2.3.9. Determination of Total Free Amino Acids

For the extraction and identification of free amino acids, 1.75 ± 0.1 g of sample was mixed with 15 mL of 15% trichloroacetic acid (TCA). The pH of the mixture was adjusted to 2.2 (isoelectric precipitation point of the proteins) and the extract was further diluted to 25 mL with 15% trichloroacetic acid. Then, the supernatant was collected and filtered using 0.45 μm microfilters. A total of 100 μL of filtered supernatant was subjected to the determination of organic components, using the EZfaast GC-MS kit (Phenomenex, Torrance, CA, USA), following the protocol given by the manufacturer. Identification and separation of free amino acids was performed using a gas chromatograph coupled with a mass spectrometer (MS) equipped with a Zebron TM ZB-AAA column (10 m × 0.25 mm, film thickness: 0.25 μm). Injection: split 1:15, carrier gas: helium 1.1 mL/min, oven program: 30 ◦C/min from 110 ◦C to 320 ◦C. The MS parameters: source temperatrure 240 ◦C, scan range 45–450 *m*/*z*, sampling rate 3.5 scans/s. The identification of each amino acid was performed by calculating the area of each "peak" and comparing it with a standard consisting of 33 amino acids (alanine, sarcosine, glycine, α-aminobutyric acid, valine, βamino isobutyric acid, internal standard, leucine, allo-isoleucine, isoleucine, threonine, serine, proline, asparagine, thioproline, aspartic acid, methionine, 3-hydroxyproline/4 hydroxyproline, phenylalanine, glutamic acid, α-aminoadipic acid, α-aminopimelic acid, glutamine, ornithine, glycyl-prolizine—2 isomers, proline-hydroxyproline, histidine, lysine, tyrosine, tryptophan, cystathionine and cystine). The results are expressed as μg/mg bee pollen; all the determinations were carried out in triplicate.
