AOAC 2019 First Action Methods

(c) For homogenous samples, weigh 0.10 ± 0.01 g of well blended sample into digestion tube and record the weight. Add 2.5 mL of water and vortex well to mix. Note : The approximate protein content of the samples must be known or analyzed to determine the sample weight and dilution. For samples with very low protein, increase the sample weight to gain sensitivity, e.g., weigh 0.30 g of sample for samples with not more than 5% protein. Samples with protein content of not less than 30%, take two samples from slurry – one with 1.0 g for low level amino acids and one with 0.50 g for high level amino acids. Make up the total volume to 2.6 mL with water. (d) To each tube, add: 1 mL 1% DTDPA solution in 0.2 M NaOH; 1 mL 0.2M HCl; 400 µL norvaline stock internal standard solution (10 mM) and 5 mL 0.1% phenol in 12 M HCl solution. (e) Vortex the ampoules for 5 minutes, at room temperature to remove dissolved air. (f) Flush the air space above the liquid in the digestion tubes with a gentle stream of nitrogen gas to displace air and close the digestion tubes air-tight (using a hot acetylene flame, seal the neck of the glass ampoules or close the culture tubes). (g) Place the digestion tubes in an oven at 110 ± 2°C for 24 ± 0.5 hours. (h) Remove the digestion tubes from the oven; allow hydrolysate to cool down to room temperature. Vortex the samples and open the tubes (break open the tapered sealed part of the glass ampoules or unscrew the cap of culture tubes). (i) Filter the hydrolysate using a 0.45 µm syringe filter into 30 mL disposable tubes. (j) Pipet accurately 1.0 mL of sample hydrolysate into a 30 mL disposable tube, add 1.0 mL of 6M NaOH, 2.0 mL of 0.1M HCl and mix well. G. Derivatization of Samples and AA Calibration Standards Note : The derivatization converts free amino acids into highly stable derivatives. Standards and samples are derivatized as follows: (a) Preheat the Thermoshaker heating block to 55 ± 2°C. (b) With a pipet, add 140 µL of borate buffer to a 2 mL safe-lock tubes. (c) Add 20 µL of AA calibration standards or hydrolysates into the safe-lock tubes. (d) Close the tubes and vortex for 20 seconds. (e) Open and add 40 µL of AQC derivatising reagent to each of the tubes. (f) Close and vortex mix immediately for 20 seconds. (g) Heat the tubes in the heating block with shaking set at 55 ± 2°C, 1000 rpm for 10 minutes. (h) Cool to room temperature and transfer into UHPLC vials with inserts. (i) Proceed to UHPLC analysis. H. Chromatography

Conditioning the UHPLC system: