Liquid chromatography-mass spectrometry detection method for bulbus fritillariae cirrhosae lung-heat-clearing syrup

Abstract

The invention discloses a liquid chromatography-mass spectrometry detection method of bulbus fritillariae cirrhosae lung-heat-clearing syrup, which comprises the following steps: respectively taking a reference substance and the bulbus fritillariae cirrhosae lung-heat-clearing syrup, adding methanol for dissolving to obtain a reference substance solution and a test solution, and injecting the reference substance solution and the test solution into an HPLC-QQQ / MS liquid chromatography-mass spectrometer for detection. The detection method disclosed by the invention can be used for detecting rehmannia glutinosa-D, catalpol, amygdalin, platycodin-D, ophiopogonin-C and ophiopogonin-D in the fritillaria cirrhosa lung-heat-clearing syrup, is used for identifying active ingredients in the fritillaria cirrhosa lung-heat-clearing syrup, and perfects the quality control standard of the fritillaria cirrhosa lung-heat-clearing syrup. According to methodology verification, the detection method is high in accuracy and sensitivity, can realize specific detection of the characteristic components of the bulbus fritillariae cirrhosae lung-heat-clearing syrup, and has practical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the detection method of traditional Chinese medicine compound, in particular to a kind of detection method of chuanbei qingfei syrup by liquid chromatography-mass spectrometry. BACKGROUND

[0002] Chuanbei qingfei syrup is a traditional Chinese medicine compound preparation in the national non-prescription drug directory, which is made of eight kinds of traditional Chinese medicines such as loquat leaf, bitter apricot kernel, chuanbei, ophiopogon japonicus, rehmannia glutinosa, licorice, platycodon grandiflorum and mint. Among them, chuanbei and loquat leaf are the monarch drugs for clearing lung and drying; ophiopogon japonicus and rehmannia glutinosa are the ministerial drugs for being sweet and cool and generating fluid; bitter apricot kernel and mint are the auxiliary drugs for reducing qi and expelling phlegm; platycodon grandiflorum and licorice are the ministerial drugs for detoxifying and benefiting the throat. When used together, they have the effects of clearing lung and drying, relieving cough and phlegm, and dispelling wind and relieving heat. At present, the quality standard for chuanbei qingfei syrup only has the inspection items of physical and chemical properties such as appearance, relative density and loading capacity. The method for controlling the components of chuanbei qingfei syrup also only relies on colorimetry, thin layer chromatography, ultraviolet spectrophotometry and high performance liquid chromatography. The specificity and sensitivity of these detection methods cannot meet the needs of quality control of compound preparations.

[0003] And in the existing research, the component detection of chuanbei qingfei syrup is mostly limited to a few medicinal materials such as bitter apricot kernel and licorice. There is no effective detection method for the active components of other medicinal materials such as ophiopogon japonicus and platycodon grandiflorum. In the research of <detection of adulterated ophiopogon japonicus in chuanbei qingfei syrup based on HPLC-O-TOF-MS technology> by GONG Xue-wan et al., only ophiopogonan saponins in ophiopogon japonicus were detected by liquid chromatography-mass spectrometry. Therefore, it is urgent to establish a method that is convenient and reliable, high in sensitivity and can detect more components, in order to improve the quality control level of chuanbei qingfei syrup. High performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ MS) combines the advantages of fast separation, high sensitivity and high selectivity, and can realize specific detection of target components. However, there are many types of compounds in traditional Chinese medicine compound preparations composed of multiple drugs, and the structures are complex. There are also problems such as signal interference and matrix effect. It is a big challenge to find a liquid chromatography-mass spectrometry method that can effectively separate the characteristic components in chuanbei qingfei syrup. At present, there is no report on the liquid chromatography-mass spectrometry method for simultaneous analysis and detection of multiple drugs and multiple characteristic components in chuanbei qingfei syrup. SUMMARY

[0004] To solve the above problems, the present application provides a liquid chromatography-mass spectrometry detection method for chuanbei qingfei syrup, which comprises the following steps:

[0005] Respectively take the control sample and chuanbei qingfei syrup, dissolve them with methanol as the control sample solution and the sample solution, and inject them into the HPLC-QQQ / MS liquid chromatography-mass spectrometry instrument for detection;

[0006] The chromatographic conditions are as follows:

[0007] Chromatographic column: C18 chromatographic column; mobile phase: mobile phase A is acetonitrile, mobile phase B is 0.1% formic acid solution; gradient elution conditions are as follows: 0-10 min, 2%-98% acetonitrile; 10-12 min, 98% acetonitrile; 12-13 min, 98%-2% acetonitrile; 13-15 min, 2% acetonitrile;

[0008] Mass spectrometry conditions are as follows:

[0009] Ion source: electrospray ion source; detection mode: multiple reaction monitoring in negative ion mode.

[0010] Further, the concentration of each control in the control solution is 0.5-50 μg / ml.

[0011] Still further, the control is a catalpol, rehmatannoside-D, amygdalin, platycodon saponin-D, ophiopogon saponin-C and / or ophiopogon saponin-D control.

[0012] Further, the concentration of the test product solution of the bulbus platycodi lung-clearing syrup is 10-50%, v / v.

[0013] Further, in the chromatographic conditions, the chromatographic column is a Waters XBridge BEH C18 chromatographic column with a size of 2.1 mm x 100 mm and a particle size of 2.5 μm.

[0014] Still further, in the chromatographic conditions, the column temperature is 30°C, the flow rate is 0.3 ml / min, and the injection volume is 5 μl.

[0015] Still further, in the mass spectrometry conditions, the ion source temperature is 150°C, and the capillary voltage is 2.2 KV.

[0016] Further, in the mass spectrometry conditions, the monitored qualitative ion pairs and their conditions are as follows:

[0017]

[0018] Further, in the chromatogram of the test product solution, a chromatographic peak with a retention time consistent with that of the qualitative ion pair in the chromatogram of the control solution is present, and the deviation of the relative abundance of the qualitative ion pair of the chromatographic peak from the relative abundance of the qualitative ion pair of the chromatographic peak of the control solution with a comparable concentration does not exceed the specified maximum allowable deviation range, thereby determining that the bulbus platycodi lung-clearing syrup contains the compound corresponding to the control; the specified maximum allowable deviation range is as follows:

[0019]

[0020] The "relative abundance of the qualitative ion pair" in the present application is the percentage of the ion abundance of the qualitative ion pair to the relative strongest ion abundance.

[0021] The present application obtains a new liquid chromatography-mass spectrometry detection method of Chuanbei Qingfei syrup through analysis of characteristic components of Chuanbei Qingfei syrup and exploration of liquid chromatography-mass spectrometry detection conditions. The method can detect rehymn-D, catalpol, amygdalin, platycodon saponin-D, ophiopogon saponin-C and ophiopogon saponin-D in Chuanbei Qingfei syrup through specific chromatographic conditions and mass spectrometry conditions. The detection method is verified by methodology, has high accuracy and sensitivity, and can realize specific detection of six characteristic components in Chuanbei Qingfei syrup. The method is used to identify active components in Chuanbei Qingfei syrup, improves the quality control level of Chuanbei Qingfei syrup, and has practical application value.

[0022] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present application.

[0023] The above content of the present application is further described in detail through the specific embodiments below. However, it should not be understood that the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Catalpol investigation extraction ion flow chart

[0025] Figure 2 Rehymn D investigation extraction ion flow chart

[0026] Figure 3 Amygdalin investigation extraction ion flow chart

[0027] Figure 4 Platycodon saponin D investigation extraction ion flow chart

[0028] Figure 5 Ophiopogon saponin C investigation extraction ion flow chart

[0029] Figure 6 Ophiopogon saponin D investigation extraction ion flow chart

[0030] Figure 7 Catalpol optimization extraction ion flow chart

[0031] Figure 8 Rehymn D optimization extraction ion flow chart

[0032] Figure 9 Amygdalin optimization extraction ion flow chart

[0033] Figure 10 Three batches of preparation verification extraction ion flow chart

[0034] Figure 11 Blank, bitter apricot kernel-leaf of loquat double negative preparation, bitter apricot kernel reference substance, test sample comparison chart (from top to bottom)

[0035] Figure 12 Blank, jujube negative preparation, jujube saponin D reference substance, test sample comparison chart (from top to bottom)

[0036] Figure 13 Blank, rehmannia negative preparation, catalpol, rehmannia glycoside D reference substance, test sample comparison chart (from top to bottom)

[0037] Figure 14 Blank, ophiopogon negative preparation, ophiopogon saponin D, ophiopogon saponin C reference substance, test sample comparison chart (from top to bottom).

[0038] Figure 15 Each reference substance extraction ion chromatogram. DETAILED DESCRIPTION

[0039] Example 1 Detection method of the present application

[0040] The method of the present application uses rehmannia glycoside-D and catalpol as markers of rehmannia, uses amygdalin as a marker of bitter apricot kernel, uses jujube saponin-D as a marker of jujube, and uses ophiopogon saponin-C and ophiopogon saponin-D as markers of ophiopogon.

[0041] The chromatographic conditions and system suitability test were as follows: octadecylsilane-bonded silica gel as the filler (Waters XBridge BEH C18 chromatographic column), acetonitrile as the mobile phase A, 0.1% formic acid solution as the mobile phase B, gradient elution according to the provisions in Table 1, flow rate of 0.3 ml per minute; column temperature of 30°C.

[0042] Table 1 Gradient elution procedure table

[0043]

[0044] A triple quadrupole mass spectrometer detector was used, and the ion source temperature was 150°C, the capillary voltage was 2.2KV, and the monitoring ion pair and conditions were as shown in Table 2:

[0045] Table 2 Mass spectrometry parameters

[0046]

[0047] Preparation of reference solution: The reference solution was prepared by taking appropriate amounts of the control samples of catalpol, D, amyranthin, platycodin-D, ophiopogonin-C and ophiopogonin-D, and dissolving them in methanol to prepare a control sample solution containing 10 μg of catalpol, 1 μg of D, 0.1 μg of amyranthin, 1 μg of platycodin-D, 1 μg of ophiopogonin-C and 1 μg of ophiopogonin-D per 1 ml of the solution.

[0048] Preparation of test sample solution: 1 ml of Chuanbei Qingfei Sugar Syrup was precisely measured and placed in a 5 ml volumetric flask, which was diluted with methanol and made up to the mark, shaken and centrifuged (12000 r / min) for 5 minutes, filtered (0.22 μm filter membrane), and the filtrate was taken to obtain the test sample solution.

[0049] Determination: 5 μl of the control sample solution and the test sample solution were precisely taken and injected into the high performance liquid chromatography-mass spectrometry instrument for determination.

[0050] The test sample solution and the control sample solution were determined according to the high performance liquid chromatography-mass spectrometry conditions, the retention time of each compound in the test sample solution and the control sample solution was recorded, the relative abundance of the qualitative ion pair was taken as the percentage of the most abundant ion, the relative ion abundance of each compound in the test sample solution and the control sample solution with the same concentration was recorded, when the retention time of each compound in the sample was consistent with the chromatographic peak of the control sample (the change range was within ± 2.5%), and the relative ion abundance error was not more than the range specified in Table 3, the compound could be determined to be detected.

[0051] Table 3 Maximum error allowed for qualitative confirmation of relative ion abundance

[0052]

[0053] The beneficial effects of the present application are illustrated by the following test examples:

[0054] Test Example 1 Investigation and verification of the methodology of the present application

[0055] I. Instruments and reagents

[0056] (1) Instruments

[0057] Table 4 Instrument list

[0058]

[0059] (2) Reagents

[0060] Table 5 Reagent information

[0061]

[0062] (3) Control samples

[0063] Table 6 Control sample information

[0064]

[0065] (4) Drug testing

[0066] Table 7 Sample information of Chuanbei Qingfei Syrup and negative-labeled finished products

[0067]

[0068] Samples No. 1-15: Fritillaria cirrhosa lung-clearing syrup; Samples No. 16-20: negative samples.

[0069] II. Methodological Examination

[0070] (1) Exploration of chromatographic conditions and investigation of indicative components

[0071] The acetonitrile-formic acid system was selected as the chromatographic conditions. The optimization objectives were to reduce the co-elution of indicative components and to avoid the failure of competing ions to separate from the target ions. The retention time of indicative components was examined by comparing with reference standards and by combining the extracted ion chromatograms. The gradient elution program of the mobile phase was explored and determined (see Table 1).

[0072] Take appropriate amounts of catalpol, rehmannia glutinosa glycoside-D, ophiopogonin-D, ophiopogonin-C, platycodonin-D, and amygdalin reference solutions, and inject them into a liquid chromatography-tandem mass spectrometer. Based on the previously determined chromatographic conditions, further refine the mass spectrometer parameters. See the specific results below. Figures 1-6 And Table 8.

[0073] Table 8 Preliminary parameters for mass spectrometry

[0074]

[0075] (2) Optimization of control standard analysis

[0076] according to Figures 1-6 Based on the mass spectrometry results in Table 8, the reference solutions for catalpol, rehmannia glutinosa-D, and amygdalin were diluted to optimize peak shapes. Components were identified through multi-stage mass spectrometry analysis, ultimately determining the daughter ions, cone pore energies, and collision energies that could be evaluated in the form of ion chromatograms. The results are shown in Table 8. Figures 7-9 And Table 9. The optimized qualitative evaluation method based on ion chromatogram extraction is simpler to operate and easier to analyze in actual detection and analysis compared to detection and identification by extracting multi-stage mass spectrometry information.

[0077] Table 9 shows the optimized mass spectrometry parameters.

[0078]

[0079] (3) Formulation validation

[0080] Take three batches of chuanbei qingfei syrup products, use the pretreatment method in Example 1 and the optimized mass spectrometry parameters in this test example to machine, determine the optimized mass spectrometry parameters applicable to preparation analysis, the results are shown in Figure 10 .

[0081] III. Methodology verification

[0082] (1) Specificity test

[0083] The components in the reference solution are consistent with the solution of the test product (batch number: 1911176), and the blank and negative have no interference. It shows that this method has good specificity. The results are shown in Figures 11-14 .

[0084] (2) Detection limit

[0085] The detection limit of this method for catalpol is 300 ng / ml, the detection limit of rehmatannin-D is 40 ng / ml, the detection limit of amygdalin is 1 ng / ml, the detection limit of platycodon saponin-D is 10 ng / ml, the detection limit of ophiopogon saponin-C is 2 ng / ml, and the detection limit of ophiopogon saponin-D is 2 ng / ml. The results are shown in Figure 15 Note: The ion abundance ratio error should be compared with the reference solution and the test product solution in the same range.

[0086] (3) Sample detection

[0087] Take 15 batches of chuanbei qingfei syrup samples, and prepare the test product solution as described above. Qualitative confirmation of catalpol, platycodon saponin-D, ophiopogon saponin-C, ophiopogon saponin-D, amygdalin, and rehmatannin-D in 15 batches of chuanbei qingfei syrup is carried out. The experimental results are shown in Table 10 (marking with a check mark means detection).

[0088] Table 10 Qualitative results of chuanbei qingfei syrup

[0089]

[0090] If the content of each compound in the sample is higher than the detection limit of the method, it is detected.

[0091] In summary, the detection method of the present application has high accuracy and sensitivity after methodology verification, and can realize specific detection of six characteristic components in chuanbei qingfei syrup. The method of the present application is used to identify the active ingredients in chuanbei qingfei syrup, which improves the quality control level of chuanbei qingfei syrup. At the same time, the detection technology is simple and easy to analyze, and has practical application value.

Claims

1. A method for detecting Chuanbei Qingfei syrup by liquid chromatography-mass spectrometry, characterized in that: The method comprises the following steps: The control sample and the Chuanbei Qingfei syrup are respectively dissolved in methanol to obtain a control sample solution and a test sample solution, which are injected into an HPLC-QQQ / MS liquid chromatograph-mass spectrometer for detection; The chromatographic conditions are as follows: The chromatographic column is a C18 chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is 0.1% formic acid solution, the gradient elution conditions are as follows: 0-10 min, 2%-98% acetonitrile; 10-12 min, 98% acetonitrile; 12-13 min, 98%-2% acetonitrile; 13-15 min, 2% acetonitrile; The mass spectrometry conditions are as follows: The ion source is an electrospray ion source, and the detection mode is a multiple reaction monitoring in a negative ion mode.

2. The method according to claim 1, wherein: The concentration of each control sample in the control sample solution is 0.5-50 μg / ml. 3.The method according to claim 1 or 2, characterized in that: The control sample is a control sample of catalpol, rehmannioside-D, amygdalin, platycodon saponin-D, ophiopogon saponin-C and / or ophiopogon saponin-D.

4. The method according to claim 1, wherein: The concentration of the Chuanbei Qingfei syrup in the test sample solution is 10-50%, v / v.

5. The method according to claim 1, wherein: In the chromatographic conditions, the chromatographic column is a Waters XBridge BEH C18 chromatographic column.

6. The method according to claim 5, wherein: The specification of the Waters XBridge BEH C18 chromatographic column is 2.1 mm x 100 mm, 2.5 μm.

7. The method according to claim 5, wherein: In the chromatographic conditions, the column temperature is 30°C, the flow rate is 0.3 ml / min, and the injection volume is 5 μl.

8. The method according to any one of claims 1-7, wherein: In the mass spectrometry conditions, the ion source temperature is 150°C, and the capillary voltage is 2.2 KV.

9. The method according to claim 8, wherein: In the mass spectrometry conditions, the monitored qualitative ion pairs and conditions are as follows:

10. The method of claim 1, wherein: In the chromatogram of the test sample solution, there is a chromatographic peak with a retention time consistent with that of the qualitative ion pair in the chromatogram of the control sample solution, and the relative abundance of the qualitative ion pair of the chromatographic peak deviates from the relative abundance of the qualitative ion pair of the chromatographic peak of the control sample solution with a concentration equivalent to the maximum allowable deviation range, so that it is determined that the Chuanbei Qingfei syrup contains the compound corresponding to the control sample; the maximum allowable deviation range is as follows:

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