阅读说明：本技术 一种小儿咽扁颗粒中麦冬掺伪检验方法 (Method for detecting adulteration of radix ophiopogonis in children throat and tonsil particles ) 是由 胡亮 罗疆南 周明 文庆 王湘波 李瑞莲 于 2021-08-31 设计创作，主要内容包括：本发明采用超高效液相色谱和质谱的联用的方法检测小儿咽扁颗粒中是否含有麦冬掺伪成分山麦冬皂苷B和短葶山麦冬皂苷C。具体过程如下：分别制备一定浓度的供试品溶液和对照品溶液,并分别采用超高效液相色谱-质谱联用仪进行检测,并分别获得供试品溶液和对照品溶液的选择离子色谱图,然后进行掺伪分析；当供试品溶液和对照品溶液的选择离子色谱图中,出峰时间相同且峰面积比一致时视为掺伪。本发明所述的检测方法能排除小儿咽扁颗粒中其他7种药味的干扰；并同时兼具专属性强、灵敏度较好、精密度好、重复性好、稳定性强及回收率高的特点；且该方法能够检测出市面上掺伪10％以上的山麦冬。(The method provided by the invention adopts a method of combining ultra-high performance liquid chromatography and mass spectrometry to detect whether the children pharyngeal and prolapsus granule contains liriope spicata saponin B and liriope muscari saponin C which are pseudo-doped ingredients of radix ophiopogonis. The specific process is as follows: respectively preparing a test solution and a reference solution with certain concentrations, respectively detecting by using an ultra-high performance liquid chromatography-mass spectrometer, respectively obtaining selective ion chromatograms of the test solution and the reference solution, and then performing adulteration analysis; and when the peak emergence time is the same and the peak area ratio is consistent in the selective ion chromatograms of the test solution and the reference solution, the sample solution and the reference solution are considered to be adulterated. The detection method can eliminate the interference of other 7 medicinal flavors in the children throat and flat granules; meanwhile, the method has the characteristics of strong specificity, good sensitivity, good precision, good repeatability, strong stability and high recovery rate; and the method can detect the wild ophiopogon root which is adulterated by more than 10% in the market.)

1. A method for detecting adulteration of radix ophiopogonis in children pharyngeal and pharyngeal paradoxical granules is characterized by comprising the following steps:

preparation of S1 sample

Preparation of a test solution: dissolving XIAO ER YAN BIAN KE LI in methanol, performing ultrasonic treatment for 20-60min, and filtering to obtain test solution;

preparation of control solutions: respectively preparing a liriope spicata saponin B reference substance solution and a liriope spicata saponin C reference substance solution;

liriope spicata saponin B reference solution: precisely weighing radix liriopes saponin B reference substance, and dissolving in methanol solution to obtain radix liriopes saponin B reference substance solution;

liriope muscari baily saponins C reference solution: precisely weighing the liriope muscari baily saponins C reference substance, and dissolving the liriope muscari baily saponins C reference substance in a methanol solution to obtain a liriope muscari baily saponins C reference substance solution;

s2 adulteration analysis: respectively detecting the test solution and the reference solution obtained in the step S1 by using an ultra-high performance liquid chromatography-mass spectrometer, respectively obtaining selective ion chromatograms of the test solution and the reference solution, and then performing adulteration analysis;

the adulteration analysis specifically comprises the following steps: and when the peak emergence time of the ion pairs corresponding to the test solution and the reference solution is the same and the peak area ratio of the test solution to the reference solution is consistent in the selective ion chromatograms of the test solution and the reference solution, the test solution and the reference solution are considered to be adulteration.

2. The method for detecting the adulteration of radix Ophiopogonis in Xiao' er Yan Jian Flat granule according to claim 1, wherein the concentration of the test solution in S1 is 3-5X 10⁵Mu g/ml, the concentration of the reference substance solution is 1-3 mu g/ml, and the dosage of the test solution and the reference substance solution in S2 is 2-5 mu l during detection.

3. The method for detecting adulteration of radix ophiopogonis in children' S pharyngeal and flatus granules according to claim 1, wherein in S2, the detection parameters of the high performance liquid chromatography-mass spectrometer are set as follows:

the instrument comprises the following steps: thermo Scientific Dionex UltiMate 3000 ultra high performance liquid chromatograph, TSQ Endura triple quadrupole mass spectrometry system;

chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; taking ammonium acetate solution with concentration of 10mmol/L as mobile phase A and acetonitrile as mobile phase B, performing gradient elution according to the following table, wherein the flow rates of the mobile phase A and the mobile phase B are both 0.2-0.3 ml/min;

time (min) Mobile phase A Mobile phase B 0～1.5 55 45 1.5～8 55→5 45→95

Mass spectrum conditions: the ionization source conditions were set as follows: the ion source is in a negative ion mode ESI-, the spray voltage is 2500-:

screening ingredients Parent ion Daughter ions Liriope spicata saponin B 721.2 575.3、89.1 Liriope muscari baily saponins C 870.2 738.3、576.2

4. The method for detecting adulteration of radix ophiopogonis in children's pharyngeal and oblastic throat granules according to claim 3, wherein the spraying voltage is 3500V, and the collision energy of m/z 575.3 and 89.1 ions of liriope spicata saponin B is 23.0V and 32.6V respectively; the collision energy of the liriope muscari baily saponins C seed ions m/z 738.3 and 576.2 is 26.5V and 32.9V respectively.

5. The method for detecting the adulteration of radix ophiopogonis in the children' S pharyngeal and prolapsed granules according to claim 1, wherein in S2, the adulteration analysis is specifically as follows: when the peak emergence time of the ion pairs corresponding to the test solution and the reference solution is the same and the peak area ratio of the test solution to the reference solution is consistent in the selective ion chromatograms of the test solution and the reference solution, the test solution and the reference solution are considered to be adulterated, and the specific reference standard is as follows:

6. the method for detecting the adulteration of radix ophiopogonis in the infantile pharyngeal paranoid granule as claimed in claim 1, wherein the preparation of the test solution in the step S1 further comprises a pretreatment, wherein the pretreatment is a pre-grinding treatment of the infantile pharyngeal paranoid granule.

7. The method for detecting the adulteration of radix ophiopogonis in the infantile pharyngeal flattening granules according to any one of claims 1 to 6, wherein the infantile pharyngeal flattening granules are the infantile pharyngeal flattening granules recorded in the first P568 of 2020 edition of Chinese pharmacopoeia.

Technical Field

The invention relates to a method for detecting adulteration of radix ophiopogonis, in particular to a method for detecting adulteration of radix ophiopogonis in children throat and tonsil particles.

Background

The child throat and tongue granules are class A non-prescription medicines, the prescription consists of eight medicines of honeysuckle, blackberrylily rhizome, tinospora root, platycodon root, figwort root, dwarf lilyturf tuber, artificial bezoar (calculus bovis cultivated in vitro) and borneol, the prescription amount of the dwarf lilyturf tuber is 78.1g, and the prescription amount in the preparation is the third. The honeysuckle flower in the formula has the effects of clearing away heat and toxic materials, dispersing and dispersing the qi and the blood gently, and eliminating the blackberry lily and the phlegm and relieving sore throat, and is a monarch drug together; radix tinosporae is capable of clearing and relieving the throat and pain, platycodon grandiflorum is capable of ventilating lung qi, reducing phlegm and relieving sore throat, radix scrophulariae and radix ophiopogonis are capable of nourishing yin and moistening dryness, and eliminating stagnation and relieving sore throat, and are used as ministerial drugs together; artificial bezoar (calculus bovis cultivated in vitro) has effects of clearing heat and toxic materials, eliminating phlegm, inducing resuscitation, and Borneolum has effects of clearing heat, relieving pain, and inducing resuscitation; the medicines are combined to play the roles of clearing heat and relieving sore throat, and detoxifying and relieving pain.

At present, the pseudo-mixture of radix ophiopogonis in the market is mainly liriope spicata which is collected in the first part of 2020 edition of Chinese pharmacopoeia together with radix ophiopogonis, has the same functions and main indications, and has the functions of nourishing yin, promoting the secretion of saliva or body fluid, moistening lung and clearing heart. Wherein radix Ophiopogonis is dried root tuber of Ophiopogon japonica (L.f) Ker-Gawl. of Liliaceae; liriope spicata (Thunb.) Liriope spicata (dried.) of Liriope Hubei Liriope spicata (Liliaceae) dried root tuber of Lour. var. prolifera Y. T. Ma or Baily of Liriope muscari (Decne.) Baily. According to relevant documents, the main components of the radix ophiopogonis and the liriope spicata are steroid saponins, the radix ophiopogonis mainly contains ophiopogonin A, ophiopogonin D and the like, the Hubei radix ophiopogonis mainly contains liriope spicata saponin B, and the liriope muscari mainly contains liriope muscari saponin C. Therefore, the detection of radix ophiopogonis adulteration is also used for essentially detecting whether the sample contains liriope muscari saponin B and liriope muscari saponin C. The method has some defects in the existing detection method, (1) when the characteristic components of liriope spicata are detected by adopting a thin-layer chromatography, the method has the defects of poor sensitivity and specificity, false positive caused by interference of spots, and the method cannot accurately judge whether the liriope spicata adulterated condition exists in the children cigarette flat particles; (2) when the characteristic components of liriope spicata are detected by a high performance liquid chromatography evaporative light spectroscopy (HPLC-ELSD) method, the sensitivity is low, the specificity is poor, and the characteristic components of liriope spicata cannot be detected, so that a false negative result is caused.

In addition, the detection difficulty and the detection method of the ophiopogon root are different in different medical products due to different interference components. Therefore, the method for detecting the adulteration of the radix ophiopogonis in the throat and tonsil particles of the children is especially important for researching a method which has the advantages of strong specificity, good sensitivity, good precision, good repeatability, strong stability and high recovery rate and is suitable for detecting the adulteration of the radix ophiopogonis in the throat and tonsil particles of the children.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for detecting the adulteration of radix ophiopogonis in children pharyngeal and tongue granules, which can eliminate the interference of eight and 7 medicines of honeysuckle, blackberrykiky rhizome, radix tinosporae, platycodon grandiflorum, radix scrophulariae, artificial bezoar (bezoar cultivated in vitro) and borneol, and has the advantages of strong specificity, good sensitivity, good precision, good repeatability, strong stability and high recovery rate.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for detecting adulteration of radix ophiopogonis in children pharyngeal and pharyngeal paradoxical granules comprises the following steps:

preparation of S1 sample:

preparation of a test solution: dissolving XIAO ER YAN BIAN KE LI in methanol, performing ultrasonic treatment for 20-60min, and filtering to obtain test solution;

preparation of control solutions: respectively preparing a liriope spicata saponin B reference substance solution and a liriope spicata saponin C reference substance solution;

liriope spicata saponin B reference solution: precisely weighing radix liriopes saponin B reference substance, and dissolving in methanol solution to obtain radix liriopes saponin B reference substance solution;

liriope muscari baily saponins C reference solution: precisely weighing the liriope muscari baily saponins C reference substance, and dissolving the liriope muscari baily saponins C reference substance in a methanol solution to obtain a liriope muscari baily saponins C reference substance solution;

s2 adulteration analysis: respectively detecting the test solution and the reference solution obtained in the step S1 by using an ultra-high performance liquid chromatography-mass spectrometer to respectively obtain selective ion chromatograms of the test solution and the reference solution, and then carrying out adulteration analysis;

the adulteration analysis specifically comprises the following steps: and when the peak emergence time of the ion pairs corresponding to the test solution and the reference solution is the same and the peak area ratio of the test solution to the reference solution is consistent in the selective ion chromatograms of the test solution and the reference solution, the test solution and the reference solution are considered to be adulteration.

Further, the preparation of the test solution in S1 further comprises a pretreatment of pre-grinding the pediatric pharyngeal and flatus particles.

Further, the concentration of the sample solution in S1 is 3-5X 10⁵Mu g/ml, the concentration of the reference substance solution is 1-3 mu g/ml, and the dosage of the test solution and the reference substance solution in S2 is 2-5 mu l during detection.

Further, in S2, the detection parameters of the high performance liquid chromatography-mass spectrometer are set as follows:

the instrument comprises the following steps: thermo Scientific Dionex UltiMate 3000 ultra high performance liquid chromatograph, TSQ Endura triple quadrupole mass spectrometry system;

chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; taking ammonium acetate solution with concentration of 10mmol/L as mobile phase A and acetonitrile as mobile phase B, performing gradient elution according to the following table, wherein the flow rates of the mobile phase A and the mobile phase B are both 0.2-0.3 ml/min;

time (min)	Mobile phase A	Mobile phase B
			0～1.5	55	45
1.5～8	55→5	45→95

Mass spectrum conditions: the ionization source conditions were set as follows: the ion source is in a negative ion mode ESI-, the spray voltage is 2500-:

screening ingredients	Parent ion	Daughter ions
			Liriope spicata saponin B	721.2	575.3、89.1
Liriope muscari baily saponins C	870.2	738.3、576.2

Further, the spraying voltage is preferably 3500V, the collision energy of liriope muscari saponin B ion m/z 575.3 and 89.1 is 23.0V and 32.6V respectively, and the collision energy of liriope muscari saponin C ion m/z 738.3 and 576.2 is 26.5V and 32.9V respectively.

Further, in S2, the adulteration analysis specifically includes: and when the peak emergence time of the ion pairs corresponding to the test solution and the reference solution is the same and the peak area ratio of the test solution to the reference solution is consistent, the test solution and the reference solution are regarded as adulteration, and the specific reference standard is as follows:

further, the preparation of the test solution in S1 further comprises a pretreatment of pre-grinding the pediatric pharyngeal and flatus particles.

Furthermore, the infantile pharyngeal flattening granules detected by the detection method for adulteration of radix ophiopogonis in any one of the infantile pharyngeal flattening granules are the infantile pharyngeal flattening granules recorded in P568I 2020 edition of Chinese pharmacopoeia.

In the invention, the object for detecting liriope spicata saponin B and liriope muscari saponin C is infantile Yanbian granules, and in the prescription of the patent reagent, 7 main medicine interferences are existed, namely honeysuckle, blackberrykiky rhizome, tinospora root, platycodon root, figwort root, artificial bezoar (in vitro cultured bezoar) and borneol eight; the method adopts the combination of high performance liquid chromatography and mass spectrometry to compare whether the peak emergence time and the peak area ratio of characteristic components liriope muscari saponin B and liriope muscari saponin C are consistent at the same time, and if the peak emergence time and the peak area ratio are consistent at the same time, the characteristic components are regarded as containing corresponding characteristic components; and when any one of liriope muscari saponin B and liriope muscari saponin C in the sample is detected, the adulteration is determined. The method can detect the liriope spicata which is adulterated by more than 10% in the market through the determination of the judgment indexes and the limit.

Compared with the prior art, the invention has the beneficial effects that: (1) can eliminate the interference of eight 7 medicinal flavors of honeysuckle, blackberrykiky rhizome, radix tinosporae, platycodon grandiflorum, figwort root, artificial bezoar (in vitro cultured bezoar) and borneol in the throat and tonsil particles of the children; (2) the detection method has the characteristics of strong specificity, good sensitivity, good precision, good repeatability, strong stability and high recovery rate; (3) the radix liriopes adulterated by more than 10% in the market can be detected.

Drawings

FIG. 1 is a selective ion chromatogram of liriope spicata saponin B in the test solution X in example 1.

FIG. 2 is a selective ion chromatogram of liriope muscari baily saponins C in the test solution Y in example 1.

FIG. 3 is a selective ion chromatogram of a Liriope spicata saponin B control solution in example 1.

FIG. 4 is a selective ion chromatogram of a liriope muscari baily saponins C control solution in example 1.

FIG. 5 is a selective ion chromatogram of a ophiopogonin D control solution in example 1.

FIG. 6 is a selective ion chromatogram of liriope spicata saponin B in a mobile phase of 0.1% formic acid solution.

FIG. 7 is a selective ion chromatogram of liriope spicata saponin B in a mobile phase of 10mmol/L ammonium acetate solution.

FIG. 8 is the ion optimization chart of ophiopogonin D.

FIG. 9 is the ion intensity response diagram of ophiopogonin D [ m/z 721.3(a), 575.3(b), 707.33(c) ].

FIG. 10 is a graph of the collision energy optimization of ophiopogonin D daughter ions [ m/z 721.3(a), 575.3(b) ].

FIG. 11 is the ion optimization chart of ophiopogonin B.

FIG. 12 is the ion intensity response diagram of ophiopogonin B seed [ m/z 575.3(a), 103.05(B), 89.1(c) ].

FIG. 13 is a graph of the collision energy optimization of ophiopogonin B daughter ions [ m/z 575.3(a), 89.1(B) ].

FIG. 14 is the ion optimization chart of ophiopogonin C.

FIG. 15 is the ion intensity response diagram [ m/z 738.3(a), 576.2(b), 708.3(C) ] of ophiopogonin C.

FIG. 16 is a graph of the collision energy optimization of ophiopogonin C daughter ions [ m/z 738.3(a), 576.2(b) ].

FIG. 17 is a selective ion chromatogram of radix Ophiopogonis control sample No. 2, wherein (a) represents the detection of liriope spicata saponin B. Detecting, (b) shows the detection of liriope muscari baily saponins C, and (C) shows the detection of liriope muscari baily saponins D.

FIG. 18 is a selective ion chromatogram of a control drug of Liriope spicata (Hubei Liriope spicata) No. 3, wherein (a) represents the detection of liriope spicata saponin B, (B) represents the detection of liriope spicata saponin C, and (C) represents the detection of liriope spicata saponin D.

FIG. 19 is a selective ion chromatogram of sample number 6.

FIG. 20 is a selective ion chromatogram of sample No. 7.

FIG. 21 is a selective ion chromatogram of liriope spicata saponin B in a negative control solution in a specificity experiment.

FIG. 22 is a selective ion chromatogram of liriope muscari baily saponins C in negative control solution in a specificity experiment.

FIG. 23 is a line graph of liriope spicata saponin B.

FIG. 24 is a line graph of liriope muscari saponin C.

Detailed Description

Example 1

Preparation of S1 sample:

preparation of a test solution: grinding child throat and flat particles purchased from X and Y companies, respectively dissolving 4g of the ground particles in methanol, performing ultrasonic treatment for 30min, cooling, and filtering to obtain a test solution X and a test solution Y;

preparation of control solutions:

liriope spicata saponin B reference solution: precisely weighing radix liriopes saponin B reference substance, and dissolving in methanol solution to obtain radix liriopes saponin B reference substance solution;

liriope muscari baily saponins C reference solution: precisely weighing the liriope muscari baily saponins C reference substance, and dissolving the liriope muscari baily saponins C reference substance in a methanol solution to obtain a liriope muscari baily saponins C reference substance solution;

the ophiopogonin D reference solution is prepared by precisely weighing liriope muscari baily saponins D reference, and dissolving in methanol solution to obtain liriope muscari baily saponins D reference solution;

the solubility of the three reference substance solutions is 2 mug/ml; and three controls were provided from the hospital specimen facility.

S2 adulteration analysis: respectively taking 5 mu l of the test solution and the reference solution obtained in the step S1, detecting by adopting an ultra-high performance liquid chromatography-mass spectrometer, respectively obtaining selective ion chromatograms of the test solution and the reference solution, and then carrying out adulteration analysis;

FIGS. 1-5 are selective ion chromatograms of test solution X, test solution Y, liriope spicata saponin B reference solution, liriope spicata saponin C reference solution, and liriope spicata saponin D reference solution, respectively.

Table 1 shows the reference data for three samples of control solutions from example 1

Table 1 is the measurement reference regulation of the general rules 0512 and 0431 in the chinese pharmacopoeia 2015 edition.

Table 2 shows the reference data of the two companies X and Y in example 1

Comparing the table 1 with the table 2, it can be known that in the screening process, a chromatographic peak consistent with the peak appearance time of the liriope spicata saponin B reference substance is detected in the sample of the company X, and the ratio of the monitored ion to the peak area of the sample is consistent with that of the reference substance; detecting a chromatographic peak with the same peak appearance time as a control substance of liriope muscari saponin C in a sample of a company Y, wherein the ratio of the monitored ion to the peak area of the sample is consistent with that of the control substance; therefore, the children throat and tonsil particles produced by the company X and the company Y are unqualified products with more than 10 percent adulteration.

The method provided by the invention can effectively attack the behaviors of production without production according to a production process and suspected adulteration and feeding, and provides a solid guarantee for the medication safety of people.

The following also performed parameter optimization and methodological validation for this scheme:

1. optimization of liquid chromatography conditions

For different brands of ultra-high performance liquid chromatography columns (weltch ultate C)₁₈、Thermo C₁₈) The different sample volumes of 1, 3 and 5 mul, the flow rates of 0.2 and 0.3ml/min, the column temperature of 30 ℃ and 35 ℃, the gradient elution of a mobile phase of 10mmol/L ammonium acetate solution and a 0.1 percent formic acid solution are investigated, and the results show that chromatographic columns, flow rates and column temperatures of different brands have little influence on the measurement result, but the liriope spicata saponin B reference substance is extracted and separated when the mobile phase is 0.1 percent formic acid and the sample volume of 5 mul are adoptedThe sub-chromatograms had tailing (see FIG. 6); the problem of poor peak shape of liriope spicata saponin B chromatogram can be solved well by adopting 10mmol/L ammonium acetate solution as a mobile phase (see figure 7), and the gradient elution conditions are shown in Table 3.

TABLE 3 gradient elution conditions Table

Time (min)	Mobile phase A	Mobile phase B
			0～1.5	55	45
1.5～8	55→5	45→95

2. Optimization of mass spectrometry conditions

The method adopts a uniform ion source, and adopts an anion mode ESI^-The influence of switching of different ion sources on sample detection is overcome, the spray voltage optimization range is 2500-.

In the process of optimizing the ophiopogonin component ion pair, the ophiopogonin D parent ion is m/z 853.3 to obtain 3 daughter ions which are respectively m/z 721.3, 575.3 and 707.3 (see figure 8), the response strengths of the ophiopogonin D parent ion are respectively 497801, 57551 and 11195 (see figure 9), 2 ions (m/z 721.3 and 575.3) with high ion corresponding strength are selected as the daughter ions, the collision energy (15-55V) of the daughter ions is optimized, and the result shows that the response strength is high when the collision energy is respectively 27.0V and 32.9V (see figure 10).

The liriope spicata saponin B parent ion is m/z721.2, 3 daughter ions are obtained and are respectively m/z 575.3, 103.05 and 89.1 (see figure 11), 2 ions (m/z 575.3 and 89.1) are selected as the daughter ions according to the response intensity (see figure 12), the collision energy (15-55V) of the daughter ions is optimized, and the result shows that the response intensity is high when the collision energy is 23.0V and 32.6V respectively (see figure 13).

The parent ion of liriope muscari saponin C is m/z 870.2, 3 daughter ions are obtained, wherein the parent ion is m/z 738.3, 576.2 and 708.3 (see fig. 14), 2 ions (m/z 738.3 and 576.2) are selected as daughter ions according to response intensity (see fig. 15), and the collision energy (15-55V) is optimized, and the result shows that the corresponding intensity is high when the collision energy is 26.5V and 32.9V respectively (see fig. 16).

3. Determination of judgment index and limit

In order to judge the indication and the limit of the scheme, eight samples with different numbers and batches are prepared, and the specific sample preparation process is as follows:

all sample No. 1, prepared according to the method for preparing the test article solution in example 1;

preparation of reference medicinal material (No. 2-4): precisely weighing 1g each of radix Ophiopogonis provided by enterprises and collected radix Ophiopogonis Japonici, adding 15ml methanol, ultrasonic treating for 30min (300W, 50kHz), cooling, filtering, and collecting filtrate.

Preparation of positive test solution (nos. 5 to 8):

in the regulation of the four parts of the 2015 edition of Chinese pharmacopoeia, the allowable limit of impurities in medicinal materials is 3%, and in order to avoid misjudgment, the concentration of a test solution is investigated according to a sample containing 3% of liriope spicata.

Respectively mixing radix liriopes into the formula of children throat-flattening granules according to the proportion of 3%, 6%, 10% and 100% to prepare a simulation sample containing 3%, 6%, 10% and 100% of radix liriopes.

Taking 4g of each simulated sample, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of methanol, weighing, ultrasonically treating for 30 minutes (300W, 50kHz), cooling, weighing again, adding methanol to supplement the lost weight, shaking uniformly, filtering, and taking the subsequent filtrate as a test solution.

The measurement results are shown in Table 4 and FIGS. 17 to 20.

The result shows that the ophiopogonin D is the common component of ophiopogon root and liriope spicata, and the liriope spicata saponin B is the specific component of liriope spicata. According to the Chinese pharmacopoeia and related documents, liriope muscari contains liriope muscari saponin C, so liriope muscari saponin B and liriope muscari saponin C are temporarily used as the investigation indexes in the research.

Experiments show that the liriope spicata saponin B can be detected when the doping amount of liriope spicata (Hubei liriope spicata) is more than 10%, interference of 3% of impurities allowed in medicinal materials can be eliminated, meanwhile, the liriope spicata in the liriope spicata has less circulation in the market through research, medicinal materials are not collected, and the limit of false doping inspection of liriope spicata is determined as undetected for strictly controlling the quality of the pediatric flat throat particle prescription preparation.

TABLE 4-1 reference materials and the results of each material

TABLE 4-2 sample Peak out time and Peak area ratio results Table

4. Determination of methodology

In the methodological assay, control solutions of different solubilities were used, and the preparation process is summarized in example 1, and specifically as follows:

liriope muscari baily saponins C reference solution: precisely weighing 10.96mg of liriope muscari baily saponins C reference substance, placing the liriope muscari baily saponins C reference substance into a 100ml volumetric flask, dissolving the liriope muscari baily saponins C reference substance solution with methanol and fixing the volume to obtain a liriope muscari baily saponins C reference substance solution (109.6 mu g/ml), precisely sucking 1ml of the reference substance solution, respectively placing the solution into 10ml volumetric flasks and 50ml volumetric flasks, and diluting the solution to the scale with methanol to obtain the liriope muscari baily saponins C reference substance solution (10.96 mu g/ml, 2.192 mu g/ml).

Liriope muscari baily saponins B reference solution is prepared by precisely weighing 12.94mg of Liriope muscari baily saponins B reference, placing into a 100ml volumetric flask, dissolving with methanol and fixing volume to obtain Liriope muscari baily saponins C reference solution (129.4 μ g/ml), precisely sucking 1ml of the reference solution, placing into 10ml measuring flask and 50ml measuring flask respectively, and diluting with methanol to scale to obtain Liriope muscari baily saponins C reference solution (12.94 μ g/ml, ③ 2.588 μ g/ml).

Ophiopogonin D reference solution: accurately weighing 10.41mg of ophiopogonin D reference substance, placing in a 5ml measuring flask, dissolving with methanol and fixing volume, accurately sucking 1ml of the reference substance solution into a 10ml measuring flask, diluting with methanol to scale to obtain ophiopogonin D reference substance solution (i.e., (2.082 mg/ml, ② 20.82 μ g/ml).

(I) specificity experiments

Weighing other medicines of the dwarf lilyturf tuber lack according to the prescription to prepare a negative control sample of the dwarf lilyturf tuber lack, and preparing a negative control solution of the dwarf lilyturf tuber lack by the same method. Precisely sucking 5 mu l of a liriope muscari saponin C reference substance solution, a liriope muscari saponin B reference substance solution and a negative reference solution, and determining the result shown in the figures 21-22. No corresponding ion current exists at the positions where the chromatographic peak times of the ophiopogon root lacking negative control solution, the liriope muscari saponin C control and the liriope muscari saponin B control are the same, which indicates that other medicines in the prescription have no interference to the measurement result and have strong specificity.

(II) sensitivity

Detection limit of liriope spicata saponin B

M/z721.2 → 89.11 is used as a detection ion pair for detection, 1 mu l of liriope spicata saponin B reference substance solution is precisely absorbed and injected into a UPLC-MS-MS instrument, the signal-to-noise ratio of a characteristic peak is 23, the detection limit is calculated to be 0.28ng, and the instrument sensitivity is good.

Detection limit of liriope muscari baily saponins C

M/z 870.2 → 576.2 is used as a detection ion pair for detection, 1 mu l of liriope muscari baily saponins C reference substance solution is precisely absorbed and injected into a UPLC-MS-MS instrument, the signal-to-noise ratio of a characteristic peak is 12, the detection limit is calculated to be 0.65ng, and the instrument sensitivity is good.

(III) precision test

Precision of Liriope spicata saponin B

Precisely absorbing 5 mu l of liriopesides B reference solution, injecting into UPLC-MS-MS instrument, continuously injecting 6 needles, and measuring to obtain the result shown in Table 5. In the ion flow chromatogram of the test sample extracted with m/z721.2 → 575.3, the peak area value RSD is 4.2%, and the precision is good.

Precision of liriope muscari baily saponins C

Precisely sucking 5 mu l of liriope muscari baily saponins C reference solution, injecting into UPLC-MS-MS instrument, continuously injecting sample of 6 needles, and measuring, the result is shown in Table 5. In the ion flow chromatogram of the test sample extracted with m/z 870.2 → 738.3, the peak area value RSD is 3.1%, and the precision is good.

TABLE 5 results of precision test

(IV) repeatability test

Liriope spicata saponin B

The same batch of positive test sample (the batch number of Hebei Chengde Yanfeng pharmaceutical company Limited is 19852001) was used to prepare 6 test sample solutions according to the method, and the results are shown in Table 6. The RSD of liriope spicata saponin B in the test sample is 5.80%.

TABLE 6 repeatability test results (m/z721.2 → 575.3)

Liriope muscari baily saponins C

A positive test sample (manufactured by Shanxi Meidi pharmaceutical Co., Ltd.: 521802) was taken, 6 test sample solutions were prepared by the method, and the results of the measurements are shown in Table 7. The RSD of the liriope muscari saponin C in the test sample is 3.81 percent.

TABLE 7 repeatability test results (m/z 870.2 → 738.3)

(V) stability test

Liriope spicata saponin B

Taking a test solution I under repeatability, precisely sucking 5 mu l of the test solution respectively 0 hour, 1 hour, 2 hours, 4 hours, 8 hours and 16 hours after preparation, injecting the test solution into UPLC-MS-MS, and measuring the result, wherein the result is shown in Table 8. The test results show that the test solution is substantially stable within 16 hours after preparation.

TABLE 8 test results of solution stability of test article

Liriope muscari baily saponins C

Taking a test solution I under repeatability, precisely sucking 5 mu l of the test solution respectively 0 hour, 1 hour, 2 hours, 4 hours, 8 hours and 16 hours after preparation, injecting the test solution into UPLC-MS-MS, and measuring the result, wherein the result is shown in a table 9. The test results show that the test solution is substantially stable within 16 hours after preparation.

TABLE 9 test results of solution stability of test article

(VI) Linear relationship test

Liriope spicata saponin B

Precisely sucking 2 mul, 5 mul and 10 mul of liriope spicata saponin B reference substance solution and 1 mul, 3 mul and 5 mul of liriope spicata saponin B reference substance solution, injecting into a liquid chromatograph according to the chromatographic conditions, and measuring the peak area. And (3) taking the peak area value as a vertical coordinate and the sample amount as a horizontal coordinate, drawing a standard curve, and calculating a regression equation: y is 1.380x +1006, r is 0.9975, the sample amount of liriope spicata saponin B is in linear relation with the chromatographic peak area within the range of 2.192 ng-109.6 ng. The results are shown in Table 10 and FIG. 23.

Liriope muscari baily saponins C

Precisely sucking 2 mul, 5 mul and 10 mul of liriope muscari baily saponins C reference substance solution and 1 mul, 3 mul and 5 mul of liriope muscari baily saponins C reference substance solution, injecting into a liquid chromatograph according to the chromatographic conditions, and measuring the peak area. And (3) taking the peak area value as a vertical coordinate and the sample amount as a horizontal coordinate, drawing a standard curve, and calculating a regression equation: y is 1.169x +1006, r is 0.9975, and the sample injection amount of liriope muscari baily saponins C is in a linear relation with the chromatographic peak area within the range of 2.588 ng-129.400 ng. The results are shown in Table 10 and FIG. 24.

TABLE 10 table of results of linear relationship test

(VII) recovery test

Taking 6 parts of powder of the same negative sample, each part is about 2.0g, precisely weighing, precisely adding liriope muscari saponin B reference substance and liriope muscari C reference substance, preparing a test solution according to a method, measuring, calculating, and finding the result shown in Table 11.

TABLE 11 table of the results of the recovery test