Amino acid analysis

To prepare the samples they were transferred from the − 80 °C freezer and placed on ice to be melted. To 50 µL of sample, 20 µL norleucine (500 μM) and 200 µL of methanol (kept at − 20 °C) were added and all are mixed for five seconds. To completely deproteinate, samples were kept at − 20 °C for 2 h, then centrifuged at 19,000 g for 12 min at 4 °C. The entire supernatant was transferred to a Heidolph rotary evaporator and dried in a vacuum. These samples could be stored at 4 °C for four weeks. For HPLC analysis, previously dried samples were dissolved in 100 µl of water (containing 0.01% formic acid). According to a previously described derivatization strategy, 10 µL o-phthaldialdehyde (OPA,) (for derivatization of primary amino acids) and 10 µL fluoronylmethyl chloroformate (FMOC-Cl) (for secondary amino acid derivatization) were added to 10 µl of each sample, and 20 µL of this was then injected into the HPLC system (Fekkes, 2012; Wu et al., 2016). For the HPLC–DAD method, a Knauer system (WellChrom, Germany) equipped with a K-1001 pump, a K-2800 diode array detector, an autosampler S3900 (Midas), a K-5004 analytical degasser, and a 2301 Rheodyneinjector with a 20 µL loop was used. HPLC separation was achieved using a Eurospher C18 column (4.6 mm × 250 mm, 5 µm), with a gradient elution program at a flow rate of 1.0 ml min⁻¹. The mobile phase was composed of solution A (acetonitrile + 0.05% trifluoroacetic acid, v/v) and solution B (0.05% aqueous trifluoroacetic acid, v/v). Then, the following gradient was applied: 0–10 min, isocratic gradient 70% B; 10–30 min, linear gradient 70–40% B; 30–40 min, linear 40–20% B; 40–50 min, linear 20–0% B; 50–65 min, linear 0–70% B; 65–75 min, isocratic gradient 70% B. The chromatographic peaks of the sample solution were identified by spiking and comparing their retention times and UV spectra with those of reference standards. Quantitative analysis was carried out by integration of the peak using the standard external method. To detect primary amino acids, the fluorescence detector was set at 337 nm and 470 nm for adsorption and excitation, respectively. Also, detection of the first-type and the second-type amino acids was performed at 262 nm and 338 nm. The instrument stability of the analyses was checked during the study using quality control samples (QCs), a pool of all samples. This pool was prepared through mixing equal volumes of all samples and stored in 100 µl aliquots to avoid the repeated freeze–thaw cycles. A QC sample was injected in every six samples after being prepared under the same conditions. Measurement variability was assessed by calculating CVs based on QCs. All 22 amino acids showed acceptable repeatability with CVs < 10% in QCs. Also, as mentioned, the accuracy and precision of both derivatization and HPLC technique were performed using analyzing of five individual samples. The intra-day mean coefficient of variation (n = 3) and the inter-day mean coefficient of variation (n = 3) were within 2% and 7%, respectively.