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Evaluation of an underivatized compared with a derivatized method to quantify bovine plasma amino acids via liquid chromatography electrospray mass spectrometry.

M. Z. Toledo

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06-24-2020

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Abstract:

W105
Evaluation of an underivatized compared with a derivatized method to quantify bovine plasma amino acids via liquid chromatography electrospray mass spectrometry.
M. Z. Toledo*1, Y. Agarwal1, C. Nienow3, D. Luchini2, S. I. Arriola Apelo1, M. C. Wiltbank1. 1University of Wisconsin-Madison Madison, WI, 2Adisseo Alpharetta, GA, 3Shimadzu Scientific Instruments Inc Columbia, MD.

Objectives were to evaluate and compare a novel underivatized with a derivatized method to quantify bovine plasma AA by isotope dilution ratio via liquid chromatography electrospray single quadrupole mass spectrometry. Plasma from lactating dairy cows (n = 24; 80 DIM; 49 kg/d of milk) was isolated by centrifugation and deproteinized in 0.5 N perchloric acid. Deproteinized plasma was filtered and injected into a 50 � 2 mm column (Imtakt, OR) or extracted, derivatized, and injected into a 250 � 3 mm column (EZ:Faast, Phenomenex, CA) and analyzed. Linearity of the mass-to-charge ratio (m/z) signal was evaluated for each AA with 5-point standard curves (range: 3.9 to 500 �M). Recovery rates were evaluated using pooled plasma samples (n = 4) spiked with 10, 20, or 50 �M of each AA. Statistical analysis was performed using the TTEST and IML procedure of SAS. Both methods showed linearity within the dynamic range analyzed for all essential AA (R2 ≥ 0.995). Meanwhile, underivatized samples had poor chromatogram (signal-to-noise ratio < 3) or linearity (R2 ≤ 0.990) for cystine, Asp, Gly, Tyr, and Ser. Moreover, sensitivity for 12C AA was greater for derivatized than underivatized samples. Both methods had recovery rates ranging from 84.5% to 126.0% and none differ from 100% (P ≥ 0.05), except Gln (20 �M [85.7%] and 50 �M [87.6%], P < 0.01) and Val (50 �M [120.6%], P < 0.02) using the underivatized method. Coefficient of correlation (r) and concordance coefficient of correlation (CCC) varied from 0.42 to 0.99. Highest r and CCC was observed for Arg (r = 0.99; CCC = 0.99), Leu (r = 0.98; CCC = 0.98), Met (r = 0.99; CCC = 0.96), and Thr (r = 0.97; CCC = 0.96). In particular, the Bland-Altman plot showed that Met has a mean bias of 2.1 �M. In conclusion, derivatization had greater 12C AA sensitivity, linearity, and recovery rates, however, the underivatized method showed linearity and accurate recovery rates for most essential AA, and in particular Met quantification appears to be comparable with a derivatized method.

Keywords: amino acids, bovine, mass spectrometry.