Detection method of menadione and related substances thereof

Abstract

The invention provides a menadione and related substance detection method, which comprises: A) dissolving a to-be-detected raw material with ethanol to obtain a to-be-detected solution; b) determining the liquid to be detected by adopting a high performance liquid chromatography to obtain an HPLC chromatogram of menadione and related substances thereof; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 column; the mobile phase A is an aqueous solution, and the mobile phase B is a methanol or acetonitrile solution. According to the method, a high performance liquid chromatography is adopted, a water-methanol or acetonitrile solution is selected as a mobile phase for gradient elution, and reference substances of impurities A, J, K, L and M are taken as reference substances, so that the method for determining the contents of menadione and related substances thereof is established, and more scientific technical means are provided for controlling the quality of menadione.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting menatetrenone and related substances thereof. Background Art

[0002] Menatetrenone, also known as vitamin K2, is a naturally occurring vitamin that contains the basic structure of menaquinone and has anti-hemorrhagic effects. It has been on the market for many years, and its quality standards are included in the Japanese Pharmacopoeia and the United States Pharmacopoeia, but the relevant substances under the quality standards are all treated with isocratic methods, and known impurities are not controlled. When the control of known impurities is added, one of the known impurities completely overlaps with the main peak of menatetrenone, and other known impurities also have peak overlap, and the chromatographic conditions cannot meet the requirements of detection separation. Summary of the invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting menatetraquinone and its related substances. The present invention can well separate menatetraquinone and other impurities, and perform qualitative and quantitative analysis, and the detection method is accurate and reliable.

[0004] The present invention provides a method for detecting menatetrenone and its related substances, comprising the following steps:

[0005] A) dissolving the raw material to be tested in ethanol to obtain a solution to be tested;

[0006] B) the liquid to be tested is measured by high performance liquid chromatography to obtain an HPLC chromatogram of menatetrenone and its related substances;

[0007] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an aqueous solution, and the mobile phase B is a methanol or acetonitrile solution;

[0008] The gradient elution conditions were:

[0009] 0-5min, phase A: 60%→15%, phase B: 40→85%;

[0010] 5-50 min, phase A: 15%→2%, phase B: 85%→98%;

[0011] 50-52 min, phase A: 2%→0%, phase B: 98%→100%;

[0012] 52-70 min, phase A: 0%, phase B: 100%;

[0013] 70-71 min, phase A: 0%→60%, phase B: 100%→40%;

[0014] 71-80min, phase A: 60%, phase B: 40%.

[0015] The invention provides a method for detecting tetraene menatequinone and related substances thereof. Firstly, a raw material to be detected is dissolved in ethanol to obtain a solution to be detected.

[0016] The raw material to be tested in the present invention includes a menatetrenone preparation, and the present invention does not limit the dosage form of the preparation, such as tablets, pills, oral liquids, capsules, syrups, dripping pills or granules.

[0017] The present invention does not limit the specific operation of the dissolution, which is well known to those skilled in the art.

[0018] According to the present invention, the ratio of the mass g of the raw material to be tested to the volume mL of ethanol is 1:1000;

[0019] The above raw materials of the present invention can all be subjected to quality control and qualitative and quantitative detection by the method of the present invention.

[0020] The present invention also includes preparing a reference solution: taking menatetrenone, impurities A, J, K, L, and M respectively, dissolving them in ethanol to obtain a reference solution;

[0021] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of menatetrenone and its related substances are qualitatively determined according to the chromatogram of the reference;

[0022] According to the present invention, the specific structural formula of the impurity is as follows:

[0023]

[0024] Impurities J and K are enantiomers, and the total amount thereof is determined in the present invention; impurities L and M are enantiomers, and the total amount thereof can be determined in the present invention.

[0025] Specifically, the concentrations of the reference solution are as follows: impurity A is 0.0622 μg / ml to 12.4408 μg / ml; impurity J is 0.02 μg / ml to 0.5 μg / ml; impurity K is 0.02 μg / ml to 0.5 μg / ml; impurity L is 0.01 μg / ml to 0.5 μg / ml; impurity M is 0.01 μg / ml to 0.5 μg / ml.

[0026] The linear equation of impurity A is y=0.3700x+0.0265, and the linear correlation coefficient r is 0.9996.

[0027] The test solution is measured by high performance liquid chromatography to obtain an HPLC chromatogram of menatetrenone and its related substances.

[0028] The HPLC chromatographic conditions are:

[0029] The chromatographic column is a C18 column; the chromatographic column specification is C 18 250×4.6mm 3μm;

[0030] The column temperature is 30°C to 35°C, preferably 35°C.

[0031] The chromatographic peaks of the present invention are symmetrical in shape, have good separation and are more complete in peak emission at the above chromatographic column temperature.

[0032] The mobile phase A of the present invention is an aqueous solution, and the mobile phase B is a methanol or acetonitrile solution;

[0033] The gradient elution conditions were:

[0034] 0-5min, phase A: 60%→15%, phase B: 40→85%;

[0035] 5-50 min, phase A: 15%→2%, phase B: 85%→98%;

[0036] 50-52 min, phase A: 2%→0%, phase B: 98%→100%;

[0037] 52-70 min, phase A: 0%, phase B: 100%;

[0038] 70-71 min, phase A: 0%→60%, phase B: 100%→40%;

[0039] 71-80min, phase A: 60%, phase B: 40%.

[0040] The invention has good baseline separation under the elution gradient, good separation degree of menatetrenone and its impurities, and a stable baseline.

[0041] The flow rate of the mobile phase of the present invention is 0.8-1.2 mL / min; preferably 1.0 mL / min

[0042] The present invention found that the chromatographic peaks were well separated and the separation degree was moderate under the above flow rate, which is the most preferred solution.

[0043] The injection volume is 10 to 30 μL; preferably, the injection volume is 10 to 20 μL.

[0044] The detection wavelength of the present invention is 270nm.

[0045] The inventors found that the information content of the chromatographic peak at 270 nm is relatively large, and the peak area of ​​the chromatographic peak is relatively large.

[0046] The S / N of the quantitative limits of impurities J and K, impurities L and M, menatetrenone and impurity A of the present invention are 10.9, 13.1, 10.5 and 11.6 respectively, and the S / N of the detection limits are 3.6, 3.9, 3.5 and 4.6 respectively.

[0047] The present invention also provides a quality evaluation method for menatetrenone and its preparation, which uses the method described in the above technical solution to perform detection and analyze the detection results.

[0048] The present invention provides a method for detecting tetraene methyl quinone and its related substances, comprising the following steps: A) dissolving a raw material to be tested by ethanol to obtain a solution to be tested; B) determining the solution to be tested by high performance liquid chromatography to obtain an HPLC chromatogram of tetraene methyl quinone and its related substances; the chromatographic conditions of the high performance liquid chromatography are: a chromatographic column is a C18 column; mobile phase A is an aqueous solution, and mobile phase B is a methanol or acetonitrile solution. The present invention adopts high performance liquid chromatography, selects water-methanol or acetonitrile solution as the mobile phase for gradient elution, takes impurity A, J, K, L, M reference substances as reference substances, establishes a method for determining the content of tetraene methyl quinone and its related substances, and provides more scientific technical means for controlling the quality of tetraene methyl quinone. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The HPLC chromatogram of the contents of tetraene-methyl menatetraquinone and its related substances in Example 1 of the present invention.

[0050] Figure 2 This is the linear spectrum of impurity A. DETAILED DESCRIPTION

[0051] The present invention provides a method for detecting tetraene menaquinone and its related substances. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0052] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0053] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0054] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0055] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0056] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0057] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0058] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0059] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0060] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0061] In order to further illustrate the present invention, a method for detecting menatetrenone and its related substances provided by the present invention is described in detail below in conjunction with examples.

[0062] Example 1

[0063] Sample preparation

[0064] There are 3 batches of batch numbers of tetraenequinone raw materials: 20220305, 20220310, and 20220311.

[0065] Take an appropriate amount of tetraene quinone in the test solution, dissolve it in anhydrous ethanol or ethanol and dilute it to make a solution containing about 1 mg per 1 ml.

[0066] For the control solution, take an appropriate amount of tetraene quinone, add anhydrous ethanol or ethanol to dissolve and dilute to make a solution containing about 1 mg per 1 ml. Then accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with anhydrous ethanol, and shake well.

[0067] Impurity localization solution: Take appropriate amount of impurity A, J, K, L, and M reference substances respectively, weigh accurately, and quantitatively dilute them with anhydrous ethanol to make solutions of about 10 μg / ml for impurity A, about 1 μg / ml for impurity J, about 1 μg / ml for impurity K, about 10 g / ml for impurity L, and about 1 μg / ml for impurity M.

[0068] Chromatographic column: CAPCELL PAK C18 MG II, 4.6 mm × 250 mm, 3 μm

[0069] Column temperature: 35°C.

[0070] Flow rate: 1.0ml / min

[0071] Detection wavelength: 270nm

[0072] Injection volume: 20 μl

[0073] Mobile phase: water as mobile phase A, methanol as mobile phase B

[0074] gradient:

[0075]

[0076] The test results are shown in Table 1:

[0077] Table 1

[0078]

[0079] Table 2

[0080]

[0081]

[0082] Chromatogram Figure 1 As shown, Figure 1 The HPLC chromatogram of the contents of tetraene-methyl menatetraquinone and its related substances in Example 1 of the present invention.

[0083] Example 2

[0084] Take an appropriate amount of tetraene quinone in the test solution, dissolve it in anhydrous ethanol or ethanol and dilute it to make a solution containing about 1 mg per 1 ml.

[0085] For the control solution, take an appropriate amount of tetraene quinone, add anhydrous ethanol or ethanol to dissolve and dilute to make a solution containing about 1 mg per 1 ml. Then accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with anhydrous ethanol and shake well.

[0086] Impurity localization solution: Take appropriate amount of impurity A, J, K, L, and M reference substances respectively, weigh accurately, and quantitatively dilute them with anhydrous ethanol to make solutions of about 10 μg / ml for impurity A, about 1 μg / ml for impurity J, about 1 μg / ml for impurity K, about 10 g / ml for impurity L, and about 1 μg / ml for impurity M.

[0087] Chromatographic column: Agilent C18, 4.6mm×250mm, 3μm

[0088] Column temperature: 35°C.

[0089] Flow rate: 1.0ml / min

[0090] Detection wavelength: 270nm

[0091] Injection volume: 10 μl

[0092] Mobile phase: water as mobile phase A, methanol as mobile phase B

[0093] gradient:

[0094]

[0095] The test results are shown in Table 3:

[0096] Table 3

[0097]

[0098] Embodiment three:

[0099] Take an appropriate amount of tetraene quinone in the test solution, dissolve it in anhydrous ethanol or ethanol and dilute it to make a solution containing about 1 mg per 1 ml.

[0100] For the control solution, take an appropriate amount of tetraene quinone, add anhydrous ethanol or ethanol to dissolve and dilute to make a solution containing about 1 mg per 1 ml. Then accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with anhydrous ethanol and shake well.

[0101] Impurity localization solution: Take appropriate amount of impurity A, J, K, L, and M reference substances respectively, weigh accurately, and quantitatively dilute them with anhydrous ethanol to make solutions of about 10 μg / ml for impurity A, about 1 μg / ml for impurity J, about 1 μg / ml for impurity K, about 10 g / ml for impurity L, and about 1 μg / ml for impurity M.

[0102] Column: Welch C18, 4.6mm×250mm, 3μm

[0103] Column temperature: 35°C.

[0104] Flow rate: 0.9ml / min

[0105] Detection wavelength: 270nm

[0106] Injection volume: 30 μl

[0107] Mobile phase: water as mobile phase A, acetonitrile as mobile phase B

[0108] gradient:

[0109]

[0110]

[0111] The test results are shown in Table 4:

[0112] Table 4

[0113]

[0114] Embodiment 4:

[0115] Take an appropriate amount of tetraene quinone in the test solution, dissolve it in anhydrous ethanol or ethanol and dilute it to make a solution containing about 1 mg per 1 ml.

[0116] For the control solution, take an appropriate amount of tetraene quinone, add anhydrous ethanol or ethanol to dissolve and dilute to make a solution containing about 1 mg per 1 ml. Then accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with anhydrous ethanol and shake well.

[0117] Impurity localization solution: Take appropriate amount of impurity A, J, K, L, and M reference substances respectively, weigh accurately, and quantitatively dilute them with anhydrous ethanol to make solutions of about 10 μg / ml for impurity A, about 1 μg / ml for impurity J, about 1 μg / ml for impurity K, about 10 g / ml for impurity L, and about 1 μg / ml for impurity M.

[0118] Chromatographic column: CAPCELL C18, 4.6mm×250mm, 3μm

[0119] Column temperature: 30℃.

[0120] Flow rate: 1.2ml / min

[0121] Detection wavelength: 270nm

[0122] Injection volume: 30 μl

[0123] Mobile phase: water as mobile phase A, methanol as mobile phase B

[0124] gradient:

[0125]

[0126] The test results are shown in Table 5:

[0127] Table 5

[0128]

[0129] Verification Example

[0130] 1.1 Precision

[0131] Test solution: Take an appropriate amount of this product, dissolve it in anhydrous ethanol and dilute it to make a solution containing about 1.0 mg per 1 ml. Prepare 6 portions according to the method.

[0132] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with anhydrous ethanol, and shake well. Prepare 6 control solutions corresponding to the test solution according to the law.

[0133] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 6 below.

[0134] Table 6

[0135]

[0136] The results show that the deviations of the impurity contents and the total impurity contents (except impurity A) in the 6 test solutions from the average value are all within the range of ±0.03%. The precision of this method meets the requirements.

[0137] 2.1 Reproducibility (repeatability)

[0138] Test solution: Take an appropriate amount of this product, dissolve it in anhydrous ethanol and dilute it to make a solution containing about 1.0 mg per 1 ml. Prepare 6 portions according to the method.

[0139] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with anhydrous ethanol, and shake well. Prepare 6 control solutions corresponding to the test solution according to the law.

[0140] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 7 below.

[0141] Table 7

[0142]

[0143]

[0144] The results show that the deviations of the impurity contents and the total impurity contents (except impurity A) in the 6 test solutions from the average value are all within the range of ±0.03%. The precision of this method meets the requirements.

[0145] 3. Linearity and range

[0146] Linear stock solution: Take appropriate amount of impurity A reference substance, weigh accurately, dissolve in anhydrous ethanol and quantitatively dilute to make a mixed solution containing approximately 100 μg of impurity A per 1 ml.

[0147] Linear solution: Accurately measure 0.2ml, 0.5ml, 1.0ml, 1.2ml, and 1.5ml of the linear stock solution and place them in different 10ml volumetric flasks. Dilute to the scale with anhydrous ethanol and shake well. Use them as 20%, 50%, 100%, 120%, and 150% linear solutions respectively.

[0148] Accurately measure 20 μl of the linear solution and the quantitative limit solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. The results are shown in Table 8 below.

[0149] Table 8

[0150]

[0151] From the results, we can see that within the concentration range of impurity A of 0.0622 μg / ml to 12.4408 μg / ml (quantitative limit concentration to 1.24% of the test sample concentration), the linear equation is y=0.3700x+0.0265, and the linear correlation coefficient r is 0.9996, both of which meet the requirements.

[0152] 4. Accuracy

[0153] Reference stock solution, recovery rate stock solution: Take an appropriate amount of impurity A reference substance, weigh accurately, dissolve in anhydrous ethanol and quantitatively dilute to make a solution containing about 100 μg per 1 ml.

[0154] Reference substance solution: Accurately measure 1 ml of reference substance stock solution, place it in a 10 ml volumetric flask, dilute to the mark with anhydrous ethanol, and shake well.

[0155] Test solution: Take about 20 mg of tetraene menadione raw material, weigh accurately, place in a 20 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, shake well. Prepare 2 portions according to the method.

[0156] 50% recovery solution: Take about 20 mg of tetraene-methyl quinone raw material, weigh accurately, put it in a 20 ml volumetric bottle, add appropriate amount of anhydrous ethanol to dissolve, accurately add 1 ml of recovery stock solution, dilute to the scale with anhydrous ethanol, shake well. Prepare 3 portions according to the method.

[0157] 100% recovery solution: Take about 10 mg of tetraene-methyl quinone raw material, weigh accurately, put it in a 10 ml volumetric bottle, add appropriate amount of anhydrous ethanol to dissolve, accurately add 1 ml of recovery stock solution, dilute to the scale with anhydrous ethanol, shake well. Prepare 3 portions according to the method.

[0158] 120% recovery solution: Take about 10 mg of tetraene-methyl quinone raw material, weigh accurately, put it in a 10 ml volumetric bottle, add appropriate amount of anhydrous ethanol to dissolve, accurately add 1.2 ml of recovery stock solution, dilute to the scale with anhydrous ethanol, shake well. Prepare 3 portions according to the method.

[0159] 1% self-control solution: Accurately measure 1 ml of the test solution and recovery solution respectively, place them in different 100 ml volumetric flasks, dilute to the scale with anhydrous ethanol, and shake well.

[0160] Accurately measure 20 μl of each of the reference solution, test solution, recovery solution and 1% self-control solution, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 9 below.

[0161] Table 9

[0162]

[0163]

[0164] From the results, it can be seen that, calculated by the external standard method, the recovery rate of impurity A in 9 recovery solutions is 93.55% to 107.57%, the average recovery rate is 98.07%, and the RSD value is 4.11%, which is less than 6%.

[0165] 5. Limit of quantification and limit of detection

[0166] Impurity A, J, L stock solutions: Take appropriate amounts of impurity A, J, L reference substances respectively, weigh accurately, add anhydrous ethanol to dissolve and dilute to make solutions of about 100 μg / ml for impurity A, about 20 μg / ml for impurity J, and about 20 μg / ml for impurity L.

[0167] Quantitative limit solution of tetraenequinone and impurity A: Accurately measure appropriate amounts of tetraenequinone reference substance stock solution and impurity A stock solution, and quantitatively dilute them with anhydrous ethanol to prepare a mixed solution containing each component with a signal-to-noise ratio of approximately 10:1.

[0168] Detection limit solution of tetraene quinone and impurity A: Accurately measure 3 ml of the quantitative limit solution of tetraene quinone and impurity A, place it in a 10 ml volumetric flask, dilute to the scale with anhydrous ethanol, and shake well.

[0169] Quantitative limit solutions of impurities J, K, L and M: Accurately measure appropriate amounts of the impurity J stock solution and the impurity L stock solution, and quantitatively dilute them with anhydrous ethanol to prepare a mixed solution containing impurities J and impurity L with a signal-to-noise ratio of approximately 10:1.

[0170] Impurity J, K, L and M detection limit solution: Accurately measure 3 ml of impurity J, K, L and M quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with anhydrous ethanol, and shake well.

[0171] Accurately measure 20 μl of the quantitative limit solution and the detection limit solution, respectively, and inject them into the liquid chromatograph, and record the chromatograph. The quantitative limit solution is measured 6 times continuously, and the detection limit solution is measured 3 times continuously. The results are shown in Table 10 below.

[0172] Table 10

[0173]

[0174]

[0175] From the results, we can see that the S / N of the quantification limits of impurities J and K, impurities L and M, tetraene menadione and impurity A are 10.9, 13.1, 10.5 and 11.6 respectively, and the S / N of the detection limits are 3.6, 3.9, 3.5 and 4.6 respectively, which meet the requirements.

[0176] Comparative Examples 1 to 3

[0177]

[0178] The pretreatment is the same as in Example 1.

[0179] In the above two methods, the blank solvent does not interfere with the determination of tetraene methyl quinone related substances, the separation between the main peak of tetraene methyl quinone and the adjacent impurity peaks is greater than 1.5, the separation between the impurity peaks is greater than 0.8, and all component peaks emerge before 55 minutes. Comparative Example 2 has a long elution period.

[0180] Take 20 μl of the high-temperature destruction solution (take about 20 mg of tetraene quinone, accurately weigh it, put it in a 20 ml volumetric flask, destroy it at 90°C for 12 hours, add anhydrous ethanol to dissolve it and dilute it to the scale, and shake it well.) Inject 20 μl of it into the liquid chromatograph, and compare examples 1 and 3. The separation degree between the two high-temperature degradation impurity peaks at 31.917 min and 32.320 min in the high-temperature destruction solution of example 1 is 0.99, and the separation effect is poor.

[0181] In the high temperature destruction solution of Comparative Example 3, three high temperature degradation impurity peaks (37.617min, 38.087min and 38.453min) were separated, but the main peak was not eluted.

[0182] The methods of the above three cases are not better than the method of this patent.

[0183] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting menatetrenone and its related substances, characterized in that: The steps include: A) dissolving the raw material to be tested in ethanol to obtain a solution to be tested; B) the liquid to be tested is measured by high performance liquid chromatography to obtain an HPLC chromatogram of menatetrenone and its related substances; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an aqueous solution, and the mobile phase B is a methanol or acetonitrile solution; The gradient elution conditions were: 0-5min, phase A: 60%→15%, phase B: 40→85%; 5-50 min, phase A: 15% → 2%, phase B: 85% → 98%; 50-52 min, phase A: 2%→0%, phase B: 98%→100%; 52-70 min, phase A: 0%, phase B: 100%; 70-71 min, phase A: 0%→60%, phase B: 100%→40%; 71-80min, phase A: 60%, phase B: 40%.

2. The method according to claim 1, characterized in that The method also includes preparing a reference solution: taking tetraene methyl quinone, impurities A, J, K, L, and M, respectively, dissolving them in ethanol to obtain a reference solution; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of menatetrenone and its related substances are qualitatively determined according to the chromatogram of the reference; 3. The method according to claim 2, characterized in that The concentrations of the reference solution are as follows: impurity A is 0.0622 μg / ml to 12.4408 μg / ml; impurity J is 0.02 μg / ml to 0.5 μg / ml; impurity K is 0.02 μg / ml to 0.5 μg / ml; impurity L is 0.01 μg / ml to 0.5 μg / ml; impurity M is 0.01 μg / ml to 0.5 μg / ml.

4. The method according to claim 1, characterized in that: The chromatographic column is C 18 250×4.6mm3μm; column temperature is 30℃~35℃.

5. The method according to claim 4, characterized in that The flow rate of the mobile phase is 0.8-1.2 mL / min; the injection volume is 10-30 μL.

6. The method according to claim 4, characterized in that The detection wavelength is 270 nm.

7. The method according to claim 5, characterized in that The column temperature is 35° C.; the mobile phase flow rate is 1.0 mL / min, and the injection volume is 10 μL to 20 μL.

8. The method according to claim 2, characterized in that: The S / N of the quantitative limits of impurities J and K, impurities L and M, tetraene menaquinone, and impurity A are 10.9, 13.1, 10.5, and 11.6, respectively, and the S / N of the detection limits are 3.6, 3.9, 3.5, and 4.6, respectively.

9. The method according to claim 8, characterized in that The ratio of the mass g of the raw material to be tested to the volume mL of ethanol is 1:1000; The raw materials to be tested are menatetrenone and its impurities.

10. A method for evaluating the quality of menatetrenone and its preparation, comprising: performing detection by the method according to any one of claims 1 to 9 and analyzing the detection results.

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