Determination of Free Amino Acids in Milk, Colostrum and Plasma of Swine via Liquid Chromatography with Fluorescence and UV Detection

Abstract

Amino acids are ubiquitous components of mammalian milk and greatly contribute to its nutritional value. The compositional analysis of free amino acids is poorly reported in the literature even though their determination in the biological fluids of livestock animals is necessary to establish possible nutritional interventions. In the present study, the free amino acid profiles in mature swine milk, colostrum and plasma were assessed using a targeted metabolomics approach. In particular, 20 amino acids were identified and quantified via two alternative and complementary reversed-phase HPLC methods, involving two stationary phases based on core-shell technology, i.e., Kinetex C18 and Kinetex F5, and two detection systems, i.e., a diode array detector (DAD) and a fluorescence detector (FLD). The sample preparation involved a de-proteinization step, followed by pre-chromatographic derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl). The two optimized methods were validated for specificity, linearity, sensitivity, matrix effect, accuracy and precision and the analytical performances were compared. The analytical methods proved to be suitable for free amino acid profiling in different matrices with high sensitivity and specificity. The correlations among amino acid levels in different biological fluids can be useful for the evaluation of physio-pathological status and to monitor the effects of therapeutic or nutritional interventions in humans and animals.

Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high‐performance liquid chromatography‐ultraviolet detection method

“…Also, it enabled the simultaneous determination of ionophoric coccidiostats and AAs in one run, in contrast to previous reports. The reported HPLC methods necessitate a gradient mobile phase system [14,16,[29][30][31][32][33], long run time [30,33] or temperature controlling [26][27][28][29][30][31]. Preand/or post-column derivatization reactions were resorted [29][30][31][32][33].…”

“…The reported HPLC methods necessitate a gradient mobile phase system [14,16,[29][30][31][32][33], long run time [30,33] or temperature controlling [26][27][28][29][30][31]. Preand/or post-column derivatization reactions were resorted [29][30][31][32][33]. Additionally, the use of MS detection is generally not preferred due to its high cost, maintenance, operational requirements, limited sample throughput, and lack of portability [15][16][17].…”

“…Regarding AA analysis, it is considered a classic fundamental tool in life science that has long been used in different fields of applications such as protein analysis, food chemistry, disease diagnosis, physiology, and pharmacology [28]. Most of these methods are based upon derivatization to enable AAs to be detected with fluorescence or UV detectors [29][30][31][32][33]. Although most of these derivatization-based AAs quantification methods are validated, inter-laboratory studies showed that the derivatization step can represent a source of variability in the obtained results [34].…”

Overview of chromatographic and electrophoretic methods for the determination of branched‐chain amino acids

“…In conclusion, a pre‐column derivatization is a crucial tool for analyzing BCAAs due to its ability to enhance detection sensitivity and its versatility in accommodating various sample types and detectors. Table 1 shows a compilation of 19 commonly used derivatization reagents, along with a summary of their respective advantages and disadvantages [33–55]. The different reactions with AAs are described in Figure 2.…”