Liquid phase detection method for related substances of nafamostat mesylate

Abstract

The invention discloses a detection method for separating and detecting related substances of nafamostat mesylate, and belongs to the technical field of medicine detection.A specific sample pretreatment method is adopted for treating a sample to be detected, and a specific method liquid chromatography is adopted for liquid detection; the detection method comprises the following steps: carrying out gradient elution under a certain condition by adopting an octadecyl silane bonded silica gel chromatographic column and taking a sodium heptanesulfonate solution and an organic phase as mobile phases, can effectively separate, analyze and detect nafamostat mesilate and six impurities thereof, and is convenient, simple, high in sensitivity, good in separation degree and accurate in result; the method can be used for detecting related substances in nafamostat mesylate.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and relates to a detection method for separating and detecting nafamostat mesylate related substances. Background Art

[0002] Nafamostat mesylate is chemically named 4-guanidinobenzoic acid-6-amidino-2-naphthyl ester-dimesylate, and its molecular formula is C 19 H 17 N5O 22 CH4O3S, molecular weight of 539.58, CAS number 82956-11-4, this product is a broad-spectrum, effective protease inhibitor, with strong selective inhibition of thrombin, trypsin, kallikrein, plasmin and trypsin-like serine proteases of the classical pathway of the complement system, and also has an inhibitory effect on phospholipase A2. It also has an in vitro inhibitory effect on trypsin bound to α2-macroglobulin, just like free trypsin, and is used to treat acute pancreatitis, acute exacerbation of chronic pancreatitis, disseminated intravascular coagulation, and prevent blood coagulation during extracorporeal circulation. It can be synthesized from p-guanidinobenzoic acid, 6-cyano-2-naphthol and other pathways, and the access routes are simple and diverse, opening up new uses in the pharmaceutical industry. Since it is mainly used in oral preparations and injections in medicine, it is more important to establish a set of precise quality control methods to control product quality.

[0003] In the prior art, there are few detection methods for nafamostat mesylate. At present, regarding the detection of related substances of nafamostat mesylate, after consulting the literature, only the Japanese Pharmacopoeia (JP) has included nafamostat mesylate and controlled the related substances therein, but the Pharmacopoeia only controls the separation of degradation impurities 3 and the main component, and the remaining 5 impurities cannot be effectively controlled and separated, and miscellaneous peaks are prone to appear near the main peak, which is difficult to separate, and is prone to peak tailing or incomplete impurity separation. In order to further ensure the quality of drugs and prevent drug safety, this method is developed to establish strict quality standards, control impurities within a safe limit, and achieve controllable drug quality, which is of great significance to ensuring the safe production of drugs. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a simple, efficient and suitable high performance liquid chromatography method for nafamostat mesylate related substances, wherein the chromatographic column stationary phase is an octadecylsilane bonded silica gel chromatographic column, the mobile phase is composed of phase A and phase B, phase A is a sodium heptane sulfonate solution, phase B is acetonitrile, and elution is performed according to a gradient program, and the high performance liquid chromatography method is equipped with an ultraviolet detector.

[0005] In the preferred technical scheme of the present invention, the octadecylsilane bonded silica gel chromatographic column is commercially available, preferably an octadecylsilane bonded silica gel chromatographic column produced by Thermofishier, Waters, Agilent, Kromasil, and Agela, preferably a series produced by Thermofishier and Waters, more preferably Waters.

[0006] In the preferred technical scheme of the present invention, the octadecylsilane bonded silica gel chromatographic column is selected from any one of Kromasi1100-5-C18, YMC Triat C18, Phenomenex kinetex C18, Titank C18 ES-C18, EpicC18, ZORBAXSB-C18, ZORBAX 300SB-C18, XDB-C18, Waters XBridge C18, Venusil HLPC C18, Venusil C18Plus, Venusil MP C18, AgilentMicrospherC18, and Thermofishier Hypersil GOLD C18 or a combination thereof.

[0007] In the preferred technical solution of the present invention, the preferred solution of the octadecylsilane bonded silica gel chromatographic column is any one of Waters XBridge C18 and Thermofishier Hypersil GOLD C18.

[0008] In the preferred technical solution of the present invention, the preferred solution of the octadecylsilane bonded silica gel chromatographic column is Thermofishier Hypersil GOLD C18 4.6mm×250mm, 5μm.

[0009] In the preferred technical solution of the present invention, the column temperature of the chromatographic column is 35-45°C, preferably 37-43°C, and more preferably 39-41°C.

[0010] In the preferred technical solution of the present invention, the sample preparation solvent is acetonitrile, acetone, water or a mixed solution thereof in different proportions, preferably acetone: water (V:V) = 30:70 and acetonitrile: water (V:V) = 20:80.

[0011] In the preferred technical solution of the present invention, the mobile phase A is a sodium heptane sulfonate solution, which is prepared by weighing 6.07 g of 1-sodium heptane sulfonate, adding an appropriate amount of water to dissolve it, adding 6 ml of acetic acid, and diluting it with water to 1000 ml.

[0012] In the preferred technical solution of the present invention, the flow rate of the mobile phase is 0.8 ml / min-1.2 ml / min, preferably 0.9 ml / min-1.1 ml / min, and more preferably 1.0 ml / min.

[0013] In the preferred technical solution of the present invention, the detection wavelength of the ultraviolet detector is 245nm-265nm, preferably 250nm-260nm, and more preferably 254nm.

[0014] In the preferred technical solution of the present invention, the injection volume is 5 μl-10 μl, preferably 10 μl.

[0015] In the preferred technical solution of the present invention, the related substance is selected from any one of impurities 1 to 6 or a combination thereof.

[0016]

[0017]

[0019] In the preferred technical solution of the present invention, the order of the impurity peaks of the related substances is impurity 2, impurity 3, impurity 5, impurity 4, impurity 6, impurity 1, and nafamostat mesylate.

[0020] In the preferred technical solution of the present invention, the high performance liquid chromatography method contains an elution sample, and the elution sample includes any one or a combination of a reference solution, a test solution and a separation solution.

[0021] In the preferred technical solution of the present invention, the concentration of the test solution is 0.2 mg / ml-1 mg / ml, preferably 0.4 mg / ml-0.8 mg / ml, and more preferably 0.5 mg / ml.

[0022] In the preferred technical solution of the present invention, the high performance liquid chromatography method comprises the following steps:

[0023] 1) Solution preparation:

[0024] Solvent: acetonitrile:water (V:V) = 20:80 and acetone:water (V:V) = 30:70.

[0025] Localization solution: Take an appropriate amount of each impurity reference substance, dissolve it in acetonitrile: water (V:V) = 20:80 and dilute it to prepare a single localization solution with a concentration of 500 μg / ml.

[0026] Mixed separation solution: Take appropriate amounts of each impurity location solution and nafamostat mesylate reference substance, dissolve and dilute with acetone: water (V:V) = 30:70 to prepare a mixed solution with each impurity concentration of 0.5 μg / ml and nafamostat mesylate concentration of 0.5 mg / ml.

[0027] Test solution: Take an appropriate amount of nafamostat mesylate, dissolve and dilute with acetone: water (V:V) = 30:70 to prepare a test solution with a concentration of 0.5 mg / ml.

[0028] 2) Chromatographic conditions:

[0029] Octadecylsilane bonded silica gel column Thermofishier Hypersil GOLD C18

[0030] 4.6mm×250mm, 5μm; use sodium heptane sulfonate solution (weigh 6.07g of 1-heptane sulfonate sodium, add appropriate amount of water to dissolve, add 6ml of acetic acid, and dilute with water to 1000ml) as mobile phase A; use acetonitrile as mobile phase B, flow rate is 1.0ml per minute, linear gradient elution, detection wavelength is 254nm; column temperature is 40℃. Accurately measure 10μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the HPLC chromatogram of the mixed separation solution;

[0032] Figure 2 This is the HPLC chromatogram of the nafamostat mesylate test product;

[0033] Figure 3 is the linear spectrum of nafamostat mesylate impurity 1;

[0034] Figure 4 is the linear spectrum of nafamostat mesylate impurity 2;

[0035] Figure 5 is the linear spectrum of nafamostat mesylate impurity 3;

[0036] Figure 6 is the linear spectrum of nafamostat mesylate impurity 4;

[0037] Figure 7 is the linear spectrum of nafamostat mesylate impurity 5;

[0038] Figure 8 is the linear spectrum of nafamostat mesylate impurity 6;

[0039] Fig. 9 This is the linear spectrum of nafamostat mesylate. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention will be described in detail below. The test methods for specific conditions not specified in the preferred embodiments are usually carried out under conventional conditions. The described embodiments are only part of the embodiments of the present invention, which can better illustrate the contents of the present invention, but are not all embodiments. Based on the embodiments of the present invention, non-essential improvements and adjustments made by technicians in this field to the implementation scheme are all within the scope of protection of the present invention.

[0041] Example 1 Sample detection

[0042] Instrument and HPLC conditions:

[0043] High performance liquid chromatography: Thermo Vanquish Core high performance liquid chromatography system and workstation;

[0044] Chromatographic column: Thermofishier Hypersil GOLD C18, (250 mm × 4.6 mm, 5 μm) octadecylsilane bonded silica gel chromatographic column;

[0045] Mobile phase: Sodium heptane sulfonate solution (weigh 6.07 g of 1-sodium heptane sulfonate, add appropriate amount of water to dissolve, then add 6 ml of acetic acid, and dilute with water to 1000 ml) is used as mobile phase A; acetonitrile is used as mobile phase B.

[0046] Elution procedure:

[0047]

[0048] Detection wavelength: 254nm;

[0049] Flow rate: 1.0ml / min;

[0050] Column temperature: 40℃.

[0051] Experimental steps:

[0052] Take an appropriate amount of nafamostat mesylate raw material, dissolve it in acetone: water (V:V) = 30:70 and dilute it into a 0.5 mg / ml solution as the test solution.

[0053] Take an appropriate amount of the test solution, dilute it 200 times with 30% acetone aqueous solution, shake well, and use it as the control solution.

[0054] Take an appropriate amount of nafamostat mesylate reference substance, dissolve it in acetone: water (V:V) = 30:70, add an appropriate amount of impurity 1 location solution and dilute to a mixed solution of nafamostat mesylate 0.5 mg / ml and impurity 1 0.5 μg / ml as the system suitability solution.

[0055] Take an appropriate amount of nafamostat mesylate, dissolve it with a solvent and dilute it into a 0.5 mg / ml solution as the test solution.

[0056] Take 10 μl of each of the above solutions, inject them into liquid chromatograph, record the chromatogram, and the results are shown in the attached figure.

[0057] Example 2 Method Verification

[0058] Instrument and HPLC conditions: Same as Example 1

[0059] 1. Exclusivity

[0060] Experimental steps:

[0061] Accurately weigh appropriate amounts of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, and impurity 6, dissolve and dilute them into reference substance stock solutions containing 0.1 mg / ml each using acetonitrile:water (V:V) = 20:80.

[0062] Accurately weigh appropriate amounts of reference substance stock solutions of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, and impurity 6, and dissolve and dilute them into individual localization solutions each containing 0.5 μg / ml using acetone: water (V:V) = 30:70.

[0063] Accurately weigh an appropriate amount of nafamostat mesylate reference substance, add an appropriate amount of each impurity stock solution, dissolve and dilute with acetone: water (V:V) = 30:70 to form a mixed solution containing 0.5 mg / ml of nafamostat mesylate and 0.5 μg / ml of each of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, and impurity 6 as the test solution.

[0064] Take 10 μl of each positioning solution and test solution, inject them into the liquid chromatograph, record the chromatogram, and the results are shown in the table.

[0065]

[0066] The results showed that each impurity and nafamostat mesylate could be effectively detected, the separation between each impurity was better than 1.3, the separation between adjacent impurity peaks of nafamostat mesylate was greater than 1.5, the method of the present invention had good sensitivity and the theoretical plate number of the nafamostat mesylate peak was greater than 10000. The test results showed that the method had good system applicability.

[0067] 2. Limit of detection and limit of quantification

[0068] The impurity reference stock solution was diluted stepwise. The quantification limit was determined when the signal-to-noise ratio was approximately 10:1, and the detection limit was determined when the signal-to-noise ratio was approximately 3:1. The results are shown in the table.

[0069]

[0070]

[0072] Experimental results: The quantitative limit concentration of each impurity is between 0.0399μg / ml and 0.1054μg / ml, which is 0.008% to 0.021% relative to the concentration of the test solution. The results are good and meet the detection sensitivity requirements.

[0073] 3. Linearity and correction factors

[0074] Take the impurity reference stock solution to prepare different concentration linear relationship solutions, calculate the linear relationship equation, and use the slope value to calculate the impurity correction factor (f = K 主 / K 杂 ), and the results are shown in the table.

[0075] Linear relationship and correction factor test results

[0076] name Concentration range (μg / ml) Linear relationship equation Correlation coefficient (r) Correction Factor Impurity 2 0.0496—3.9680 y=0.7191x+0.0165 0.9997 0.74 Impurity 3 0.0399—3.9920 y=1.5095x+0.0091 0.9999 0.35 Impurity 5 0.1054—4.2160 y=0.3536x+0.0082 0.9980 1.50 Impurity 4 0.0992—3.9680 y=0.4071x+0.0306 0.9999 1.30 Impurity 6 0.1010—4.0400 y=0.6242x+0.0532 0.9998 0.85 Impurity 1 0.0994—3.9760 y=1.3217x+0.0278 0.9988 0.40 Main Peak 0.1004—4.0176 y=0.5307x-0.0317 0.9994 -

[0077] .

[0078] Test results: Each component has a good linear relationship in the range of quantitative limit concentration to limit concentration 200%, and the correlation coefficient is greater than 0.99; except for impurities 4 and 5, the correction factors of the remaining impurities are less than 1.1, so the main component self-control method with correction factors is used to calculate the content.

[0079] 4. Accuracy experiment

[0080] Take the stock solution of impurity reference substance, prepare the test solution with limit concentration of 50%, 100% and 150%, respectively, calculate the measured amount and added amount of 3 recovery solutions at each concentration by the principal component self-control method with correction factor, calculate the original amount of impurities in the test solution, calculate the recovery rate according to the added amount, original amount and measured amount, and calculate the mean of 3 recoveries at the quantitative limit concentration, the mean of 9 recoveries at other concentrations and relative standard deviation (RSD). The test results are shown in the table.

[0081] Accuracy test results

[0082]

[0083]

[0084] Experimental results: The method of the present invention performs recovery test, and the recovery of each impurity at different concentrations is in the range of 80% to 110%, and the RSD (%) of the recovery of each impurity is in the range of 10%. The method has good accuracy, and the detection results of impurities are accurate and reliable.

[0085] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. For those skilled in the art, other different forms of changes or modifications can be made based on the above description, and the obvious changes or modifications extended therefrom are still within the protection scope of the present invention.

Claims

1. A detection method for separating and detecting related substances of nafamostat mesylate, characterized in that: High performance liquid chromatography was used and the chromatographic conditions were as follows: Chromatographic column packing: octadecylsilane bonded silica gel chromatographic column; UV detector; Column temperature: 35-45°C; Sample preparation solvents: acetonitrile, acetone, water; Flow rate: 0.8ml / min-1.2ml / min; Mobile phase: Sodium heptane sulfonate solution was used as mobile phase A; acetonitrile was used as mobile phase B; Elution conditions: 0-60 min, gradient conditions with A phase / B phase ratio from high to low; Test solution: dissolve the nafamostat mesylate raw material in a diluent to obtain a test solution; Reference solution: Dissolve the nafamostat mesylate test sample in a diluent as the reference solution; System suitability solution: Take an appropriate amount of nafamostat mesylate reference substance, dissolve it in acetone: water (V:V) = 30:70, add an appropriate amount of impurity 1 location solution and dilute to a mixed solution of 0.5 mg / ml nafamostat mesylate and 10.5 μg / ml impurity as the system suitability solution. The method is used for detecting and analyzing nafamostat mesylate and related substances; in, The structure of nafamostat mesylate is as follows: The structural formula of the relevant substances is as follows:

2. The high performance liquid chromatography method according to claim 1, characterized in that: The sodium heptane sulfonate solution is prepared by weighing 6.07 g of 1-heptane sulfonate sodium, adding an appropriate amount of water to dissolve it, then adding 6 ml of acetic acid, and diluting it with water to 1000 ml.

3. The high performance liquid chromatography method according to claim 1, characterized in that: The concentration of the test solution is 0.5 mg / ml, and the concentration of the reference solution is 0.5 μg / ml.

4. The high performance liquid chromatography method according to claim 1, characterized in that: The elution procedure is as follows:

5. The high performance liquid chromatography method according to claim 1, characterized in that: The injection volume is 5 μl-10 μl.

6. The high performance liquid chromatography method according to claim 1, characterized in that: The wavelength of the UV detector is 245nm-265nm.

7. The high performance liquid chromatography method according to claim 1, characterized in that: The particle size of the chromatographic column is ≤5μm and the column length is 150mm~250mm.

8. The high performance liquid chromatography method according to claim 1, characterized in that: During the sample preparation process, one or a mixture of different proportions of various solvents such as acetonitrile, acetone, water, etc. may be used depending on the nature of the sample.

9. The high performance liquid chromatography method according to claim 1, characterized in that: Ultrasonic vibration is required to dissolve the sample completely.

10. The high performance liquid chromatography method according to claim 1, characterized in that: The flow rate is 0.8ml / min-1.2ml / min.

11. The high performance liquid chromatography method according to claim 1, characterized in that: The separation between impurities and impurity chromatographic peaks is greater than 1.3, and the separation between impurities and the main peak is greater than 1.

5.

12. The high performance liquid chromatography method according to claim 1, characterized in that: The theoretical plate number of the peak area of ​​nafamostat mesylate is greater than 10,000.

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