AOAC SPIFAN Nutrients ERP (December 7, 2022)

HMO-02 (Analysis Paper) 10-2022 FOR ERP USE ONLY DO NOT DISTRIBUTE

Table 1: Scheme for preparation of working standard solutions.

Vol. of Stock Solution [E(a – g)] (µL)

Final Vol. (mL)

Calibration Level

2’FL

3FL

DFL

3’SL

6’SL

LNT

LNnT

1 2 3 4 5 6

50

50

50 70 70

40 75 80

40 75 80

40 70 70

50 60 60 90

50 10

110 110 170 240 300

110 110 170 240 300

5 5 5 5

110 150 200

130 180 240

130 180 240

110 150 200

130 170

(i) Laminaritriose internal standard stock solution (1.0 mg/mL): Transfer about 50 mg of Laminaritriose [D(h)] into a 50-mL volumetric flask and complete to the mark with water. This solution can be stored for up to 1 year at -18°C (j) Laminaritriose internal standard working solution (0.4 mg/mL): Transfer 10 mL of the laminaritriose stock solution [E(i)] into a 25-mL volumetric flask and make up to the mark with water. This solution can be stored for up to 1 year at -18°C (k) Sodium acetate buffer (0.2 M, pH 4.5 ): Into a beaker containing 400 mL of demineralised water [D(a)], pipette 5.8 mL of glacial acetic acid [D(f)]. Adjust to pH 4.5 with sodium hydroxide solution 1 M [D(k)]. Transfer the solution to a 500-mL volumetric flask and make up to the mark with water. This solution can be stored for up to 1 month at 4-8°C. (l) Amyloglucosidase (AMG) solution (70 U/mL in 0.2 M sodium acetate buffer pH 4.5): Weigh into a glass tube an amount of amyloglucosidase [D(m)] corresponding to 14 U ± 2 U of AMG per test sample and add sodium acetate buffer (0.2 M pH 4.5[E(k)]) to reach a final concentration of 70 U/mL. Dissolve by mixing on a vortex mixer. This solution is prepared on the day of use and kept at 4 °C until use. (m) Mixed Enzyme Solution ( β -galactosidase + Amyloglucosidase): Depending on the number of tests to perform (100 µL per test), prepare a 8:2 ratio mixture of AMG (70 U/mL in Na acetate buffer 0.2 mol/L pH 4.5 [E(l)] and beta-galactosidase enzyme suspension (4000 U/mL) [D(l)]. (n) Water: acetonitrile solution (25:75): Add 125 mL ± 2 mL of water [D8a)] to 375 mL ± 2 mL of acetonitrile [D(g)] in a glass bottle and mix. This solution can be stored for up to 3 months at room temperature. (o) 2AB labelling reagent: 2AB [0.35 mol/L] – Picoline Borane [1.0 mol/L] in DMSO:acetic acid [70:30] solution: Pipette the volume of dimethylsulfoxide (DMSO) [D(b)] and glacial acetic [D(f)] acid in a 50-mL glass tube according to the number of tests to perform (Table 2). Mix the solution using a vortex mixer. Weigh the amount of 2-aminobenzamide (2AB) [D(c)] and picoline borane [D(d)] (Table 2) in another 50-mL glass tube, then add the corresponding volume of DMSO:acetic acid (Table 2). Mix using a vortex mixer. The labelling reagent solution can be prepared on the day of use or can be stored at – 18 °C protected from moisture for up to three months.

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