A method for detecting monacolins in monascus extract

Abstract

This invention provides a method for detecting cephalosporin in monkey earring extract. The method establishes a method for detecting cephalosporin in monkey earring extract, using cephalosporin I as a reference standard, and simultaneously determining the content of three components—cephalosporin I, cephalosporin II, and cephalosporin III—in monkey earring medicinal materials / extracts / preparations. The chromatographic conditions employed not only improve analytical efficiency and shorten running time, but also utilize a mild 0.1% formic acid-water-methanol system, which helps protect the chromatographic column and extend its service life.

Description

Technical Field

[0001] This invention belongs to the field of drug extract analysis technology, specifically relating to a method for detecting cephalosporin in monkey earring extract. Background Technology

[0002] Monkey earrings ( Archidendron clypearia (Jack) IC Nielsen, formerly known as Pithecellobium clypearia (Jack) Benth. is a plant belonging to the genus *Heliotropium* in the legume family (Fabaceae), also known as the chicken heart tree or the birth tree. The leaves, branches (below 2cm), and flowers are used medicinally. They possess properties of clearing heat and detoxifying, cooling the blood and reducing swelling, stopping diarrhea, and removing dampness and astringing sores. Clinically, they are mainly used to treat upper respiratory tract infections, acute pharyngitis, acute tonsillitis, and acute gastroenteritis. *Heliotropium* mainly contains flavonoids and flavans, phenylpropanoids, organic phenolic acids, triterpenes, and steroidal compounds. Several galloyl-substituted flavans are characteristic chemical components of archidylcholine, among which archidylcholine I (7-O-Galloyltricetiffavan), archidylcholine II (7,3′-O-di-galloyltricetiffavan), and archidylcholine III (7,4′-O-di-galloyltricetiffavan) are the most abundant.

[0003] Monkey earwort possesses antioxidant, anti-inflammatory, antiviral, and hypoglycemic effects, and its flavanoids (such as cephalosporin I, II, and III) are considered key pharmacologically active substances. However, existing technologies for quality control of monkey earwort have significant limitations, severely restricting the accurate evaluation and improvement of the quality of its raw materials and preparations. Currently available detection methods mainly focus on two categories: one is the determination of conventional components such as quercetin and gallic acid (e.g., CN109946397A, a method for testing monkey earwort raw materials), which lacks the specificity of monkey earwort raw materials and fails to target the characteristic flavanoid active components of monkey earwort; the other, although it can detect characteristic components, is limited to a single indicator, cephalosporin I (e.g., CN 101596229A, a quality control method for monkey earwort extracts and their preparations). Because monocyclonine I, II, and III are structurally highly similar, all being galloyl-substituted flavanes, they are prone to mutual interference in chromatographic analysis. Furthermore, monocyclonine II and III are interconvertible, and reference standards are difficult to obtain. As a result, there has long been a lack of detection methods that can simultaneously, rapidly, and accurately determine the above three flavanes.

[0004] The lack of specific or single quality control components cannot fully reflect the overall pharmacodynamic material basis of monkey ear fungus, and it is even more difficult to scientifically assess the quality uniformity and stability of different batches and origins of the medicinal material. Therefore, developing a method to simultaneously determine the content of monkey ear fungus I, II, and III without the need for multiple reference standards has become an urgent technical requirement to improve the quality control level of monkey ear fungus and ensure its clinical efficacy and safety. Summary of the Invention

[0005] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide a method for detecting cephalosporin in monkey earring extract.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for detecting cephalosporin in monkey earring extract, using high performance liquid chromatography to determine the content of 7-galloyltertrin (cephalosporin I), 7,3′-galloyltertrin (cephalosporin II) and 7,4′-galloyltertrin (cephalosporin III) in the monkey earring sample; Includes the following steps: Prepare a test solution for monkey earring samples and a reference solution of 7-galloylterlithronane. The chromatographic conditions were as follows: the column was a column packed with octadecylsilane-bonded silica gel; mobile phase A was methanol, and mobile phase B was a 0.1%-0.3% formic acid aqueous solution. A gradient elution was used between mobile phases A and B, and the gradient elution program was as follows, where all mobile phase proportions were volume percentages: 0–8 min, mobile phase A linearly changed from 28%–32% to 36%–40%, and mobile phase B linearly changed from 68%–72% to 60%–64%; 8–18 min, mobile phase A was maintained at 36%–40%, and mobile phase B was maintained at 60%–64%; 18–34 min, mobile phase A linearly changed from 36%–40% to 44%–48%, and mobile phase B linearly changed from 60%–64% to 52%–56%. Inject the reference solution and the test solution into the liquid chromatograph separately, determine the peak area value and calculate the content according to the above chromatographic conditions.

[0007] The structural formula of monkey ear cyclophosphine I is shown in Formula A: Formula A.

[0008] The structural formula of monkey ear cyclophosphamide II is shown in Formula B: Formula B.

[0009] The structural formula of monkey ear cyclophosphine III is shown in Formula C: Formula C.

[0010] Preferably, the monkey earring sample comprises monkey earring medicinal material, monkey earring extract, and a pharmaceutical composition containing monkey earring extract.

[0011] More preferably, the pharmaceutical composition is a formulation containing at least one of simianocycline I, simianocycline II, and simianocycline III.

[0012] More preferably, the dosage form of the preparation includes any one of oral preparations, injectable preparations, respiratory preparations, and transdermal preparations.

[0013] For example, the monkey earring preparations include tablets, capsules, granules, and oral liquids.

[0014] Preferably, the preparation method of the monkey earring sample test solution includes the following steps: The monkey earring herb / extract / preparation is dissolved in an alcohol solvent to obtain the monkey earring extract test solution.

[0015] More preferably, the monkey earring medicinal material includes monkey earring branches, leaves, flowers, and fruits.

[0016] More preferably, the preparation method of the monkey earring extract includes the following steps: The monkey earring raw material was extracted with water or an aqueous ethanol solution and concentrated into an extract to obtain the monkey earring extract.

[0017] More preferably, the raw material for monkey earrings is crushed monkey earring branches and leaves or monkey earring flowers.

[0018] More preferably, the raw material for monkey earrings is extracted using an 80%-100% ethanol aqueous solution.

[0019] More preferably, the extraction is performed at room temperature.

[0020] More preferably, the alcohol solvent is selected from methanol, methanol aqueous solution, ethanol or ethanol aqueous solution.

[0021] Preferably, the preparation method of the 7-galloylterlithronane reference solution includes the following steps: 7-Galloylterylcryoflavin was added to methanol to prepare a reference solution of 7-galloylterylcryoflavin.

[0022] Preferably, the particle size of the chromatographic column is selected from 3.5 or 5 μm, the column length is selected from 100, 150 or 250 mm, and the inner diameter is selected from 2.1 or 4.6 mm.

[0023] More preferably, the chromatographic column is an Agilent ZORBAX Eclipse XDB-C18 column.

[0024] Preferably, the gradient elution program is as follows, wherein the proportions of the mobile phases are all volume percentages: 0~8 min, mobile phase A linearly changes from 30% to 38%, and mobile phase B linearly changes from 70% to 62%; 8~18 min, mobile phase A is maintained at 38%, and mobile phase B is maintained at 62%; 18~34 min, mobile phase A linearly changes from 38% to 46%, and mobile phase B linearly changes from 62% to 54%.

[0025] Preferably, the chromatographic conditions further include a column temperature of 40°C.

[0026] Preferably, the chromatographic conditions further include a detection wavelength of 278 nm.

[0027] Preferably, the chromatographic conditions further include: a flow rate of 0.5-2 mL / min and an injection volume of 5 mL / min. 25μL.

[0028] The beneficial effects of this invention are: This invention establishes a method for detecting cephalosporin in *Eriocaulon buergerianum* medicinal materials / extracts / preparations. Using cephalosporin I as a reference standard, this method simultaneously determines the content of three components—cephalosporin I, cephalosporin II, and cephalosporin III—in *Eriocaulon buergerianum* extracts. The chromatographic conditions employed not only improve analytical efficiency and shorten run time, but also utilize a specific elution program (adjusting the mobile phase ratio) to separate the three cephalosporins. Furthermore, the use of a mild 0.1% formic acid-water-methanol system helps protect the chromatographic column and extend its lifespan. The method for determining cephalosporin content in this invention has undergone methodological studies including repeatability, precision, and stability, verifying its reliability and scientific validity. Attached Figure Description

[0029] Figure 1 The total ion chromatogram of the ethyl acetate component of monkey earrings; Figure 2 This is the first-order mass spectrum of monkey cloxacin I; Figure 3 This is a secondary mass spectrum of monkey cloxacillin I; Figure 4 This is the first-order mass spectrum of monkey cloxacin II; Figure 5 This is a secondary mass spectrum of monkey cloxacin II; Figure 6 This is the first-order mass spectrum of monkey cloxacin III; Figure 7 This is a secondary mass spectrum of monkey cloxacin III; Figure 8This is a chromatogram of the reference standard for the HPLC content determination method of the present invention; Figure 9 This is a chromatogram of the test sample for the HPLC content determination method of the present invention; Figure 10 This is a blank solvent chromatogram of the HPLC content determination method of the present invention. Detailed Implementation

[0030] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0031] The following information regarding the source and model of the instruments, reagents, and samples used in the examples is as follows: Instruments: Ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); High performance liquid chromatograph (Shimadzu Corporation, Japan); Quadrupole time-of-flight high resolution mass spectrometer (AB SCIEX, Singapore); Electronic balance (0.0001 g / L, Sartorius, Germany).

[0032] Reagents: Cyclocycline reference standard (laboratory-prepared, purity >98%); anhydrous ethanol (Annegi Shanghai Pharmaceutical Chemical Co., Ltd.); methanol (Beijing Opsen Technology Co., Ltd.); formic acid (Tianjin Kemio Chemical Reagent Co., Ltd.).

[0033] The water used in the experiment was ultrapure water, methanol and phosphoric acid were of chromatographic grade, and all other reagents were of analytical grade.

[0034] The samples of the medicinal herb Archidendron clypearia (branches, leaves, and flowers) were collected from the Guangzhou Laitai Pharmaceutical Archidendron Base in Maogang Village, Zhongluotan Town, Baiyun District, Guangzhou. They were identified by Professor Yang Depo of the School of Pharmaceutical Sciences, Sun Yat-sen University, as Archidendron clypearia (Jack) IC Nielsen, a plant belonging to the genus Archidendron of the Fabaceae family.

[0035] Monkey Earring Granules, sourced from Guangzhou Laitai Pharmaceutical Co., Ltd., product batch number: 2408053.

[0036] Monkey Earring Oral Solution, prepared by the Pharmacognosy Laboratory of the School of Pharmacy, Sun Yat-sen University.

[0037] Example 1: Chemical composition study of monkey earring extract 1. Extract preparation A suitable amount of dried monkey ear fungus leaves and tender branches (<2cm) were pulverized using a pulverizer and passed through a 24-mesh sieve. 5kg of the herbal powder was weighed and extracted with 95% ethanol at room temperature (7 days / time, 20L each time, 3 extractions). The extracted liquids were combined and evaporated under reduced pressure to obtain a total extract of 900g. The extract was dissolved and dispersed in an appropriate amount of water using ultrasound to form a suspension. The suspension was transferred to a 5L separatory funnel and extracted three times with petroleum ether to obtain 124.15g of the petroleum ether extract. The remaining suspension was extracted three times with ethyl acetate to obtain 562.10g of the ethyl acetate extract. Similarly, the remaining suspension was further extracted three times with n-butanol to obtain 158.10g of the n-butanol extract.

[0038] 2. Preparation of the test sample: Accurately weigh 0.1 g of the ethyl acetate extract of monkey ear ring, add methanol and sonicate until no obvious particles are visible, transfer to a 250 mL volumetric flask, and dilute to the mark. Filter the sample through a 0.22 μm filter membrane to obtain the final sample.

[0039] 3. Sample injection and detection Take the test solution and blank solvent (methanol) and perform UPLC-MS detection.

[0040] Chromatographic conditions: Waters ACQUITY UPLC BEH C18 column (150mm*2.1mm, 1.7μm); mobile phase: 0.1% formic acid acetonitrile (B) - 0.1% formic acid (A), gradient elution (0-40min, 5%-95% (v / v) B); flow rate: 0.3mL / min; column temperature: 40℃; injection volume: 2μL.

[0041] Table 1. UPLC-MS mobile phase gradient

[0042] 4. Experimental Results Based on database analysis and literature comparison, the main components are inferred as follows: Table 2 Main components of the ethyl acetate fraction of monkey earring extract

[0043] Compound 1: such as Figure 2 , 3 As shown, the quasi-molecular ion peak [MH] in negative ion mode - For m / z441.0806 (C 22 H 18 0 10 The retention time was 17.90 min. The fragment ion in the secondary mass spectrum was 289.0676 m / z, which was obtained by removing galloyl group (m / z 151.04) from monocyclonol I. Based on database analysis and literature comparison, it is speculated that it is monocyclonol I.

[0044] Compound 2: such as Figure 4 , 5 As shown, the quasi-molecular ion peak [MH] in negative ion mode - For m / z593.0905 (C 29 H 22 0 14 The retention time was 20.32 min. The fragment ions in the secondary mass spectrum were m / z 441.0799 and 289.0675, which were obtained by removing one or two galloyl groups (m / z 151) from monocyclocycline II. Based on database analysis and literature comparison, it is speculated that it is monocyclocycline II.

[0045] Compound 3: such as Figure 6 , 7 As shown, the quasi-molecular ion peak [MH] in negative ion mode - For m / z593.0907 (C 29 H 22 0 14 The retention time was 27.13 min. The fragment ions in the secondary mass spectrum were m / z 441.0795 and 289.0679, which were obtained by removing one or two galloyl groups (m / z 151) from monocyclocycline III. Based on database analysis and literature comparison, it is speculated that it is monocyclocycline III.

[0046] Example 2: Determination of the content of monkey earring extract 1. Extraction preparation: Pulverize an appropriate amount of dried monkey ear ring leaves and tender branches (<2cm) using a pulverizer and pass them through a 24-mesh sieve. Weigh 5kg of the herbal powder and extract it by soaking in 95% ethanol at room temperature (7 days / time, 20L each time, 3 extractions). Combine the obtained extracts and evaporate them under reduced pressure to obtain a total extract of 900g.

[0047] 2. Preparation of reference solution: Accurately weigh 2.5 mg of monocyclocarpine and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to the mark to prepare a solution with a mass concentration of 0.25 mg / mL. The sample is then filtered through a 0.22 μm filter membrane.

[0048] 3. Preparation of the test sample: Accurately weigh 1g of monkey earring extract, add methanol and sonicate until no obvious particles are visible, transfer to a 250mL volumetric flask and dilute to the mark. Filter the sample through a 0.22μm filter membrane to obtain the final sample.

[0049] 4. Sample injection and detection: Take the test solution, reference solution and blank solvent (methanol) and inject them for testing.

[0050] Chromatographic conditions: Agilent Eclipse XDB-C18 (4.6) The mobile phase consisted of methanol (A) and 0.1% formic acid water (B), with a flow rate of 0.8 mL / min, a detection wavelength of 278 nm, and an injection volume of 10 μl.

[0051] The elution procedure is shown in Table 3.

[0052] Table 3. Elution Procedure for Determination of Monkey Earring Content

[0053] 5. Test Results and Conclusions: Under these testing conditions, the results of the injection of the reference solution, the test solution, and the blank solvent are as follows: Figure 8 , 9 As shown in Figure 10, the chromatograms show that the blank solvent did not interfere with the detection of the analytes. The retention time of cyrtocycline I in the reference solution and the test solution was consistent, at 15 min. The retention times of cyrtocycline II and cyrtocycline III were 16 min and 26 min, respectively. The separation of each component was good, all greater than 1.5, indicating good separation under these conditions. The reference solution (cyrtocycline I 0.2500 mg / mL) was injected five times consecutively, and the peak areas were recorded. The RSD of the peak areas was 0.51%, indicating good injection repeatability of this method. The average contents of cyrtocycline II and cyrtocycline III were 0.17 g / g, 0.22 g / g, and 0.11 g / g, respectively.

[0054] Example 3: Determination of the content of monkey earring extract 1. Crush an appropriate amount of dried monkey ear flower using a pulverizer and pass it through a 24-mesh sieve. Weigh 50g of the herbal powder and extract it by soaking it in 50% ethanol at room temperature (1 day / time, 200mL each time, 3 extractions). Combine the resulting extracts and evaporate them under reduced pressure to obtain a total extract of 9.5g.

[0055] 2. Preparation of reference solution: Accurately weigh 2.5 mg of monocyclocarpine and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to the mark to prepare a solution with a mass concentration of 0.25 mg / mL. The sample is then filtered through a 0.22 μm filter membrane.

[0056] 3. Preparation of the test sample: Accurately weigh 1g of monkey earring extract, add methanol and sonicate until no obvious particles are visible, transfer to a 250mL volumetric flask and dilute to the mark. Filter the sample through a 0.22μm filter membrane to obtain the final sample.

[0057] 4. Sample injection and detection: Take the test solution, reference solution and blank solvent (methanol), inject them for detection, and use the same chromatographic conditions as in Example 2.

[0058] 5. Test Results and Conclusions: Under these testing conditions, the average contents of simian cyclophosphine I, simian cyclophosphine II, and simian cyclophosphine III were 0.23 g / g, 0.30 g / g, and 0.15 g / g, respectively.

[0059] Example 4: Determination of the content of monkey earring medicinal material 1. Preparation of the test solution: Pulverize an appropriate amount of dried monkey ear fungus leaves and tender branches (<2cm) using a pulverizer and pass through a 24-mesh sieve. Accurately weigh 1g of monkey ear fungus powder and place it in a stoppered conical flask. Accurately add 30mL of 50% ethanol, seal tightly, weigh, and sonicate for 30min. Cool. Weigh again, replenish the lost mass with solvent, shake well, filter through filter paper, and collect the filtrate. Filter through a 0.22μm filter membrane to obtain the final solution.

[0060] 2. Preparation of reference solution: Accurately weigh 2.5 mg of monocyclocarpine and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to the mark to prepare a solution with a mass concentration of 0.25 mg / mL. The sample is then filtered through a 0.22 μm filter membrane.

[0061] 3. Sample injection and detection: Take the test solution, reference solution and blank solvent (50% ethanol), inject them for detection, and use the same chromatographic conditions as in Example 2.

[0062] 4. Test Results and Conclusions: Under these testing conditions, the average contents of simian cyclophosphine I, simian cyclophosphine II, and simian cyclophosphine III were 42.26 mg / g, 53.88 mg / g, and 26.94 mg / g, respectively.

[0063] Example 5: Determination of the content of monkey earring granules 1. Preparation of test solution: Grind the monkey ear ring granules into fine powder, accurately weigh 0.5 g of the powder, place it in a stoppered conical flask, accurately add 30 mL of methanol, weigh the sample, sonicate for 30 min, cool, weigh the sample again, replenish the lost mass with methanol, shake well, filter, take the filtrate, and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0064] 2. Preparation of reference solution: Accurately weigh 2.5 mg of monocyclocarpine and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to the mark to prepare a solution with a mass concentration of 0.25 mg / mL. The sample is then filtered through a 0.22 μm filter membrane.

[0065] 3. Sample injection and detection: Take the test solution, reference solution and blank solvent, and inject them for detection. The chromatographic conditions are the same as in Example 2.

[0066] 4. Test Results and Conclusions: Under these testing conditions, the average contents of simian cyclophosphine I, simian cyclophosphine II, and simian cyclophosphine III were 62.55 mg / g, 70.21 mg / g, and 35.10 mg / g, respectively.

[0067] Example 6: Determination of the content of monkey earring oral liquid 1. Preparation of test solution: Accurately measure 2 mL of sample, place it in a 10 mL volumetric flask, add methanol to make up to volume, shake well, filter, and take the filtrate to obtain the test solution.

[0068] 2. Preparation of reference solution: Accurately weigh 2.5 mg of monocyclocarpine and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to the mark to prepare a solution with a mass concentration of 0.25 mg / mL. The sample is then filtered through a 0.22 μm filter membrane.

[0069] 3. Sample injection and detection: Take the test solution, reference solution and blank solvent, and inject them for detection. The chromatographic conditions are the same as in Example 2.

[0070] 4. Test Results and Conclusions: Under these testing conditions, the average contents of simian cyclophosphine I, simian cyclophosphine II, and simian cyclophosphine III were 407.99 μg / mL, 411.22 μg / mL, and 205.63 μg / mL, respectively.

[0071] Example 7: Linear Range Examination A series of standard solutions of different concentrations were prepared according to the method in Example 2, and each concentration of standard solution was injected three times. Linear regression was performed with peak area (Y) as the ordinate and compound concentration (X, mg / mL) as the abscissa. The linear equation for simian cyclophosphamide I was Y = 22266553X - 520415, r = 0.9994, and the linear range was 0.1 mg / mL to 1 mg / mL. The results show that the reference standard simian cyclophosphamide I exhibits good linearity within the linear range.

[0072] Example 8 Precision Test The reference solution from Example 2 was injected into the chromatograph and analyzed under the chromatographic conditions of Example 2. Five consecutive injections were performed, and the peak areas of the components were recorded. The RSD value of the peak area was 0.51%, indicating that the method has good precision.

[0073] Example 9 Repeatability Test Six test solutions were prepared according to the method in Example 2, injected into the chromatograph, and analyzed under the chromatographic conditions of Example 2. Each solution was injected twice. The control solution was injected under the same chromatographic conditions. The average contents of cyclodextrin I, cyclodextrin II, and cyclodextrin III obtained from the six test solutions prepared from the same monkey earring sample were 0.17 g / g, 0.22 g / g, and 0.11 g / g, respectively; the RSDs were 0.87%, 0.87%, and 1.17%, respectively. The RSDs of all three components were less than 2.0%, indicating that the method has good repeatability.

[0074] Table 4. Peak Area RSD in Repeatability Tests

[0075] Example 10 Stability Test The test solution from Example 2 was placed at room temperature for 0 h, 3 h, 6 h, 10 h, 12 h, and 24 h before being injected into a liquid chromatograph. The chromatographic conditions of Example 2 were used for detection, and the peak area RSD was calculated. The peak area RSDs of monocyclonone I, monocyclonone II, and monocyclonone III were 0.32%, 0.75%, and 1.91%, respectively, indicating that the prepared monocyclonone samples remained stable within 24 h.

[0076] Example 11 Accuracy Test The determination was performed using the standard spiking method. Approximately 0.5 g of the extract was accurately weighed and placed in conical flasks, for a total of nine portions. Three levels of spiking were performed: low, medium, and high levels, representing 50%, 100%, and 150% of the sample amount, respectively. Each concentration was repeated three times. The test solution was prepared according to the method in Example 2, injected into the liquid chromatograph, and detected under the chromatographic conditions of Example 2. The recovery rate was calculated using the following formula: R = (CB) / A × 100% In the formula, R is the recovery rate (%), C is the measured value after adding the standard substance (mg), B is the amount of the analyte in the sample (mg), and A is the mass of the added standard substance (mg).

[0077] The results show that the average recovery rate of monococcal I was 102.73% and the RSD was 1.09%, indicating that the method has good accuracy.

[0078] Table 5 Results of the recovery test

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting cephalosporin in monkey earring extract, characterized in that, The contents of 7-galloylteryl thiazoline, 7,3′-galloylteryl thiazoline and 7,4′-galloylteryl thiazoline in monkey earring samples were determined by high performance liquid chromatography. Includes the following steps: Prepare a test solution for monkey earring samples and a reference solution of 7-galloylterlithronane. The chromatographic conditions were as follows: the column was a column packed with octadecylsilane-bonded silica gel; mobile phase A was methanol, and mobile phase B was a 0.1%-0.3% formic acid aqueous solution. A gradient elution was used between mobile phases A and B, and the gradient elution program was as follows, where all mobile phase proportions were volume percentages: 0–8 min, mobile phase A linearly changed from 28%–32% to 36%–40%, and mobile phase B linearly changed from 68%–72% to 60%–64%; 8–18 min, mobile phase A was maintained at 36%–40%, and mobile phase B was maintained at 60%–64%; 18–34 min, mobile phase A linearly changed from 36%–40% to 44%–48%, and mobile phase B linearly changed from 60%–64% to 52%–56%. Inject the reference solution and the test solution into the liquid chromatograph separately, determine the peak area value and calculate the content according to the above chromatographic conditions.

2. The method for detecting cephalosporin in monkey earring extract according to claim 1, characterized in that, The monkey earring sample includes monkey earring medicinal materials, monkey earring extract, and a pharmaceutical composition containing monkey earring extract.

3. The method for detecting cephalosporin in monkey earring extract according to claim 1, characterized in that, The particle size of the chromatographic column is selected from 3.5 or 5 μm, the column length is selected from 100, 150 or 250 mm, and the inner diameter is selected from 2.1 or 4.6 mm.

4. The method for detecting cephalosporin in monkey earring extract according to claim 1 or 2, characterized in that, The chromatographic column was an Agilent ZORBAX Eclipse XDB-C18 column.

5. The method for detecting cephalosporin in monkey earring extract according to claim 1, characterized in that, The preparation method of the monkey earring sample test solution includes the following steps: The monkey earring sample was dissolved in an alcohol solvent to prepare the monkey earring extract test solution.

6. The method for detecting cephalosporin in monkey earring extract according to claim 5, characterized in that, The alcohol solvent is selected from methanol, methanol aqueous solution, ethanol or ethanol aqueous solution.

7. The method for detecting cephalosporin in monkey earring extract according to claim 1, characterized in that, The gradient elution program is as follows, where all mobile phase proportions are volume percentages: 0-8 min, mobile phase A linearly changes from 30% to 38%, and mobile phase B linearly changes from 70% to 62%; 8-18 min, mobile phase A is maintained at 38%, and mobile phase B is maintained at 62%; 18-34 min, mobile phase A linearly changes from 38% to 46%, and mobile phase B linearly changes from 62% to 54%.

8. The method for detecting cephalosporin in monkey earring extract according to claim 1, characterized in that, The chromatographic conditions also include a column temperature of 40°C.

9. The method for detecting cephalosporin in monkey earring extract according to claim 1, characterized in that, The chromatographic conditions also include a detection wavelength of 278 nm.

10. The method for detecting cephalosporin in monkey earring extract according to claim 1, characterized in that, The chromatographic conditions also include: a flow rate of 0.5-2 mL / min and an injection volume of 5 mL / min. 25μL.

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