AOAC 2019 First Action Methods

(a)

Prime solvent lines for 5 minutes.

(b)

Prime wash syringes for 4 cycles.

(c) Allow the column to equilibrate for at least 30 minutes (the initial composition is 99.9% mobile phase A and 0.1% mobile phase B).

(d) Allow the chromatographic system to stabilise before injecting the standards and samples.

(e)

Before starting the analyses, inject two blanks to condition the column.

Chromatographic conditions:

(a) (b) (c) (d) (e) (f) (g)

Column: Acquity UPLC HSS T3 100 x 2.1 mm x 1.8 µm

Column temperature: 45 ± 1°C Needle wash: 5% Acetonitrile

Injection volume: 1 µL PDA detector: 260 nm

Fluorescence detector: Ex: 350nm, Em: 450nm (optional)

Run time: 20 minutes

(h) Mobile phase A: 984 mL water, 10 mL acetonitrile, 6 mL formic acid and mix well. Filter and degas. (i) Mobile phase B: 100 % Acetonitrile. (j) Gradient program (Table 2019.09C ) I. Calculation and Expression of Results The calibration curve is established using blank and five calibration standards for each amino acid by plotting the peak area ratio of analyte to internal standard against concentration of analyte in mg/100 mL. The concentration of internal standard is kept constant in standards and samples and so the concentration is entered as 1. When a Chromatography Software is used, the software will perform the calculation as shown below. The software will automatically calculate results in mg/100 g, under following setup. (a) Set the standard method to internal standard L-norvaline. Calibration type is Linear with Offset except for Methionine which is Linear, forced through origin.

(b) Identify the internal standard (Norvaline) peak in the software and the concentration is entered as 1.

(c) Enter calculated amino acid analyte amount (mg/100 mL) for five levels of calibration and enter concentration as zero for blank.

(d)

Enter sample weight or calculated sample weight where slurry is used in grams.

(e) Enter the dilution factor, 40. The sample is finally diluted 40 times during sample preparation (digestion involves a 10 times and neutralization steps involves 4 times dilution). (f) The dilution in the final derivatization step with AQC is not taken into consideration as the standards are also derivatized as samples.