Abstract

RESTREPO OSORIO, Jaime. Validation of a Chromatography Method to Evaluate Protein Quality of Food and the Impact in Plasmatic Levels. rev. cienc. [online]. 2019, vol.23, n.1, pp.43-62. ISSN 0121-1935.  https://doi.org/10.25100/rc.v23i1.8621.

A reversed-phase liquid chromatography method for separation of 18 phenylthiocarbamyl derivatives of amino acids in human plasma in more or less 20 min. is described and validated. Influence of liophiolization in pretreatment of the sample and the standard solutions was studied. In the phase I, we used a precolumn derivatization with phenil-isotil-ocianate (PICT) and separation in column C18 PicoTag column thermostated at 38 °C, and UV detection at 254 nm. The gradient elution with two solvents (A: 0.150 M sodium acetate (pH 6.40)-acetonitrile 96:6 v/v, and B: water-acetonitrile 40:60 v/v), the timing, column temperature, flow rate, and mobile phase composition were optimized. The method was validated by accuracy (90 % recovery), precision (% RSD < 5%) and linearity (r=0.996). Detection and quantification limit ranged from 0.46 to 1.54 µM for cysteine (Cys), and 1.42 to 4.57 µM for leucine (Leu), respectively. In the phase II, we compared post prandial and basal levels in plasma amino-acid and also serum levels of total protein, albumin, cholesterol, cholesterol-HDL, triglycerides and glucose in nine young healthy individuals (five men and four women) after the ingestion of three types of traditional Colombian food (corn flour pancakes, corn flour pancake with Mozzarella cheese and hard-boiled egg). There were no statistical differences in cholesterol, cholesterol HDL, triglycerides and glucose in the subjects after consuming each of the three meals, however, we observed statistically significant changes (P<0.01) in plasma aminoacid levels, seric total protein and seric albumin, being the meals with corn flour and mozzarella cheese pancake the ones that made the difference (18.8 ± 19.1 % recovery in total amino acid levels, 21.7 ± 12.1 % protein total recovery and 21.2 ± 14.8 % albumin recovery). The validated method allows us to perform amino acid analysis, and we could make inferences about the protein quality of food and the effect on bioavailability in humans.

Keywords : high-performance liquid chromatography; human plasma amino acids; phenylthiocarbamyl derivatives; lyophilization.

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