CN104865319B - Ultra performance liquid chromatographic dual-wavelength multi-index content determination method for six-ingredient glutinous rehmannia preparation - Google Patents

Abstract

The invention discloses an ultra performance liquid chromatographic dual-wavelength multi-index content determination method for a six-ingredient glutinous rehmannia preparation. The method comprises the following steps: (1) taking the six-ingredient glutinous rehmannia preparation and dissolving the six-ingredient glutinous rehmannia preparation in methanol so as to obtain a six-ingredient glutinous rehmannia preparation solution with a concentration of 1 g/10-30 ml; (2) dissolving a standard substance in methanol so as to obtain a standard substance solution with a concentration of 20-30 [mu]g/10-30 ml; (3) respectively acquiring the chromatograms of the six-ingredient glutinous rehmannia preparation solution and the standard substance solution under same detection conditions by using ultra performance liquid chromatography, wherein the detection conditions are that octadecyl silane bonded silica glue is used as a filler for a chromatographic column, gradient elution is carried out, a mobile phase A is an aqueous phosphoric acid solution with a concentration of 0.08 ml/100 ml, a mobile phase B is acetonitrile, and detection wavelengths are in a range of 200 to 220 nm and a range of 238 to 250 nm, respectively; and (4) calculating the contents of components in the six-ingredient glutinous rehmannia preparation corresponding to components in the standard substance according to the concentration and chromatogram of the standard substance. The method provided by the invention can realize rapid and comprehensive detection of main components in the six-ingredient glutinous rehmannia preparation and has the advantages of easy availability of instruments, simplicity and good operationality.

Description

Ultra-high performance liquid phase dual-wavelength multi-index content determination method for Liuwei Dihuang preparation

technical field

The invention relates to the technical field of traditional Chinese medicine testing, and more specifically relates to an ultra-high-efficiency liquid-phase dual-wavelength multi-index content determination method for Liuwei Dihuang preparation.

Background technique

As a representative prescription of "nourishing kidney yin", Liuwei Dihuang's curative effect is unquestionable. There are many Liuwei Dihuang preparations on the market today, so only by ensuring its quality can this classic ancient prescription give full play to its therapeutic advantages. The content determination stipulated in the 2010 edition of the Chinese Pharmacopoeia: For the three dosage forms of Shuimiwan, Xiaomiwan and Damiwan, the content of Moutan cortex is calculated as paeonol, and Shuimiwan must not be less than 0.90mg per 1g; Xiaomiwan must not be less than 1g per 1g. Less than 0.70mg; each big honeyed pill should not be less than 6.3mg. Cornus cornus containing wine is calculated as loganin, water honeyed pills shall not be less than 0.70 mg per lg; small honeyed pills shall not be less than 0.50 mg per lg; large honeyed pills shall not be less than 4.5 mg per lg. For concentrated pills, every 1g of wine cornel should not be less than 1.4mg, calculated as loganin, and per 1g of Moutan cortex, calculated as paeonol, should not be less than 1.8mg. Each capsule contains Moutan cortex, calculated as paeonol, the specification (1) shall not be less than 3.0mg; specification (2) shall not be less than 1.5mg, and wine cornus shall be calculated as ursolic acid, and the specification (1) shall not be less than 0.60 mg; specification (2) shall not be less than 0.30mg. Wherein paeonol and loganin are detected by high-performance liquid chromatography, and the chromatographic conditions are different, and the detection wavelength is 274nm and 236nm respectively, and ursolic acid is determined by thin-layer chromatography, and the operation steps are cumbersome and time-consuming. It is beneficial to fast and efficient detection and cannot evaluate and control the quality of Liuwei Dihuang whole prescription from the whole.

In the past, the research reports on the quality control of Liuwei Dihuang preparations were mostly about high performance liquid chromatography coupled with ultraviolet detectors, but often fewer components can be detected at the same time, and quality control cannot be carried out as a whole. Micellar electrokinetic capillary chromatography is used to determine the content of multiple components in Liuwei Dihuang Pills, and all four components can be detected simultaneously. However, the popularization of this method is limited by the lack of instruments. It is also reported in the literature that high performance liquid chromatography coupled with diode array and evaporative light scattering detector can simultaneously detect eight components in Liuwei Dihuang Pills, but the detection process using two detectors is cumbersome and time-consuming. Ultra-high performance liquid chromatography (UHPLC) adopts a small particle packing column and an ultra-high pressure system, which can significantly improve the resolution and detection sensitivity of chromatographic peaks, and can significantly shorten the analysis cycle and reduce reagent consumption. It is equipped with a diode array detector (DAD ), the detection range is wider. Therefore, the use of ultra-high performance liquid chromatography and DAD full-wavelength scanning to determine the appropriate detection wavelength can quickly and comprehensively detect the main components in Liuwei Dihuang pills and capsules. The instrument is easy to obtain and the method is simple and operable. A powerful tool for quality control of compound preparations.

Contents of the invention

The object of the present invention is to provide an ultra-high performance liquid-phase dual-wavelength content determination method for the multi-component Liuwei Dihuang preparation in view of the defects in the prior art. The method provided by the invention can quickly and comprehensively detect the main components in the Liuwei Dihuang preparation, has easy-to-obtain instruments, is simple and convenient, and has strong operability.

The invention provides an ultra-high performance liquid-phase dual-wavelength multi-index content determination method for Liuwei Dihuang preparations. The method specifically includes the following steps:

(1) Take the Liuwei Dihuang preparation, pulverize it, weigh it accurately, dissolve it in methanol at a concentration of 1g/10-30ml, extract it by ultrasonic, centrifuge, take the supernatant and filter it, and obtain the Liuwei Dihuang preparation solution, which is set aside;

(2) Accurately weigh the standard substance, dissolve it in methanol at a concentration of 20-30 μg/ml, and obtain the standard substance solution, and set aside;

(3) Using ultra-high performance liquid chromatography, obtain the chromatograms of the Liuwei Dihuang preparation solution and the standard solution under the same detection conditions; the detection conditions include:

The column filler is octadecylsilane bonded silica gel;

Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～5min, 2～25% mobile phase B; 5～8min, 25～70 % mobile phase B; 8 ~ 8.1min, 70-95% mobile phase B; 8.1 ~ 13min, 95% mobile phase B;

The detection wavelength is 200-220nm and 238-250nm;

(4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance .

The Liuwei Dihuang preparation of the present invention is Liuwei Dihuang water honeyed pills, large honeyed pills, concentrated pills or capsules.

The step (1) of the present invention optimizes the extraction of the Liuwei Dihuang preparation and the preparation method of the solution, thereby increasing the detection accuracy. Specifically, the ultrasonic frequency of the ultrasonic extraction is preferably 35000-45000 Hz, and the extraction time is preferably 20-40 min. Under the above extraction conditions, the centrifugation speed is preferably 5000-20000 rpm, and the centrifugation time is 5-15 min. After centrifugation, the supernatant can be filtered using a filter membrane with a pore size of 0.2-0.3 μm. By optimizing the above conditions, the accuracy of detection in subsequent steps can be improved.

As a preferred solution, the step (1) of the present invention is specifically: take Liuwei Dihuang preparation, crush it and weigh it accurately, dissolve it in methanol at a concentration of 1g/10ml, extract it with an ultrasonic wave with a frequency of 40000Hz for 30min, and extract it with a frequency of 40000Hz for 30min. Centrifuge at a high speed for 10 minutes, take the supernatant, and filter it with a filter membrane with a pore size of 0.22 μm to obtain the Liuwei Dihuang preparation solution.

The standard product described in the step (2) of the present invention is selected according to the active ingredients in the Liuwei Dihuang preparation and the detection ingredients specified in the Pharmacopoeia, including gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose , Paeonol, 5-Hydroxymethylfurfural, Morroniside, Loganin, Swaticurin, Paeoniflorin, Benzoic Acid, and Benzoyl Paeoniflorin. In the present invention, the concentration of the standard substance refers to the concentration of each standard substance. In actual operation, various standard substances can be accurately weighed respectively, combined, dissolved in a specific amount of methanol, and mixed Uniformly, a mixed standard solution is obtained. The concentration of the standard product is preferably: gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin , swertigin, paeoniflorin, benzoic acid and benzoylpaeoniflorin concentrations were 25μg/ml.

Since different components have different peaks at different wavelengths, the present invention preferably detects gallic acid, protocatechuic acid, and 1,2,3,4,6-pentagalloyl under the condition that the detection wavelength is 200-220nm. Glucose, paeonol; under the detection wavelength of 238-250nm, detect 5-hydroxymethylfurfural, morroniside, loganin, swertigin, paeoniflorin, benzoic acid, benzoylpaeoniflorin.

The wavelengths detected in step (3) of the present invention are preferably 210 nm and 238 nm. Using these two wavelengths to detect the above components can achieve the best analysis results.

The meaning of the phosphoric acid aqueous solution concentration of 0.08ml/100ml in the step (3) of the present invention is: every 100ml solution contains 0.08ml phosphoric acid. In the gradient elution procedure, the percentage of mobile phase B refers to the volume percentage of mobile phase B in the sum of the volumes of the two phases.

The step (3) of the present invention may also include the following detection conditions: the specification of the chromatographic column is 100-250mm×2.1-4.6mm, preferably 100mm×2.1mm. The particle size of the chromatography column filler is 1.8-5.0 μm, preferably 1.8-2.0 μm, more preferably 1.8 μm. The filler can be selected from Agilent SB C ₁₈ purchased from Agilent Technologies. The column temperature of the chromatographic column is 30-40°C, preferably 35°C. The flow rate of the mobile phase is 0.1-1.0 ml/min, preferably 0.4 ml/min. The injection volume is 1-5 μl, preferably 1-2 μl, more preferably 1 μl.

Because the detection conditions of the standard solution and the Liuwei Dihuang preparation solution are the same, in the chromatogram, the retention time and peak shape of the peaks corresponding to the standard components in the Liuwei Dihuang preparation should be basically the same as the standard product. The retention time judges the corresponding components in the two chromatograms, and the confirmation is assisted by the shape of the peak. Step (4) of the present invention is by comparing the peak area of the corresponding component in the standard solution chromatogram and the Liuwei Dihuang preparation solution chromatogram, according to the known concentration of the standard product, calculates the corresponding component in the Liuwei Dihuang preparation solution and the standard product. Concentration, and further calculate the content of the components corresponding to the standard in the Liuwei Dihuang preparation.

The method of the invention adopts ultra-high performance liquid phase dual-wavelength multi-index for chromatographic detection, and can detect and quantify at least 11 main components in Liuwei Dihuang pills and capsules in less than 10 minutes. Compared with traditional high-performance liquid chromatography, it can save more than 3/4 of the analysis time, and at the same time, save reagent consumption. The method provided by the invention adopts dual-wavelength detection, thereby covering most components with relatively high content in the Liuwei Dihuang preparation, and the detection method is accurate and reliable.

The ultra-high-efficiency liquid-phase dual-wavelength multi-index content determination method provided by the invention can accurately, simply and quickly detect the Liuwei Dihuang preparation, and is beneficial to evaluate the quality of the Liuwei Dihuang preparation as a whole to ensure its clinical efficacy.

Description of drawings

Fig. 1 is the chromatogram of the mixed standard substance and Liuweidihuang concentrated pill sample (No. 34 sample) under the wavelength of 210nm; Marks in the figure: a, mixed standard substance, b, Liuweidihuang concentrated pill sample; 1, gallic acid, 2, original Catechin, 3, 1,2,3,4,6-pentagalloylglucose, 4, paeonol.

Figure 2 is the chromatogram of the mixed standard and Liuwei Dihuang Concentrated Pill sample (No. 34) at a wavelength of 238nm; in the figure: c, mixed standard, d, Liuwei Dihuang Concentrated Pill sample; 5,5-Hydroxymethylfurfural , 6, morroniside, 7, loganin, 8, swertigin, 9, paeoniflorin, 10, benzoic acid, 11, benzoyl paeoniflorin.

detailed description

Embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but should not be used to limit the scope of the present invention.

The instruments and reagents used in the embodiments of the present invention include: Agilent 1290 ultra-high performance liquid chromatograph, prepared G4204A quaternary pump, G4226A autosampler, G1316C temperature control box, G4212A diode array detector, 1/10,000 electronic balance (ME204E, Mettler-Toledo), ultrapure water (Milli-Q, Millipore), acetonitrile (chromatographically pure, Merk), phosphoric acid (chromatographically pure, DikmaPure). Standard products: gallic acid (Beijing Saibaicao Co., Ltd., 130718), protocatechuic acid (Beijing Saibaicao Co., Ltd., 130106), 1,2,3,4,6-pentagalloyl glucose (Beijing Saibaicao Co., Ltd., 130508), paeonol (China Food and Drug Administration, 110708-200506), 5-Hydroxymethylfurfural (Beijing Saibaicao Co., Ltd., 131124), morroniside (Beijing Saibaicao Co., Ltd., 130912), horse money Glycoside (Beijing Saibaicao Co., Ltd., 1201204), swertigin (Beijing Saibaicao Co., Ltd., 140425), paeoniflorin (Beijing Saibaicao Co., Ltd., 130726), benzoic acid (Beijing Saibaicao Co., Ltd.), benzoyl Paeoniflorin (Beijing Saibaicao Co., Ltd., 131108).

Example 1

Follow the steps below to test the Liuwei Dihuang preparation:

(1) Take the Liuwei Dihuang preparation, crush it, weigh it accurately, dissolve it in methanol at a concentration of 1g/10ml, extract it with ultrasonic waves with a frequency of 40000Hz for 30min, centrifuge at a speed of 10000rpm for 10min, take the supernatant, and filter it with a pore size of 0.22 Filter through a filter membrane of μm to obtain the Liuweidihuang preparation solution, which is set aside;

(2) Accurately weigh gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swerve Brain, paeoniflorin, benzoic acid, and benzoylpaeoniflorin were prepared into standard solutions with a concentration of 25 μg/mL, respectively, using methanol as a solvent, and set aside;

(3) Using ultra-high performance liquid chromatography, obtain the chromatograms of Liuwei Dihuang concentrated pill solution and standard solution respectively under the same detection conditions; the detection conditions include:

The column filler is octadecylsilane bonded silica gel Agilent SB C ₁₈ with a particle size of 1.8 μm; the column specification is 100mm×2.1mm;

Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～5min, 2～25% mobile phase B; 5～8min, 25～70 % mobile phase B; 8 ~ 8.1min, 70 ~ 95% mobile phase B; 8.1 ~ 13min, 95% mobile phase B;

The detection wavelength is 210nm and 238nm;

The column temperature is 35°C;

The mobile phase flow rate is 0.4mL/min;

The injection volume is 1 μL;

(4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance .

According to the method described in this example, 81 kinds of Liuwei Dihuang preparations (including 34 kinds of Liuwei Dihuang Concentrated Pills, 17 kinds of Liuwei Dihuang Honey Pills, 11 kinds of Liuwei Dihuang Honey Pills and 10 kinds of Liuwei Dihuang Capsules) on the market were tested. The test results are shown in Table 1.

Among them, the chromatogram of sample No. 34 (namely Liuwei Dihuang Concentrated Pill No. 34) at a wavelength of 210nm is shown in Figure 1, and the chromatogram at a wavelength of 238nm is shown in Figure 2.

Table 1: Contents of main components of Liuwei Dihuang preparation (ND = not detected)

Example 2

Follow the steps below to test the Liuwei Dihuang preparation:

(1) Take the Liuwei Dihuang preparation, crush it and weigh it accurately, dissolve it in methanol at a concentration of 1g/15ml, extract it with an ultrasonic wave with a frequency of 35000Hz for 40min, centrifuge it at a speed of 5000rpm for 15min, take the supernatant, and filter it with a pore size of 0.2 Filter through a filter membrane of μm to obtain the Liuweidihuang preparation solution, which is set aside;

(2) Accurately weigh gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swerve Brain, paeoniflorin, benzoic acid, and benzoylpaeoniflorin were prepared into standard solutions with a concentration of 25 μg/mL, respectively, using methanol as a solvent, and set aside;

(3) Using ultra-high performance liquid chromatography, obtain the chromatograms of Liuwei Dihuang concentrated pill solution and standard solution respectively under the same detection conditions; the detection conditions include:

The column filler is octadecylsilane bonded silica gel Agilent SB C ₁₈ with a particle size of 2.0 μm; the column specification is 100mm×4.6mm;

Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～5min, 2～25% mobile phase B; 5～8min, 25～70 % mobile phase B; 8 ~ 8.1min, 70 ~ 95% mobile phase B; 8.1 ~ 13min, 95% mobile phase B;

The detection wavelength is 200nm and 238nm;

The column temperature is 30°C;

The mobile phase flow rate is 0.1mL/min;

The injection volume is 2 μL;

(4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance .

Example 3

Follow the steps below to test the Liuwei Dihuang preparation:

(1) Take Liuwei Dihuang preparation, crush it and weigh it accurately, dissolve it in methanol at a concentration of 1g/10ml, extract it with ultrasonic waves with a frequency of 45000Hz for 20min, centrifuge at a speed of 20000r/min for 5min, take the supernatant, and filter it with a pore size of 0.3 Filter through a filter membrane of μm to obtain the Liuweidihuang preparation solution, which is set aside;

(2) Accurately weigh gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swerve Brain, paeoniflorin, benzoic acid, and benzoylpaeoniflorin were prepared into standard solutions with a concentration of 25 μg/mL, respectively, using methanol as a solvent, and set aside;

(3) Using ultra-high performance liquid chromatography, obtain the chromatograms of Liuwei Dihuang concentrated pill solution and standard solution respectively under the same detection conditions; the detection conditions include:

The column filler is octadecylsilane bonded silica gel Agilent SB C ₁₈ with a particle size of 5 μm; the column specification is 250mm×4.6mm;

Gradient elution is adopted; among them, the mobile phase A is phosphoric acid aqueous solution with a concentration of 0.08ml/100ml, and the mobile phase B is acetonitrile; the elution procedure is: 0～5min, 2～25% mobile phase B; 5～8min, 25～70 % mobile phase B; 8 ~ 8.1min, 70 ~ 95% mobile phase B; 8.1 ~ 13min, 95% mobile phase B;

The detection wavelength is 220nm and 250nm;

The column temperature is 40°C;

The mobile phase flow rate is 1mL/min;

The injection volume is 5 μL;

(4) According to the concentration of the standard substance, the peak area of the standard substance in the chromatogram and the peak area of the corresponding component in the Liuwei Dihuang preparation in the chromatogram, calculate the content of the corresponding component in the Liuwei Dihuang preparation and the standard substance .

After comparison, among the three embodiments provided by the present invention, embodiment 1 has the best comprehensive effects such as accuracy.

Experimental example: methodological investigation

1. Linear relationship investigation: Take the standard product, prepare five different concentrations of the standard product solution from high to low, draw the standard curve with the standard product concentration as the abscissa, and the chromatographic peak area as the ordinate to obtain the regression equation. The results show that the 11 kinds of standard products have good linear relationship at the above concentrations, and the linear regression equations of each standard product and the linear range of the concentration are shown in Table 2. The concentration of the standard substance used in the present invention is within the linear range.

Table 2: Results of Linear Relationship Investigation

compound Detection wavelength standard curve line Correlation coefficient (r) Linear range (μg/mL) gallic acid 210nm y=20730.713x-11.3787 0.9994 9.70-203.70 5-Hydroxymethylfurfural 238nm y=1927.059x+22.9385 0.9997 19.62-1418.00 protocatechuic acid 210nm y=15846.456x+0.6091 0.9998 2.52-25.25 Morroniside 238nm y=3743.123x-7.5733 0.9992 20.00-120.00 Loganin 238nm y=4151.934x-5.1161 1.0000 15.15-303.00 Swertiin 238nm y=1033.950x-0.4331 0.9998 5.05-171.70 Paeoniflorin 238nm y=2528.373x+6.6397 0.9991 20.00-141.40 1,2,3,4,6-pentagalloylglucose 210nm y=11472.582x+4.2163 0.9999 4.95-123.70 benzoic acid 238nm y=8079.100x+0.4757 0.9998 5.00-25.00 Benzoyl Paeoniflorin 238nm y=3760.100x+2.2759 0.9999 9.90-128.70 Paeonol 210nm y=11710.942x+35.3310 0.9994 18.25-351.70

2. Precision test: Take the Liuwei Dihuang Concentrated Pill sample (Sample No. 34) to prepare 1 part of Liuwei Dihuang preparation solution according to the method provided in Example 1, and perform 6 tests on the solution according to the method provided in Example 1. According to the results of 6 tests, the relative standard deviation (RSD) values of the 11 components are not more than 1.6% (as shown in Table 3), indicating that the precision of the instrument is good.

Table 3: Precision test results

compound RSD(%) gallic acid 1.07 5-Hydroxymethylfurfural 0.27

protocatechuic acid 0.45 Morroniside 0.91 Loganin 0.42 Swertiin 1.36 Paeoniflorin 0.33 1,2,3,4,6-pentagalloylglucose 0.17 benzoic acid 0.20 Benzoyl Paeoniflorin 1.60 Paeonol 0.28

3. Repeatability experiment: Take the Liuwei Dihuang Concentrated Pill sample (Sample No. 34) to prepare 6 parts of Liuwei Dihuang preparation solutions according to the method provided in Example 1, and test the 6 parts of the solutions according to the method provided in Example 1. According to the test results, gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swertigin The RSD values of paeoniflorin, paeoniflorin, benzoic acid and benzoylpaeoniflorin were all less than 1.75%, indicating that the method has good repeatability.

4. Stability test: Take the Liuwei Dihuang Concentrated Pill sample (Sample No. 34) to prepare 1 part of Liuwei Dihuang preparation solution according to the method provided in Example 1, and take the Liuwei Dihuang preparation solution at 0, 2, 4, 6, 8, 12 and 24 hours respectively. Equivalent solution is detected according to the method provided in Example 1. According to the test results, gallic acid, protocatechuic acid, 1,2,3,4,6-pentagalloyl glucose, paeonol, 5-hydroxymethylfurfural, morroniside, loganin, and swertigin The RSD values of paeoniflorin, paeoniflorin, benzoic acid and benzoylpaeoniflorin were all less than 1.99%, which proved that the sample was stable within 24 hours.

5. Accuracy test: Take a sample of Liuwei Dihuang Concentrated Pills with known content of each component, add a standard substance equal to the corresponding component in the sample, and then detect the content of each component in the sample after adding the sample, and add a sample for each standard substance 6 times, and detected according to the method provided in Example 1. The sample recovery results are shown in Table 4. The sample recovery rates of each standard product were all between 95.25% and 104.64%, indicating that the content determination method is accurate and reliable.

Table 4: Results of sample recovery test

The above experimental results show that the method provided by the present invention has good repeatability, stability, precision and accuracy, and it is feasible to use the ultra-high performance liquid phase dual-wavelength multi-index content determination method provided by the present invention to carry out quality control of Liuwei Dihuang Pill preparations of.

The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that various combinations, modifications or equivalent replacements of the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and all should cover Within the scope of the claims of the present invention.

Claims (6)

1. a kind of ultra high efficiency liquid phase dual wavelength multi objective content assaying method of Liuwei Dihuang preparation, it is characterised in that the method Including step：

(1) Liuwei Dihuang preparation is taken, precision is weighed after crushing, is dissolved in methanol with concentration 1g/10～30ml, supersound extraction, Centrifugation, takes supernatant liquid filtering, obtains final product Liuwei Dihuang preparation solution, standby；

(2) precision weighs standard substance, is dissolved in methanol with 20～30 μ g/ml of concentration, obtains final product standard solution, standby；It is described Standard substance include gallic acid, protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol, 5 hydroxymethyl furfural, Morroniside, loganin, sweroside, peoniflorin, benzoic acid and benzoylpaeoniflorin；

(3) ultra-performance liquid chromatography is adopted, Liuwei Dihuang preparation solution and standard is obtained under identical testing conditions respectively The chromatogram of product solution；The testing conditions include：

Chromatograph column packing is octadecylsilane chemically bonded silica；The specification of the chromatographic column be 100～250mm × 2.1～ 4.6mm；The particle diameter of filler is 1.8～5 μm；

Using gradient elution；Wherein, phosphate aqueous solution of the mobile phase A for concentration 0.08ml/100ml, Mobile phase B is acetonitrile；Wash De- program is：0～5min, 2～25% Mobile phase Bs；5～8min, 25～70% Mobile phase Bs；8～8.1min, 70～95% streams Dynamic phase B；8.1～13min, 95% Mobile phase B；

Detection wavelength is 200-220nm and 238-250nm；Detection wavelength be 200-220nm under conditions of detect gallic acid, Protocatechuic acid, 1,2,3,4,6- Penta-O-galloyl-D-glucopyranoses, paeonol；Under conditions of Detection wavelength is 238-250nm, detection 5 hydroxymethyl furfural, morroniside, loganin, sweroside, peoniflorin, benzoic acid, benzoylpaeoniflorin；

The testing conditions also include：The column temperature of chromatographic column is 30～40 DEG C；Flow rate of mobile phase is 0.1～1.0ml/min；Sample introduction Measure as 1～5 μ l；

(4) with standard condition in peak area and Liuwei Dihuang preparation of the concentration, standard substance according to standard substance in chromatogram Peak area of the tie element in chromatogram, calculates the content of composition corresponding with standard substance in Liuwei Dihuang preparation.

2. method according to claim 1, it is characterised in that the Detection wavelength described in step (3) is 210nm and 238nm.

3. method according to claim 1 and 2, it is characterised in that in step (1) supersound extraction, ultrasonic frequency For 35000～45000Hz, extraction time is 20～40min.

4. method according to claim 3, it is characterised in that in step (1) centrifugation, the speed of centrifugation is 5000～ 20000 revs/min, centrifugation time is 5～15min.

5. method according to claim 4, it is characterised in that step (1) is described to be filtered using 0.2～0.3 μm of aperture Membrane filtration.

6. method according to claim 1 and 2, it is characterised in that the dosage form of the Liuwei Dihuang preparation is：Water-honeyed pill, Big honeyed pills, concentrated pill or capsule.