1. A method for measuring the content of ferulic acid and catalpol in spleen-tonifying and waist-strengthening medicinal liquor comprises the following steps:

(1) preparation of ferulic acid control solution: taking a proper amount of ferulic acid reference substance, precisely weighing, and adding methanol to dissolve to prepare a solution;

(2) preparation of a test solution: sucking the spleen-invigorating and waist-strengthening medicinal liquor, and filtering to obtain the medicinal liquor;

(3) preparation of negative solution: preparing a negative solution without the angelica medicinal material according to the medicinal liquor prescription, and filtering to obtain the medicinal liquor;

(4) and (3) determination: precisely absorbing the negative solution, the ferulic acid reference solution and the test solution respectively, injecting into a liquid chromatograph to obtain a chromatogram, and calculating the content of ferulic acid according to the chromatogram.

2. The method of claim 1, comprising the steps of:

(1) preparation of ferulic acid control solution: taking a proper amount of ferulic acid reference substance, precisely weighing, adding 60-800% methanol to dissolve to obtain solution containing 15-25 μ g per 1ml, filtering, and collecting the subsequent filtrate;

(2) preparation of a test solution: sucking the spleen invigorating and waist strengthening medicinal liquor, filtering, and collecting the subsequent filtrate;

(3) preparation of negative solution: preparing a negative solution without the angelica medicinal material according to the medicinal liquor prescription, filtering, and taking a subsequent filtrate to obtain the medicinal liquor;

(4) and (3) determination: precisely absorbing the negative solution, ferulic acid reference solution and test solution by 5-20 μ l respectively, injecting into a liquid chromatograph to obtain chromatogram, and calculating ferulic acid content according to the chromatogram.

3. The method of claim 1, comprising the steps of:

(1) preparation of ferulic acid control solution: taking a proper amount of ferulic acid reference substance, precisely weighing, dissolving with 70% methanol to obtain a solution containing 20 μ g per 1ml, filtering with 0.22 μm filter membrane, and collecting the filtrate;

(2) preparation of a test solution: sucking 3ml of spleen invigorating and waist strengthening medicinal liquor, filtering with 0.22 μm filter membrane, and collecting the filtrate;

(3) preparation of negative solution: preparing a negative solution without radix Angelicae sinensis according to the prescription of the medicated liquor, filtering with a 0.22 μm filter membrane, and collecting the filtrate;

(4) and (3) determination: precisely absorbing 10 μ l of each of the negative solution, the ferulic acid reference solution and the test solution, injecting into a liquid chromatograph to obtain a chromatogram, and calculating the content of ferulic acid according to the chromatogram.

4. The method of claim 1, wherein the chromatographic conditions are as follows:

a chromatographic column: agilent ZORBAX SB-C18(4.6 mm 5 μm); column temperature: 35 ℃; sample introduction amount: 10 mu l of the mixture; detection wavelength: 316 nm; flow rate: 1 ml/min; mobile phase: acetonitrile-0.1% phosphoric acid (16:84), ferulic acid should have a separation of >1.5 from the adjacent chromatographic peak; operating time: and (5) 50 min.

5. The method as claimed in claim 1, wherein the method for measuring the catalpol content in the spleen-invigorating and waist-strengthening medicinal liquor comprises the following steps:

(1) catalpol reference solution preparation: taking a proper amount of catalpol reference substance, precisely weighing, and adding phosphoric acid to prepare a solution;

(2) preparation of a test solution: taking a proper amount of the spleen-invigorating and waist-strengthening medicinal liquor, and filtering to obtain the medicinal liquor;

(3) preparation of negative solution: preparing a negative solution without rehmannia root according to a medicinal liquor prescription, and filtering to obtain the medicinal liquor;

(4) and (3) determination: and (3) determination: respectively and precisely absorbing the negative solution, the catalpol reference solution and the sample solution, injecting into a liquid chromatograph to obtain a chromatogram, and calculating the catalpol content according to the chromatogram.

6. The method of claim 5, comprising the steps of:

(1) catalpol reference solution preparation: weighing appropriate amount of catalpol as reference, adding 0.08-0.12% phosphoric acid to obtain solution containing 0.08-0.12mg per 1 ml;

(2) preparation of a test solution: taking a proper amount of the spleen-invigorating and waist-strengthening medicinal liquor, filtering, and taking a subsequent filtrate to obtain the medicinal liquor;

(3) preparation of negative solution: preparing a negative solution without rehmannia root according to a medicinal liquor prescription, filtering, and taking a subsequent filtrate to obtain the medicinal liquor;

(4) and (3) determination: and (3) determination: precisely absorbing the negative solution, catalpol reference solution and sample solution respectively by 5-20 μ l, injecting into liquid chromatograph to obtain chromatogram, and calculating catalpol content according to the chromatogram.

7. The method of claim 5, comprising the steps of:

(1) catalpol reference solution preparation: taking a proper amount of catalpol reference substance, precisely weighing, and adding 0.1% phosphoric acid to prepare a solution containing about 0.1mg per 1 ml;

(2) preparation of a test solution: filtering appropriate amount of the spleen invigorating and waist strengthening medicated liquor with 0.22 μm filter membrane, and collecting the filtrate;

(3) preparation of negative solution: preparing a negative solution without rehmannia root according to a medicinal liquor prescription, filtering the negative solution with a filter membrane of 0.22 mu m, and taking a subsequent filtrate to obtain the medicinal liquor;

(4) and (3) determination: and (3) determination: precisely absorbing the negative solution, catalpol reference solution and sample solution respectively by 10 μ l, injecting into a liquid chromatograph to obtain chromatogram, and calculating catalpol content according to the chromatogram.

8. The method of claim 5, wherein the chromatographic conditions are as follows:

a chromatographic column: agilent ZORBAX SB-C18(4.6 mm 5 μm); column temperature: 30 ℃; sample introduction amount: 10 mu l of the mixture; wavelength: 210 nm; flow rate: 0.8 ml/min; mobile phase: methanol-0.1% phosphoric acid (1: 99).

Background

A medicinal liquor for strengthening the spleen and strengthening the waist,

[ prescription ] licorice root 10g safflower 10g Chinese yam 30g rehmannia root 60g dried longan pulp 30g astragalus root 50g jujube 80g prepared fleece flower root 40g Chinese angelica root 30g achyranthes root 50g dipsacus root 60g pilose asiabell root 40g poria 40g eucommia bark 40g

[ PREPARATION METHOD ] soaking above fourteen Chinese medicinal herbs in 4500g and 4500g of Chinese liquor twice every 2 weeks, collecting supernatant, filtering, and mixing filtrates. And heating and dissolving a small amount of white spirit, adding the medicinal liquor, stirring uniformly, standing and precipitating for 2-3 weeks, taking supernate, and filtering to obtain 9450 g.

[ PROPERTIES ] the product is a reddish brown clarified liquid; sweet and slightly bitter.

The spleen-invigorating and waist-strengthening medicinal liquor has the effects of benefiting qi, nourishing blood, invigorating spleen and kidney, and clearing and activating channels and collaterals. Can be used for treating deficiency of qi and blood, poor appetite, soreness of waist and legs, listlessness, asthenia, insomnia and amnesia. The oral preparation is taken 20-30 ml at a time, and is taken once in the morning and before bedtime.

The existing quality standards of the spleen-tonifying and waist-strengthening medicinal liquor only comprise a liquorice, angelica, safflower and teasel root thin-layer identification method and an ethanol content detection method, a content determination method is not established, and along with the improvement of the quality control requirements of the traditional Chinese medicine, the requirements for detecting the effective components in the medicine are also increased, so that the research on the effective components of the traditional Chinese medicine in the spleen-tonifying and waist-strengthening medicinal liquor is necessary and the content determination method is established.

In the existing literature reports, only thin-layer chromatography identification research is carried out on liquorice, safflower, astragalus, angelica, teasel root and the like which are main medicinal materials in the spleen-tonifying and waist-strengthening medicinal liquor by using old rock. Sunying and the like research the content determination method of catalpol and ferulic acid in the Yijing pill.

The prior art does not disclose a method for measuring the content of ferulic acid and catalpol in the medicinal liquor for strengthening spleen and loins. The method for measuring the content of ferulic acid and catalpol in the spleen-tonifying and waist-strengthening medicinal liquor disclosed by the invention is simple to operate, strong in specificity, high in accuracy and reliable in result, and can be used as a quality control standard of the spleen-tonifying and waist-strengthening medicinal liquor.

Disclosure of Invention

The invention aims to provide a method for measuring the content of ferulic acid and catalpol in medicinal liquor for strengthening spleen and loins. Method 1, the method for measuring the content of ferulic acid in the spleen-tonifying and waist-strengthening medicinal liquor comprises the following steps:

(1) preparation of ferulic acid control solution: taking a proper amount of ferulic acid reference substance, precisely weighing, and adding methanol to dissolve to prepare a solution;

(2) preparation of a test solution: sucking the spleen-invigorating and waist-strengthening medicinal liquor, and filtering to obtain the medicinal liquor;

(3) preparation of negative solution: preparing a negative solution without the angelica medicinal material according to the medicinal liquor prescription, and filtering to obtain the medicinal liquor;

(4) and (3) determination: precisely absorbing the negative solution, the ferulic acid reference solution and the test solution respectively, injecting into a liquid chromatograph to obtain a chromatogram, and calculating the content of ferulic acid according to the chromatogram.

Preferred method 1, comprising the steps of:

(1) preparation of ferulic acid control solution: taking a proper amount of ferulic acid reference substance, precisely weighing, adding 60-800% methanol to dissolve to obtain solution containing 15-25 μ g per 1ml, filtering, and collecting the subsequent filtrate;

(2) preparation of a test solution: sucking the spleen invigorating and waist strengthening medicinal liquor, filtering, and collecting the subsequent filtrate;

(3) preparation of negative solution: preparing a negative solution without the angelica medicinal material according to the medicinal liquor prescription, filtering, and taking a subsequent filtrate to obtain the medicinal liquor;

(4) and (3) determination: precisely absorbing the negative solution, ferulic acid reference solution and test solution by 5-20 μ l respectively, injecting into a liquid chromatograph to obtain chromatogram, and calculating ferulic acid content according to the chromatogram.

Most preferred method 1, comprises the steps of:

(1) preparation of ferulic acid control solution: taking a proper amount of ferulic acid reference substance, precisely weighing, dissolving with 70% methanol to obtain a solution containing 20 μ g per 1ml, filtering with 0.22 μm filter membrane, and collecting the filtrate;

(2) preparation of a test solution: sucking 3ml of spleen invigorating and waist strengthening medicinal liquor, filtering with 0.22 μm filter membrane, and collecting the filtrate;

(3) preparation of negative solution: preparing a negative solution without radix Angelicae sinensis according to the prescription of the medicated liquor, filtering with a 0.22 μm filter membrane, and collecting the filtrate;

(4) and (3) determination: precisely absorbing 10 μ l of each of the negative solution, the ferulic acid reference solution and the test solution, injecting into a liquid chromatograph to obtain a chromatogram, and calculating the content of ferulic acid according to the chromatogram.

Chromatographic conditions are as follows:

a chromatographic column: agilent ZORBAX SB-C18(4.6 mm 5 μm); column temperature: 35 ℃; sample introduction amount: 10 mu l of the mixture; detection wavelength: 316 nm; flow rate: 1 ml/min; mobile phase: acetonitrile-0.1% phosphoric acid (16:84), ferulic acid should have a separation of >1.5 from the adjacent chromatographic peak; operating time: and (5) 50 min.

Method 2, the catalpol content determination method in the spleen-invigorating and waist-strengthening medicinal liquor comprises the following steps:

(1) catalpol reference solution preparation: taking a proper amount of catalpol reference substance, precisely weighing, and adding phosphoric acid to prepare a solution;

(2) preparation of a test solution: taking a proper amount of the spleen-invigorating and waist-strengthening medicinal liquor, and filtering to obtain the medicinal liquor;

(3) preparation of negative solution: preparing a negative solution without rehmannia root according to a medicinal liquor prescription, and filtering to obtain the medicinal liquor; (4) and (3) determination: and (3) determination: respectively and precisely absorbing the negative solution, the catalpol reference solution and the sample solution, injecting into a liquid chromatograph to obtain a chromatogram, and calculating the catalpol content according to the chromatogram.

Preferred method 2, comprises the steps of:

(1) catalpol reference solution preparation: taking appropriate amount of catalpol reference substance, precisely weighing, adding 0.08-0.12% phosphoric acid to obtain solution containing 0.08-0.12mg per 1 ml.

(2) Preparation of a test solution: taking a proper amount of the spleen-invigorating and waist-strengthening medicinal liquor, filtering, and taking a subsequent filtrate to obtain the medicinal liquor;

(3) preparation of negative solution: preparing a negative solution without rehmannia root according to a medicinal liquor prescription, filtering, and taking a subsequent filtrate to obtain the medicinal liquor;

(4) and (3) determination: and (3) determination: precisely absorbing the negative solution, catalpol reference solution and sample solution respectively by 5-20 μ l, injecting into liquid chromatograph to obtain chromatogram, and calculating catalpol content according to the chromatogram.

The most preferred method 2, comprises the steps of:

(1) catalpol reference solution preparation: taking a proper amount of catalpol reference substance, precisely weighing, and adding 0.1% phosphoric acid to prepare a solution containing about 0.1mg per 1 ml.

(2) Preparation of a test solution: filtering appropriate amount of the spleen invigorating and waist strengthening medicated liquor with 0.22 μm filter membrane, and collecting the filtrate;

(3) preparation of negative solution: preparing a negative solution without rehmannia root according to a medicinal liquor prescription, filtering the negative solution with a filter membrane of 0.22 mu m, and taking a subsequent filtrate to obtain the medicinal liquor;

(4) and (3) determination: and (3) determination: precisely absorbing the negative solution, catalpol reference solution and sample solution respectively by 10 μ l, injecting into a liquid chromatograph to obtain chromatogram, and calculating catalpol content according to the chromatogram.

Chromatographic conditions are as follows:

a chromatographic column: agilent ZORBAX SB-C18(4.6 mm 5 μm); column temperature: 30 ℃; sample introduction amount: 10 mu l of the mixture; wavelength: 210 nm; flow rate: 0.8 ml/min; mobile phase: methanol-0.1% phosphoric acid (1: 99). The invention is obtained by screening, and the screening process is as follows:

determination of ferulic acid content: the mobile phase is screened and replaced by acetonitrile-0.1% phosphoric acid from different proportions of acetonitrile-water, then the proportion of acetonitrile and 0.1% phosphoric acid is screened and finally optimized and determined to be 17:83 from 12:88,15:85 and 16:84, and good separation of ferulic acid and other interfering components is obtained.

Catalpol content determination: the extraction of the test sample solution is screened, firstly ultrasonic extraction is adopted, 0.22 mu m microporous filter membrane filtration is adopted, then 4000 r/min is adopted, centrifugation is carried out for 10min, then 0.22 mu m microporous filter membrane filtration is adopted, and finally the mode that the test sample is directly filtered and injected by the 0.22 mu m microporous filter membrane is adopted, the result shows that the results of the former two treatment methods are basically consistent with the result of the last method, and finally the simplest mode that the test sample is directly injected after the test sample is filtered by the 0.22 mu m microporous filter membrane is adopted.

The present invention does not directly employ prior art methods, such as those described in the following references:

HPLC (high performance liquid chromatography) for simultaneously determining catalpol and ferulic acid content in Yijing pill

High performance liquid chromatography for determining catalpol content in rehmannia glutinosa leaves

RP-HPLC-ELSD for simultaneously determining the contents of ferulic acid, stachydrine hydrochloride, catalpol and acteoside in compound motherwort herb capsule

High performance liquid chromatography for determining catalpol content in SHENKANGNING pill

Dual-wavelength HPLC method for simultaneously determining content of 4 components in kidney-tonifying and rheum-derived pill

High performance liquid chromatography for determining catalpol content in YI-JIAN granule

High performance liquid chromatography for determining catalpol content in rehmannia root decoction pieces in different regions

Solid phase extraction-high performance liquid chromatography for determining the content of ferulic acid in the oral liquid of Taitai and a method in pharmacopoeia.

But the method of the prior art is greatly improved, and the main improvement points are as follows:

1. the difference of ultraviolet absorption wavelengths of the two components is large, and the two components are respectively quantitatively measured by adopting different maximum absorption wavelengths.

2. The method for treating the test sample is simple, and complex treatment modes such as ultrasonic treatment, macroporous resin purification, rotary evaporation and the like are not adopted in literature reports.

The improved method greatly improves the detection accuracy, shortens the sample processing time and greatly improves the repeatability of the measurement result.

The method is superior to the prior art in the following aspects:

1. the method provided by the invention is simple to operate, and the sample and the reference substance do not need to be processed in a complex way and are directly filtered and injected;

2. the isocratic elution is adopted, so that the interference of other components in the medicinal liquor can be removed simultaneously, and the accuracy and reliability of a measurement result are ensured;

3. the quality of the product can be controlled by measuring the content of the main active ingredients by high performance liquid chromatography.

Drawings

FIG. 1 blank solution chromatogram peak (70% methanol);

FIG. 2 blank solution chromatogram Peak (0.1% phosphoric acid)

FIG. 3 shows the HPLC chromatogram of the negative solution with the angelica root removed;

FIG. 4 HPLC chromatogram of ferulic acid control solution;

FIG. 5 shows HPLC chromatogram of catalpol reference solution;

FIG. 6 is an HPLC chromatogram of a test solution for measuring ferulic acid in the spleen-invigorating and waist-strengthening medicinal liquor of the embodiment of the invention;

FIG. 7 is an HPLC chromatogram of a test solution for catalpol determination in the spleen-invigorating and waist-strengthening medicinal liquor of the embodiment of the invention;

Detailed Description

The invention is further explained below with reference to the drawings and the detailed description.

Example 1

A method for determining ferulic acid content in spleen invigorating and waist strengthening medicinal liquor comprises precisely weighing 10.21mg of ferulic acid reference substance, placing in a 50ml measuring flask, adding 70% methanol for dissolving and diluting to scale, shaking to obtain ferulic acid reference substance stock solution, precisely sucking 1ml of ferulic acid reference substance stock solution, adding 70% methanol for constant volume to 10ml, filtering with 0.22 μm filter membrane, and collecting filtrate as ferulic acid reference substance solution; sucking 3ml of medicinal liquor, filtering with a 0.22 μm filter membrane, collecting the filtrate to obtain a sample solution, and preparing 6 parts in parallel;

the optimal determination method of ferulic acid in the test solution comprises the following steps:

a chromatographic column: agilent ZORBAX SB-C18(4.6 mm 5 μm); column temperature: 35 ℃; sample introduction amount: 10 mu l of the mixture; detection wavelength: 316 nm; flow rate: 1 ml/min; mobile phase: acetonitrile-0.1% phosphoric acid (16:84), ferulic acid should have a separation of >1.5 from the adjacent chromatographic peak; operating time: and (5) 50 min.

And (3) determination: respectively sucking 10 μ l each of ferulic acid reference solution and test solution, injecting into liquid chromatograph, and measuring.

The following table shows the determination results of the ferulic acid content in the spleen-invigorating and waist-strengthening medicinal liquor.

Example 2

A method for determining catalpol content in spleen invigorating and waist strengthening medicated liquor comprises precisely weighing catalpol reference substance 10.83mg, placing into 10ml measuring flask, adding 0.1% phosphoric acid to dissolve and dilute to scale, shaking, filtering with 0.22 μm filter membrane, and collecting the filtrate to obtain reference substance solution; sucking appropriate amount of medicated liquor, filtering with 0.22 μm filter membrane, collecting filtrate to obtain test solution, and preparing 6 parts in parallel;

the method for determining the optimal content of catalpol in the test solution comprises the following steps:

a chromatographic column: agilent ZORBAX SB-C18(4.6 mm 5 μm); column temperature: 30 ℃; sample introduction amount: 10 mu l of the mixture; wavelength: 210 nm; flow rate: 0.8 ml/min; mobile phase: methanol-0.1% phosphoric acid (1: 99).

And (3) determination: respectively and precisely sucking 10 μ l of catalpol reference solution and sample solution, injecting into liquid chromatograph, measuring, and calculating catalpol content in the medicinal liquor for invigorating spleen and strengthening waist according to external standard method.

The following table shows the catalpol content determination results in the spleen-invigorating and waist-strengthening medicinal liquor.

The above embodiments of the present invention are to be considered as illustrative and not restrictive, and any minor modifications of the chromatographic conditions and others to the above examples, which are in accordance with the spirit of the present invention, are within the scope of the present invention.