Quantitative detection method for alkaloid in bulbus fritillariae cirrhosae

Abstract

The invention discloses a quantitative detection method for alkaloid in bulbus fritillariae cirrhosae. The quantitative detection method comprises the following steps: a, preparing a reference substance solution; b, preparing a test solution; and c, respectively sucking the series of mixed reference substance solutions and the test solution, and injecting the solutions into a high performance liquid chromatography-triple quadrupole tandem mass spectrometer for detection. According to the method, alkaloid components are extracted from the bulbus fritillariae cirrhosae only by using the concentrated ammonia test solution-methanol solution, so that the method is friendly to the environment and human bodies. Although a large amount of water-soluble impurities in the bulbus fritillariae cirrhosae are dissolved out by taking a concentrated ammonia test solution-methanol solution as an extraction solvent, the impurities and alkaloid components are effectively separated through a specific chromatographic column and an elution procedure in an instrument analysis stage, so that the interference of the impurities on alkaloid quantification during mass spectrometric detection is avoided, and the detection accuracy is improved. The accurate quantification of the five alkaloid components in the bulbus fritillariae cirrhosae is realized.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a quantitative detection method for alkaloids in Fritillaria cirrhosa. Background Technology

[0002] Fritillaria cirrhosa is the dried bulb of various Fritillaria cirrhosa species, including Fritillaria thunbergii, Fritillaria nigra, Fritillaria kansui, and Fritillaria spathulata. It is bitter, sweet, and slightly cold in nature, primarily affecting the lung and heart meridians. Its cold nature clears lung heat, its bitter taste lowers lung qi, and its sweet taste moistens the lungs. It is mainly used to treat dry cough due to lung heat. The antitussive and anti-inflammatory pharmacological effects of Fritillaria cirrhosa are closely related to its alkaloid content, including isosteroidal and steroidal alkaloids, which are the key material basis for its efficacy. The Chinese Pharmacopoeia clearly stipulates the alkaloid content in Fritillaria cirrhosa, but there are significant differences in alkaloid content among different commercial specifications and source species. Quantitative analysis can ensure that the quality of Fritillaria cirrhosa products meets national standards, avoid quality problems caused by substandard components, and provide a reliable basis for scientific research and clinical application.

[0003] Currently, there are numerous methods for quantifying alkaloids in Fritillaria cirrhosa, including colorimetric methods, two-phase titration, non-aqueous titration, thin-layer chromatography, high-performance liquid chromatography-evaporative light scattering (HPLC-SLS), and HPLC-tandem mass spectrometry (HPLC-MS / MS). HPLC-MS / MS is widely used because it can simultaneously quantify multiple major pharmaceutical components in Fritillaria cirrhosa. However, when using HPLC-MS / MS to quantify alkaloids in Fritillaria cirrhosa, chloroform-methanol is required as the extraction solvent for pretreatment. Chloroform is a chemical substance with various toxicities and is classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). To reduce the harm to the health of analytical personnel, it is necessary to develop a method for quantifying Fritillaria cirrhosa alkaloids using HPLC-MS / MS without requiring chloroform pretreatment, thus achieving both quality control and environmental friendliness. Summary of the Invention

[0004] To address the above problems, this invention provides a method for the quantitative detection of alkaloids in Fritillaria cirrhosa, comprising the following steps:

[0005] a. Preparation of reference solution: Take alkaloid reference standard, dissolve and dilute it with methanol to prepare a series of reference solution concentrations;

[0006] b. Preparation of test solution: Take the fine powder of Fritillaria cirrhosa to be tested, extract it with concentrated ammonia test solution-methanol solution, filter the extract, and take the filtrate to obtain the test solution;

[0007] c. Inject the series of mixed reference solutions and test solutions separately into the high-performance liquid chromatography-triple quadrupole tandem mass spectrometer; chromatographic conditions:

[0008] Column: C 18 Chromatographic column; Mobile phase: 0.1% formic acid solution as mobile phase A, acetonitrile as mobile phase B; Gradient elution program:

[0009]

[0010] Mass spectrometry conditions: ionization mode was electrospray ionization source, monitoring mode was multiple reaction monitoring mode, and scanning mode was positive ion scanning mode.

[0011] Furthermore, each 1L of the reference solution described in step a contains 1–1000 μg of each alkaloid reference standard.

[0012] Furthermore, the alkaloid reference standards are fritillary alkaloid reference standard, fritillary alkaloid glycoside reference standard, fritillary alkaloid A reference standard, fritillary alkaloid B reference standard and fritillary alkaloid sine reference standard.

[0013] Further, in step b, the mass-to-volume ratio of the Fritillaria cirrhosa powder to the concentrated ammonia solution-methanol is 0.1g:10-50ml, preferably 0.1g:20ml.

[0014] Furthermore, the volume ratio of the concentrated ammonia solution to methanol is 4:6.

[0015] Further, in step c, the chromatographic conditions are as follows: the column is an Agilent Poroshell 120EC-C18, 2.1×100mm, 1.9μm; the flow rate is 0.3ml / min; the column temperature is 40℃; and the injection volume is 1μl.

[0016] Furthermore, in step c, the mass spectrometry conditions are as follows: ion source temperature: 325℃, spray voltage: 4000+, atomizing gas flow rate: 7L / min; sheath gas temperature: 350℃, and curtain gas pressure: 35psi.

[0017] Furthermore, the mass spectrometry parameters of the chemical components corresponding to the reference standard in the mass spectrometry conditions described in step c are as follows:

[0018]

[0019]

[0020] Furthermore, the quantitative ion pairs for the cisperidin are m / z 592.4→m / z 96.1, the quantitative ion pairs for fritillary cinnamic acid are m / z 428.3→m / z 67.2, the quantitative ion pairs for cisperidin are m / z 430.3→m / z 96.1, the quantitative ion pairs for fritillary acetyl are m / z 432.3→m / z 112.1, and the quantitative ion pairs for fritillary acetyl are m / z 430.3→m / z 98.1.

[0021] Furthermore, it uses the external standard curve method to calculate the alkaloid content in Fritillaria cirrhosa.

[0022] The quantitative detection method for alkaloids in Fritillaria cirrhosa provided by this invention uses only concentrated ammonia solution-methanol solution for alkaloid extraction during the pretreatment of Fritillaria cirrhosa, which is environmentally and human-friendly. Although using concentrated ammonia solution-methanol solution as the extraction solvent will cause a large number of water-soluble impurities in Fritillaria cirrhosa to dissolve, interfering with instrumental quantitative analysis, the detection method of this invention effectively separates impurities from alkaloids through specific chromatographic columns and elution programs during the instrumental analysis stage. This avoids the interference of impurities on alkaloid quantification during mass spectrometry detection, achieving accurate quantification of five alkaloids in Fritillaria cirrhosa. This method meets the requirements of Fritillaria cirrhosa quality control while being environmentally and human-friendly, and has practical application value.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 TIC diagram of mixed standard solution in mobile phase gradient elution program ①;

[0026] Figure 2 TIC plot of mixed standard solution in mobile phase gradient elution program ②;

[0027] Figure 3 TIC diagram of mixed standard solution in mobile phase gradient elution program ③;

[0028] Figure 4 TIC plot of mixed standard solution in mobile phase gradient elution program ④;

[0029] Figure 5 TIC plot of mixed standard solution in mobile phase gradient elution program ⑤;

[0030] Figure 6 TIC diagram of reference solution (1) and test solution (2) in mobile phase gradient elution program ①;

[0031] Figure 7 MRM diagrams of different reference standard components;

[0032] Figure 8TIC chromatograms of different columns (①Agilent poroshell 120EC-C) 18 , 2.1×100mm, 1.9μm; ②Shim-pack Velox C 18 , 2.1×100mm, 2.7μm; ③Agilent poroshell 120SB-Aq, 2.1×100mm, 2.7μm). Detailed Implementation

[0033] Example 1: Determination of alkaloid content in Fritillaria cirrhosa according to the present invention

[0034] a. Preparation of reference solutions: Take the reference standards of fritillary, fritillary glycoside, fritillary A, fritillary B, and fritillary cinnamic acid, and dissolve them in methanol to prepare a solution containing 1 μg of each per ml as the reference stock solution. Accurately measure the reference stock solutions and dissolve them in methanol to prepare a series of mixed reference solutions containing 1000 μg, 500 μg, 200 μg, 100 μg, 40 μg, 20 μg, 10 μg, 4 μg, 2 μg, and 1 μg per L, respectively.

[0035] b. Preparation of test solution: Take 0.1g of fine powder of Fritillaria cirrhosa, place it in a stoppered conical flask, accurately add 20ml of concentrated ammonia test solution-methanol (4:6) solution, weigh it, heat it under reflux in a water bath for 1h, cool it to room temperature, make up the lost weight with concentrated ammonia test solution-methanol (4:6) solution, filter it, and take the filtrate to obtain the test solution.

[0036] c. Inject the series of mixed reference solutions and test solutions separately into the high-performance liquid chromatography-triple quadrupole tandem mass spectrometer; chromatographic conditions:

[0037] Chromatographic column: Agilent Poroshell 120EC-C18 (2.1×100mm, 1.9μm); Mobile phase: 0.1% formic acid solution as mobile phase A, acetonitrile as mobile phase B; Flow rate: 0.3ml / min; Column temperature: 40℃; Injection volume: 1μl; Gradient elution program:

[0038]

[0039] Mass spectrometry conditions: Ionization method: electrospray ionization source; Monitoring mode: multiple reaction monitoring (MRM); Scanning mode: positive ion scan; Ion source temperature: 325℃; Spray voltage: 4000(+); Nebulizer gas flow rate: 7 L / min; Sheath gas temperature: 350℃; Curtain gas pressure: 35 psi. Monitoring ion pairs and collision voltages for each alkaloid:

[0040]

[0041]

[0042] * represents the quantitative ion.

[0043] d. Calculate the alkaloid content in Fritillaria cirrhosa using the external standard curve method.

[0044] The following experimental examples illustrate the beneficial effects of the present invention.

[0045] Experimental Example 1: Study on the determination method of alkaloid content in Fritillaria cirrhosa

[0046] This study established a multi-index quantitative analysis method using UPLC-MS / MS to simultaneously determine alkaloids such as fritillary glycoside, fritillary alkaloid, and fritillary alkaloid in Fritillaria cirrhosa. The method was applied to 56 batches of Fritillaria cirrhosa samples from 12 companies across the country, including Sichuan Shushan Pharmaceutical Co., Ltd., Sichuan Shufeng Daodi Medicinal Herbs Co., Ltd., Pingyi Hongcheng Traditional Chinese Medicine Co., Ltd., Shengfa Pharmacy of Chengdu Hehuachi Traditional Chinese Medicine Professional Market in Jinniu District (Chengdu), Mianyang Santai County Guohe Traditional Chinese Medicine Planting Co., Ltd., and Anhui Jucheng Pharmaceutical Co., Ltd.

[0047] 1. Instruments and reagents

[0048] Instruments: Agilent 1290-6495 liquid chromatography-triple quadrupole mass spectrometer (Agilent Technologies, Inc., USA); ME204E electronic balance and XPE26 electronic balance (Mettler-Toledo, Switzerland).

[0049] Reagents: Methanol, acetonitrile (Thermo Fisher Scientific, chromatographic grade); formic acid (Fisher, mass spectrometry grade); ultrapure water was used in the experiment.

[0050] Reference standards: Fritillary, Fritillary glycoside, Fritillary A, Fritillary B, and Fritillary sine were all provided by the China National Institutes for Food and Drug Control.

[0051] 2 Chromatographic conditions

[0052] Agilent poroshell 120EC-C 18 (2.1×100mm, 1.9μm) chromatographic column; gradient elution with 0.1% formic acid solution as mobile phase A and acetonitrile as mobile phase B; flow rate 0.3ml / min, column temperature 40℃, injection volume 1μl.

[0053] 3. Mass Spectrometry Conditions

[0054] Electrospray ionization (ESI) was used; multiple reaction monitoring (MRM) mode; positive ion scanning mode; relevant parameters were: ion source temperature: 325℃, spray voltage: 4000(+), atomizing gas flow rate: 7L / min; sheath gas temperature: 350℃, curtain gas pressure: 35psi. The monitored ion pairs and collision voltages (CE) of each compound are shown in Table 1.

[0055] Table 1. Monitoring of ion pairs and collision voltage (CE)

[0056]

[0057]

[0058] Note: * indicates quantitative ions.

[0059] 4. Solution Preparation

[0060] 4.1 Preparation of reference solution

[0061] Accurately weigh the reference standards and dissolve them in methanol to prepare a solution containing 1 μg of each standard per 1 ml as a reference standard stock solution. Accurately measure appropriate amounts of the above reference standard stock solutions and dissolve them in methanol to prepare a series of mixed reference standard solutions containing 1000 μg, 500 μg, 200 μg, 100 μg, 40 μg, 20 μg, 10 μg, 4 μg, 2 μg, and 1 μg per 1 L.

[0062] 4.2 Preparation of blank solution

[0063] A blank solution was prepared according to the method for preparing the test solution. Testing showed that the blank solution caused no interference.

[0064] 4.3 Preparation of the test solution

[0065] Take 0.1g of fine powder of Fritillaria cirrhosa and place it in a stoppered conical flask. Accurately add 20ml of concentrated ammonia test solution-methanol (4:6) solution, weigh it, heat it under reflux in a water bath for 1h, cool it to room temperature, make up the lost weight with concentrated ammonia test solution-methanol (4:6) solution, filter it, and take the filtrate to obtain the product.

[0066] 5. Determination method

[0067] Accurately pipette 1 μl of the reference solution and the test solution into the liquid chromatography-tandem mass spectrometry instrument, and calculate according to the external standard curve method.

[0068] 6. Selection of chromatographic conditions

[0069] 6.1 Selection of elution program

[0070] Take the mixed reference solution prepared under “4.1” and analyze it according to the chromatographic conditions under “2”, where the gradient elution programs are shown in Tables 2 to 6, and the mass spectrometry conditions under “3”.

[0071] Table 2 Mobile Phase Gradient Elution Program ①

[0072]

[0073]

[0074] Table 3 Mobile Phase Gradient Elution Program ②

[0075]

[0076] Table 4 Mobile Phase Gradient Elution Program ③

[0077]

[0078] Table 5 Mobile Phase Gradient Elution Program ④

[0079]

[0080] Table 6 Mobile Phase Gradient Elution Program ⑤

[0081]

[0082] TIC chromatograms of the reference solutions obtained from different gradient elution programs are shown below. Figures 1-5 As can be seen from the TIC results, under the mobile phase gradient elution program ①, the five alkaloid components can be effectively separated, avoiding the decrease in ionization efficiency of the target alkaloid molecules due to interference from competing molecules during ionization, which would lead to an underestimation of the target alkaloid molecule's quantitative result. Under the mobile phase gradient elution programs ② to ⑤, the five alkaloid components cannot be effectively separated, interfering with subsequent mass spectrometry detection and making it impossible to accurately quantify the alkaloid component content.

[0083] The TIC and MRM diagrams obtained under the mobile phase gradient elution program ① are shown below. Figures 6-7 Based on the results of the TIC and MRM plots, it was finally determined that the elution program was the mobile phase gradient elution program ①, which could accurately quantify the five alkaloids. Therefore, the elution program was selected as the mobile phase gradient elution program ①.

[0084] 6.2 Column Investigation

[0085] Take a sample of the mixed control solution (100 μg / L) from section “4.1” and analyze it according to the chromatographic conditions in section “2”, where the elution program is the mobile phase gradient elution program ①, and the mass spectrometry conditions in section “3”. Analyze ①Agilent poroshell 120EC-C 18(2.1×100mm, 1.9μm)②Shim-pack Velox C 18 Three chromatographic columns were investigated: ① 2.1 × 100 mm, 2.7 μm; ② Agilent poroshell 120SB-Aq (2.1 × 100 mm, 2.7 μm); ③ Agilent poroshell 120SB-Aq (2.1 × 100 mm, 2.7 μm). The TIC chromatograms for column investigation are shown below. Figure 8 As can be seen from the results: Agilent poroshell 120EC-C 18 The (2.1×100mm, 1.9μm) column can effectively separate five alkaloid molecules from competing molecules, avoiding interference from the mass spectrometry stage. Therefore, the Agilent Poroshell 120EC-C column was selected. 18 (2.1×100mm, 1.9μm).

[0086] 7. Selection of the method for preparing the test solution

[0087] 7.1 Selection of Extraction Solvent

[0088] 0.1 g of fine powder of Fritillaria cirrhosa (sample number: CD-1) was taken and a test solution was prepared according to "4.3". Concentrated ammonia solution-methanol (2:8), concentrated ammonia solution-methanol (3:7), concentrated ammonia solution-methanol (4:6), and concentrated ammonia solution-methanol (5:5) were selected as extraction solvents for investigation. Analysis was performed according to "2" (with the elution program being the mobile phase gradient elution program ①) and "3" (mass spectrometry). The results showed that the content extracted by concentrated ammonia solution-methanol (4:6) was the highest among the four solvents. Therefore, concentrated ammonia solution-methanol (4:6) was selected as the extraction solvent. See Table 7.

[0089] Table 7. Effects of different solvent extraction methods on content determination results

[0090]

[0091] 7.2 Investigation on the amount of extraction solvent used

[0092] 0.1 g of finely powdered Fritillaria cirrhosa (sample number: CD-1) was used to prepare a test solution according to section 4.3. 10 ml, 20 ml, and 40 ml of concentrated ammonia solution-methanol (4:6) were added for analysis. Analysis under the chromatographic conditions of section 2 (elution program: gradient elution program ①) and section 3 (mass spectrometry conditions) showed that the content was significantly lower when the extraction solvent volume was 10 ml. The content of fritillaria cirrhosaine differed when the extraction solvent volumes were 20 ml and 40 ml, with the content higher at 20 ml than at 40 ml. Therefore, 20 ml was selected as the extraction solvent volume. The results are shown in Table 8.

[0093] Table 8. Effect of different solvent dosages on content determination results

[0094]

[0095] 7.3 Examination of Different Extraction Methods

[0096] Method 1: Take 0.1g of the fine powder of Fritillaria cirrhosa (No.: CD-1), place it in a stoppered conical flask, accurately add 20ml of concentrated ammonia test solution-methanol (4:6) solution, weigh it, sonicate for 1h, let it cool to room temperature, replenish the lost weight with concentrated ammonia test solution-methanol (4:6) solution, filter, and take the filtrate to obtain the product.

[0097] Method 2: Take 0.1g of the fine powder of Fritillaria cirrhosa (No.: CD-1), place it in a stoppered conical flask, accurately add 20ml of concentrated ammonia test solution-methanol (4:6) solution, weigh it, soak it overnight, heat it under reflux in a water bath for 1h, cool it to room temperature, make up the lost weight with concentrated ammonia test solution-methanol (4:6) solution, filter it, and take the filtrate to obtain the product.

[0098] Method 3: Take 0.1g of Fritillaria cirrhosa test sample (No.: CD-1), place it in a stoppered conical flask, accurately add 20ml of concentrated ammonia test solution-methanol (4:6) solution, weigh it, heat it under reflux in a water bath for 1h, cool it to room temperature, make up the lost weight with concentrated ammonia test solution-methanol (4:6) solution, filter it, and take the filtrate to obtain the sample.

[0099] According to item "2", the chromatographic conditions of the mobile phase gradient elution program ① and the mass spectrometry conditions under item "3" were analyzed. The results are shown in Table 9. The content of each component of Fritillaria cirrhosa was significantly lower in method 1. There was no significant difference in the content of each component of Fritillaria cirrhosa in method 2 and method 3. From the perspective of saving time, method 3 was selected as the extraction method for preparing the test sample.

[0100] Table 9. Effects of different extraction methods on content determination results

[0101]

[0102] 7.4 Examination of extraction time

[0103] 0.1 g of fine powder of Fritillaria cirrhosa (sample number: CD-1) was taken and the sample solution was prepared according to "4.3". The solution was heated under reflux for 0.5 h, 1 h and 2 h respectively. The chromatographic conditions of "2" (with the elution program being the mobile phase gradient elution program ①) and the mass spectrometry conditions of "3" were analyzed and determined. The results showed that the content gradually increased with the increase of extraction time. When the extraction time reached 1 h, the difference in the content determination results was not significant. Therefore, heating under reflux for 1 h was selected. The results are shown in Table 10.

[0104] Table 10 Effect of different extraction times on content determination results

[0105]

[0106] 7.5 Examination of the sample size of the test sample

[0107] 0.05 g, 0.1 g, and 0.2 g of fine powder of Fritillaria cirrhosa (sample number: CD-1) were taken respectively, and test solutions were prepared according to "4.3". The results showed that different sampling amounts had no significant effect on the content determination results. See Table 11.

[0108] Table 11 Effect of different sampling amounts on content determination results

[0109]

[0110] 8. Methodological Investigation

[0111] 8.1 Linear

[0112] Accurately pipette 1 μl of the series of mixed control solutions under section "4.1" and inject them into the chromatographic conditions under section "2", where the elution program is the mobile phase gradient elution program ①, and the mass spectrometry conditions under section "3". A standard curve was plotted with the concentration of the target compound on the x-axis (X) and the peak area on the y-axis (Y). The results show that the correlation coefficient R of each target compound is greater than 0.99, indicating that each target compound exhibits a good linear relationship within the corresponding range. The results are shown in Table 12.

[0113] Table 12 Results of Linear Relationship

[0114]

[0115] 8.2 Precision

[0116] The mixed reference solution (100 μg / L) from section "4.1" was injected six times consecutively. The RSD was calculated according to the chromatographic conditions in section "2" (where the elution program is the mobile phase gradient elution program ①) and the mass spectrometry conditions in section "3". All results were less than 2.0%, indicating good instrument precision. The results are shown in Table 13.

[0117] Table 13 Precision Results

[0118]

[0119] 8.3 Stability

[0120] The mixed reference solution (100 μg / L) from section “4.1” was injected at 0 h, 8 h, 18 h, 28 h, 38 h, and 48 h, respectively. The RSD was calculated according to the chromatographic conditions in section “2” (where the elution program is the mobile phase gradient elution program ①) and the mass spectrometry conditions in section “3”. All results were less than 2.0%, indicating that the reference solution was stable within 48 h. The results are shown in Table 14.

[0121] Table 14 Stability Results

[0122]

[0123]

[0124] 8.4 Accuracy

[0125] Six portions (approximately 0.05 g) of the fine powder of the test sample (No.: CD-1) were accurately weighed and added to the respective reference solutions. The test solution was prepared according to section "4.3". Then, the analysis was performed according to section "2", with the elution program being the mobile phase gradient elution program ①, and the mass spectrometry conditions under section "3". The recoveries were calculated. The recoveries of fritillary glycoside, fritillary cinnamic acid, fritillary alkaloid, fritillary alkaloid A, and fritillary alkaloid B were 97.86%, 93.45%, 106.29%, 100.85%, and 99.06%, respectively, with RSDs of 2.79%, 2.01%, 2.45%, 2.24%, and 2.14%, respectively. This indicates that the method has good accuracy. The results are shown in Table 15.

[0126] Table 15 Accuracy Results

[0127]

[0128]

[0129] 8.5 Limit of Detection and Limit of Quantification

[0130] The limits of detection for fritillary alkaloids, fritillary alkaloids, fritillary alkaloids, fritillary alkaloid A, and fritillary alkaloid B were 0.41 μg / kg, 1.35 μg / kg, 0.20 μg / kg, 0.11 μg / kg, and 0.12 μg / kg, respectively; and the limits of quantitation were 1.36 μg / kg, 4.49 μg / kg, 0.65 μg / kg, 0.37 μg / kg, and 0.39 μg / kg, respectively.

[0131] 8.6 Testing and Verification of Commercially Available Fritillaria Cirrhosae

[0132] Take 0.1 g of commercially available Fritillaria cirrhosa sample, prepare the test solution according to section "4.3", and then analyze it according to section "2", where the elution program is the mobile phase gradient elution program ①, and the mass spectrometry conditions under section "3". The results are shown in Table 16.

[0133] Table 16 Summary of Sample Content Determination Results

[0134]

[0135]

[0136]

[0137] 8.7. Setting Content Limits

[0138] During the testing of 56 batches of samples, it was found that commercially available Fritillaria cirrhosa may have been adulterated or mixed with other substances, resulting in low levels of Fritillariaein A and B, which could not be integrated. Therefore, no limits were set for these substances based on this method.

[0139] Since there were many mixed samples in this monitoring and the origin was uncertain, 25 batches of Fritillaria cirrhosa samples with known origins were collected independently (13 batches of Fritillaria cirrhosa and 12 batches of Fritillaria cirrhosa), and the results were measured according to the proposed method. The results are shown in Table 17.

[0140] Table 17 Results of content determination in 25 batches of Fritillaria cirrhosa samples

[0141]

[0142]

[0143] Fritillary alkaloids and fritillary glycosides are two forms of the same substance, namely, the aglycone and the glucoside. They may interconvert within the plant. Furthermore, the content determination results for fritillary alkaloids and fritillary glycosides differ significantly from those for fritillary cinnamic acid. Therefore, the provisional limit is: this product contains fritillary glycosides (C...). 33 H 53 NO8) and cibeneline (C 27 H 43 The total amount of NO3) shall not be less than 1.2 μg / g; the content of fritillaria cirrhosa (C 27 H 41 NO3) shall not be less than 27 μg / g.

[0144] 8.8 Sample Result Analysis

[0145] Of the 56 batches of samples monitored, 55 batches met the proposed limits. The test results of commercially available Fritillaria cirrhosa show that the detection method of this invention can achieve rapid and accurate quantification of five alkaloids in Fritillaria cirrhosa.

Claims

1. A method for quantitative detection of alkaloids in Fritillaria cirrhosa, characterized in that: It includes the following steps: a. Preparation of reference solution: Take alkaloid reference standard, dissolve and dilute it with methanol to prepare a series of reference solution concentrations; b. Preparation of test solution: Take the fine powder of Fritillaria cirrhosa to be tested, extract it with concentrated ammonia test solution-methanol solution, filter the extract, and take the filtrate to obtain the test solution; c. Inject the series of mixed reference solutions and test solutions separately into the high-performance liquid chromatography-triple quadrupole tandem mass spectrometer; chromatographic conditions: Column: C 18 Chromatographic column; Mobile phase: 0.1% formic acid solution as mobile phase A, acetonitrile as mobile phase B; Gradient elution program: Mass spectrometry conditions: ionization mode was electrospray ionization source, monitoring mode was multiple reaction monitoring mode, and scanning mode was positive ion scanning mode.

2. The quantitative detection method according to claim 1, characterized in that: The reference solution described in step a contains 1 to 1000 μg of each alkaloid reference standard per 1 L.

3. The quantitative detection method according to claim 1 or 2, characterized in that: The alkaloid reference standards are fritillary alkaloid reference standard, fritillary alkaloid glycoside reference standard, fritillary alkaloid A reference standard, fritillary alkaloid B reference standard and fritillary alkaloid sine reference standard.

4. The quantitative detection method according to claim 1, characterized in that: The mass-to-volume ratio of the Fritillaria cirrhosa powder to concentrated ammonia solution-methanol in step b is 0.1g:10-50ml, preferably 0.1g:20ml.

5. The quantitative detection method according to claim 1 or 4, characterized in that: The volume ratio of concentrated ammonia solution to methanol is 4:

6.

6. The quantitative detection method according to claim 1, characterized in that: In step c, the chromatographic conditions are as follows: the column is an Agilent Poroshell 120EC-C18, 2.1×100mm, 1.9μm; the flow rate is 0.3ml / min; the column temperature is 40℃; and the injection volume is 1μl.

7. The quantitative detection method according to claim 1, characterized in that: In step c, the mass spectrometry conditions are as follows: ion source temperature: 325℃, spray voltage: 4000+, nebulizer gas flow rate: 7L / min; sheath gas temperature: 350℃, curtain gas pressure: 35psi.

8. The quantitative detection method according to claim 1, characterized in that: The mass spectrometry parameters of the alkaloids corresponding to the reference standard in the mass spectrometry conditions described in step c are as follows:

9. The quantitative detection method according to any one of claims 1 to 8, characterized in that: It uses the external standard curve method to calculate the alkaloid content in Fritillaria cirrhosa.

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