Basis: GB/T 22388-2008
Principle: The sample is extracted with trichloroacetic acid-acetonitrile, purified by cation exchange solid phase extraction column, and determined by high performance liquid chromatography, quantified by external standard method.
Reagents and materials: Unless otherwise stated, all reagents were of analytical grade and the water was first-grade water specified in GB/T 6682.
3.1 Methanol: chromatographically pure;
3.2 Acetonitrile: chromatographically pure;
3.3 Ammonia water: content is 25% ~ 28%;
3.4 Trichloroacetic acid;
3.5 Citric acid.
3.6 Sodium octane sulfonate: chromatographically pure;
3.7 Aqueous methanol solution: accurately measure 50 mL of methanol and 50 mL of water, mix and reserve;
3.8 Trichloroacetic acid solution (1%): Accurately weigh 10 g of trichloroacetic acid in a 1 L volumetric flask, dissolve in water and dilute to volume, mix and set aside;
3.9 Ammoniated methanol solution (5%): accurately measure 5 mL of ammonia water and 95 mL of methanol, mix and reserve;
3.10 Ion pair reagent buffer: Accurately weigh 2.10 g of citric acid and 2.16 g of sodium octane sulfonate, add about 980 mL of water to dissolve, adjust the pH to 3.0, and dilute to 1 L for use.
3.11 Melamine standard: CAS 108-78-01, purity greater than 99.0%;
3.12 Melamine standard stock solution: Accurately weigh 100 mg (accurate to 0.1 mg) melamine standard in a 100 mL volumetric flask, dissolve it with methanol aqueous solution (3.7) and dilute to volume to prepare a standard of 1 mg/mL. Store stock at 4°C protected from light.
3.13 Cation Exchange SPE: Mixed cation-exchange solid phase extraction column, benzenesulfonic matrix is polystyrene-divinyl benzene polymer, 60 mg, 3 mL, or equivalent.
3.14 Qualitative filter paper.
3.15 Microporous membrane: 0.2 μm, organic phase.
3.16 Nitrogen: purity greater than or equal to 99.999%.
Instruments and equipment
4.1 High Performance Liquid Chromatography (HPLC): equipped with a UV detector or a diode array detector.
4.2 Analytical balance: sensitivities are 0.00001 g and 0.01 g.
4.3 Centrifuge: speed not less than 10000 r/min.
4.4 Tianjin Hengao ultrasonic extractor. HS, HU series
4.5 Tianjin Hengao solid phase extraction device. HSE-12D
4.6 Tianjin Heng Ao Nitrogen Blowing Instrument. HGC, HSC series
4.7 Tianjin Hengao vortex oscillator. HMS-350
4.8 Tianjin Hengao vacuum pump. HPD-25
4.9 Tianjin Hengao precision gas steady flow regulating valve.
4.10 Stoppered plastic centrifuge tube: 50 mL.
Sample processing
5.1 Extraction
Weigh 2 g (accurate to 0.01 g) sample in a 50 mL stoppered plastic centrifuge tube, add 15 mL of trichloroacetic acid solution (3.8) and 5 mL of acetonitrile. Ultrasonic extraction for 10 min, then shaking for 10 min, centrifuged at not less than 10000 r/min for 30 min. After the supernatant was filtered through a filter paper moistened with trichloroacetic acid solution, the volume was adjusted to 25 mL with a trichloroacetic acid solution, 5 mL of the filtrate was removed, and 5 mL of water was added to mix and be purified.
Note: If the fat content of the sample is high, it can be degreased with hexane liquid-saturated with trichloroacetic acid solution and then purified by SPE column.
5.2 Activation
A cation exchange solid phase extraction column was activated (3.13) with 3 mL of methanol and 5 mL of water in that order. Precision gas steady flow control valve before rotating solid phase extraction device to make the washing liquid flow rate not exceed 1 mL/min
5.3 Loading
5.1 liquid to be purified is transferred to a solid phase extraction column (5.2) in.
5.4 Rinse
Wash with 3 mL of water and 3 mL of methanol in turn, and draw to near dryness.
5.5 Elution
Elute with 6 mL of ammoniated methanol solution (3.9) and collect the eluate in a test tube. The flow rate of the whole solid phase extraction process does not exceed 1 mL/min.
5.6 Concentration
The eluate was dried with nitrogen at 50°C. The residue (corresponding to 0.4 g of sample) was made to volume with 1 mL of mobile phase, vortexed for 1 min, and passed through a microporous membrane for HPLC.
High performance liquid chromatography
HPLC reference conditions
a) Column: C8 column, 250 mm × 4.6 mm (id), 5 μm, or equivalent; C18 column, 250 mm × 4.6 mm (id), 5 μm, or equivalent.
b) Mobile phase: C8 column, ion pair reagent buffer (3.2.10) - acetonitrile (85+15, volume ratio), mix. C18 column, ion pair reagent buffer (3.2.10) - acetonitrile (90+10, volume ratio), mix.
c) Flow rate: 1.0 mL/min.
d) Column temperature: 40°C.
e) Wavelength: 240 nm.
f) Injection volume: 20 μL.
Analysis
With GB / T 22388-2008 standard test method for analysis of a sample measured using the device of constant Tianjin Otis recovery results were as follows:
Add level (mg/Kg) Recovery rate
blank
2 116%
4 108%
6 92%
8 96%
It can be seen from the above table that using Tianjin Hengao equipment to process samples can not only improve the speed of analyzing samples, but also obtain satisfactory recovery.
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