CN105651903A - Determination method of siraitia grosvenorii saponin V in siraitia grosvenorii - Google Patents

Abstract

The invention discloses a method for determining mogroside V in Luo Han Guo, which comprises the following steps: step 1: crushing Luo Han Guo and extracting it with an ASE extraction method to obtain an extract; step 2: centrifuging the extract to obtain a supernatant; Step 3: The supernatant was analyzed by liquid chromatography. The invention provides a method for determining mogroside V in Luo Han Guo, which has simple operation and high detection precision.

Description

A kind of assay method of mogroside V in Luo Han Guo

technical field

The invention relates to the field of laboratory detection, in particular to a method for determining mogroside V in Luo Han Guo.

Background technique

In 2010, the Pharmacopoeia recorded the method of extracting Mogroside V from Luo Han Guo: Take about 0.5g of the powder of this product (passed through a No. 4 sieve), accurately weigh it, put it in a stoppered Erlenmeyer flask, add 50ml of methanol precisely, seal it tightly, and weigh it Weight, heat to reflux for 2 hours, let cool, weigh again, make up the lost weight with methanol, shake well, and filter. Precisely measure 20ml of the continued filtrate, recover the solvent to dryness, add 10ml of water to dissolve, pass through the macroporous adsorption resin column AB-8 (inner diameter is 1cm, column height is 10cm), elute with 100ml of water, discard the water, and then use 20 Elute with 100ml of % ethanol, discard the eluent, continue to elute with 100ml of dilute ethanol, collect the eluent, recover the solvent to dryness, dissolve the residue with mobile phase, transfer to a 10ml measuring bottle, add mobile phase to the mark, shake Uniform, that is.

The parameters of the liquid chromatography are: octadecylsilane bonded silica gel is used as filler; acetonitrile-water (23:77) is used as mobile phase; detection wavelength is 203nm. The number of theoretical plates should not be less than 3000 based on the mogroside V peak.

The operation process of the pharmacopoeia method is complicated, and requires high operational skills of the operators.

Contents of the invention

The object of the present invention is to provide a method for determining mogroside V in Luo Han Guo, which is simple to operate and has high detection accuracy.

The technical scheme provided by the invention is: a method for assaying mogroside V in Luo Han Guo, comprising the following steps:

Step 1: crushing Luo Han Guo and extracting by ASE extraction method to obtain an extract;

Step 2: centrifuging the extract to obtain a supernatant;

Step 3: analyzing the supernatant by liquid chromatography;

Wherein, the analysis condition of liquid chromatography is:

a) Instrument: double ternary liquid phase

b) Column specification: 3*100mm3um

c) Column temperature: 40°C

d) Flow rate: 0.3mL/min

e) Mobile phase: acetonitrile-water, volume ratio 23:77

f) Detection wavelength: 203nm.

In the assay method of mogroside V in the above-mentioned Luo Han Guo, the described step 1 is specifically:

S11: crush the Luo Han Guo and sieve;

S12: Weigh 0.5 parts by weight of Luo Han Guo powder, mix it with 1 part by weight of diatomaceous earth and set aside;

S13: Add 2 parts by weight of silica gel to the extraction tank with the filter membrane in advance, mix well and then add an appropriate amount of diatomaceous earth until it reaches the pool mouth of the extraction tank; cover the lid of the extraction tank for static extraction; after the extraction is completed, the extraction is obtained liquid;

The extraction conditions are as follows: the extraction solvent is methanol; the extraction temperature is 100° C.; the static extraction time is 5 min; the washing volume is 100%; the static cycle number is 1 time.

In the above method for determining mogroside V in Luo Han Guo, the volume of the extraction pool is 10 ml.

In the above method for determining mogroside V in Luo Han Guo, the pressure of the static extraction is 1500 psi.

In the above method for determining mogroside V in Luo Han Guo, the purging time of the static extraction is 60s.

In the above-mentioned method for determining mogroside V in Luo Han Guo, step 2 specifically includes: centrifuging the extract for 3 min under the condition of 15000 r/min, and taking the supernatant.

In the above method for determining mogroside V in Luo Han Guo, the instrument is a ThermoU3000UHPLC liquid chromatograph.

In the above method for determining mogroside V in Luo Han Guo, the chromatographic column is ThermoSyncronis C18.

In the above method for determining mogroside V in Luo Han Guo, the instrument used in the ASE extraction method is an ASE350 fast solvent extractor.

After the present invention adopts above-mentioned technical scheme, the beneficial effect that it has is:

The detection method of the invention is simple to operate, has the same precision as the pharmacopoeia method, and has good repeatability.

Description of drawings

Fig. 1 is the Cartesian coordinate diagram of the effectiveness verification of chromatographic analysis of the present invention;

Fig. 2 is the chromatogram of mogroside V reference substance of the present invention;

Fig. 3 is the chromatogram that the present invention adopts pharmacopoeia method test to obtain;

Fig. 4 is the chromatogram obtained by the extraction and testing method of the present invention.

detailed description

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments, but this does not constitute any limitation to the present invention.

Example 1:

Grind Luo Han Guo through a grinder, pass through a No. 3 sieve, about 0.5g, weigh it accurately, mix it with 1g of diatomaceous earth, set aside, put the filter membrane in advance First add 2g of silica gel to the 10ml extraction pool, then add the well-mixed sample, then add an appropriate amount of diatomaceous earth, shake gently to make it on the same level as the pool mouth, and tighten the top cover of the extraction pool. After the extraction, the extract was transferred to a 25ml volumetric flask, diluted to the mark with methanol, centrifuged at 15000r/min for 3min, and the supernatant was taken for LC determination.

The extraction conditions (see table 1) are:

Table 1

close to Pharmacopoeia Extraction cell volume (ml) 10 extraction solvent Methanol pressure (psi) 1500 temperature(℃) 100 Static extraction time (min) 5 Static cycle times 1 Flushing volume (%) 100 Purge time (s) 60

Analysis method is LC liquid chromatography, liquid chromatography analysis conditions:

a) Instrument: Thermo double ternary liquid phase (U-3000);

b) Chromatographic column: ThermoSyncronis C183*100mm3um, that is, a carbon-18 chromatographic column with a diameter of 3mm, a length of 100mm, and a particle size of 3 microns;

c) Column temperature: 40°C;

d) Flow rate: 0.3mL/min;

e) Mobile phase: acetonitrile-water (volume ratio 23:77);

f) Detection wavelength: 203nm;

Validation of chromatographic analysis:

Take an appropriate amount of mogroside V reference substance, weigh it accurately, add mobile phase to make a solution containing 0.2 mg per 1 ml, and obtain the mogroside V reference substance.

Take an appropriate amount of Mogroside V reference substance, weigh it accurately, put it in a brown measuring bottle, add 50% acetonitrile to make a solution containing 880.4 μg of isoferulic acid per 1 ml, and then accurately draw 0.1 μl, 0.2 μl, and 0.5 μl of the solution , 0.8 μl, 1.0 μl enter LC measurement, and measure according to above-mentioned LC liquid chromatography, the results are shown in Table 2; Accompanying drawing 1 corresponds to Table 2, and accompanying drawing 1 abscissa represents concentration, and ordinate represents peak area. Accompanying drawing 2 is the chromatogram of the mogroside V reference substance measured by the above-mentioned LC liquid chromatography.

Table 2

1.1 The reproducibility verification of the extraction method of embodiment 1:

Take 0.5g of the sample (batch number: 1510180) of the same batch number, 6 parts in total, accurately weighed, extract the solution of the test product according to the ASE extraction method, the injection volume is 1 μL, and use the above-mentioned chromatographic conditions in parallel experiments to measure the concentration of Luo Han Guo in the sample. The content of saponin V is shown in Table 2, and the RSD is 1.46%. The test shows that the ASE extraction method has good repeatability, and the results are shown in Table 3.

table 3

1.2 The accuracy test of the extraction method of embodiment 1

The ASE extraction method of Example 1 and the extraction method of the Pharmacopoeia were used to extract Luo Han Guo, and there were 3 batches of Luo Han Guo, and the batch numbers were 1508166, 1510180, and 150723 respectively;

The ASE extraction method in Example 1 was tested twice in parallel on the same batch of Luo Han Guo, which are represented by ASE1 and ASE2 respectively.

Fig. 3 is the chromatogram that sample 1510180 obtains according to the method recorded in Pharmacopoeia;

Fig. 4 is the chromatogram that sample 1510180 obtains according to the ASE extraction method of the present invention and LC liquid chromatography test (ASE2).

The specific test results are shown in Table 4 below;

Table 4

The above are only preferred embodiments of the present invention, and any modifications, equivalent replacements and improvements made within the spirit and scope of the present invention shall be included within the protection scope of the present invention.

Claims (9)

1. an assay method for mogroside V in Momordica grosvenori, is characterized in that, comprises the following steps: Step 1: crushing Luo Han Guo and extracting by ASE extraction method to obtain an extract; Step 2: centrifuging the extract to obtain a supernatant; Step 3: analyzing the supernatant by liquid chromatography; Wherein, the analysis condition of liquid chromatography is: a) Instrument: double ternary liquid chromatograph b) Chromatographic column specifications: carbon 18 chromatographic column, 3*100mm3um c) Column temperature: 40°C d) Flow rate: 0.3mL/min e) Mobile phase: acetonitrile-water, volume ratio 23:77 f) Detection wavelength: 203nm. 2. the assay method of mogroside V in Luo Han Guo according to claim 1, is characterized in that, described step 1 is specially: S11: crush the Luo Han Guo and sieve; S12: Weigh 0.5 parts by weight of Luo Han Guo powder, mix it with 1 part by weight of diatomaceous earth and set aside; S13: Add 2 parts by weight of silica gel to the extraction tank with the filter membrane in advance, mix well and then add an appropriate amount of diatomaceous earth until it reaches the pool mouth of the extraction tank; cover the lid of the extraction tank for static extraction; after the extraction is completed, the extraction is obtained liquid; The extraction conditions are as follows: the extraction solvent is methanol; the extraction temperature is 100° C.; the static extraction time is 5 min; the washing volume is 100%; the static cycle number is 1 time. 3. the assay method of mogroside V in Luo Han Guo according to claim 2, is characterized in that, the volume of described extraction pool is 10ml. 4. the assay method of mogroside V in Luo Han Guo according to claim 2, is characterized in that, the pressure of described static extraction is 1500psi. 5. the assay method of mogroside V in Luo Han Guo according to claim 2, is characterized in that, the purging time of described static extraction is 60s. 6 . The method for determining mogroside V in Luo Han Guo according to claim 1 , wherein step 2 is specifically: centrifuging the extract at 15,000 r/min for 3 min, and taking the supernatant. 7. the assay method of mogroside V in Luo Han Guo according to claim 1, is characterized in that, described instrument is ThermoU3000UHPLC liquid chromatograph. 8. the assay method of mogroside V in Luo Han Guo according to claim 7, is characterized in that, described chromatographic column is ThermoSyncronisC18. 9. the assay method of mogroside V in Luo Han Guo according to claim 1, is characterized in that, the instrument that described ASE extraction method adopts is ASE350 fast solvent extractor.