GB 5009.286-2022National food safety standard - Determination of Natamycin in foods (English PDF)

3.1 Reagents

3.1.1 Methanol (CH4O). chromatographically pure.

3.1.2 Glacial acetic acid (C2H4O2).

3.2 Reference Substance

Natamycin (C33H47NO13, CAS. 7681-93-8). purity  98%, or reference substances certified by the state and awarded with a reference substance certificate.

3.3 Preparation of Standard Solutions

3.3.1 Natamycin standard stock solution (250 mg/L). accurately weigh-take 25 mg of

natamycin reference substance (accurate to 0.1 mg); add an appropriate amount of methanol and 1 mL of glacial acetic acid to perform ultrasonic dissolution; use methanol to reach a Weigh-take 5.0 g of sample (accurate to 0.01 g); place it in a 50 mL centrifuge tube; add 15.0 mL of methanol to perform ultrasonic extraction for 20 min. Then, add 5.0 mL of water to mix it up. At 4,000 r/min, centrifuge it for 10 min. In a refrigerator at 20 C, freeze it for 1 h. The supernatant is filtered through 0.22 m organic syringe-type filter. Collect about 2 mL of the filtrate and determine it on the instrument.

5.2.2 Pastry samples Weigh-take 5.0 g of sample (accurate to 0.01 g); place it in a 50 mL centrifuge tube; add 15.0 mL of methanol to perform ultrasonic extraction for 20 min. Then, add 5.0 mL of water to mix it up. At 4,000 r/min, centrifuge it for 10 min. The supernatant is filtered through 0.22 m organic syringe-type filter. Collect about 2 mL of the filtrate and determine it on the instrument.

5.2.3 Fruit and vegetable juice (pulp), fruit juice drinks Weigh-take 5.0 g of sample (accurate to 0.01 g); place it in a 50 mL centrifuge tube; add 15.0 mL of methanol to perform ultrasonic extraction for 20 min. At 4,000 r/min, centrifuge it for 10 min. The supernatant is quantitatively transferred to a 25 mL volumetric flask. Use water to reach a constant volume and mix it up. Use 0.22 m organic syringe-type filter to filter it.

Collect about 2 mL of the filtrate and determine it on the instrument.

5.2.4 Fermented wines Weigh-take an appropriate amount of the wine sample. Use 0.22 m syringe-type filter to filter it. Collect about 2 mL of the filtrate and determine it on the instrument.

5.3 Instrument Reference Conditions

5.3.1 Chromatographic column. C18 chromatographic column (4.6 mm  250 mm, 5 m), or equivalent.

5.3.2 Mobile phase. A. methanol, B. acetic acid + water = 5 + 95 (volume ratio).

5.3.3 Flow rate. 1.0 mL/min.

5.3.4 Detection wavelength. 305 nm.

5.3.5 Column temperature. 30 C.

5.3.6 Injection volume. 10 L.

5.3.7 The gradient elution procedure is shown in Table 1.