CN112964805A

CN112964805A - Method for measuring contents of atractylenolide II and atractylenolide III in atractylenolide medicinal material

Info

Publication number: CN112964805A
Application number: CN202110241897.7A
Authority: CN
Inventors: 郭静; 张学花; 邢会香; 王飞; 刘红; 余梅; 王周霞; 曹得辉; 杨恋
Original assignee: Qinghai Plateau Pharmaceutical Co ltd
Current assignee: Qinghai Plateau Pharmaceutical Co ltd
Priority date: 2021-03-04
Filing date: 2021-03-04
Publication date: 2021-06-15

Abstract

The invention relates to a method for measuring contents of atractylenolide II and atractylenolide III in an atractylenolide medicinal material, which comprises the following steps: taking a white atractylodes rhizome medicinal material, extracting with 70% methanol, performing gradient elution by adopting an acetonitrile-0.1% phosphoric acid water system, and determining the content of atractylenolide II and atractylenolide III in the white atractylodes rhizome medicinal material. On the basis of the alisma decoction detection method, by optimizing the gradient of the flowing phase ratio, the detection wavelength, the extraction solvent, the extraction mode, the extraction time and the series of researches on a chromatographic column, the method shows that the peak time is fast, the peak shape is good, the separation degree and the peak purity are qualified, the accuracy is high, the stability is good, the operation is simple, convenient and fast, the quality detection method of the atractylodes macrocephala medicinal material is perfected, and the quality is better controlled.

Description

Method for measuring contents of atractylenolide II and atractylenolide III in atractylenolide medicinal material

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a content determination method for atractylenolide II and atractylenolide III in an atractylenolide medicinal material.

Background

The bighead atractylodes rhizome is a rhizome of a perennial herb bighead atractylodes rhizome in the family of compositae, is a tonifying traditional Chinese medicine which is very commonly used in clinical traditional Chinese medicine, has a beautiful name of ' southern art and northern ginseng ', and is originally recorded in ancient books such as Shanhai Jing and Erya '. The white atractylodes rhizome is warm in nature, sweet in taste, slightly pungent and bitter in taste, enters spleen channels and stomach channels, is mainly used for tonifying spleen qi clinically, has the functions of strengthening spleen and tonifying qi, eliminating dampness and promoting diuresis, stopping sweating and preventing abortion, and is used for treating diseases such as spleen deficiency and poor appetite, abdominal distension and diarrhea, phlegm and fluid retention, palpitation and dizziness, spontaneous perspiration, threatened abortion and the like. Research in recent decades shows that the white atractylodes rhizome has some functions in diuresis, tumor resistance, antibiosis, anti-inflammation, diabetes treatment, aging resistance and other aspects, has certain effect on the nervous system, the digestive tract and the uterine smooth muscle, and can also regulate the immune function. The atractylodes macrocephala koidz has the functions of strengthening spleen, tonifying qi and regulating cecal motion, can promote the contraction motion of smooth muscle of jejunum, and has obvious inhibiting effect on the autonomic contraction motion of cecal. The effects of Atractylodis rhizoma on immune system are mainly anti-inflammatory, anti-tumor and antioxidant. The Atractylodis rhizoma can effectively inhibit the growth of tumor cells, wherein atractylenolide and volatile oil are effective antitumor active ingredients. The antioxidation of the atractylodes macrocephala koidz can effectively inhibit the peroxidation of lipid, reduce the content of lipid peroxide in tissues and avoid further damage of harmful substances to the structure and the function of tissue cells. The processed products which are commonly used clinically mainly comprise raw rhizoma atractylodis macrocephalae and fried rhizoma atractylodis macrocephalae, the fried rhizoma atractylodis macrocephalae is mainly used for strengthening the spleen, and the rhizoma atractylodis macrocephalae fried with bran and the rhizoma atractylodis macrocephalae fried with soil can be selected according to different symptoms.

The bighead atractylodes rhizome has high clinical application value and wide market demand, but the quality of the bighead atractylodes rhizome cannot be comprehensively evaluated because no content measurement item is collected in the bighead atractylodes rhizome medicinal material in 2015 edition of Chinese pharmacopoeia. At present, the content of atractylenolide in atractylenolide reported in literature research is mostly separated by adopting an HPLC method and a gradient elution procedure, wherein the gradient elution is to mix two or more solvents with different polarities and mutual solubility according to a certain proportion along with the change of time so as to continuously change the polarity of a flushing liquid and obtain good separation of complex components, but the following technical problems exist in the result of the gradient elution procedure: long peak-out time, poor resolution, poor peak shape, poor reproducibility, poor stability, large solvent consumption, low accuracy and the like.

Application number CN201910299616.6, entitled construction method and detection method of UPLC characteristic spectrum of Atractylodis rhizoma medicinal material, discloses preparation of reference substance solution with chlorogenic acid as reference substance; preparing a reference solution of a reference medicinal material by using a bighead atractylodes rhizome reference medicinal material; respectively taking the medicinal materials of the largehead atractylodes rhizome, the largehead atractylodes rhizome decoction pieces fried with bran, the standard largehead atractylodes rhizome decoction pieces and the standard largehead atractylodes rhizome decoction fried with bran, adding an extraction solvent for extraction, filtering, and taking subsequent filtrates as test solution respectively; and (3) performing gradient elution by taking the mobile phase A as acetonitrile and the mobile phase B as 0.05-0.15% phosphoric acid solution: the volume fraction of the mobile phase A is increased from 1% to 6% and the volume fraction of the mobile phase B is reduced from 99% to 94% in 0-4 min; 4-10min, the volume fraction of the mobile phase A is increased from 6% to 9%, and the volume fraction of the mobile phase B is reduced from 94% to 91%; 10-16min, the volume fraction of the mobile phase A is increased from 9% to 18%, and the volume fraction of the mobile phase B is reduced from 91% to 82%; 16-22min, the volume fraction of the mobile phase A is increased from 18% to 20%, and the volume fraction of the mobile phase B is decreased from 82% to 80%; and (4) for 22-23min, the volume fraction of the mobile phase A is increased from 20% to 90%, and the volume fraction of the mobile phase B is decreased from 80% to 10%. Taking the column temperature as (25 +/-3) DEG C; the flow rate is (0.25 plus or minus 0.1) ml/min; the detection wavelength was (325. + -.40) nm, and the measurement was carried out.

The technical problem of application No. CN201910299616.6 is: the method is used for researching the characteristic maps of the largehead atractylodes rhizome decoction pieces, the largehead atractylodes rhizome decoction pieces fried with bran, the largehead atractylodes rhizome standard decoction pieces and the largehead atractylodes rhizome standard decoction fried with bran, and the content determination of the largehead atractylodes rhizome lactone II and the largehead atractylodes rhizome lactone III is not analyzed, and the method has the advantages of long peak emergence time, poor peak shape, poor separation degree and poor stability.

Application number CN201910725324.4, entitled UPLC method for detecting atractylenolide I and atractylenolide III in Atractylodes macrocephala, discloses that Atractylodes macrocephala is crushed and then placed in a triangular flask, methanol is added, and then the mixture is placed in an ultrasonic instrument for ultrasonic treatment and centrifugation, and finally filtered; injecting the standard rhizoma atractylodis macrocephalae sample solution into an ultra-high performance liquid chromatograph, and purifying by using a chromatographic column: BEH C18 column (2.1 μm. times.100 mm,1.7 mm); flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 5 mu L of the solution; ultraviolet detection wavelength: 221 nm; mobile phase: and (3) acetonitrile and water, wherein the volume ratio of the acetonitrile to the water is 54:46, and the prepared bighead atractylodes rhizome sample to be detected is quantified through the UPLC characteristic spectrum of the obtained standard bighead atractylodes rhizome sample and the UPLC characteristic spectrum of the bighead atractylodes rhizome sample to be detected.

The technical problem of application No. CN201910725324.4 is: the method aims at the detection of atractylenolide I and atractylenolide III, atractylenolide II is not analyzed, and the method has the advantages of poor target peak separation degree, poor reproducibility and low accuracy.

Application No. CN202010678723.2, entitled method for measuring contents of effective components atractylenolide III and atractylenolide I in rhizoma Atractylodis Macrocephalae, discloses soaking rhizoma Atractylodis Macrocephalae powder in ethyl acetate; filtering, evaporating to dryness, and dissolving in methanol; filtering with filter membrane to obtain filtrate; precisely weighing atractylenolide III and atractylenolide I reference substances; adding methanol to obtain solution containing 0.1mg per 1 ml; precisely sucking 10 μ L of each of the reference solution and the sample solution, and respectively injecting into a liquid chromatograph for determination; performing chromatography by using a chromatographic column: a C18 column (150 mm. times.4.6); column temperature: 35 ℃; mobile phase: methanol and water, the ratio of methanol to water being 72: 28; flow rate: 1.0 ml/min; detection wavelength: 220 nm; sample introduction amount: 10 mu L of the chromatogram map is recorded, and the contents of atractylenolide III and atractylenolide I are calculated by an external standard method.

The technical problem of application No. CN202010678723.2 is: the method aims at the detection of atractylenolide I and atractylenolide III, atractylenolide II is not analyzed, and the method is complex in operation, poor in chromatographic peak separation, slow in peak-producing time, poor in stability, large in solvent consumption and low in accuracy.

Aiming at the problems, the inventor obtains a detection method for detecting the contents of atractylenolide II and atractylenolide III in rhizoma alismatis decoction, which is a product of the company, by researching a series of optimization of mobile phase gradient, detection wavelength, extraction solvent, extraction mode, extraction time and chromatographic columns, and the method has the advantages of good peak shape, qualified separation degree and peak purity, high accuracy, short peak-out time and simple operation, perfects the quality detection method of the rhizoma atractylodis macrocephalae and better controls the quality.

Disclosure of Invention

The invention provides a content determination method for atractylenolide II and atractylenolide III in an atractylenolide medicinal material, which comprises the following steps:

(1) preparation of a test solution: weighing 0.5g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 30-70% methanol, weighing, ultrasonically treating or refluxing, cooling, weighing again, supplementing the lost weight with 30-70% methanol, shaking, centrifuging for 2-8min, filtering, and collecting the subsequent filtrate;

(2) preparation of control solutions: accurately weighing appropriate amount of atractylenolide II and atractylenolide III, and adding 70% methanol to obtain solutions containing atractylenolide II 10ug and atractylenolide III 10ug per 1ml respectively;

(3) chromatographic conditions and system applicability test: the chromatographic column is CORTECS UPLC T31.6um; the column temperature is 39-41 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.09-0.11% phosphoric acid solution as a mobile phase B; the detection wavelength is 210-230 nm; the flow rate is 0.34-0.36ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, wherein the mobile phase A is 44% and the mobile phase B is 56%;

(4) the determination method comprises the following steps: precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Preferably, the content determination method comprises the following steps:

(1) preparation of a test solution: weighing 0.5g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating or refluxing, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking, centrifuging for 5min, filtering, and collecting the subsequent filtrate;

(3) chromatographic conditions and system applicability test: the chromatographic column is CORTECS UPLC T31.6um; the column temperature was 40 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; the detection wavelength is 220 nm; the flow rate is 0.35ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, wherein the mobile phase A is 44% and the mobile phase B is 56%;

The methanol in the step (1) may be any one of acetonitrile and ethanol.

The concentration of the acetonitrile and the ethanol are respectively 50 percent.

The centrifugal frequency is 8000-12000 r/min.

Preferably, the centrifugal frequency is 10000 r/min.

The ultrasonic treatment or reflux treatment time is 10-30 min.

The ultrasonic or reflux treatment time is 10 min.

The content determination method comprises the following steps:

(1) preparation of a test solution: weighing 0.5g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and collecting the subsequent filtrate;

(3) chromatographic conditions and system applicability test: the chromatographic column is CORTECS UPLC T31.6um; column temperature: 40 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; the detection wavelength is 220 nm; the flow rate is 0.35ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, wherein the mobile phase A is 44% and the mobile phase B is 56%;

The content determination method is applied to detection of the medicinal material of the white atractylodes rhizome, the processed product of the white atractylodes rhizome and the preparation containing the medicinal material of the white atractylodes rhizome.

The invention has the following advantages:

1. the content determination method has the advantages of good peak shape, qualified separation degree and peak purity, high accuracy, short peak-producing time, good reproducibility and simple operation.

2. On the basis of the alisma decoction detection method, the reliability and rationality of the method are further explained by optimizing the gradient of the flowing phase ratio, the detection wavelength, the extraction solvent, the extraction mode, the extraction time and the series of researches on chromatographic columns, so that the quality detection method of the medicinal materials is more scientific and perfect, and the quality of the medicinal materials is better controlled.

3. The content determination method of the invention performs the screening of chromatographic conditions and gradient elution by the optimization method before and the optimization method I, the optimization method II, the optimization method III and the optimization method IV to obtain the content determination method of atractylenolide II and atractylenolide III in the medicinal materials of atractylenolide, and the optimization scheme of the chromatographic conditions and the system applicability test is as follows: the chromatographic column is CORTECS UPLC T31.6um; column temperature: 40 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; the detection wavelength is 220 nm; the flow rate is 0.35ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, wherein the mobile phase A is 44% and the mobile phase B is 56%; the method is further proved to be reasonable and reliable.

4. In a methodological verification test, a system applicability test result shows that the Relative Standard Deviation (RSD) of the areas of main peaks (atractylenolide II and atractylenolide III) of the method is less than 2.0 percent; a Relative Standard Deviation (RSD) of retention time of less than 1.0%; the theoretical plate numbers are all more than 4500; the separation degrees are all more than 1.5; therefore, the content determination method of atractylenolide II and atractylenolide III meets the requirement of system applicability.

5. In a methodological verification test, a special investigation result shows that the blank solvent has no interference on peak positions of all peaks, and the method determines that the contents of atractylenolide III and atractylenolide II in the medicinal material of the atractylenolide are special; and (3) performing scanning detection at 210 nm-400 nm by using a PDA (personal digital assistant) detector according to specified chromatographic conditions, and calculating the peak purity, wherein the result shows that the purity angle of each peak to be detected is smaller than the purity threshold value, and the purity meets the requirement.

6. In a methodological verification test, the result of precision investigation shows that the average recovery rate of the atractylenolide III reference substance is 105 percent, and the RSD of the determination result of nine samples is 1.0 percent; the average recovery rate of the atractylenolide II reference substance is 102 percent, and the RSD of the determination result of the nine samples is 0.69 percent, which both meet the requirements.

7. In a methodological verification test, a linear relation investigation result shows that the highest and lowest batch content measurement response value ranges can be covered by the concentration range of the atractylenolide II of 0.4212ug/ml-52.65ug/ml and the concentration range of the atractylenolide III of 0.4164ug/ml-52.05ug/ml, and that the precision, accuracy and linearity requirements can be met when the concentration ranges of the atractylenolide II and the atractylenolide III are within the ranges.

8. Methodological verification tests carried out by examining the durability of the mobile phase composition change, the column temperature change, the flow rate change, different chromatographic columns and the stability of the tested solution show that the tested ACQUITY UPLC HSS T31.8um (100X 2.1mm) chromatographic column is not suitable for the method, and the tested ACQUITY UPLC T31.6um (100X 2.1mm) chromatographic column and the tested ACQUITY UPLC BEH C181.7um (100X 2.1mm) chromatographic column are suitable for the method; the other small variation of the measurement condition can meet the requirement of the system applicability test; RSD in the peak area of the atractylenolide II and atractylenolide III of the solution to be detected is less than 2% within 24h, so that the solution to be detected is stable within 24 h.

9. The method for detecting the contents of atractylenolide II and atractylenolide III in the medicinal material of atractylenolide provided by the invention passes through a comparison test before and after optimization, and the result shows that the separation degree of atractylenolide II and III peaks and adjacent peaks in the optimization scheme is more than 2.0, so that baseline separation is achieved, the symmetry factor is obviously reduced, the peak shape is better, and the purity angles of atractylenolide II and III peaks are both smaller than the purity threshold, so that the method is more suitable for detecting the contents of atractylenolide II and III in different producing areas, and the product quality is improved.

Drawings

FIG. 1 is UPLC spectrum of optimized content method of Atractylodis rhizoma;

FIG. 2 UPLC spectrum of medicinal material content method of Atractylodis rhizoma (optimization method one);

FIG. 3 UPLC spectrum of medicinal material content method of Atractylodis rhizoma (optimization method II);

FIG. 4 shows UPLC spectrum of medicinal material content method of Atractylodis rhizoma (optimization method III);

FIG. 5 shows UPLC spectrum of medicinal material content method of Atractylodis rhizoma (optimized method IV);

FIG. 6 shows the maximum absorption patterns of atractylenolide II and atractylenolide III;

FIG. 7 UPLC spectra of contents of atractylenolide II and atractylenolide III in Atractylodis rhizoma;

FIG. 8 UPLC spectrum of contents of atractylenolide II and atractylenolide III in Atractylodis rhizoma (solvent 70% methanol);

FIG. 9 UPLC spectrum of contents of atractylenolide II and atractylenolide III in Atractylodis rhizoma (solvent 50% methanol);

FIG. 10 UPLC spectrum of contents of atractylenolide II and atractylenolide III in Atractylodis rhizoma (solvent 30% methanol);

FIG. 11 UPLC spectrum of contents of atractylenolide II and atractylenolide III in Atractylodis rhizoma (solvent is 50% acetonitrile);

FIG. 12 UPLC spectrum (solvent is 50% ethanol) of contents of atractylenolide II and atractylenolide III in Atractylodis rhizoma;

FIG. 13 HPLC chromatogram of atractylenolide II and atractylenolide III (ultrasonic extraction for 10 min);

FIG. 14 HPLC chromatogram of atractylenolide II and atractylenolide III of Atractylodes macrocephala (ultrasound extraction for 20 min);

FIG. 15 HPLC chromatogram of atractylenolide II and atractylenolide III (ultrasonic extraction for 30 min);

FIG. 16 HPLC chromatogram of atractylenolide II and atractylenolide III of Atractylodes macrocephala (reflux extraction for 10 min);

FIG. 17 HPLC chromatogram of Atractylodes macrocephala koidz lactone II and Atractylodes macrocephala koidz lactone III (reflux extraction for 20 min);

FIG. 18 HPLC chromatogram of Atractylodes macrocephala koidz lactone II and Atractylodes macrocephala koidz lactone III (reflux extraction for 30 min);

FIG. 19 HPLC chromatogram of contents of atractylenolide II and atractylenolide III [ column ACQUITY UPLC HSS T3(100 mm. times.2.1 mm,1.8 μm) MVK ];

FIG. 20 HPLC chromatogram of contents of atractylenolide II and atractylenolide III [ ACQUITY UPLC BEH (100 mm. times.2.1 mm,1.7 μm) ];

FIG. 21 HPLC chromatogram of contents of atractylenolide II and atractylenolide III [ CORTECS UPLC T3(100 mm. times.2.1 mm,1.6 μm) ];

FIG. 22 chromatogram of content method of Atractylodis rhizoma materials in different producing areas (before optimization);

FIG. 23 is a chromatogram of the content method of Atractylodis rhizoma in different places (after optimization);

FIG. 24A diagram of content determination specificity investigation;

FIG. 25 is a chart showing the spectra of atractylenolide III and atractylenolide II in a control solution;

FIG. 26 is a chart showing the spectra of atractylenolide III and atractylenolide II in the test solution

FIG. 27 is a linear relationship of the measurement method for the content of atractylenolide II;

FIG. 28 is a linear relationship of the measurement method for the content of atractylenolide III;

FIG. 29 different mobile phase survey chromatograms;

FIG. 30 different column temperature chromatogram for investigation;

FIG. 31 is a chromatogram for examining different flow rate variations;

FIG. 32 different columns examine chromatograms.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

[ measurement of Atractylodes lactone II and Atractylodes lactone III ] by high performance liquid chromatography (general rule 0512)

Preparation of a test solution: weighing 0.5g of Atractylodis rhizoma powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and collecting the subsequent filtrate;

preparation of control solutions: accurately weighing appropriate amount of atractylenolide II and atractylenolide III, and adding 70% methanol to obtain solutions containing atractylenolide II 10ug and atractylenolide III 10ug per 1ml respectively;

chromatographic conditions and system applicability test: the chromatographic column is CORTECS UPLC T31.6um; column temperature: 40 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; the detection wavelength is 220 nm; the flow rate is 0.35ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, wherein the mobile phase A is 44% and the mobile phase B is 56%;

the determination method comprises the following steps: precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Example 2

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 30% methanol, weighing, ultrasonically treating for 20min, cooling, weighing again, supplementing the lost weight with 30% methanol, shaking, centrifuging at a frequency of 8000r/min for 2min, filtering, and taking the subsequent filtrate;

chromatographic conditions and system applicability test: the chromatographic column is CORTECS UPLC T31.6um; column temperature: 39 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.09 percent phosphoric acid solution as a mobile phase B; the detection wavelength is 210 nm; the flow rate is 0.34ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, wherein the mobile phase A is 44% and the mobile phase B is 56%;

Example 3

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, centrifuging at a frequency of 12000r/min for 8min, filtering, and taking the subsequent filtrate;

chromatographic conditions and system applicability test: the chromatographic column is CORTECS UPLC T31.6um; column temperature: 41 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.11% phosphoric acid solution as a mobile phase B; the detection wavelength is 230 nm; the flow rate is 0.36ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, wherein the mobile phase A is 44% and the mobile phase B is 56%;

Example 4

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, reflux-extracting for 10min, cooling, weighing again, supplementing lost weight with 70% methanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and taking the subsequent filtrate;

Example 5

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 30% methanol, weighing, reflux-extracting for 20min, cooling, weighing again, supplementing lost weight with 30% methanol, shaking, centrifuging at a frequency of 8000r/min for 2min, filtering, and taking the subsequent filtrate;

Example 6

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% methanol, weighing, reflux-extracting for 30min, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, centrifuging at a frequency of 12000r/min for 8min, filtering, and taking the subsequent filtrate;

Example 7

Preparation of a test solution: taking 0.5g of rhizoma atractylodis macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% acetonitrile, weighing, ultrasonically treating for 10min, cooling, weighing again, complementing the loss weight with 50% acetonitrile, shaking uniformly, centrifuging for 5min at a frequency of 10000r/min, filtering, and taking the subsequent filtrate;

Example 8

Preparation of a test solution: taking 0.5g of rhizoma atractylodis macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% acetonitrile, weighing, ultrasonically treating for 20min, cooling, weighing again, supplementing the lost weight with 50% acetonitrile, shaking up, centrifuging at a frequency of 8000r/min for 2min, filtering, and taking the subsequent filtrate;

Example 9

Preparation of a test solution: taking 0.5g of rhizoma atractylodis macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% acetonitrile, weighing, ultrasonically treating for 30min, cooling, weighing again, complementing the loss weight with 50% acetonitrile, shaking uniformly, centrifuging for 8min at the frequency of 12000r/min, filtering, and taking the subsequent filtrate;

Example 10

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% ethanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with 50% ethanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and taking the subsequent filtrate;

Example 11

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% ethanol, weighing, ultrasonically treating for 20min, cooling, weighing again, supplementing the lost weight with 50% ethanol, shaking, centrifuging at a frequency of 8000r/min for 2min, filtering, and taking the subsequent filtrate;

Example 12

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% ethanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the lost weight with 50% ethanol, shaking, centrifuging at a frequency of 12000r/min for 8min, filtering, and taking the subsequent filtrate;

Example 13

Example 14

Preparation of a test solution: taking 0.5g of rhizoma atractylodis macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% acetonitrile, weighing, reflux extracting for 20min, cooling, weighing again, supplementing the lost weight with 50% acetonitrile, shaking up, centrifuging at a frequency of 8000r/min for 2min, filtering, and taking the subsequent filtrate;

Example 15

Preparation of a test solution: taking 0.5g of rhizoma atractylodis macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% acetonitrile, weighing, reflux extracting for 30min, cooling, weighing again, complementing the loss weight with 50% acetonitrile, shaking up, centrifuging for 8min at the frequency of 12000r/min, filtering, and taking the subsequent filtrate;

Example 16

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% ethanol, weighing, reflux-extracting for 10min, cooling, weighing again, supplementing the lost weight with 50% ethanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and taking the subsequent filtrate;

Example 17

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% ethanol, weighing, reflux-extracting for 20min, cooling, weighing again, supplementing the lost weight with 50% ethanol, shaking, centrifuging at a frequency of 8000r/min for 2min, filtering, and taking the subsequent filtrate;

Example 18

Preparation of a test solution: taking 0.5g of rhizoma Atractylodis Macrocephalae powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 50% ethanol, weighing, reflux-extracting for 30min, cooling, weighing again, supplementing the lost weight with 50% ethanol, shaking, centrifuging at a frequency of 12000r/min for 8min, filtering, and taking the subsequent filtrate;

Experimental example: to prove the scientificity and rationality of the invention, the following experimental studies of methodology were carried out:

materials, devices, reagents and controls

1.1 materials

1.2 apparatus

1.3 reagents

1.4 control

2 screening of method for detecting content of rhizoma atractylodis macrocephalae medicinal material

2.1 selection of mobile phase and gradient of the content method of Atractylodis rhizoma

2.1.1 before optimization, referring to the chromatographic conditions of contents of alisma rhizome decoction atractylenolide II and atractylenolide III of my company, a chromatographic column: ACQUITY UPLC HSS T31.8um (100X 2.1mm), eluted with acetonitrile-0.1% phosphoric acid water system, acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, column temperature: 30 ℃; sample introduction volume: 5 mu l of the solution; flow rate: 0.30 ml/min; gradient elution was performed as specified in Table 1 and the results are shown in FIG. 1.

TABLE 1 gradient elution Table

As a result: as can be seen from FIG. 1, when the contents of atractylenolide II and atractylenolide III in the Atractylodis rhizoma medicinal material are detected by the gradient elution procedure of Alismatis rhizoma decoction, the problems of poor peak separation degree, poor peak shape, poor accuracy, poor stability, etc. are found.

2.1.2 aiming at the problem of '2.1.1', the flow rate, the column temperature, the gradient proportion of mobile phase, a chromatographic column and the like in the content methods of atractylenolide II and atractylenolide III of the largehead atractylodes rhizome are examined, and the following concrete steps are carried out:

the optimization method comprises the following steps: a chromatographic column: ACQUITY UPLC HSS T31.8um (100X 2.1 mm); acetonitrile is taken as a mobile phase A, and 0.1% phosphoric acid water is taken as a mobile phase B; column temperature: 30 ℃; sample introduction volume: 5 mu l of the solution; flow rate: 0.30 ml/min; gradient elution was performed as specified in Table 2, and the results are shown in FIG. 2.

TABLE 2 gradient elution Table (optimization method one)

As a result: as can be seen from FIG. 2, the peak shape and peak purity of the method are good, but when the contents of atractylenolide II and atractylenolide III in different production areas are examined, the method has poor stability, and the separation degree of adjacent peaks is poor individually.

And the second optimization method comprises the following steps: a chromatographic column: CORTECS UPLC T31.6um (100X 2.1 mm); acetonitrile is used as a mobile phase A, 0.1% phosphoric acid water is used as a mobile phase B, and the column temperature is as follows: 40 ℃; sample introduction volume: 5 mu l of the solution; flow rate: 0.35 ml/min; gradient elution was performed as specified in Table 3 and the results are shown in FIG. 3.

TABLE 3 gradient elution Table (optimization method two)

As a result: the column temperature, flow rate and mobile phase gradient are changed, the atractylenolide II and adjacent peaks form M peak, and the separation degree is poor.

And the optimization method comprises the following steps: a chromatographic column: CORTECS UPLC T31.6um (100X 2.1 mm); acetonitrile is used as a mobile phase A, 0.1% phosphoric acid water is used as a mobile phase B, and the column temperature is as follows: 30 ℃; sample introduction volume: 5 mu l of the solution; flow rate: 0.35 ml/min; gradient elution was performed as specified in Table 4 and the results are shown in FIG. 4.

TABLE 4 gradient elution Table (optimization method III)

As a result: by changing the proportion of the mobile phase, the atractylenolide II and III can not reach baseline separation from the adjacent peaks.

The optimization method comprises the following steps: a chromatographic column: CORTECS UPLC T31.6um (100X 2.1 mm); acetonitrile mobile phase A, and 0.1% phosphoric acid water as mobile phase B; column temperature: 40 ℃; flow rate: 0.35 mL/min; sample introduction volume: 5 mu L of the solution; the optimized gradient is shown in Table 5, and the detection result is shown in FIG. 5.

TABLE 5 gradient elution Table (optimization method four)

As a result: the optimization method IV detects the contents of atractylenolide II and atractylenolide III in the rhizoma atractylodis macrocephalae, has good separation degree, peak shape and peak purity, and is suitable for the detection of the rhizoma atractylodis macrocephalae in different producing areas, so the optimization method IV is used as the optimal scheme for detecting chromatographic conditions of the contents of the atractylenolide II and atractylenolide III in the rhizoma atractylodis macrocephalae.

2.2 determination of the wavelength by the method for determining the content of Atractylodis rhizoma

Selecting detection wavelength according to 2.1.2' optimization method four

Precisely weighing 0.4056g of largehead atractylodes rhizome medicinal material (batch number: BZY02) powder, placing the powder into a conical flask, precisely adding 50ml of 70% methanol, carrying out ultrasonic treatment for 10min, cooling, weighing, complementing the loss weight with 70% methanol, shaking uniformly, centrifuging for 5min at the frequency of 10000r/min, filtering, taking the subsequent filtrate, and recording the absorption spectrum within the range of 190-400 nm, as shown in figure 6 and figure 7.

The result shows that the maximum absorption of alisma decoction atractylenolide II and atractylenolide III is about 220nm, the peak response is high, and the base line is stable, so that the 220nm is selected as the content detection wavelength of alisma decoction atractylenolide II and atractylenolide III.

2.3 investigation of the content of Atractylodis rhizoma in the medicinal material by solvent extraction

Selecting the extraction solvent according to 2.1.2 "optimization method one

Precisely weighing 0.4056g, 0.4014g, 0.4040g, 0.4045g, 0.4001g, 0.4042g, 0.4000g, 0.4006g, 0.4000g and 0.3944g of powder of a medicinal material (batch number: BZY02) of the Chinese atractylodes, respectively placing the powder into a 50ml conical flask, respectively and precisely adding 70% of methanol, 50% of methanol, 30% of methanol, 50% of acetonitrile and 50% of ethanol into the conical flask, respectively and ultrasonically treating the mixture for 20min, cooling the mixture, weighing the mixture, complementing the lost weight by using the same solvent, shaking the mixture uniformly, centrifuging the mixture for 5min at the frequency of 10000r/min, filtering the mixture, and taking the subsequent filtrate to obtain the Chinese medicinal powder, wherein the Chinese medicinal powder is measured according to the chromatographic conditions, and is shown in the figures 8, 9, 10, 11, 12 and 6.

TABLE 6 Largehead Atractylodes rhizome medicinal material content method solvent investigation table

As a result: the sample is treated by 70% methanol, 50% acetonitrile and 50% ethanol, the peak areas are not very different, and the methods of adopting 70% methanol and contents of rhizoma alismatis soup atractylenolide II and atractylenolide III are kept consistent, so 70% methanol is selected as a sample treatment solvent for the method of the contents of rhizoma atractylodis macrocephalae.

2.4 investigation of solvent extraction method and extraction time of medicinal material content method of Atractylodis rhizoma

Selecting solvent extraction mode and extraction time according to 2.1.2' optimization method I

Precisely weighing powder 0.4003g, 0.4062g, 0.4073g, 0.4099g, 0.4032g, 0.4023g, 0.4005g, 0.4054g, 0.4031g, 0.4014g, 0.4003g and 0.4010g of rhizoma Atractylodis Macrocephalae (batch number: BZY02), respectively placing in a conical flask, precisely adding 50ml of 70% methanol, respectively performing ultrasonic treatment for 10min, 20min and 30min, performing reflux extraction for 10min, 20min and 30min, cooling, weighing, supplementing lost weight with 70% methanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and collecting filtrate, wherein the measurement is performed according to the chromatographic conditions shown in the figure 13, figure 14 and figure 15,

Fig. 16, 17, and 18: table 7.

TABLE 7 Largehead Atractylodes rhizome medicinal material content method solvent investigation table

As a result: ultrasonic treatment is adopted for 10min, 20min and 30min, reflux extraction is carried out for 10min, 20min and 30min, and the results of atractylenolide II and atractylenolide III obtained by reflux extraction for 30min have no obvious difference, so that the ultrasonic treatment for 10min is selected to treat the content of atractylenolide in the sample.

2.5 investigation of the crude drug content of Atractylodis rhizoma by chromatographic column

Selecting a chromatography column according to 2.1.2 "optimization method one

Weighing 0.4012g of powder of Bingbaishu medicinal material (lot number: BZY02), placing in a conical flask, precisely adding 50ml of 70% methanol, ultrasonically extracting for 10min, cooling, weighing again, supplementing lost weight with 70% methanol, shaking up, centrifuging at a frequency of 10000r/min for 5min, filtering, and taking subsequent filtrate to obtain the final product, wherein the subsequent filtrate is respectively used:

CORTECS UPLC T3(100mm×2.1mm,1.6μm)；

ACQUITY UPLC BEH(100mm×2.1mm,1.7μm)；

ACQUITY UPLC HSST 3(100 mm. times.2.1 mm,1.8 μm) MVK, determined according to the chromatographic conditions described above: the results are shown in FIG. 19, FIG. 20, FIG. 21; tables 8, 9 and 10.

TABLE 8 chromatographic column investigation of Atractylodis rhizoma medicinal material content

TABLE 9 chromatographic column investigation of Atractylodis rhizoma medicinal material content

TABLE 10 chromatographic column investigation of Atractylodis rhizoma medicinal material content

As a result: the chromatographic conditions of the content of the white atractylodes rhizome in optimization are inspected by chromatographic columns with different specifications, the chromatographic column CORTECS UPLC T3(2.1 × 100mm, 1.6um) has better peak shape, and the separation degree and the peak purity are qualified, so the content detection method of the white atractylodes rhizome adopts a CORTECS UPLC T3(2.1 × 100mm, 1.6um) chromatographic column.

And (4) conclusion: through the screening experiments, the preferable method for measuring the contents of atractylenolide II and atractylenolide III in the medicinal material of the atractylenolide is as follows:

the preferable method for chromatographic condition and system applicability test is as follows: the chromatographic column is CORTECS UPLC T31.6um; the column temperature was 40 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; the detection wavelength is 220 nm; the flow rate is 0.35ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, mobile phase A is 44%, and mobile phase B is 56%.

The preferable preparation method of the test solution comprises the following steps: weighing 0.5g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and collecting the subsequent filtrate.

2.6 content detection method of Atractylodes macrocephala lactone II and Atractylodes macrocephala lactone III in Atractylodes macrocephala medicinal material before and after optimization

Taking 12 batches of rhizoma Atractylodis Macrocephalae from different producing areas, and performing content detection according to the methods before optimization of "2.1.1" and after optimization of "2.2.2 optimization method four", respectively, with the results shown in FIG. 22 and FIG. 23; table 11, table 12.

TABLE 11 comparison of results before and after optimization of the method for measuring the content of Atractylodis rhizoma

TABLE 12 comparative investigation of results before and after optimization of the method for measuring the content of Atractylodis rhizoma

And (4) conclusion: in the method before optimization of '2.1.1', the separation degrees of atractylenolide II and III peaks and adjacent peaks are both greater than 1.5, but atractylenolide II cannot be separated from adjacent peaks to reach a baseline, and the purity angles of atractylenolide II and III peaks of atractylenolide medicinal materials in different producing areas are not both smaller than a purity threshold; in the optimization method IV of 2.2.2, the separation degrees of the atractylenolide II and III peaks and adjacent peaks are both more than 2.0, so that the baseline separation is achieved, the symmetry factor is obviously reduced, the peak shape is better, and the purity angles of the atractylenolide II and III peaks are both less than the purity threshold, therefore, the optimization method IV is more suitable for the content detection of atractylenolide II and III in the medicinal materials of atractylenolide in different production places.

3 verification and investigation of bighead atractylodes rhizome medicinal material content detection methodology

3.1 preparation of sample for determining content of Atractylodes macrocephala lactone II and Atractylodes macrocephala lactone III in Atractylodes macrocephala medicinal material and method thereof

Chromatographic conditions and system applicability test: the chromatographic column is CORTECS UPLC T31.6um; column temperature: 40 ℃; acetonitrile was used as mobile phase a, and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in table 13; the detection wavelength is 220 nm; the flow rate was 0.35 ml/min. The number of theoretical plates is not less than 4500 calculated according to the peak of atractylenolide III.

TABLE 13 gradient elution Table

Accurately weighing appropriate amount of II control and Atractylodes macrocephala lactone III control, and adding 70% methanol to obtain 10ug of solution containing Atractylodes macrocephala lactone II and 10ug of Atractylodes macrocephala lactone III per 1ml respectively.

Preparation of a test solution: weighing 0.5g of the powder (sieved by a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, centrifuging (10000r/min, 5min), filtering, and collecting the subsequent filtrate.

The determination method comprises the following steps: precisely sucking 5 μ l of the reference solution and 5 μ l of the sample solution, respectively, injecting into a liquid chromatograph, and measuring.

3.2 high Performance liquid chromatography System suitability validation

3.2.1 Atractylodes lactone II Atractylodes lactone III content determination method System applicability determination method:

precisely sucking 5 μ l of control solution under item 3.1, and continuously injecting for 6 times; and 5 mul of the test solution is subjected to 1 sample injection measurement, and the chromatogram is recorded.

3.2.2 Atractylodes lactone II Atractylodes lactone III content determination method the system applicability determination acceptance criteria:

3.2.2.1 the Relative Standard Deviation (RSD) of the area of the main peak (atractylenolide II and atractylenolide III) is not more than 2.0%;

3.2.2.2 Relative Standard Deviation (RSD) of retention time of main peak (atractylenolide II and atractylenolide III) is not more than 1.0%;

3.2.2.3 the number of theoretical plates of the main peak (atractylenolide II and atractylenolide III) is more than 4500 according to the peak of atractylenolide III;

3.2.2.4 the resolution of the main peak (Atractylodes lactone II and Atractylodes lactone III) should be greater than 1.5;

3.2.3 System suitability verification results, see tables 14, 15.

TABLE 14 System suitability confirmation result information Table

TABLE 15 System suitability confirmation result information Table

As a result: the Relative Standard Deviation (RSD) of the area of the main peak (atractylenolide II and atractylenolide III) is less than 2.0 percent; a Relative Standard Deviation (RSD) of retention time of less than 1.0%; the theoretical plate numbers are all more than 4500; the separation degrees are all more than 1.5; therefore, the content determination method of atractylenolide II and atractylenolide III meets the requirement of system applicability.

3.3 specialization examination

3.3.1 method for determining the content of Atractylodes lactone II and Atractylodes lactone III:

precisely absorbing 5ul of atractylenolide II and atractylenolide III reference solution under item 3.1, injecting 5ul of each of the test solution and the sample extraction solvent into a liquid chromatograph, and recording a chromatogram; and performing peak spectrum scanning and purity detection on the chromatographic peaks of atractylenolide II and atractylenolide III in the reference solution and the test solution by using a PDA detector.

3.3.2 method confirmation acceptance criteria:

3.3.2.1 the retention time of main peak of test solution is consistent with that of contrast solution;

3.3.2.2 the sample extraction solvent should be a straight baseline at the retention time of atractylenolide II and atractylenolide III, and no absorption peak;

3.3.2.3 Atractylodes macrocephalic lactone II and Atractylodes macrocephalic lactone III should have the purity angle of the reference solution peak and the to-be-detected peak of the sample solution smaller than the purity threshold.

3.3.3 results of the specificity study, see Table 16, FIG. 24.

TABLE 16 results of the specificity experiment

3.3.4 chromatographic Peak purities, see Table 17, FIGS. 25, 26.

TABLE 17 chromatographic Peak purity Angle and purity threshold List

As a result: the result shows that the blank solvent has no interference to the peak position of each peak, and the method has specificity in determining the contents of atractylenolide III and atractylenolide II in the medicinal material of the atractylenolide; scanning and detecting at 210-400 nm with PDA detector according to the specified chromatographic condition, calculating peak purity, and displaying that the purity angle of each peak to be detected is less than the purity threshold value

The degree meets the requirements.

3.4 precision

3.4.1 repeatability test

6 parts of sample are respectively weighed by the same person, sample test solution is prepared according to the preparation method of the test solution under 3.1 items, the content is measured under the set chromatographic condition, and RSD is calculated, wherein RSD is not more than 3%. The results are shown in Table 18:

TABLE 18 results of repeated experiments

3.4.2 intermediate precision investigation

Weighing 6 parts of sample by two experimenters respectively, preparing sample test solution according to the preparation method of the sample solution under item 3.1, injecting 5 mul of sample on the same instrument in the same laboratory on different days respectively, measuring the peak area value, calculating the content and RSD, wherein the RSD is not more than 6%. The results are shown in Table 19.

TABLE 19 results of intermediate precision experiments

As a result: the results show that the repeatability inspection and the intermediate precision both meet the requirements.

3.5. Accuracy (sample recovery test)

3.5.1 determination of accuracy of the method for determining the content of atractylenolide II atractylenolide III:

precisely weighing 0.25g of the powder, placing the powder in a 50ml volumetric flask, precisely adding a proper amount of atractylenolide II and atractylenolide III reference substance solution (the ratio of the addition amount of the reference substance solution to the amount of the component to be measured in the sample is 1.5:1,1:1, 0.5: 1; 3 parts of the sample solution is prepared for each concentration), adding a proper amount of 70% methanol, carrying out ultrasonic treatment for 10min, cooling to fix the volume and shaking up, centrifuging (10000r/min, 5min), filtering, taking subsequent filtrate, precisely sucking 5 mu l of the subsequent filtrate, injecting the subsequent filtrate into a liquid chromatograph, measuring according to a set method, calculating RSD according to the measurement result of 9 parts of the sample, and evaluating the RSD to be less than 2.0%.

Calculating the formula:

in the formula: a. the_{Sample (A)}The peak area of the solution was tested for recovery;

c is the concentration of the control solution (ug/ml);

A_{to pair}Peak area of the control solution;

V_{to pair}The sample injection volume of the reference solution;

V_{sample (A)}Test the sample volume of the solution for recovery;

3.5.2 accuracy (sample recovery test) the results are shown in tables 20 and 21.

TABLE 20 determination of accuracy (sample recovery) of atractylenolide III

TABLE 21 determination of accuracy (sample recovery) of atractylenolide II

As a result: the average recovery rate of the atractylenolide III reference substance is 105 percent, and the RSD of the determination result of nine samples is 1.0 percent; the average recovery rate of the atractylenolide II reference substance is 102 percent, and the RSD of the determination result of the nine samples is 0.69 percent, which both meet the requirements.

3.6. Investigation of Linear relationships

3.6.1 determination of Linear relationship and Linear Range of Atractylodes lactone II Atractylodes lactone III content determination method:

3.6.1.1 Linear relationship test solution preparation:

atractylenolide iii control linear solution: precisely weighing 0.01042g of atractylenolide III reference substance, adding 70% methanol to prepare 104.1ug/ml atractylenolide III reference substance solution (storage), precisely transferring 10ml of atractylenolide III reference substance solution (storage) to a 100ml volumetric flask, adding 70% methanol to constant volume to scale, and preparing solution I; precisely transferring 3ml and 5ml of atractylenolide III reference substance solution (storage) into a 10ml volumetric flask, adding 70% methanol to constant volume to reach a scale to prepare a solution II; precisely transferring 1ml and 5ml of the solution into a 25ml volumetric flask respectively, and adding 70% methanol to the volume until the volume is scaled to prepare a fifth solution. Taking the solution from the first step to the second step to the fourth step, continuously injecting the solution into a sample 2 needles, and measuring the average value of the peak area of the atractylenolide III.

Atractylenolide ii control linear solution: precisely weighing 0.01054g of atractylenolide II reference substance, adding 70% methanol to prepare 105.3ug/ml atractylenolide II reference substance solution (storage), precisely transferring 10ml of atractylenolide II reference substance solution (storage) to a 100ml volumetric flask, adding 70% methanol to constant volume to scale, and preparing solution I; precisely transferring 3ml and 5ml of atractylenolide II reference substance solution (storage) into a 10ml volumetric flask, adding 70% methanol to constant volume to scale to prepare solution II; precisely transferring 1ml and 5ml of the solution into a 25ml volumetric flask respectively, and adding 70% methanol to the volume until the volume is scaled to prepare a fifth solution. Taking the solution from the first step to the second step to the fourth step, continuously injecting the solution into a sample 2 needles, and measuring the average value of the peak area of the atractylenolide II.

3.6.1.2 calculating atractylenolide II and atractylenolide III concentration of each test solution, calculating linear regression equation and regression coefficient R with the concentration as abscissa and the average value of atractylenolide II and atractylenolide III peak area as ordinate²Linear range.

3.6.2 method confirmation acceptance criteria:

3.6.2.1 the concentration of atractylenolide II and atractylenolide III in the test solution has a good linear relationship with the peak area, and the regression coefficient R²：R²≥0.9990；

3.6.2.2 Atractylodes macrocephalic lactone II Atractylodes macrocephalic lactone III concentration range: the range should meet precision, accuracy and linearity requirements.

3.6.3 results of the linear relationship test, see FIGS. 27, 28; table 22, table 23.

TABLE 22 determination of Atractylodes macrocephala lactone II content- -determination of Linear relationship and Linear Range

TABLE 23 determination of Atractylodes macrocephala lactone III content- -determination of Linear relationship and Linear Range

As a result: the linear relation between the concentration range of atractylenolide II from 0.4212ug/ml to 52.65ug/ml and the concentration range of atractylenolide III from 0.4164ug/ml to 52.05ug/ml can cover the highest and lowest batch content measurement response value range, which shows that the precision, accuracy and linearity requirements can be reached when the upper and lower limit concentration ranges of atractylenolide II and atractylenolide III are within the above ranges.

3.7 Atractylodes lactone II Atractylodes lactone III content determination method durability investigation

3.7.1 test for variation of mobile phase composition: preparing a sample according to the content determination method under item 3.1, preparing mobile phases with different concentrations for detection, and proving that the small change of the mobile phase composition can meet the requirement of a system applicability test, and the result is shown in a table 24; FIG. 29.

TABLE 24 test results for mobile phase composition changes

3.7.2 column temperature Change test: the sample is prepared according to the content determination method under item 3.1, the column temperature is changed for detection, and the small change of the column temperature is proved to meet the requirement of the system applicability test. The results are shown in Table 25, FIG. 30.

TABLE 25 column temperature Change test results

3.7.3 test for variation in flow rate: the sample is prepared according to the content determination method under item 3.1, the flow rate is changed for detection, and the small change of the flow rate is proved, so that the system applicability test requirement can be met. The results are shown in Table 26, FIG. 31.

TABLE 26 flow Rate Change test results

3.7.4 different column tests: preparing samples according to the content determination method under item 3.1, and detecting by using chromatographic columns of different types and different batches, thereby proving that the chromatographic columns of different batches can meet the test requirement of system applicability. The results are shown in Table 27, FIG. 32.

TABLE 27 variation of different column types

3.7.5 stability test of the test solutions: preparing a sample according to the content determination method under item 3.1, testing the peak areas of the solution to be detected at different time points, and calculating RSD to investigate the stability of the solution to be detected. The results are shown in Table 28.

TABLE 28 solution stability Studies

As a result: tests carried out on an ACQUITY UPLC HSS T31.8um (100X 2.1mm) column not suitable for this method, and CORTECS UPLC T31.6um (100X 2.1mm) and ACQUITY UPLC BEH C181.7um (100X 2.1mm) columns suitable for this method; the other small variation of the measurement condition can meet the requirement of the system applicability test; RSD in the peak area of the atractylenolide II and atractylenolide III of the solution to be detected is less than 2% within 24h, so that the solution to be detected is stable within 24 h.

And (4) conclusion: through the above methodology experimental study, the preferable method for measuring the contents of atractylenolide II and atractylenolide III in the rhizoma atractylodis macrocephalae medicinal material is as follows: weighing 0.5g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and collecting the subsequent filtrate. Taking appropriate amount of atractylenolide II reference substance and atractylenolide III reference substance, precisely weighing, and adding 70% methanol to obtain solutions containing atractylenolide II 10ug and atractylenolide III 10ug per 1ml respectively. The chromatographic column is CORTECS UPLC T31.6um; column temperature: 40 ℃; performing gradient elution by using acetonitrile as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; the detection wavelength is 220 nm; the flow rate is 0.35ml/min, and the number of theoretical plates is not less than 4500 according to the peak of atractylenolide III; the gradient elution is specifically as follows: for 0-1min, the mobile phase A is 44%, and the mobile phase B is 56%; 1-2min, mobile phase A is 44 → 36%, mobile phase B is 56 → 64%; 2-3.5min, the mobile phase A is 36%, and the mobile phase B is 64%; 3.5-4.0min, mobile phase A is 36 → 43%, mobile phase B is 64 → 57%; 4-8min, wherein the mobile phase A is 43 percent, and the mobile phase B is 57 percent; 8-9min, the mobile phase A is 43 → 90%, and the mobile phase B is 57 → 10%; 9-12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; 12-13min, the mobile phase A is 90 → 44%, and the mobile phase B is 10 → 56%; 13-16min, mobile phase A is 44%, and mobile phase B is 56%. Precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. The method has the advantages of fast peak-producing time, good peak shape, qualified separation degree and peak purity, high accuracy, good stability and simple and fast operation.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for measuring the contents of atractylenolide II and atractylenolide III in an atractylenolide medicinal material is characterized in that the method for measuring the contents comprises the following steps:

2. The content measurement method according to claim 1, characterized in that the content measurement method is:

3. The content measurement method according to any one of claims 1 to 2, wherein the methanol in step (1) may be any one of acetonitrile and ethanol.

4. The method according to claim 3, wherein the concentrations of acetonitrile and ethanol are 50% respectively.

5. The assay method according to any one of claims 1-2, wherein the centrifugation in step (1) is performed at a frequency of 8000-.

6. The assay according to any one of claims 1-2, wherein the centrifugation in step (1) is carried out at a frequency of 10000 r/min.

7. The assay method according to any one of claims 1 to 2, wherein the ultrasonic treatment or the reflux treatment in the step (1) is carried out for 10 to 30 min.

8. The assay according to any one of claims 1 to 2, wherein the sonication or reflux treatment in step (1) is carried out for 10 min.

9. The content measurement method according to any one of claims 1 to 2, characterized in that the content measurement method is:

(1) preparation of a test solution: weighing 0.5g of the powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating or refluxing for 10min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, centrifuging at a frequency of 10000r/min for 5min, filtering, and collecting the subsequent filtrate;

10. The detection method according to claim 9, wherein the content determination method is applied to detection of the medicinal material Atractylodis Macrocephalae, processed product of the medicinal material Atractylodis and preparation containing the medicinal material Atractylodis.