CN110907550B

CN110907550B - Method for simultaneously detecting contents of six components in infant lung-ventilating and cough-relieving granules

Info

Publication number: CN110907550B
Application number: CN201911190492.4A
Authority: CN
Inventors: 吴木琴; 李霞; 赵刚; 陈鹏; 向阳; 熊登科; 朱立彬; 陈骞; 杨颃; 高凯; 李丽娟
Original assignee: Jianmin Pharmaceutical Groups Corp ltd
Current assignee: Jianmin Pharmaceutical Groups Corp ltd
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2022-04-05
Anticipated expiration: 2039-11-28
Also published as: CN110907550A

Abstract

The invention discloses a method for simultaneously detecting the contents of six components of a child lung-ventilating and cough-relieving granule, wherein the six components comprise ephedrine hydrochloride, baicalin, amygdalin, astragaloside IV, liquiritin and cimicidin. Compared with the prior art, the invention has the advantages of less detection time consumption, high speed, low cost, less instruments and the like, greatly improves the detection efficiency, and can more comprehensively control the internal quality of the traditional Chinese medicine.

Description

Method for simultaneously detecting contents of six components in infant lung-ventilating and cough-relieving granules

Technical Field

The invention belongs to the field of traditional Chinese medicine detection, and relates to a method for detecting six component contents of infant lung-ventilating and cough-relieving granules.

Background

CN 102284029A discloses a pediatric lung-ventilating and cough-relieving granule composition, a preparation method and a quality control method thereof, the composition is prepared from twelve traditional Chinese medicines of ephedra, bamboo leaf and divaricate saposhnikovia root, southwest baical skullcap root, platycodon root, mustard seed, bitter apricot seed, semen lepidii, Indian kalimeris herb, membranous milkvetch root, common yam rhizome, hawthorn fruit and liquoric root, has the efficacies of ventilating the lung and relieving exterior syndrome, clearing heat and reducing phlegm, and is mainly used for cough and excessive phlegm, yellow and sticky phlegm and unsmooth expectoration caused by exogenous cough and phlegm-heat obstructing lung of children.

The infantile lung-ventilating and cough-relieving granules are common medicines for infantile exogenous cough, have definite clinical curative effect and large market demand, but have simple quality standard, only carry out thin-layer qualitative identification on 3 medicinal materials of semen lepidii, semen brassicae and hawthorn, as well as ephedrine hydrochloride and baicalein, and carry out content determination on baicalin by adopting an HPLC method. The above quality control method has the following disadvantages: (1) products need to be extracted and detected for multiple times, so that the detection period is long, and a large amount of manpower, material resources and detection equipment are needed; (2) only quantitative detection is carried out on baicalin, other components can only be qualitative, the internal quality of the product cannot be comprehensively controlled, and the consistency of the quality of the traditional Chinese medicine cannot be objectively reflected and evaluated. Therefore, it is urgently needed to establish a new quality control method for the infantile lung-ventilating and cough-relieving granules so as to establish a traceable whole-process quality control system highly related to clinical curative effect.

Disclosure of Invention

The invention aims to establish a method for simultaneously detecting the contents of six components in children's granules for freeing lung and relieving cough, and aims to control the product quality more efficiently and reliably.

In order to achieve the purpose, the invention adopts the following technical means:

method for simultaneously detecting six component contents of infant lung ventilating and cough relieving granulesThe lung cough relieving granules are prepared from twelve traditional Chinese medicinal materials including ephedra, saposhnikovia divaricata, scutellaria baicalensis, platycodon grandiflorum, mustard seeds, bitter almonds, lepidium seeds, kalimeris, astragalus membranaceus, Chinese yams, hawthorns and liquorice, the six components are ephedrine hydrochloride, baicalin, amygdalin, astragaloside IV, liquiritin and cimicidin, the detection method comprises the steps of extracting the lung ventilating and cough relieving granules for children by using an extraction solvent, detecting the extracting solution by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the detection wavelength of the ultraviolet detector is set to be 205-230nm, and the stationary phase of the high performance liquid chromatograph is C₁₈Chromatographic column, mobile phase A is acetonitrile containing 0.05-0.5% (weight) sodium dodecyl benzene sulfonate, mobile phase B is 0.05-0.5% (volume) formic acid solution, gradient elution is adopted, the flow rate is 0.5-2ml/min, and the column temperature is 25-35 ℃.

Preferably, the extraction solvent is methanol containing 1% by volume of phosphoric acid.

Preferably, the set detection wavelength of the ultraviolet detector is 210 nm.

Preferably, the mobile phase a is acetonitrile containing 0.1% sodium dodecylbenzenesulfonate.

Preferably, the mobile phase B is a 0.1% formic acid solution.

Preferably, the gradient elution time is 50min, divided into 3 time periods, the volume change of mobile phases A and B in each time period is shown in Table 1 below,

TABLE 1 gradient elution procedure

Time min	Mobile phase a (% by volume)	Mobile phase B (% by volume)
			0-10	1-15	99-85
10-40	15-30	85-70
			40-50	30-40	70-60

Preferably, the flow rate is 0.8 ml/min.

Preferably, the column temperature is 30 ℃.

The invention has the beneficial effects that:

(1) the invention can simultaneously detect the contents of six substances in the infant lung-ventilating and cough-relieving granules by one-time sample preparation and sample introduction, the detection time is not more than 2 hours, compared with the prior art, the invention has the advantages of less detection time consumption, high speed, low cost, few instruments and the like, and the detection efficiency is greatly improved.

(2) The invention realizes the quantitative detection of six components in the infantile lung-ventilating and cough-relieving granules, compared with the existing quantitative detection of single component combined with other components, the invention can more comprehensively control the internal quality of the traditional Chinese medicine, thoroughly eradicates the phenomena of adulteration and faking of the traditional Chinese medicine, objectively reflects the consistency of the quality of the traditional Chinese medicine, and is more beneficial to monitoring the links of feeding the medicinal materials and the like in each stage of the traditional Chinese medicine production, thereby establishing a traceability whole-process quality control system highly related to clinical curative effect.

(3) According to the invention, through screening a large number of HPLC detection conditions, the chromatographic peak separation effect of six components to be detected is good, the interference is less, the peak appearance time is short, and the peak area is large, so that the accuracy and the sensitivity of a detection result are improved, and the quality of the infant lung-ventilating and cough-relieving granules is controllable.

Drawings

FIG. 1 is an HPLC chromatogram of six mixed controls.

Fig. 2 is an HPLC standard map of the infantile lung-ventilating and cough-relieving granule.

FIG. 3 is an HPLC chromatogram of a sample to be tested after 50% methanol ultrasonic extraction.

FIG. 4 is an HPLC chromatogram of a sample to be tested after 50% ethanol ultrasonic extraction.

FIG. 5 is the HPLC chromatogram of the sample to be tested obtained at the detection wavelength of 230 nm.

FIG. 6 is an HPLC chromatogram of a sample to be tested obtained at a detection wavelength of 250 nm.

FIG. 7 is an HPLC chromatogram of a sample to be tested obtained at a detection wavelength of 280 nm.

FIG. 8 is an HPLC chromatogram of a sample to be tested obtained by a system with acetonitrile-0.05 mol/L potassium dihydrogen phosphate as a mobile phase.

FIG. 9 is an HPLC chromatogram of a sample to be tested obtained in example 4 under the gradient elution condition (2).

FIG. 10 is an HPLC chromatogram of a sample to be tested obtained in example 4 under the gradient elution condition (3).

Detailed Description

The present invention will be described in detail below with reference to specific examples.

The infantile lung-ventilating and cough-relieving granules used in the following examples were produced by Jianmin pharmaceutical industry group GmbH.

Prescription: 105g of ephedra, 210g of saposhnikovia divaricata, 210g of scutellaria baicalensis, 105g of platycodon grandiflorum, 105g of mustard, 105g of bitter apricot kernel, 105g of semen lepidii, 210g of kalimeris, 210g of astragalus mongholicus, 105g of Chinese yam, 210g of hawthorn and 105g of liquorice.

The preparation method comprises the following steps: pulverizing herba Ephedrae, extracting with 80% ethanol under reflux for three times (each for 1.5 hr), mixing extractive solutions, recovering ethanol under reduced pressure, and concentrating to relative density of 1.08(55-60 deg.C); pulverizing the rest materials, soaking in 90 deg.C water for three times, each for 1 hr, filtering, mixing filtrates, concentrating under reduced pressure to relative density of 1.05(55-60 deg.C), cooling, adding ethanol until ethanol content reaches 60%, stirring, standing for 24 hr, collecting supernatant, recovering ethanol, concentrating to relative density of 1.21(55-60 deg.C), mixing with the above herba Ephedrae extract, concentrating under reduced pressure to relative density of 1.31(55-60 deg.C), adding appropriate amount of sucrose powder and dextrin, mixing, granulating, and drying to obtain 1000 g.

Reagent testing: infant Lung-ventilating and cough-relieving granule (batch No. 190801, provided by Jianmin pharmaceutical industry group, Yekai Thailand medicine Co., Ltd.); the reference substance ephedrine hydrochloride (for containing detection, purchased by China food and drug testing research institute, with the batch number of 171241-; acetonitrile is chromatographically pure, phosphoric acid is analytically pure, methanol is analytically pure alcohol, and water is ultrapure water.

EXAMPLE 1 preparation of test solutions

Before HPLC content measurement is carried out, the medicine to be measured needs to be extracted, and the purpose of extraction is to reduce detection impurities in the preparation, including extraction components irrelevant to the detection components in the traditional Chinese medicine and auxiliary materials in the preparation, so that the interference of the impurities on the peak shape of the characteristic peak of the object to be measured is reduced as much as possible, and the characteristic peak has better resolution; secondly, the component to be detected is extracted from the medicine as fully as possible, so that the peak area of the characteristic peak is improved, and the detection accuracy is ensured. According to the prescription and the preparation characteristics of the infant lung ventilating and cough relieving granules, the influence of three extraction solvents on the detection effect is respectively investigated by combining the solubility, polarity and stability of the medicinal components.

The preparation method of the test solution comprises the following steps: 1. mixing the above materials in different amount, grinding, weighing 0.5g, precisely weighing, precisely adding 50ml methanol containing 1% (volume) phosphoric acid, ultrasonic treating (power 250W, frequency 20kHz) for 20 min, filtering, and collecting the filtrate.

2. Mixing the above materials in different amount, grinding, weighing 0.5g, precisely weighing, precisely adding 50% (volume) methanol 50ml, ultrasonic treating (power 250W, frequency 20kHz) for 20 min, filtering, and collecting the filtrate.

3. Mixing the above materials in different amount, grinding, weighing 0.5g, precisely weighing, precisely adding 50% ethanol (volume) 50ml, ultrasonic treating (power 250W, frequency 20kHz) for 20 min, filtering, and collecting the filtrate.

The instrument comprises the following steps: a Waters e2695 high performance liquid chromatograph, a quaternary pump, an online vacuum degassing system, an automatic sample injector, a column incubator and an ultraviolet detector; AB204-E electronic balance (Shanghai Merle-Torledo instruments, Inc.).

Mobile phase: acetonitrile containing 0.1% sodium dodecyl benzene sulfonate is used as a mobile phase A; 0.1% formic acid solution as mobile phase B.

Gradient elution conditions: see table 1.

Column temperature: 30 ℃; detection wavelength: 210 nm; flow rate: 0.8 ml/min; sample introduction amount: 10ul of

As a result: as shown in fig. 2, 3 and 4, the sample solution obtained by extracting with 1% phosphoric acid in methanol can comprehensively reflect the main components in the finished product, and has a large peak area and a good separation degree (fig. 2); the peak areas of the main peaks in fig. 3 are all reduced, which indicates that the component to be detected is insufficiently extracted, the detection accuracy and precision cannot be achieved, and the liquiritin of 39.3min is not completely separated; in FIG. 4, not only the peak area is smaller, but also amygdalin at 13min is not completely separated, and liquiritin at 39.3min has no chromatographic peak, so that the content determination of 6 components cannot be realized. Finally, methanol containing 1% phosphoric acid was determined as the extraction solvent.

EXAMPLE 2 selection of detection wavelength

Detection wavelength: 210nm, 230nm, 250nm, 280nm

Other conditions were the same as in example 1.

As a result: as can be seen from FIGS. 2, 5, 6 and 7, when the wavelength is 230nm (FIG. 5), the peak areas of the other components are obviously smaller than the peak area of 210nm except that the chromatographic peak areas of amygdalin and baicalin are equivalent to the peak area of 210 nm; when the wavelength is 250nm (figure 6), the peak areas of the six components are obviously smaller than the peak area of 210 nm; at a wavelength of 280nm (FIG. 7), the chromatographic peak of ephedrine hydrochloride at 22.8min is not completely separated, and the peak areas of astragaloside IV at 30.5min and glycyrrhizin at 39.3min are obviously reduced. In general, 210nm is preferred as the optimum wavelength, and the six components have good peak resolution and large peak area.

EXAMPLE 3 selection of Mobile phase

(1) Acetonitrile A of 0.1% sodium dodecylbenzenesulfonate and acetonitrile B of 0.1% formic acid solution

(2) Acetonitrile A of 0.01% sodium dodecylbenzenesulfonate and acetonitrile B of 0.1% formic acid solution

(3) Acetonitrile A of 0.5% sodium dodecylbenzenesulfonate and acetonitrile B of 0.1% formic acid solution

(4) A is acetonitrile-B is 0.1% formic acid solution

(5) A is acetonitrile-B is 0.05mol/L potassium dihydrogen phosphate

Detection wavelength: 210nm

Other conditions were the same as in example 1.

Table 2 shows the six-component chromatographic peak separation degrees calculated from the HPLC profiles of the mobile phases (1) to (4), and the HPLC profile of the mobile phase (5) is shown in FIG. 8.

TABLE 2 chromatographic Peak resolution of six Components

As can be seen from the results in table 2, when the mobile phase a does not contain sodium dodecylbenzenesulfonate, the three peaks of linarin, amygdalin and ephedrine hydrochloride are not completely separated (Rf value is less than 1.5); when the mobile phase A contains 0.01 percent of sodium dodecyl benzene sulfonate, the cimicidine and the ephedrine hydrochloride are not completely separated; when the mobile phase A contains 0.5 percent of sodium dodecyl benzene sulfonate, the linarin and the amygdalin are not completely separated; only when the mobile phase A contains 0.1 percent of sodium dodecyl benzene sulfonate, the six components to be tested have higher separation degrees (the Rf value is more than 1.5).

As can be seen from FIG. 8, when the mobile phase is acetonitrile-0.05 mol/L potassium dihydrogen phosphate, the peak appearance time of the chromatographic peak is too fast, the baseline is severely shifted, and the chromatographic peak separation degree is poor.

Finally, acetonitrile with 0.1 percent of sodium dodecyl benzene sulfonate as a mobile phase A is selected; b is 0.1% formic acid solution.

Example 4 selection of conditions for mobile phase gradient elution

Mobile phase: 0.1% acetonitrile a of sodium dodecylbenzenesulfonate: 0.1% formic acid solution as mobile phase B

Gradient elution conditions:

as a result: as shown in fig. 2, 9 and 10, in fig. 9, the peak-appearance time is too fast, and the target peaks except the baicalin chromatographic peak are not completely separated; in FIG. 10, ephedrine hydrochloride and astragaloside IV are not completely separated. Therefore, the gradient elution conditions were selected as (1).

Example 5 method verification

1. Location of six substances

Mobile phase: acetonitrile of 0.1 percent of sodium dodecyl benzene sulfonate is used as a mobile phase A; 0.1% formic acid solution as mobile phase B

Gradient elution conditions:

Preparing a reference substance solution: accurately weighing appropriate amount of ephedrine hydrochloride, baicalin, amygdalin, astragaloside IV, linarin and liquiritin reference substances respectively, and making into mixed reference substance solution containing ephedrine hydrochloride, baicalin, amygdalin, astragaloside IV, linarin, liquiritin 20ug, 80ug, 30ug, 10ug, 20ug and 30ug per 1 ml.

The results are shown in Table 3, and the HPLC chromatogram of the mixed control is shown in FIG. 1.

TABLE 3 test result table for positioning and system adaptability of six substances

Name of substance	Corresponding peak	Retention time	Relative retention time	Degree of separation
					Girardinic acid D-L-ephedrine glycoside	1 peak	9.745	0.359	2.87
Amygdalin	2 peak of	13.083	0.482	2.19
					Ephedrine hydrochloride	3 peak of	22.902	0.843	2.25
Baicalin (S)	4 peak (B)	27.162	1	2.89
					Astragaloside IV	5 Peak	30.373	1.118	2.67
Liquiritin	6 Peak	39.235	1.444	2.48

2. Linearity

Accurately weighing the linarin glycoside R₁3.25mg of amygdalin Rg₁6.27mg, ephedrine hydrochloride 5.56mg, baicalin, 8.48mg, astragaloside 0.85mg and liquiritin 1.14mg are put into a 25ml measuring flask, and 1% of phosphoric acid methanol is added to dissolve and fix the volume to the scale, and the mother liquor of the mixed reference substance is obtained after shaking up. Precisely sucking 0.5, 1, 2, 3, 4 and 10ml of the mother liquor of the reference substance respectively, putting the mother liquor into a 10ml measuring flask, adding 1 percent of methanol of phosphoric acid to dilute the mother liquor to a scale, shaking the mother liquor uniformly, measuring according to the chromatographic conditions, performing linear regression on the sample volume (X: ug) by using the peak integral area (Y: mAU min), and taking 6 concentration points for research in the range from the quantitative limit concentration to the index concentration not higher than 150 percent. Linear relationship as a function of measured response signal (peak area) to analyte concentrationPlotting the number, linear regression by least square method, and correlation coefficient R²The linear regression coefficient R is required to confirm a good linear relationship²Should be no less than 0.990, the results are shown in Table 4.

TABLE 4 Standard curves, correlation coefficients and correction factors for six substances

3. Repeatability of

6 solutions of the infant lung-ventilating and cough-relieving granule sample (batch number: 190801) with the same concentration are prepared and tested, 2 needles of each solution are injected, and the relative standard deviation of 6 content measurement results is required to be not more than 2.0%, and the results are shown in Table 5.

TABLE 5 repeatability test results for the determination of the contents of six substances

Sample (I)

Girardinic acid D-L-ephedrine glycoside

Amygdalin

Ephedrine hydrochloride

Baicalin

Astragaloside IV

Liquiritin

RSD

1.72％

1.01％

1.90％

1.33％

1.42％

1.08％

The results show that: the RSD value of each component is in a required range, which shows that the repeatability result of the detection method is good.

4. Accuracy of

Taking 0.25g of infant lung-ventilating and cough-relieving granules (190801 batches, known amount is calculated according to repeated results) with known content, precisely weighing six parts, respectively placing into conical bottles with stoppers, precisely adding 50ml of reference substance mixed solution (ephedrine hydrochloride 0.02mg/ml, baicalin 0.08mg/ml, amygdalin 0.03mg/ml, astragaloside 0.01mg/ml, cimicidin 0.02mg/ml, liquiritin 0.03mg/ml solvent: 1% methanol of phosphoric acid), weighing, ultrasonically treating (power 250W, frequency 20kHz) for 20 minutes, cooling, weighing, adding 1% methanol of phosphoric acid, shaking, and filtering to obtain sample solution for accuracy determination; the measurement was carried out according to the text sample content measurement method, and the calculation accuracy is shown in Table 7. The results show that: the method has good accuracy, the recovery rate of each component is between 95.0% and 105.0%, and the result is shown in Table 6.

TABLE 6 results of recovery measurement of six substances

Component to be measured

Girardinic acid D-L-ephedrine glycoside

Amygdalin

Ephedrine hydrochloride

Baicalin

Astragaloside IV

Liquiritin

Average recovery rate

97.69％

98.52％

97.78％

100.24％

97.86％

98.24％

The results show that: the method has good accuracy in measuring various related substances.

EXAMPLE 6 sample determination

(1) Preparation of a test solution: mixing the above materials, grinding, weighing 0.5g, precisely weighing, precisely adding 50ml methanol containing 1% phosphoric acid, ultrasonic treating (power 250W, frequency 20kHz) for 20 min, filtering, and collecting the filtrate.

(2) Preparation of control solutions: accurately weighing appropriate amount of ephedrine hydrochloride, baicalin, amygdalin, astragaloside IV, linarin and liquiritin reference substances, and preparing into mixed solution containing ephedrine hydrochloride, baicalin, amygdalin, astragaloside IV, linarin and liquiritin 20ug, 80ug, 30ug, 10ug, 20ug and 30ug, respectively, per 1ml with 1% methanol of phosphoric acid.

(3) Chromatographic conditions and system adaptability: the instrument comprises the following steps: a Waters e2695 high performance liquid chromatograph, a quaternary pump, an online vacuum degassing system, an automatic sample injector, a column incubator and an ultraviolet detector;

Gradient elution conditions: see table 1.

Column temperature: 30 ℃;

detection wavelength: 210 nm;

flow rate: 0.8 ml/min;

sample introduction amount: 10ul of

(4) And (3) determination: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring.

(5) Formula for calculation

C pair is concentration of control solution (mg/ml)

Peak area of A pair as control solution

Sample A is sample peak area

W sample is sample weighing (g)

(6) As a result: the contents of the respective components thus measured are shown in Table 7.

TABLE 75 contents of six ingredients in batches of samples

Batches of

Girardinic acid D-L-ephedrine glycoside

Amygdalin

Ephedra hydrochlorideAlkali

Baicalin

Astragaloside IV

Liquiritin

190801

0.14％

0.33％

0.21％

0.89％

0.004％

0.044％

190802

015％

0.38％

0.22％

0.88％

0.006％

0.041％

190803

0.16％

0.31％

0.24％

0.91％

0.004％

0.043％

190804

0.14％

0.34％

0.25％

0.94％

0.006％

0.045％

190805

0.13％

0.35％

0.20％

0.87％

0.005％

0.042％

Claims

1. A method for simultaneously detecting six component contents of infant lung-ventilating and cough-relieving granules is characterized in that the infant lung-ventilating and cough-relieving granules are prepared from twelve traditional Chinese medicinal materials including ephedra, bamboo leaf and divaricate saposhnikovia root, southwest baical skullcap root, platycodon root, mustard seed, bitter apricot seed, semen lepidii, Indian kalimeris herb, membranous milkvetch root, common yam rhizome, hawthorn fruit and liquoric root, and are characterized in that: the detection method comprises the steps of extracting the infant lung-ventilating and cough-relieving granules with methanol containing 1% phosphoric acid, and detecting an extracting solution by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph contains an ultraviolet detector, the detection wavelength of the ultraviolet detector is set to be 210nm, and the stationary phase of the high performance liquid chromatograph is C₁₈A chromatographic column, wherein a mobile phase A is acetonitrile containing 0.1 percent of sodium dodecyl benzene sulfonate, a mobile phase B is 0.1 percent of formic acid solution, gradient elution is adopted, the flow rate is 0.5-2ml/min, the column temperature is 25-35 ℃, the time of the gradient elution is 50min, the gradient elution is divided into 3 time periods, the volume change of the mobile phases A and B in each time period is shown in the following table,

。

2. The method for simultaneously detecting the contents of six components in the infantile lung-ventilating and cough-relieving granule as claimed in claim 1, wherein the method comprises the following steps: the flow rate was 0.8 ml/min.

3. The method for simultaneously detecting the contents of six components in the infantile lung-ventilating and cough-relieving granule as claimed in claim 1, wherein the method comprises the following steps: the column temperature was 30 ℃.