nine AA in plasma of pigs following a bolus infusion of [U-¹³C, U-¹⁵N] AA mixture. The isotopic enrichment mean was 0, 0.29 ±0.060, 0.17 ±0.035, 0.12 ±0.025, 0.09 ±0.020, 0.07 ±0.015, 0.05 ±0.012, 0.03 ±0.009, and 0.02 ±0.007 at 0, 2.5, 5, 7.5, 10, 15, 20, 30, and 45 min post infusion, respectively. The method utilizes differences in mass between labeled and unlabeled AA. This method provides a fast and easy to use alternative for an accurate measurement of native and isotopically-labeled AA in plasma and other physiological fluids in pigs. Supported by NPB # 13-082.