GB 5009.150-2016National food safety standard - Determination of Monascus Pigments in Foods (English PDF)

3.1 Reagents

3.1.1 Methanol (CH3OH): chromatographic pure.

3.1.2 Absolute ethanol (CH3CH2OH): chromatographic pure.

3.1.3 Glacial acetic acid (CH3COOH).

3.1.4 Ammonia acetate (CH3COONH4).

3.2 Preparation of reagents

3.2.1 Acetic acid-ammonium acetate solution (pH=5): accurately weigh 0.77 g of

ammonium acetate in a 1 L volumetric flask; add 900 mL of water to dissolve; use glacial acetic acid to adjust pH=5.0; add water to fix-volume to 1 L; mix well. Use it after it is filtered through a 0.45 µm microporous membrane.

4.5 High-speed pulverizer.

4.6 Solid-phase extraction device.

4.7 Organic-phase microporous membrane: pore size of 0.45 µm.

5.1 Sample preparation and storage

5.1.1 Liquid sample

Shake and pack the samples such as fruit juice, fruit juice drink and fruity carbonated drink; seal at room temperature or refrigerate to store.

5.1.2 Semi-solid sample

For samples such as jelly and flavor fermented milk, take the edible portion to homogenate; stir evenly; package; seal; refrigerate or freeze to store.

5.1.3 Solid sample

For samples of low-water content, such as biscuits, cakes and cooked meat products, use the high-speed pulverizer to pulverize; package; seal and store at room temperature out of the sun. For powdery samples with a uniform shape such as solid beverage, package directly; seal and store at room temperature out of the sun.

5.2 Sample processing

5.2.1 Liquid sample

Weigh 5 g (accurate to 0.01 g) of uniform sample (if the sample contains carbon dioxide; first heat to remove it) in a beaker; use an appropriate amount of absolute ethanol to dissolve it; transfer it to a 50 mL volumetric flask; add absolute ethanol to fix-volume; shake well. Accurately transfer 20 mL of extract which is mentioned above to a 100 mL pear-shaped bottle; add 10 mL of absolute ethanol to the pear-shaped bottle; rotate-concentrate the mixture to near dry at 60°C ± 2°C; use 2 mL of 20% methanol solution to wash the pear-shaped bottle twice.

Transfer all the above-mentioned extracts to a pre-activated HLB solid-phase extraction column; control the flow rate at 1 mL/min ~ 2 mL/min; discard the effluent.

Use 5 mL of 40% methanol solution to rinse the purification column; discard the effluent;

then use 5 mL of methanol to elute; control the flow rate at 1 mL/min ~ 2 mL/min;

collect the eluate in a 25 mL graduated centrifuge tube. Use nitrogen to concentrate the eluent at 45°C; then, use 2 mL of 40% methanol solution to dissolve the residue;

filter it through a 0.45 µm organic filter membrane; test it later.

5.2.2 Semi-solid and solid samples

Weigh 5 g (accurate to 0.01 g) of uniform sample; put it into a 50 mL plastic centrifuge tube; add 20 mL of 80% ethanol solution; homogenize at 15 000 r/min for 2 min and at 4 000 r/min for 3 min; transfer the supernatant to a 50 mL volumetric flask; add 20 mL of 80% ethanol solution to the residue and repeat the extraction once; combine the extracts in the same volumetric flask; use absolute ethanol to fix-volume; shake well.

Accurately transfer 20 mL of extract which is mentioned above to a 100 mL pear- shaped bottle; add 10 mL of absolute ethanol to the pear-shaped bottle; rotate- concentrate the mixture to near dry at 60°C ± 2°C; use 2 mL of 20% methanol solution to wash the pear-shaped bottle twice. Transfer all the above-mentioned purification liquor to a pre-activated HLB solid-phase extraction column; control and the flow rate at 1 mL/min ~ 2 mL/min; discard the effluent. Use 5 mL of 40% methanol solution to rinse the purification column; discard the effluent; then use 5 mL of methanol to elute;

control the flow rate at 1 mL/min ~ 2 mL/min; collect the eluate in a 25 mL graduated centrifuge tube. Use nitrogen to concentrate the eluent at 45°C; then, use 2 mL of 20% methanol solution to dissolve the residue; filter it through a 0.45 µm organic filter membrane; test it later.

5.3 Apparatus reference conditions

Apparatus reference conditions are listed as below:

a) Chromatographic column: high-purity spherical porous silica gel multi-point bonding

C18 amino column, with a column length of 250 mm, an inner diameter of 4.6mm, a particle size of 5 µm, OR columns of equivalent performance;

b) Mobile phase: A: acetic acid-ammonium acetate solution (pH=5); B: methanol, gradient elution is shown in Table 1;

c) Column temperature: 40°C;

d) Injection volume: 20 µL;

e) Detection wavelength: that for monascorubramine is 264 nm, and for monascorubrin and monacolin is 390 nm.

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