Method for detecting cis-isomer in menadione medicine

Abstract

The invention provides a high performance liquid chromatography detection method for a cis-isomer in menadione, and the method adopts a silica gel column of which the surface is covalently bonded with amylose-tri (3, 5-dichlorophenyl carbamate) and a mobile phase consisting of normal hexane and an ethanol solvent. According to the method, the combination of a chromatographic column and a mobile phase is optimized for the first time, the cis-isomer in menadione is efficiently separated and accurately detected, the quality standard of menadione is improved, and the safety and effectiveness of drugs are ensured. The detection method is better in sensitivity and specificity, simple and convenient to operate, high in repeatability and suitable for quality control of menadione products.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and relates to a method for detecting cis-isomer impurities in a menatetrenone drug. Background Art

[0002] As human life expectancy increases, population aging has brought new problems to human health. Osteoporosis and the fractures it causes are ranked seventh among the most common diseases.

[0003] Menatetrenone is a compound belonging to the vitamin K2 family. Its unique structure gives it many unique biological activities. Compared with other vitamin K2 subtypes, menatetrenone is more easily absorbed and utilized in the human body, which makes it have great potential in maintaining health and preventing diseases. In recent years, more and more studies have shown that menatetrenone plays a key role in bone health, cardiovascular health, intestinal health and many other aspects. Among them, the role of menatetrenone in promoting bone health is particularly noteworthy. Studies have found that menatetrenone can effectively promote the absorption of calcium, thereby helping the body maintain strong bones. At the same time, menatetrenone can also reduce the risk of fractures, which is especially important for high-risk groups with reduced bone density such as the elderly and menopausal women.

[0004] In the production and application of tetraene methyl quinone, controlling its purity and detecting its impurity content are crucial to ensure efficacy and safety. The quality standards for vitamin K drugs in the Chinese Pharmacopoeia point out that the cis isomer, as a known impurity, is an important indicator for the quality control of this type of drug. Tetraene methyl quinone is an all-trans structure. Due to the characteristics of its spatial structure, the double bond at the 2nd position is more likely to change and form the cis isomer PZA. In contrast, the possibility of the double bond at the 6th position changing to form the cis isomer PZB is lower. Therefore, compared with PZA, the content of PZB in the finished product is lower, resulting in different difficulties in separating the two. The detection of tetraene methyl quinone cis isomers in the Japanese Pharmacopoeia adopts thin layer chromatography. However, this method has the following limitations: poor specificity, low precision, inability to accurately detect and quantify, and thus inability to effectively control the content of cis isomers in drugs. Therefore, a new method that is rapid, sensitive, accurate and suitable for the detection of tetraene methyl quinone cis isomers is urgently needed to better control the quality of drugs. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for detecting cis-isomer impurities in tetraene quinone drugs, particularly a method for detecting cis-isomer impurities PZA and PZB, aiming to improve the quality standard of tetraene quinone and ensure the safety and effectiveness of the drugs.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] A method for detecting cis isomers in tetraene menaquinone, wherein the method adopts high performance liquid chromatography detection, and the separation degree between each chromatographic peak is not less than 1.5;

[0008] The chromatographic conditions of the detection method are set as:

[0009] Chromatographic column: IE, specifications are 4.6mm×250mm, 5μm;

[0010] Mobile phase: a mixed solution of n-hexane and ethanol;

[0011] Column temperature: 30-40°C;

[0012] Flow rate: 0.1~1.0mL / min;

[0013] Injection volume: 5-20 μL;

[0014] Detection wavelength: 250~300nm.

[0015] According to an embodiment of the present invention, the cis isomers are PZA and PZB.

[0016]

[0017] According to an embodiment of the present invention, the method comprises the following steps:

[0018] (1) Instrument and chromatographic conditions:

[0019] Chromatographic column: Silica gel column with amylose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface IE, specifications are 4.6mm×250mm, 5μm;

[0020] Mobile phase: a mixed solution of n-hexane and ethanol;

[0021] Column temperature: 30-40°C;

[0022] Flow rate: 0.1~1.0mL / min;

[0023] Injection volume: 5-20 μL;

[0024] Detection wavelength: 250~300nm,

[0025] (2) Sample configuration:

[0026] Preparation of test solution: Take an appropriate amount of menatetrenone, weigh accurately, dissolve and dilute with diluent to make a solution containing about 0.5 mg per 1 mL;

[0027] Preparation of reference solution: Take an appropriate amount of menatetrenone reference substance, weigh accurately, dissolve with solvent and quantitatively dilute to make a solution containing about 0.75 μg per 1 mL;

[0028] Preparation of system suitability solution: Take 2.5 mg of PZA and PZB respectively, accurately weigh them, place them in a 25 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well; accurately measure 2 mL, place them in a 20 mL volumetric flask, dilute to the mark with diluent, shake well, as the stock solution; take about 10 mg of menatetrenone reference substance, accurately weigh them, place them in a 20 mL volumetric flask, accurately add 1.5 mL of stock solution, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready;

[0029] (4) Detection and calculation:

[0030] Accurately measure 20 μL of the system suitability solution and inject it into the liquid chromatograph. Then accurately measure 10 μL each of the control solution and the test solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0031] According to an embodiment of the present invention, the diluent is a mixed solution of n-hexane and ethanol, and the volume ratio is 90-99:10-1; preferably 98:2.

[0032] According to an embodiment of the present invention, the mobile phase is a mixed solution of n-hexane and ethanol in a volume ratio of 98:2.

[0033] According to an embodiment of the present invention, the column temperature is 35°C.

[0034] According to an embodiment of the present invention, the flow rate is 1.0 mL / min.

[0035] According to an embodiment of the present invention, the injection volume is 10 μL.

[0036] According to an embodiment of the present invention, the wavelength is 270 nm.

[0037] According to the embodiment of the present invention, the detection limit of the impurities is: calculated according to the main component external standard method, PZA should not exceed 0.3%, and PZB should not exceed 0.15%.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. Improve detection accuracy: Compared with traditional thin-layer chromatography, the high-performance liquid chromatography technology used in the present invention can more accurately quantify and separate the cis-isomers in tetraenylquinone, thereby providing more accurate drug quality control and improving the quality standard of tetraenylquinone.

[0040] 2. Improved separation efficiency: Through optimized chromatographic conditions, such as the use of a specific chromatographic column and mobile phase combination, the present invention can achieve complete separation of the cis-isomer from the main peak, solving the problem of incomplete separation in existing methods, thereby avoiding potential misjudgment and quality control risks.

[0041] 3. Easy and fast operation: The detection method described in the present invention is not only easy to operate, but also has a short reaction time and can quickly obtain the test results, which is very important for drug production and quality control, especially when the quality of the drug needs to be quickly judged. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the chromatogram of the separation of tetraene methyl quinone impurities in Example 1.

[0043] Figure 2 It is the chromatogram of the separation of tetraene methyl quinone impurities in Example 2.

[0044] Figure 3 It is the chromatogram of the separation of tetraene methyl quinone impurities in Example 3.

[0045] Figure 4 It is the chromatogram of the separation of tetraene methyl quinone impurities in Example 4.

[0046] Figure 5 It is the chromatogram of the separation of tetraene methyl quinone impurities in Example 5.

[0047] Figure 6 It is the chromatogram of the separation of tetraene methyl quinone impurities in Example 6.

[0048] Figure 7 This is the linear trend diagram of menatetrenone in Experimental Example 4. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto. Unless otherwise specified, the raw materials used can be obtained from the market or homemade.

[0050] Example 1

[0051] (1) Instruments and chromatographic conditions: Chromatographic column: Waters Nova-Pak SiLica (3.9×150 mm, 4 μm); column temperature: 30°C; flow rate: 1.0 mL / min; mobile phase: n-hexane, isocratic run for 30 min.

[0052] (2) Preparation of test solution: Take appropriate amounts of each impurity and menatetrenone, weigh accurately, and add isopropanol to make a solution containing approximately 0.1 mg of PZA, PZB and menatetrenone per 1 mL.

[0053] (3) Detection: Accurately measure 20 μL of the test solution and inject it into the liquid chromatograph, detect at a wavelength of 270 nm, and record the chromatogram.

[0054] The results are as follows Figure 1 As shown in the figure, the sample showed two chromatographic peaks at about 2.5 min, the front peak was PZA, and the back peak was a mixed peak of PZB and tetraene methyl quinone. PZB and tetraene methyl quinone could not be separated. That is, the chromatographic column could not separate PZB from the main peak. Considering that PZB contains benzene rings in its structure, we tried to use a phenyl column with the reverse phase method.

[0055] Example 2

[0056] (1) Instrument and chromatographic conditions: Chromatographic column: AgiLent Poroshell 120PFP (4.6×100 mm, 2.7 μm); Column temperature: 40°C; Flow rate: 1.0 mL / min; Mobile phase: Mobile phase A is water, mobile phase B is acetonitrile, gradient elution according to Table 1 below:

[0057] Table 1

[0058] Time (min) A％ B％ 0 50 50 10 50 50 30 0 100 35 0 100 35.1 50 50 40 50 50

[0059] (2) Preparation of test solution: Take appropriate amounts of each impurity and menatetrenone, weigh accurately, and add isopropanol to make a solution containing approximately 20 μg each of PZA and PZB and approximately 40 μg of menatetrenone per 1 mL.

[0060] (3) Detection: Accurately measure 20 μL of the test solution and inject it into the liquid chromatograph, detect at a wavelength of 270 nm, and record the chromatogram.

[0061] The results are as follows Figure 2 As shown in the figure, PZA has peaked at 21.844, and the sample has two chromatographic peaks at about 24 minutes, and the peak shape is poor and only weakly separated. That is, under this condition, PZB and menatetrenone are still not effectively separated. The experiment shows that the reverse phase method can only separate two peaks, but the separation effect is poor, so try to explore with the normal phase method.

[0062] Example 3

[0063] (1) Instruments and chromatographic conditions: Chromatographic column: CHIRALCEL OD-H (4.6×250 mm, 5 μm); column temperature: 30°C; flow rate: 1.0 mL / min; mobile phase: n-hexane: n-butyl ether: ethanol: n-pentanol = 950:50:1:0.5, isocratic operation for 30 min.

[0064] (2) Preparation of test solution: Take appropriate amounts of each impurity and menatetrenone, weigh accurately, and add isopropanol to make a solution containing approximately 0.1 mg of PZA, PZB and menatetrenone per 1 mL.

[0065] (3) Detection: Accurately measure 20 μL of the test solution and inject it into the liquid chromatograph, detect at a wavelength of 270 nm, and record the chromatogram.

[0066] The results are as follows Figure 3 As shown, the sample has 4 chromatographic peaks, peak 1 is an impurity peak, peaks 2, 3, and 4 are PZA, PZB, and tetraene menadione peaks, respectively. At this concentration, the separation degree between peak 2 and peak 3 is 1.79, and the separation degree between peak 3 and peak 4 is 1.88. The separation degree is good, and the sample is detected by this method.

[0067] Example 4

[0068] (1) Instruments and chromatographic conditions: Chromatographic column: CHIRALCEL OD-H (4.6×250 mm, 5 μm); column temperature: 30°C; flow rate: 1.0 mL / min; mobile phase: n-hexane: n-butyl ether: ethanol: n-pentanol = 950:50:1:0.5, isocratic operation for 30 min.

[0069] (2) Preparation of test solution: Weigh an appropriate amount of menatetrenone, dissolve it in n-hexane and quantitatively dilute it to make a solution containing approximately 0.1 mg per 1 mL.

[0070] (3) Detection: Accurately measure 20 μL of the test solution and inject it into the liquid chromatograph, detect at a wavelength of 270 nm, and record the chromatogram.

[0071] The results are as follows Figure 4 As shown in the figure, this method detects sample P-24021901, the main peak is split and the peak is wide, wrapping PZB. Therefore, this method is not suitable for the detection of tetraene menaquinone samples. Try to change the chromatographic column. Considering that the sample itself has good ethanol solubility and is easy to elute, n-hexane and ethanol are used as the corresponding eluent and mobile phase.

[0072] Example 5

[0073] (1) Instruments and chromatographic conditions: Chromatographic column: Silica gel column with amylose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface IE, specification: 4.6mm×250mm, 5μm; column temperature: 35℃; flow rate: 1.0mL / min; mobile phase: n-hexane-ethanol (98:2).

[0074] (2) Preparation of test solution: Take an appropriate amount of menatetrenone, weigh accurately, dissolve and dilute with a diluent to make a solution containing approximately 0.5 mg per 1 mL.

[0075] (3) Preparation of system suitability solution: Take 2.5 mg of PZA and PZB, accurately weigh them, place them in a 25 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well; accurately measure 2 mL, place them in a 20 mL volumetric flask, dilute to the mark with diluent, shake well, and use it as the stock solution. Take about 10 mg of menatetrenone reference substance, accurately weigh it, place it in a 20 mL volumetric flask, accurately add 1.5 mL of the stock solution, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0076] (4) Detection: Accurately measure 5 μL of the system suitability solution and inject it into the liquid chromatograph. Then accurately measure 5 μL of the test solution and inject it into the liquid chromatograph. Record the chromatogram.

[0077] The results are as follows Figure 5 It showed that the test samples PZA and PZB were well separated from the main peak, and the sensitivity and response were good. This method was used as the final method to detect samples.

[0078] Example 6

[0079] (1) Instruments and chromatographic conditions: Chromatographic column: Silica gel column with amylose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface IE, specification: 4.6mm×250mm, 5μm; column temperature: 35℃; flow rate: 1.0mL / min; mobile phase: n-hexane-ethanol-diethylamine (98:2),

[0080] (2) Preparation of test solution: Take an appropriate amount of menatetrenone, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 0.5 mg per 1 mL.

[0081] (3) Preparation of reference solution: Take an appropriate amount of menatetrenone reference substance, weigh it accurately, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 0.75 μg per 1 mL.

[0082] (4) Preparation of system suitability solution: Take 2.5 mg each of PZA and PZB, accurately weigh them, place them in a 25 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well; accurately measure 2 mL, place them in a 20 mL volumetric flask, dilute to the mark with diluent, and shake well to prepare the stock solution.

[0083] Take about 10 mg of tetraene-methyl menatequinone reference substance, weigh it accurately, put it in a 20 mL volumetric flask, accurately add 1.5 mL of stock solution, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0084] (5) Detection: Accurately measure 20 μL of the system suitability solution and inject it into the liquid chromatograph. Then accurately measure 10 μL of the control solution and the test solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0085] The results are as follows Figure 6 As shown, this method was used to detect sample P-24032101. Under this method, PZA, PZB and the main peak were well separated. This method is suitable for the detection of tetraene menatetraquinone samples.

[0086] Test Example 1 Specificity

[0087] Solution preparation: Blank solution / diluent: n-hexane-ethanol (98:2), measure 980 mL of n-hexane and 20 mL of ethanol, mix, and shake well to obtain.

[0088] PZA / PZB stock solution: Take about 10 mg of PZA / PZB reference substance, weigh accurately, place in a 100 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0089] PZA / PZB positioning solution: Accurately measure 2 mL of PZA / PZB stock solution, place it in a 20 mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0090] System suitability solution: Take about 10 mg of tetraene menadione, weigh accurately, put it in a 20 mL volumetric flask, accurately add 1.5 mL of PZA / PZB positioning solution, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0091] Test solution: Take about 10 mg of tetraene menadione, weigh accurately, place in a 20 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0092] The above solutions were respectively injected into the liquid chromatograph to investigate whether the blank solvent / diluent interfered with the main peak and PZA / PZB, and the separation between PZA / PZB and the main peak.

[0093] Test Example 2 Injection Precision

[0094] Solution preparation: Blank solution / diluent: n-hexane-ethanol (98:2), measure 980 mL of n-hexane and 20 mL of ethanol, mix, and shake well to obtain.

[0095] Reference solution: Take about 2.5 mg of tetraene quinone reference substance, accurately weigh it, place it in a 25 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well; accurately measure 2 mL, place it in a 20 mL volumetric flask, dilute to the scale with diluent, shake well; accurately measure 1.5 mL, place it in a 20 mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0096] The above solutions were respectively injected into the liquid chromatograph to examine the repeatability of this method.

[0097] Test Example 3 Limit of Quantitation and Limit of Detection

[0098] Solution preparation: blank solution / diluent and PZA / PZB stock solution were prepared in the same manner as in Experimental Example 1.

[0099] Quantitative limit solution: Accurately measure an appropriate amount of PZA / PZB stock solution and dilute it step by step with diluent until the S / N of each component peak is ≥ 10. (Reference preparation method: Accurately measure 1 mL of PZA / PZB positioning solution, place it in a 100 mL volumetric bottle, dilute it to the scale with diluent, shake well, and the solution is ready.)

[0100] Detection limit solution: Accurately measure 3 mL of the quantitative limit solution, place it in a 10 mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0101] Test Example 4 Linearity and Range

[0102] Solution preparation: Blank solution / diluent were prepared in the same manner as in Test Example 1.

[0103] Linear stock solution: Take about 10 mg of PZA / PZB and tetraene quinone reference substance, place them in the same 100 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0104] Linear solution -0.02%: Accurately measure 1 mL of linear solution -0.4% and place it in a 20 mL volumetric flask. Add diluent to dilute to the scale and shake well.

[0105] Linear solution -0.2%: Accurately measure 1 mL of linear stock solution, place in a 100 mL volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0106] Linear solution -0.4%: Accurately measure 2 mL of linear stock solution, place in a 100 mL volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0107] Linear solution -1%: Accurately measure 1 mL of linear stock solution, place in a 20 mL volumetric flask, add diluent to dilute to the scale, shake well, and the solution is ready.

[0108] Linear solution -2%: Accurately measure 2 mL of linear stock solution, place in a 20 mL volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0109] Linear solution -3%: Accurately measure 3 mL of linear stock solution, place it in a 20 mL volumetric flask, add diluent to dilute to the scale, shake well, and the solution is ready.

[0110] The above linear solutions of different concentrations were respectively injected into the liquid chromatograph to examine the linear relationship between concentration and peak response.

[0111] Test Example 5 Accuracy

[0112] Solution preparation: blank solution / diluent, reference solution, PZA / PZB stock solution, PZA / PZB positioning solution, and test solution were prepared in the same manner as in Test Example 1 and Test Example 2.

[0113] Quantitative limit accuracy solution: Take about 10 mg of tetraene menadione, weigh accurately, put it in a 20 mL volumetric flask, accurately add 0.2 mL of PZA / PZB positioning solution, add diluent to dissolve and dilute to the scale, shake well, and prepare 3 copies in parallel.

[0114] 0.15% limit accuracy solution: Take about 10 mg of tetraene menadione, weigh accurately, put into a 20 mL volumetric flask, accurately add 1.5 mL of PZA / PZB positioning solution, add diluent to dissolve and dilute to the scale, shake well, and prepare 3 copies in parallel.

[0115] 0.3% limit accuracy solution: Take about 10 mg of tetraene menadione, weigh accurately, put into a 20 mL volumetric flask, accurately add 3 mL of PZA / PZB positioning solution, add diluent to dissolve and dilute to the scale, shake well, and prepare 3 copies in parallel.

[0116] 3% limit accuracy solution: Take about 10 mg of tetraene menadione, weigh accurately, put into a 20 mL volumetric flask, accurately add 3 mL of PZA / PZB stock solution, add diluent to dissolve and dilute to scale, shake well, and prepare 3 copies in parallel.

[0117] Test Example 6 Repeatability

[0118] Solution preparation: Blank solution / diluent and reference solution were prepared in the same manner as in Test Example 1 and Test Example 2.

[0119] Spiked test solution: Prepare the same solution as the 0.15% limit accuracy solution in Test Example 5. Prepare 6 copies in parallel.

[0120] Take blank solution, control solution and spiked test solution and inject them into liquid chromatograph respectively.

[0121] Test Example 7 Intermediate Precision

[0122] Solution preparation: Prepare 6 portions of spiked test sample solutions according to the solution preparation method under Test Example 6.

[0123] Take blank solution, control solution and spiked test solution and inject them into liquid chromatograph respectively.

[0124] Test Example 8 Solution Stability

[0125] Solution preparation: Blank solution / diluent and reference solution were prepared as in Test Examples 1 and 2. Spiked test solution was prepared as in Test Example 6.

[0126] Take blank solution, control solution and spiked test solution and inject them into liquid chromatograph respectively.

[0127] Test Example 9 Durability

[0128] Solution preparation: Blank solution / diluent and reference solution were prepared as in Test Examples 1 and 2. Spiked test solution was prepared as in Test Example 6.

[0129] The flow rate (range: ±0.1 mL / min), column temperature (range: ±2°C), mobile phase ratio (minimum relative change of component ratio ±2%), detection wavelength (±2 nm) and other conditions were fine-tuned to examine the durability of the method.

[0130] Take blank solution, control solution and spiked test solution and inject them into liquid chromatograph respectively.

[0131] The present invention verifies the detection method from the perspectives of specificity, injection precision, quantitative limit and detection limit, linearity and range, accuracy, repeatability, intermediate precision, solution stability and durability. The results are shown in Table 2, indicating that the method can be used for the detection of cis-isomers of tetraenylmenadione.

[0132] Table 2

[0133]

[0134]

Claims

1. A method for detecting cis isomers in menatetrenone, characterized in that: The method adopts high performance liquid chromatography detection, and the separation degree between each chromatographic peak is not less than 1.5; The chromatographic conditions of the detection method are set as: Chromatographic column: IE, specifications are 4.6mm×250mm, 5μm; Mobile phase: a mixed solution of n-hexane and ethanol; Column temperature: 30-40°C; Flow rate: 0.1~1.0mL / min; Injection volume: 5-20 μL; Detection wavelength: 250~300nm.

2. The method according to claim 1, characterized in that: The cis isomers are PZA, PZB 3. The method according to claim 1, characterized in that The following steps are involved: (1) Instrument and chromatographic conditions: Chromatographic column: Silica gel column with amylose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface IE, specifications are 4.6mm×250mm, 5μm; Mobile phase: a mixed solution of n-hexane and ethanol; Column temperature: 30-40°C; Flow rate: 0.1~1.0mL / min; Injection volume: 5-20 μL; Detection wavelength: 250~300nm, (2) Sample configuration: Preparation of test solution: Take an appropriate amount of menatetrenone, weigh accurately, dissolve and dilute with diluent to make a solution containing about 0.5 mg per 1 mL; Preparation of reference solution: Take an appropriate amount of menatetrenone reference substance, weigh accurately, dissolve with solvent and quantitatively dilute to make a solution containing about 0.75 μg per 1 mL; Preparation of system suitability solution: Take 2.5 mg of PZA and PZB respectively, accurately weigh them, place them in a 25 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well; accurately measure 2 mL, place them in a 20 mL volumetric flask, dilute to the mark with diluent, shake well, as the stock solution; take about 10 mg of menatetrenone reference substance, accurately weigh them, place them in a 20 mL volumetric flask, accurately add 1.5 mL of stock solution, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready; (3) Detection and calculation: Accurately measure 20 μL of the system suitability solution and inject it into the liquid chromatograph. Then accurately measure 10 μL each of the control solution and the test solution and inject them into the liquid chromatograph respectively, and record the chromatogram.

4. The method according to claim 3, characterized in that The diluent is a mixed solution of n-hexane and ethanol, with a volume ratio of 90-99:10-1; preferably 98:

2.

5. The method according to claim 3, characterized in that: The mobile phase is a mixed solution of n-hexane and ethanol with a volume ratio of 98:

2.

6. The method according to claim 3, characterized in that The column temperature was 35°C.

7. The method according to claim 3, characterized in that The flow rate was 1.0 mL / min.

8. The method according to claim 3, characterized in that The injection volume was 10 μL.

9. The method according to claim 3, characterized in that: The wavelength is 270 nm.

10. The method according to claim 3, characterized in that: The impurity detection limits are: calculated according to the main component external standard method, PZA should not exceed 0.3%, and PZB should not exceed 0.15%.

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