GB/T 23884-2021Determination of biogenic amines in animal-derived feeds—High performance liquid chromatography (English PDF)

5.3 Acetone

1476-39-7, purity. >=99%), 13 mg of phenethylamine (CAS No.. 64-04-0, purity. >=98%), 18 mg of putrescine dihydrochloride (CAS No.. 333-93-7, purity. >=98%), 10 mg of cadaverine (CAS No.. 462-94-2, purity. >=98.8%), 10 mg of histamine (CAS No.. 51-45-5, purity. >=97%), 12 mg of octopamine hydrochloride (CAS No.. 770-05-9, purity. >=99.3%), 10 mg of tyramine (CAS No.. 51-67-2, purity. >=98%), 17 mg of spermidine trihydrochloride (CAS No.. 334-50-9, purity.

>=99.9%), and 10 mg of spermine (CAS No.. 71-44-3, purity. >=98%) (weighed to the nearest 0.01 mg). Transfer each substance individually into a separate 10 mL amber volumetric flask, dissolve in the hydrochloric acid solution (5.10), and dilute to the mark. Store at temperatures below -18°C; the shelf life is 6 months.

NOTE: The concentration of the prepared standard solutions is calculated based on the free base form of each biogenic amine; when weighing the standards, the contribution of the hydrochloride salt component must be taken into account. Once prepared, all standard solutions shall be transferred to airtight amber containers and stored away from light.

0.25 µg/mL, 0.5 µg/mL, 5.0 µg/mL, 10.0 µg/mL, 25.0 µg/mL, 50.0 µg/mL, and 100.0 µg/mL. Prepare immediately before use.

7.1 Solid Samples

Prepare the sample in accordance with GB/T 20195. Take a minimum of 100 g of sample, pulverize it so that it completely passes through a 0.42 mm analytical sieve, mix thoroughly, transfer it into a ground-glass stoppered bottle, and store it away from light for future use.

7.2 Paste or Slurry Samples

Prepare the sample in accordance with GB/T 20195. Take a minimum of 100 g of sample, and mix it using a mechanical stirrer or homogenizer. While shaking, transfer 100 g or 100 mL of the sample into a ground-glass stoppered bottle using a spoon or a wide-mouth pipette; store it away from light for future use.

8.1 Extraction

Perform two parallel determinations. Weigh out 1 g of the test sample, accurate to 0.000 1 g, and place it into a 50 mL centrifuge tube. Accurately add 10 mL of the perchloric acid extraction solution (5.7), and mix thoroughly using a vortex mixer (for paste or slurry samples, homogenize for 30 s). Place the tube on a shaker and oscillate at 200 r/min at room temperature for 10 min. Centrifuge at 5 000 r/min for 10 min. Decant the supernatant into another 50 mL centrifuge tube. Extract the residue again with 10 mL of the perchloric acid extraction solution (5.7), repeat the above procedure, and combine the supernatants for future use.

8.2 Derivatization

Accurately pipette 1 mL of the supernatant (see 8.1) into a 15 mL stoppered centrifuge tube.

Sequentially add 200 µL of sodium hydroxide solution (5.8), 300 µL of saturated sodium bicarbonate solution (5.9), and 2 mL of dansyl chloride derivatization solution (5.11). Mix thoroughly using a vortex mixer, then place the tube on a shaker and allow the reaction to proceed at 45°C in the dark for 45 min. Add 100 µL of aqueous ammonia (5.4) to terminate the reaction, mix thoroughly, and let stand for 15 min. At 45°C, use a stream of nitrogen gas to evaporate the acetone. Dilute to a final volume of 5 mL with acetonitrile (5.2), sonicate to redissolve, and mix thoroughly. Filter the solution through a 0.22 µm organic microporous filter membrane (5.15) and set aside for future use.

Accurately pipette 1 mL of each mixed standard series solution (5.14) and subject them to derivatization simultaneously with the test sample solution.

8.3 Reference Conditions for Liquid Chromatography

Chromatographic column. C18 column, 250 mm in length, 4.6 mm in inner diameter, 5 µm in particle size, or one of equivalent performance. Column temperature. 30°C. Detection wavelength. 254 nm.

Mobile phase. Ammonium acetate solution (5.6) and acetonitrile (5.2) (degassed by sonication prior to use). Flow rate. 1.0 mL/min. Injection volume. 10 µL. The gradient elution program is presented in Table 1.