Fast and Simple Determination of Free Amino Acids in Milk

Amino acids are of vital importance for human life. They serve as transport or storage media for metabolic pathways. Amino acids are components of protein-containing foods such as milk and milk products. Eight amino acids are thought to be essential for humans, meaning that they must be absorbed from foods containing animal proteins or a suitable combination of plant proteins, because the human body can’t synthesize them.
Determination of the composition and content of amino acids in milk products is therefore an important measure in ensuring quality and nutritional value.

Materials and Methods
There are many ways to analyze amino acids. In this study we used the Shimadzu UF-Amino station for automated pre-column derivatization and on-line reversed-phase LC separation. MS detection was performed using an LCMS 2020 single quadrupole MS. The chemicals used were an amino acid analysis reagent (AminoTag®), amino acids mixed standard and internal standard solutions, and the AminoTag Eluent, all from Wako Chemicals GmbH, and MS-grade acetonitrile from Promochem.

Figure 1: Flow diagram for derivatization and injection in the UF-Amino system.

A series of 10 calibration standards with concentrations between 500 and 2.5 µmol/L were prepared and various milk samples were collected from different sources, such as fresh cow milk, pasteurized milk from the supermarket, and follow-on milk for infants from the drug store.

For sample preparation, 50 µL probe (water for the blank), 50 µL of internal standard solution (water for the blank), and 100 µL of acetonitrile were filled in deep well plate tubes and mixed thoroughly. Sample pretreatment was performed automatically in the autosampler, where the probe was combined with borate buffer solution and the AminoTag^® reagent before injection. Derivatization was completed in the reaction unit at 60 °C before forwarding the amino acid derivatives obtained onto the analytical column (100 × 2.1 mm, 2.0-µm Shimpack UF-Amino) for separation and subsequent detection using the LCMS-2020 single-quadrupole mass spectrometer.

Using the overlap-injection feature, derivatization of a second sample was performed during analysis of the first probe, resulting in considerable time-saving during the course of the experiment.

Results
Calibration curves for each analyte of interest were created automatically according to the preset reprocessing method in the AmiNavi® Software.  Integration of the individual compounds was verified and corrected manually where necessary. The results of the quantification of the 38 amino acids included in the analytical method in the 11 milk samples is shown in an overview table as well as a display of individual chromatographic data (Figure 2; Table 1).

Figure 2: Processing – Calibration curve of the individual substances.

Amino acid content of 11 milk samples from different sources was determined and is displayed in Table 1. The milk differed largely in amino acid pattern. The highest total amino acid content was found in defatted and skimmed milk (471 µmol/L and 489 µmol/L), followed by fresh and pasteurized milk with a fat content of 3.8% and 3.5%, respectively (391 µmol/L and 306 µmol/L). The smallest amount of free amino acids with 22 µmol/L was found in an untreated milk sample from a penicillin treated cow. Taurin content was elevated in the follow-on milk from the drug store, as well as in a cow’s colostrum sample. Glutamic acid was the most abundant amino acid in the milk samples from the supermarket, while it was no more concentrated than other amino acids in untreated whole milk.

Table 1: Results of the determination of amino acids in milk samples (concentration in μmol/L). [Click Image to Enlarge].

Conclusion
A rapid and reliable HPLC–MS method for the determination of up to 38 amino acids in 9 min (12 min total run time), using an automated pre-column derivatization approach has been established. The assay showed good reproducibility of the derivatization procedure, as can be seen from the 10-point calibration curves that all fielded a linearity of R2 ≥ 0.99. Total content of amino acids in the 11 samples tested varied between 22–489 µmol/L, with significant variability in the amino acid distribution in the different types of milk.

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