CN113777205A

CN113777205A - Method for detecting adulteration of radix ophiopogonis in pulse-activating decoction

Info

Publication number: CN113777205A
Application number: CN202111014319.6A
Authority: CN
Inventors: 胡亮; 周明; 王湘波; 文庆; 罗疆南; 孙辉
Original assignee: Hunan Drug Inspection Research Institute (hunan Pharmaceutical Adjuvant Inspection And Detection Center)
Current assignee: Hunan Drug Inspection Research Institute (hunan Pharmaceutical Adjuvant Inspection And Detection Center)
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-12-10

Abstract

The method adopts a method of combining ultra-high performance liquid chromatography and mass spectrometry to detect whether the pulse-activating decoction contains pseudo-components of liriope muscari baily saponin B and liriope muscari baily saponin C in the liriope. 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 selected ion chromatograms of the test solution and the reference solution simultaneously meet the conditions that the peak emergence time is the same, the peak area ratio is consistent, and the peak area of the test solution is larger than that of the reference solution, the test solution is considered to be adulterated. The detection method can eliminate the interference of other 2 medicinal flavors in the pulse-activating decoction; 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 liriope spicata which is adulterated by more than 3% in the market.

Description

Method for detecting adulteration of radix ophiopogonis in pulse-activating decoction

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 pulse-activating decoction.

Background

The pulse-activating decoction is prepared from 3 medicinal materials of red ginseng, dwarf lilyturf tuber and Chinese magnoliavine fruit, and has the effects of tonifying qi, restoring pulse, nourishing yin and promoting the production of body fluid. The current quality standard is 185 production enterprises in China in the first part of the 'Chinese pharmacopoeia' 2020 edition, and 188 approved characters are provided.

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 research of the method has the advantages of strong specificity, good sensitivity, good precision, good repeatability, strong stability and high recovery rate, and is particularly important for the detection method of the adulteration of the radix ophiopogonis in the pulse-activating decoction.

Disclosure of Invention

Aiming at the defects in the prior art, the invention solves the technical problem of providing the method for detecting the adulteration of the radix ophiopogonis in the pulse-activating decoction, which can eliminate the interference of the two medicines of the red ginseng and the schisandra chinensis 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 pulse-activating decoction comprises the following steps:

preparation of S1 sample

Preparation of a test solution: dissolving the pulse-activating decoction in methanol, shaking, and filtering to obtain a 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;

specifically, in the selective ion chromatograms of the test solution and the reference solution, the adulteration analysis is considered to be adulteration when the following three conditions are simultaneously met:

the peak output time of the ion pairs corresponding to the test solution and the reference solution is the same;

(II) the peak area ratio of the test solution is consistent with the peak area ratio of the reference solution;

and (III) the peak area of the test solution is larger than the peak area ratio of the control solution.

Further, the specific preparation method of the test solution in S1 is as follows: precisely measuring 5ml of pulse-activating decoction, dissolving in 10-20ml of methanol, shaking, and filtering to obtain a test solution; the concentration of the reference solution in S1 is 0.5-2.1 mug/ml; the dosage of the test solution and the reference solution in the S2 is 2-5 mul during detection.

Further, in S2, the detection parameters of the hplc-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 3500V, the collision energy of the liriope muscari saponin B ion m/z 575.3 and 89.1 is 23.0V and 32.6V respectively, and the collision energy of the 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: meanwhile, the ion pair corresponding to the test solution and the reference solution has the same peak-out time, the peak area ratio of the test solution is consistent with that of the reference solution, and the adulteration is considered when the peak area of the test solution is larger than that of the reference solution, and the specific reference standard is as follows:

further, the preparation of the test solution in S1 further includes a pretreatment, wherein the pretreatment is to randomly mix 5-10 pulse-activating beverages uniformly, and prepare the test solution according to the preparation process of S1 after mixing uniformly.

Furthermore, the pulse-activating decoction is the pulse-activating decoction recorded in part P835 of the 2020 edition of Chinese pharmacopoeia.

In the invention, the object for detecting the liriope spicata saponin B and the liriope spicata saponin C is the pulse-activating decoction, and in the prescription of the prescription reagent, 2 main medicinal taste interferences are shown, namely red ginseng and schisandra chinensis; the method adopts the combination of high performance liquid chromatography and mass spectrometry, and when the peak emergence time of the ion pairs corresponding to the test solution and the reference solution is the same, the peak area ratio of the test solution is equal to that of the reference solution, and the peak area of the test solution is equal to that of the reference solution, the test solution is regarded as containing corresponding characteristic components in the selective ion chromatograms of the test solution and the reference solution. 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 3% 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) interference of 2 kinds of medicinal flavors of red ginseng and schisandra chinensis in the pulse-activating decoction can be eliminated; (2) the detection method has the characteristics of strong specificity, good sensitivity, good precision, good repeatability, strong stability and high recovery rate; (3) can detect the liriope spicata which is adulterated by more than 3 percent in the market.

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 liriope muscari baily saponins C in the test solution Z in example 1. .

FIG. 4 is a selective ion chromatogram of a Liriope spicata saponin B control solution in example 1.

FIG. 5 is a selective ion chromatogram of a liriope muscari baily saponins C 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 a negative control solution, wherein panel (a) shows liriope spicata saponin B; FIG. (b) shows liriope muscari saponin C.

FIG. 18 is a selective ion chromatogram of a Liriope muscari baily saponins C reference solution in a specific experiment.

FIG. 19 is a selective ion chromatogram of a Liriope spicata saponin B reference solution in a specific experiment.

FIG. 20 is a line graph of liriope spicata saponin B.

FIG. 21 is a line graph of liriope muscari saponin C.

FIG. 22 is a selective ion chromatogram of a ophiopogonin D control solution.

FIG. 23 is a selective ion chromatogram of a radix Ophiopogonis control, wherein (a) shows liriope spicata saponin B; panel (b) shows liriope muscari baily saponins C, and panel (C) shows liriope muscari baily saponins D.

FIG. 24 is a selective ion chromatogram of a control drug of radix liriopes (Hubei radix Ophiopogonis), in which (a) shows liriope spicata saponin B; panel (b) shows liriope muscari baily saponins C, and panel (C) shows liriope muscari baily saponins D.

FIG. 25 is a selective ion chromatogram of Liriope muscari drug, wherein (a) shows liriope muscari saponin B; panel (b) shows liriope muscari baily saponins C, and panel (C) shows liriope muscari baily saponins D.

FIG. 26 is a selective ion chromatogram of different contents of liriope spicata saponin B adulteration, in which (a) represents liriope spicata saponin B reference substance, (B) is a positive sample adulterated by 3%, and (c) is a positive sample adulterated by 5%, and (d) is a positive sample adulterated by 10%.

FIG. 27 is a selective ion chromatogram of different contents of liriope muscari baily saponins C adulteration, wherein (a) represents a liriope muscari baily saponins C reference substance, (b) is a adulteration 3% positive sample, (C) is a adulteration 5% positive sample, and (d) is a adulteration 10% positive sample.

Detailed Description

Example 1

Preparation of S1 sample

Preparation of a test solution: uniformly mixing 5 pulse-activating beverages of the same type and the same batch purchased by an X company, precisely measuring 5ml of pulse-activating beverage, dissolving the pulse-activating beverage in 10ml of methanol, shaking uniformly, and filtering to obtain a test solution X;

the pulse-activating drink sold by company Y was prepared into a test solution Y in the same manner. A sample solution Z corresponding to Z company was prepared in the same manner.

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

in the three reference substance solutions, the concentration of the liriope muscari saponin B reference substance solution is 0.506 mug/ml, and the concentration of the liriope muscari saponin C reference substance solution is 2.056 mug/ml; the content of ophiopogonin D in the control solution is 2 μ g/ml.

FIGS. 1-5 are diagrams of a test solution X, a test solution Y, a test solution Z, a liriope spicata saponin B reference solution, and a liriope spicata saponin C 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 X, Y and Z companies in example 1

Comparing table 1, table 2 and fig. 1-5, it can be seen that in the screening process, a chromatographic peak (fig. 4) with the peak-out time of the liriope spicata saponin B reference substance (concentration 0.506ug/ml) is detected in the sample of company X, the ratio of the monitored ion-to-peak area of the sample is consistent with that of the reference substance, meanwhile, the area of the chromatographic peak of the ion current extracted from the sample is larger than that of the chromatographic peak of the ion current extracted from the reference substance, and the feeding production condition of the liriope spicata (hubei lilyturf) exists (see fig. 1); y, Z company samples detected a chromatographic peak (figure 5) consistent with the peak emergence time of a liriope muscari saponin C reference substance (concentration 2.056ug/ml), and the sample monitored ion-to-peak area ratio was consistent with the monitored ion-to-peak area ratio of the reference substance, while the sample extracted ion current chromatographic peak area was greater than the reference extracted ion current chromatographic peak area, and the liriope muscari doped feeding production condition existed (see figures 2-3). 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.

In order to investigate the adulteration of radix ophiopogonis in the pulse-activating decoction, 169 collected samples are measured, and the result shows that 6 batches of samples detect liriope muscari saponin B and 2 batches of samples detect liriope muscari saponin C; the detection range of liriope spicata saponin B is 3.72-7.49g/ml, and the detection range of liriope spicata saponin C is 9.012-12.78 g/ml.

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 mobile phase of 10mmol/L ammonium acetate solution and the 0.1 percent formic acid solution are subjected to gradient elution for examination, and the result shows that chromatographic columns, flow rates and column temperatures of different brands have small influence on the measurement result, but the tailing phenomenon exists in the extraction ion chromatogram of the liriope spicata saponin B reference substance when 0.1 percent formic acid is used as the mobile phase and the sample volume is 5 mul (see figure 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/z870.2, 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).

In all methodology determinations and determinations of judgment indicators and limits, the samples and methods of preparation employed are as follows:

preparation of control solutions

Precisely weighing 12.04mg of liriope muscari baily saponins C reference substance, placing the liriope muscari baily saponins C reference substance into a 25ml 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 (481.6 mu g/ml), precisely sucking 1ml of the reference substance solution into a 100ml volumetric flask, diluting the solution with methanol to a scale to obtain a liriope muscari baily saponins C reference substance solution (4.816 mu g/ml), precisely sucking 3ml of the reference substance solution into a 10ml volumetric flask, and diluting the solution with methanol to a scale to obtain the liriope muscari baily saponins C reference substance solution (1.4448 mu g/ml)

Precisely weighing 11.14mg of liriope spicata saponin B reference substance, placing the liriope spicata saponin B reference substance in a 25ml volumetric flask, dissolving the liriope spicata saponin B reference substance with methanol and fixing the volume to obtain a reference substance solution (445.6 mu g/ml), precisely sucking 1ml of the reference substance solution, respectively placing the reference substance solution in a 100ml volumetric flask, diluting the reference substance solution to a scale with methanol to obtain a reference substance solution (4.456 mu g/ml), precisely sucking 3ml of the reference substance solution, placing the reference substance solution in a 10ml volumetric flask, and diluting the reference substance solution to a scale with methanol to obtain a reference substance solution (1.3368 mu g/ml).

Precisely weighing 10.28mg of ophiopogonin D reference substance, placing in a 100ml measuring flask, dissolving with methanol and fixing volume to obtain reference substance solution (102.8 μ g/ml), precisely sucking 1ml of the reference substance solution into the 100ml measuring flask,

diluting with methanol to scale to obtain ophiopogonin D reference solution (1.028 μ g/ml).

Preparation of reference medicinal material and medicinal material solution

Precisely weighing 1g of radix Ophiopogonis and radix Ophiopogonis Japonici, adding 25ml of methanol, ultrasonic treating for 30 min (300W, 50kHz), cooling, and collecting filtrate.

Precisely weighing 1g each of radix Ophiopogonis provided by enterprises and collected radix Ophiopogonis Japonici, adding 25ml methanol, ultrasonic treating for 30 min (300W, 50kHz), cooling, and collecting filtrate.

Preparation of test solution

Firstly, taking 5 samples of the pulse-activating decoction, mixing uniformly, precisely measuring 5ml of the pulse-activating decoction, putting the sample into a 10ml volumetric flask, adding methanol to dilute the sample to a scale, shaking uniformly, filtering, and taking a subsequent filtrate as a test solution.

② taking 5 samples of the pulse-activating decoction, mixing uniformly, precisely measuring 5ml, shaking and extracting with water saturated n-butanol for 3 times, each time 20ml, combining n-butanol solution, evaporating to dryness, adding methanol into residue to dissolve, fixing volume to 5ml, filtering to obtain test solution.

③ taking 5 samples of the pulse-activating decoction, mixing uniformly, precisely measuring 5ml, shaking and extracting with ethyl acetate for 3 times, 20ml each time, combining ethyl acetate solutions, evaporating to dryness, dissolving residues with methanol, fixing the volume to 5ml, and filtering to obtain a test solution.

The three test samples extracted by different extraction modes are respectively measured, and the method specifically comprises the following steps: precisely sucking 3 mul of each of the reference solution and the test solution, injecting into a liquid chromatogram-tandem mass spectrometer, measuring and calculating, and the result is shown in table 4.

TABLE 4 comparison of the results of different extraction methods for liriope muscari baily saponins C

Research shows that the extraction effect of the sample by direct dilution is better, and the condition of false negative result can be avoided.

Preparation of positive control solution:

according to the regulation in the four parts of the 2015 edition of Chinese pharmacopoeia, the allowable limit of impurities in medicinal materials is 3%, in order to avoid misjudgment, the liriope spicata is added into the decoction according to the prescription and the preparation method of the pulse-activating decoction according to the proportion of 3%, 5% and 10%, and positive control samples containing 3%, 5% and 10% of liriope spicata are prepared to investigate the concentration of a test solution.

Precisely measuring 5ml of each positive control sample, placing the positive control samples in a 10ml volumetric flask, adding methanol to the scale, shaking up, filtering, and taking the subsequent filtrate as a positive control solution.

3. Determination of methodology

(1) 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 3 μ l of each of the negative control solution and the control solution, and measuring. The radix ophiopogonis lacking negative control solution (figure 17) has no corresponding ion current at the position with the same chromatographic peak time as the control of liriope muscari saponin C (figure 18) and liriope muscari saponin B (figure 19), which indicates that other medicines in the prescription have no interference to the measurement result and strong specificity.

(2) Sensitivity of the probe

Detection limit of liriope spicata saponin B

And (3) detecting by taking m/z721.2 → 89.11 as a detection ion pair, gradually diluting the liriope spicata saponin B reference substance solution, injecting the diluted solution into an UPLC-MS-MS instrument, calculating the detection limit when the signal-to-noise ratio is 3: 1, and obtaining a result of 0.12ng, wherein the instrument sensitivity is good.

Detection limit of liriope muscari baily saponins C

And (3) detecting by taking m/z870.2 → 576.2 as a detection ion pair, gradually diluting the liriope spicata saponin B reference substance solution, injecting the diluted solution into an UPLC-MS-MS instrument, calculating the detection limit when the signal-to-noise ratio is 3: 1, and obtaining a result of 0.09ng, wherein the instrument sensitivity is good.

(3) Linear relation test

Liriope spicata saponin B

Precisely sucking radix liriopes saponin B reference substance solution (3 μ l, 10 μ l) and radix liriopes saponin B reference substance solution (1 μ l, 3 μ l, 5 μ l) according to the above chromatographic conditions, injecting into a liquid chromatograph, and measuring 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 14754x-8046.4, r is 0.9999, and the sampling amount of liriope spicata saponin B and the chromatographic peak area are in linear relation within the range of 1.3368 ng-44.56 ng. The results are shown in Table 5 and FIG. 20.

Liriope muscari baily saponins C

Precisely sucking 3 mul and 10 mul of liriope muscari baily saponins C reference solution and 1 mul, 3 mul and 5 mul of liriope muscari baily saponins C reference 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: 50279x +38641 for y, 0.9989 for r, and the linear relation between the sample injection amount of liriope muscari baily saponins C and the chromatographic peak area is in the range of 1.4448 ng-48.16 ng. The results are shown in Table 5 and FIG. 21.

TABLE 5 table of results of linear relationship test

(4) Precision test

Liriope spicata saponin B

Precisely absorbing 3 mu l of liriope spicata saponin B reference substance solution, injecting into a UPLC-MS-MS instrument, continuously injecting 6 needles, and measuring, wherein the results are shown in Table 4. In the ion flow chromatogram of the test sample extracted with m/z721.2 → 575.3, the peak area value RSD is 3.27%, and the precision is good.

Liriope muscari baily saponins C

Precisely sucking 3 mu l of liriope muscari baily saponins C reference solution, injecting into a UPLC-MS-MS instrument, continuously injecting a sample of 6 needles, and measuring, wherein the result is shown in Table 6. In the ion flow chromatogram of the test sample extracted by m/z870.2 → 738.3, the peak area value RSD is 3.66%, and the precision is good.

TABLE 6 results of precision test

(5) Repeatability test

Liriope spicata saponin B

The same positive test sample (20190301 for y33 lot) was used to prepare 6 test sample solutions according to the method, and the results are shown in Table 7. The RSD of the liriope spicata saponin B in the test sample is 3.65%, and the repeatability is good.

TABLE 7 repeatability test results (m/z721.2 → 575.3)

Liriope muscari baily saponins C

A positive sample (181001 as y97 lot) was taken, and 6 test solutions were prepared according to the method, and the results were shown in Table 8. The RSD of the liriope muscari saponin C in the test sample is 3.81 percent, and the repeatability is better.

TABLE 8 results of the repeatability tests (m/z 870.2 → 738.3)

(5) Stability test

Liriope spicata saponin B

The test solution was taken out repeatedly and precisely aspirated at 0, 2, 4, 8, 12, and 18 hours after preparation, 3. mu.l of the solution was injected into UPLC-MS-MS, and the results were measured as shown in Table 9. The test results show that the test solution is substantially stable within 18 hours after preparation.

TABLE 9 test results of solution stability of test article

Liriope muscari baily saponins C

The test solution was taken repeatedly and precisely pipetted at 0, 2, 4, 8, 12, and 18 hours after preparation, 3. mu.l was injected into UPLC-MS-MS, and the results are shown in Table 10. The test results show that the test solution is substantially stable within 18 hours after preparation.

TABLE 10 test results of solution stability of test article

Time (h)	Sample peak area m/z870.2 → 738.3
		0	792447
2	769667
		4	792818
8	819091
		12	794388
18	799573
		RSD(％)	1.99

(6) Recovery test

Precisely measuring 2.5ml of negative test solution, adding 5 micrograms of each of the liriope spicata saponin B reference substance and the liriope muscari C reference substance, preparing 6 parts of the test solution in parallel according to a proposed method, and measuring and calculating to obtain a result that the average recovery rate of the liriope spicata saponin B is 105.5 percent and the average recovery rate of the liriope muscari saponin C is 95.0 percent (see table 11), wherein the method has better recovery rate.

TABLE 11 table of the results of the recovery test

4. Determination of judgment index and limit

(1) Sample determination and limit determination

Determination of radix Ophiopogonis and radix Liriopis Japonica

Precisely sucking 3 μ l of each of the reference solution and the radix Ophiopogonis solution and radix Ophiopogonis solution, injecting into a liquid chromatogram-tandem mass spectrometer, and recording chromatogram.

Precisely sucking 3 mul of each of a radix ophiopogonis reference medicinal material solution, a liriope spicata reference medicinal material solution, a radix ophiopogonis medicinal material and a liriope spicata medicinal material solution, a ophiopogonin D reference solution (shown in figure 22), a liriope muscari saponin C reference solution (shown in figure 18) and a liriope spicata saponin B reference solution (shown in figure 19), and measuring the results, wherein the results are shown in figures 18, 19 and 22-25.

The results show that ophiopogonin D is a common component of radix ophiopogonis and liriope spicata, liriope spicata saponin B is a specific component of liriope spicata, and liriope muscari saponin C is a specific component of liriope spicata, so liriope spicata saponin B and liriope spicata saponin C are used as investigation indexes in the study (see Table 12).

TABLE 12 sample Peak out time and Peak area ratio results Table

(2) Determination of judgment index and limit

Research shows that when the doping amount of the Hubei radix ophiopogonis and the Liriope muscari is 3%, the liriope muscari saponin B and the Liriope muscari saponin C can be detected (see the figures 23-24), but the collected Hubei radix ophiopogonis and the Liriope muscari in batches are less, so that the content of the liriope muscari saponin B and the Liriope muscari saponin C in the medicinal material cannot be integrally reflected. On the basis of the reference of related documents, the content of liriope spicata saponin B in the pulse-activating decoction is 0.82 mu g/ml and the content of liriope muscari saponin C in the pulse-activating decoction is 4.27 mu g/ml, which are temporarily converted according to the prescription dosage by taking the average value of 10 percent of liriope spicata as the reference (see table 13). Calculating the concentration of the reference substance in the quality standard draft according to the preparation method of the proposed test sample, namely the concentration of liriope spicata saponin B is 0.41 mug/ml (0.82 × 5/10), the concentration of liriope muscari saponin C is 2.13 mug/ml (4.27 × 5/10), so that the finally determined reference concentration is liriope spicata saponin B: 0.5 μ g/ml, liriope muscari saponin C: 2 mu g/ml, if the characteristic component of the liriope spicata is detected in the test sample and the peak area is larger than the reference, the doping is considered to be larger than 10 percent, so that a result judgment principle is established.

The result judgment principle is that (1) in the extraction ion flow chromatogram of the test sample, no chromatographic peak corresponding to the chromatogram of the liriope spicata saponin B or liriope muscari saponin C reference solution appears, and the liriope spicata is considered as not detected to be adulterated; (2) in the extraction ion flow chromatogram of the test sample, a chromatographic peak corresponding to the chromatogram of the liriope spicata saponin B or liriope spicata saponin C control solution appears, the selected monitoring ion-to-peak area ratio is consistent with the monitoring ion-to-peak area ratio of the control sample (the relative ratio is greater than 50%, the deviation of +/-20% is allowed, the relative ratio is greater than 20-50%, the deviation of +/-25% is allowed, the relative ratio is greater than 10-20%, the deviation of +/-30% is allowed, the relative ratio is less than 10%, the deviation of +/-50% is allowed), and meanwhile, the chromatographic peak area values of m/z721.2 → 575.3 and m/z870.2 → 738.3 in the chromatogram of the test sample are not greater than the corresponding peak area values in the liriope spicata saponin B or the liriope spicata saponin C control solution, the liriope spicata is regarded as undetected; (3) in the extraction ion flow chromatogram of the test sample, chromatographic peaks corresponding to the chromatogram of the liriope spicata saponin B or liriope spicata saponin C control solution appear, the selected monitoring ion-to-peak area ratio is consistent with the monitoring ion-to-peak area ratio of the control sample (relative ratio > 50%, deviation of +/-20%, relative ratio > 20% -50%, deviation of +/-25%, relative ratio > 10% -20%, deviation of +/-30%, relative ratio < 10%, deviation of +/-50%), and meanwhile, chromatographic peak area values of m/z721.2 → 575.3 and m/z870.2 → 738.3 in the chromatogram of the test sample are larger than the corresponding peak area values in the liriope spicata saponin B or the liriope spicata saponin C control solution, and the sample is regarded as the liriope spicata adulteration.

Making result judgment according to a result judgment principle, namely: in the ion flow chromatography of the sample, no chromatographic peak corresponding to the control solution chromatogram of liriope muscari saponin B or liriope muscari saponin C should be detected.

TABLE 13 content of saponins in different adulteration ratios of radix liriopes

Claims

1. A method for detecting adulteration of radix ophiopogonis in pulse-activating decoction is characterized by comprising the following steps:

preparation of S1 sample:

and (III) the peak area of the test solution is larger than that of the reference solution.

2. The method for detecting adulteration of radix ophiopogonis in pulse-activating decoction according to claim 1, wherein in S1, the specific preparation method of the test solution comprises the following steps: precisely measuring 5ml of pulse-activating decoction, dissolving in 10-20ml of methanol, shaking, and filtering to obtain a test solution; in S1, the concentration of the reference solution is 0.5-2.1 μ g/ml; in S2, the test solution and the reference solution are used in an amount of 2-5 μ l.

3. The method for detecting adulteration of radix ophiopogonis in pulse-activating decoction according to claim 1, wherein in S2, the detection parameters of the ultra-high performance liquid chromatography-mass spectrometer are set as follows:

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

4. The method for detecting adulteration of radix ophiopogonis in pulse-activating decoction according to claim 3, wherein the spraying voltage is 3500V, the collision energy of m/z 575.3 and 89.1 ions of liriope spicata saponin B is 23.0V and 32.6V respectively, and the collision energy of m/z 738.3 and 576.2 ions of liriope spicata saponin C is 26.5V and 32.9V respectively.

5. The method for detecting adulteration of radix ophiopogonis in pulse-activating decoction according to claim 1, wherein in S2, the adulteration analysis specifically comprises: meanwhile, the ion pair corresponding to the test solution and the reference solution has the same peak-out time, the peak area ratio of the test solution is consistent with that of the reference solution, and the adulteration is considered when the peak area of the test solution is larger than that of the reference solution, and the specific reference standard is as follows:

6. the method for detecting the adulteration of the radix ophiopogonis in the pulse-activating decoction as claimed in claim 1, wherein the preparation of the sample solution in the step S1 further comprises a pretreatment step of randomly mixing 5-10 pulse-activating decoction, and preparing the mixture according to the preparation process of the sample solution in the step S1.

7. The method for testing adulteration of the pulse-activating decoction according to any one of claims 1 to 6, wherein the pulse-activating decoction is the pulse-activating decoction recorded in P835 of 2020 edition of Chinese pharmacopoeia.