AOAC 2019 First Action Methods

(q)

Disposable tubes, 30 mL

(r) UHPLC system.--Thermo Scientific Dionex Ultimate 3000 RS systemwith LPG pump, diode array detector (DAD) 3000 RS with a semi-micro flow cell, 2.5 µL, SST, fluoro detector FLD 3000 RS with micro flow cell 2 µL, SST or equivalent

(s)

Acquity UPLC HSS T3 UHPLC column 100 x 2.1 mm x 1.8 µm (Waters Corp.)

(t)

Acquity UPLC HSS T3 Van Guard precolumn 5 x 2.1 mm x 1.8 µm (Waters Corp.)

E. Standard Preparation (a) Internal standard solution, stock (10 mM): In a 100mL volumetric flask, weigh 117 ± 2 mg of norvaline, dissolve and make up to volume with 0.1M HCl. Stable for a month at 2 to 8°C (fridge). (b) Internal standard solution (2.5 mM): Pipet 2.5mL of the norvaline stock internal standard solution into a 10 mL volumetric flask and make up to volume with 0.1M HCl. Stable for a month at 2 to 8°C (fridge). (c) Amino acid (AA) stock standard solution: In a 100mL volumetric flask, weigh amino acids using a 4 decimal place balance as per Table 2019.09A . Dissolve in 80 mL of 1 M HCl, sonicate to dissolve the amino acids and make up the volume with 1 M HCl. Store this solution at 2 to 8 ° C (refrigerator) for up to 1 month. (d) AA intermediate standard solution: Pipet accurately 1 mL of AA stock solution, 2 mL of water, 1 mL of 1% DTDPA, 1 mL of 0.2 M HCl and add 5 mL of 0.1% phenol in 12M HCl solution into a digestion tube. Flush the air space above the liquid in the digestion tube with a gentle stream of nitrogen gas to displace air. Using a hot acetylene flame, seal the neck of the glass ampoules and ensure there are no leaks or close the culture tubes immediately with PTFE caps. Place the digestion tubes in an oven at 110 ± 2°C for 24 ± 0.5 hours. Take the tubes out from the oven; allow hydrolysates to cool down to room temperature. Break open the tapered sealed part of the glass ampoules or open the culture tubes and filter the hydrolysate using a 0.45 µm syringe filter into 30 mL disposable tubes. (e) AA calibration standards (neutralization): Follow Table 2019.09B to neutralize and prepare 10 mL of calibration standards. Into a 10 mL volumetric flask, pipette the standards, 6M NaOH and internal standards as shown in Table 2019.09B . Make up the volume with 0.1 M HCl. Filter using 0.45 µm syringe filter. The AA calibration standards are to be prepared fresh for each run. F. Sample Preparation (a) For milk powders and infant formula weigh 10.00 ± 0.01 g into a 120 mL plastic container and record weight (W1). Without taring the balance make the total weight to 100.00 ± 0.01 g with water and record weight (W2). Mix well to make a homogenous slurry and weigh accurately 1.00 ± 0.01 g into digestion tube and record the weight (W3). Add 1.6 mL water.

Sample (powder) equivalent of slurry (g) = (W1 x W3) / W2

(b) For liquid milk use 1.00 ± 0.01 g of sample into digestion tube and record the weight. Add 1.6 mL water.