CN116482268A

CN116482268A - Method for detecting impurities in argatroban raw material or preparation

Info

Publication number: CN116482268A
Application number: CN202310490299.2A
Authority: CN
Inventors: 宋更申; 王迟; 安百峰; 习雪娥
Original assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Current assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Priority date: 2023-05-04
Filing date: 2023-05-04
Publication date: 2023-07-25

Abstract

The invention belongs to the technical field of drug detection, and particularly relates to a detection method of impurities in argatroban raw materials or preparations. The method adopts a High Performance Liquid Chromatography (HPLC) method to perform gradient elution, and can separate and detect 12 impurities in the argatroban raw material or preparation. The method has the advantages of good specificity, good separation effect, high sensitivity, accurate quantification and remarkable increase of impurity detection types.

Description

Method for detecting impurities in argatroban raw material or preparation

Technical Field

The invention relates to the technical field of drug detection, in particular to a method for detecting impurities in argatroban raw materials or preparations.

Background

Argatroban is a white or off-white crystalline powder, is readily soluble in glacial acetic acid, slightly soluble in methanol, slightly soluble in ethanol, and very slightly soluble in water. Argatroban having the chemical name (2R, 4R) -4-methyl-1- [ N ² - ((R, S) -3-methyl-1, 2,3, 4-tetrahydro-8-quinolinesulfonyl) -L-arginyl]2-piperidinecarboxylic acid monohydrate having a chemical composition of 21%R) and 21 (S) in a ratio of generally 64 to 65:36 to 35. The structural formula is as follows:

argatroban (C) ₂₃ H ₃₆ N ₆ O ₅ S·H ₂ O, 526.66) is an artificially synthesized monovalent small molecule direct thrombin inhibitor, can be selectively and reversibly combined with thrombin catalytic sites, can inactivate liquid phase thrombin and also inactivate thrombin combined with fibrin thrombus, is mainly used for heparin-induced thrombocytopenia and thrombosis, percutaneous coronary intervention treatment and cerebral apoplexy thrombolytic thrombosis diseases clinically, and is mainly used as an injection in clinical application at present.

Argatroban injection (Argatroban Injection) is an antithrombotic agent developed by Tokyo Tajia-b pharmaceutical Co., ltd., shida Bian Sanling pharmaceutical Co., ltd., and first pharmaceutical Co., ltd., shida Sanchi Co., ltd. The injection (20 mL:10mg in specification) of this variety was first marketed in Japan in 1990 for the treatment of peripheral thrombosis, approved in 1996 for the treatment of acute ischemic cerebral apoplexy, approved in U.S. FDA for menstruation in 2000 (2.5 mL:250mg in specification), marketed in China in 2002, developed by Takara Bian Sanling pharmaceutical Co., ltd. In 2005 with a specification of 10mg:2mL of the injection.

The detection method of the Argatroban injection and impurities (related substances) is the key of quality control. Reference is made to the united states pharmacopeia (USP 43) argatroban drug substance and argatroban injection quality standard, the argatroban drug substance quality standard received in japanese pharmacopeia (JP 17), the argatroban injection import registration standard (JX 20090312) and comprehensive impurity profile analysis is performed in combination with the prescribed process and degradation route of the product, wherein the control of stereoisomer impurity is one of difficulties in quality control of argatroban injection, argatroban contains 4 chiral centers, has 3 corresponding isomer impurities, is 2s,4 s-isomer, 2s,4 r-isomer and 2r,4 s-isomer respectively, wherein impurity 2s,4 s-isomer is process degradation impurity, there is a reception and research in import registration standard, impurity 2s,4 r-isomer and 2r,4 s-isomer is process impurity, and no reception and research is performed in existing standard.

By detecting patents or documents, search:

the related substance detection method of Argatroban injection is disclosed in Chinese patent CN 104237397A: the chromatographic column is preferably octadecylsilane chemically bonded silica, the mobile phase is preferably a mixture of glacial acetic acid buffer solution and methanol, and the isocratic elution is carried out, but the method can only separate and detect two impurities, namely impurity F (Japanese IF file impurity A) and impurity G (Japanese IF file impurity B), and the unreported impurities of 2S, 4R-isomer and 2R, 4S-isomer are not quantitatively analyzed and detected, other impurities reported in pharmacopoeia and literature are not involved, and the detection impurity types are few.

The method only effectively controls impurities F (Japanese IF file impurity A) and G (Japanese IF file impurity B), does not quantitatively analyze and detect unreported impurities of 2S, 4R-isomer and 2R, 4S-isomer, does not relate to other impurities reported in pharmacopoeia and literature, and has few detection impurity types.

The studies on the stereoisomerism impurities 2s,4 r-isomer and 2r,4 s-isomer have not been made in the prior art patent and literature, and the detection of the impurity species is small. Aiming at the defects and shortcomings of the prior art, the invention provides a method for detecting impurities in Argatroban injection, which can separate and detect 12 reported impurities in Argatroban injection, wherein the 12 impurities are respectively: 2 impurities (2 s,4 r-isomer, 2r,4 s-isomer) not reported and 10 impurities (impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) reported in pharmacopoeia and literature; meanwhile, 3 impurities in the Argatroban injection are separated, quantitatively analyzed and detected, wherein the 3 impurities are respectively: unreported 2 impurities (2 s,4 r-isomer, 2r,4 s-isomer) and a key degradation impurity G. The impurity detection method has the advantages of good specificity, good separation effect, high sensitivity, accurate quantification and remarkable increase of impurity detection types.

Disclosure of Invention

Along with the improvement of the quality standard of medicines, higher requirements are put forward on the accuracy of medicine detection. Aiming at the defects and shortcomings in the prior art, the application of the invention provides a detection method for impurities in an argatroban raw material or preparation, which can separate and detect 12 impurities in the argatroban raw material or preparation. The method comprises the following steps:

Firstly, 12 impurities in Argatroban injection can be separated and detected, and the 12 impurities are respectively: 2 impurities (2 s,4 r-isomer, 2r,4 s-isomer) not reported and 10 impurities (impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) reported in pharmacopoeia and literature;

wherein, 3 kinds of impurities in the Argatroban injection can be separated and quantitatively analyzed and detected, and the 3 kinds of impurities are respectively: unreported 2 impurities (2 s,4 r-isomer, 2r,4 s-isomer) and a key degradation impurity G.

The method has the advantages of (1) good specificity (12 impurities can be detected, the separation degree is larger than 1.28), good separation effect, (2) high sensitivity (the quantitative limit of each impurity is as low as 0.5104 mug/mL, which is equivalent to 0.1% of the limit concentration, (0.2092 mug/mL, which is equivalent to 0.04% of the limit concentration)), accurate quantification (3) and remarkable increase of impurity detection types (the accuracy is remarkably improved by more than 4 times), and (4) remarkable increase of impurity detection types from 2 types (impurity F and impurity G) to 12 types.

The impurity information in Argatroban injection is as follows:

the method for detecting the impurities in the argatroban raw material or the preparation comprises the following technical scheme:

The detection is carried out by adopting a high performance liquid chromatography method, and the chromatographic conditions comprise:

the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler;

the mobile phase A is a mixed solution of glacial acetic acid buffer solution and tetrahydrofuran, and the volume ratio is that of the glacial acetic acid buffer solution: tetrahydrofuran= (85-90) (15-10), and glacial acetic acid buffer solution with pH of 5.8-6.2;

the mobile phase B is a mixed solution of methanol and acetonitrile, the volume ratio of the methanol to the acetonitrile= (58-62 (42-38), and the preferable volume ratio of the methanol to the acetonitrile= (60:40);

elution mode: gradient elution.

In some preferred embodiments, wherein the chromatographic column is YMC J' sphere ODS-H18,4.6mm 250mm,4.0 μm or GL Sciences ODS-3,4.6mm 250mm,3 μm.

In some preferred embodiments, the volume ratio of the glacial acetic acid buffer solution to the tetrahydrofuran mixed solution in the mobile phase a of the mobile phase a is: glacial acetic acid buffer, tetrahydrofuran=86.5:13.5.

In some preferred embodiments, wherein the pH of the glacial acetic acid buffer in mobile phase a is 6.0.

In some preferred embodiments, wherein the gradient elution procedure is:

time/min	Mobile phase a/%	Mobile phase B/%
			0～90	95	5
90～100	95～40	5～60
			100～110	40	60
110～111	40～95	60～5
			111～130	95	5

。

In some embodiments, wherein the chromatographic conditions have a detection wavelength of 257 to 261nm, preferably 259nm.

In some embodiments, wherein the chromatographic column has a flow rate of 0.7 to 0.9mL/min, preferably 0.8mL/min.

In some embodiments, wherein the column temperature of the chromatographic column is 38-42 ℃, preferably 40 ℃.

In some embodiments, wherein the impurities in the mixed solution of argan Qu Banbing acetate buffer and tetrahydrofuran comprise one or more of:

in some embodiments, wherein the impurities in the argatroban starting material or formulation comprise impurity 2s,4 r-isomer, impurity 2r,4 s-isomer and impurity G.

In some embodiments, wherein the impurities in the argatroban starting material or formulation comprise the 2r,4 s-isomer and impurity G.

In some embodiments, wherein the impurities in the argatroban starting material or formulation comprise 2s,4 r-isomer, 2r,4 s-isomer, impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F.

In some embodiments, wherein the formulation is an injection.

The beneficial effects obtained by the invention are as follows:

The application provides a detection method of impurities in argatroban raw materials or preparations, and compared with detection methods in other existing standards or technologies, the detection method of the impurities has the advantages that:

1) By adopting the impurity analysis method, 12 impurities in the argatroban raw material or preparation can be detected:

unreported 2 impurities: 2s,4 r-isomer, 2r,4 s-isomer;

10 known impurities reported in pharmacopoeia and literature: impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F;

2) By adopting the impurity analysis method, 3 impurities in the argatroban raw material or preparation can be separated and quantitatively analyzed and detected:

unreported 2 impurities: 2s,4 r-isomer, 2r,4 s-isomer;

key degradation impurities: impurity G. The method for detecting the impurities in the argatroban injection has the advantages of (1) good specificity (12 impurities can be detected, the separation degree is more than 1.28), good separation effect, (2) high sensitivity (the quantitative limit of each impurity is as low as 0.5104 mug/mL, which is equivalent to 0.1% of the limit concentration, the detection limit is as low as 0.2092 mug/mL, which is equivalent to 0.04% of the limit concentration), accurate quantification (the accuracy is obviously improved by more than 4 times), (4) obviously increased impurity detection types (the impurity detection types are obviously increased from 2 types to 12 types).

Drawings

FIG. 1 is a typical spectrum of a system applicability solution of example 1 of the present invention;

FIG. 2 is a localization map of the impurity 2S, 4R-isomer of example 1 of the present invention;

FIG. 3 is a diagram showing the localization of impurity 2R, 4S-isomer in example 1 of the present invention;

FIG. 4 is a localization map of impurity G according to example 1 of the present invention;

FIG. 5 is a graph of a mixed impurity control solution according to example 1 of the present invention;

FIG. 6 is a localization map of impurity L in example 1 of the present invention;

FIG. 7 is a localization map of impurity K in example 1 of the present invention;

FIG. 8 is a localization map of impurity C in example 1 of the present invention;

FIG. 9 is a localization map of impurity O in example 1 of the present invention;

FIG. 10 is a localization map of impurity E in example 1 of the present invention;

FIG. 11 is a localization map of impurity 5 according to example 1 of the present invention;

FIG. 12 is a diagram showing the localization of impurity 2S, 4S-isomer in example 1 of the present invention;

FIG. 13 is a localization map of impurity F in example 1 of the present invention;

FIG. 14 is a localization map of impurity I in example 1 of the present invention;

FIG. 15 is a graph of undegraded sample solution of example 7 of the present invention;

FIG. 16 is a graph of a sample solution for acid degradation according to example 7 of the present invention;

FIG. 17 is a graph of a sample solution for alkali degradation of example 7 of the present invention;

FIG. 18 is a graph of a sample solution for oxidative degradation according to example 7 of the present invention;

FIG. 19 is a graph of a sample solution degraded at high temperature according to example 7 of the present invention;

FIG. 20 is a graph of a photodegradation test sample solution of example 7 of the present invention;

FIG. 21 is a graph of a mixed impurity control solution of comparative example 1 of comparative examples 1 to 2 according to the present invention;

FIG. 22 is a graph of the mixed impurity control solution of comparative example 2 of comparative examples 1 to 2 according to the present invention.

Detailed Description

The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

Example 1: specificity test

1-1, preparing a solution:

a diluent: mixing glacial acetic acid buffer solution with pH of 6.0 with methanol at a volume ratio of 55:45;

blank solvent: a diluent;

blank auxiliary materials: taking 10.9g of sorbitol, adding water for injection to dissolve and dilute to 200mL, and taking the mixture as a blank auxiliary material;

system applicability solution: accurately weighing a proper amount of Argatroban, 2S, 4R-isomer, 2R, 4S-isomer and impurity G reference substance, dissolving and diluting with a diluent to prepare a solution containing 0.5mg of Argatroban, 10 mug of 2S, 4R-isomer, 10 mug of 2R, 4S-isomer and 10 mug of impurity G in each 1mL, and taking the solution as a system applicability solution;

Each impurity localization solution: accurately weighing 2S, 4R-isomer, 2R, 4S-isomer and impurity G reference substances, respectively dissolving and diluting the 3 impurities with a diluent to prepare 0.5mg of impurity reference substances in each 1mL, and respectively marking as impurity positioning solutions of the 3 impurities: 2s,4 r-isomer positioning solution, 2r,4 s-isomer positioning solution, impurity G positioning solution;

argatroban and various impurity control stock solutions: argatroban, impurity L, impurity K, impurity C, impurity O, impurity E, impurity 5, impurity I, impurity G, impurity 2S, 4S-isomer and impurity F reference substances are respectively 5mg, precisely weighed, respectively placed in 50mL measuring flasks, dissolved and diluted to scale by methanol, uniformly shaken, and respectively marked as reference substance stock solutions of the impurities: impurity L control stock solution, impurity K control stock solution, impurity C control stock solution, impurity O control stock solution, impurity E control stock solution, impurity 5 control stock solution, impurity I control stock solution, impurity G control stock solution, impurity 2S, 4S-isomer control stock solution, impurity F control stock solution, argatroban control stock solution;

impurity localization solution: respectively precisely measuring 1ml of each impurity reference substance and Argatroban reference substance stock solution, placing into different 100ml measuring bottles, diluting to scale with diluent, shaking, and respectively marking as positioning solutions of the impurities: impurity L positioning solution, impurity K positioning solution, impurity C positioning solution, impurity O positioning solution, impurity E positioning solution, impurity 5 positioning solution, impurity I positioning solution, impurity 2S, 4S-isomer positioning solution, impurity F positioning solution;

Mixing an impurity reference substance solution: preparing an impurity L control stock solution, an impurity K control stock solution, an impurity C control stock solution, an impurity O control stock solution, an impurity E control stock solution, an impurity I control stock solution, an impurity G control stock solution, impurity 2S, 4S-isomer control stock solution, an impurity F control stock solution and an Argatroban control stock solution by using a solvent to prepare a solution containing 0.5mg of Argatroban and 1 mug of each impurity per 1mL of the mixed impurity control solution;

test solution: argatroban injection (namely Argatroban self-product stock solution with the specification of 10mg:20 mL) is taken as a test solution.

1-2, test conditions:

chromatographic column: YMC J' sphere ODS-H18, 4.6mm.times.250 mm,4.0 μm;

mobile phase a: the volume ratio of the glacial acetic acid buffer solution to the tetrahydrofuran mixed solution is that the glacial acetic acid buffer solution is tetrahydrofuran=86.5:13.5, and the pH value of the glacial acetic acid buffer solution is 6.0;

mobile phase B: methanol-acetonitrile (60:40);

elution mode: gradient elution.

The elution gradient is:

Detection wavelength: 259nm;

flow rate: 0.8mL/min;

column temperature: 40 ℃;

1-3, experimental steps and conclusions:

the precise measurement of 80. Mu.L of blank solvent, blank auxiliary material, system applicability solution, impurity localization solution, mixed impurity reference solution and test solution was carried out, the typical profile of the system applicability solution was shown in FIG. 1, the impurity localization profile was shown in FIG. 2 (2S, 4R-isomer), FIG. 3 (2R, 4S-isomer), FIG. 4 (impurity G), FIG. 6 (impurity L), FIG. 7 (impurity K), FIG. 8 (impurity C), FIG. 9 (impurity O), FIG. 10 (impurity E), FIG. 11 (impurity 5), FIG. 12 (2S, 4S-isomer), FIG. 13 (impurity F), the profile of the mixed impurity reference solution was shown in FIG. 5, and the results were shown in Table 1 and Table 2.

Table 1 system applicability solution and impurity localization results

Table 2 pharmacopoeia and literature report results of specific tests of 10 known impurities

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Conclusion:

the samples are separated and detected by a high performance liquid chromatograph, and the test results show that:

the blank solvent and the blank auxiliary materials do not interfere with the separation detection of the Argatroban injection and each impurity;

from the results of the system applicability solution and the impurity localization solution (table 1 and fig. 1, 2, 3, 4), the minimum value of the separation degree between the main peak and the impurity peak and between the impurity peaks is 1.28, the separation degree of each impurity is >1.0, the separation effect is remarkable, and 3 kinds of impurities can be separated and quantified: unreported 2 impurities (2 s,4 r-isomer, 2r,4 s-isomer) and key degradation impurity G;

in addition, the specificity investigation is carried out by adopting the impurity method of the invention, and 12 impurities in the Argatroban injection can be detected: the unreported 2 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer) and the 10 kinds of known impurities reported in pharmacopoeia and literature (impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) (table 2 and fig. 5, fig. 6, fig. 7, fig. 8, fig. 9, fig. 10, fig. 11, fig. 12, fig. 13, fig. 14) indicate that the measurement method provided by the present invention can detect 12 kinds of impurities.

The small knot:

the determination method provided by the invention can detect 12 impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) in the Argatroban injection, and has good specificity;

among them, the separation effect of 3 impurities (2S, 4R-isomer, 2R, 4S-isomer, impurity G) in Argatroban injection is remarkable and the amount of the impurities can be determined.

Example 2: selection of chromatographic columns

System applicability solution: as in example 1;

referring to the test conditions of example 1, the type of chromatographic column was changed:

example 1: the column is YMC J' sphere ODS-H18,4.6mm×250mm,4.0 μm;

method 1: the chromatographic column was GL Sciences ODS-3, 4.6mm.times.250mm, 3 μm;

method 2: the chromatographic column is COSMIL 5C18-PAQ,4.6mm×250mm,5.0 μm;

method 3: the chromatographic column is SVEA Opal C18,4.6mm×250mm,5.0 μm;

method 4: the column was Waters XTerra Shield RP, 4.6mm X250 mm,5.0 μm;

method 5: the chromatographic column is Agilent ZORBAX SB-C18,4.6mm×250mm,5.0 μm;

other chromatographic conditions were the same as in example 1, 80. Mu.L of the system applicability solution was precisely measured for analysis and detection, and the test results are shown in Table 3.

TABLE 3 influence of column types on System applicability solution test results

Conclusion:

when the system-applicable solution was separated by using the column YMC J' sphere ODS-H18,4.6 mm. Times.250 mm,4.0 μm (FIG. 1) of example 1 of the present invention, the separation degree between the main peak (Argatroban) and the adjacent peak was >1.0, and the separation degree was good, and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) could be separated.

When the chromatographic column GL Sciences ODS-3, 4.6mm.times.250 mm,3.6 of the method 1 of the invention is used for separating the system-applicable solution, the separation degree between the main peak (Argatroban) and the adjacent peak is >1.0, and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) can be separated, and the separation effect is good.

The separation degree between the system applicability solution impurity G and the main peak was <1.0 by using the column type in the methods 2 to 5 of example 2, and 12 impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) could not be separated, especially impurity G, and the separation effect was poor.

The small knot:

1) The separation effect is good only when the octadecylsilane chemically bonded silica gel of the present invention is used as a filler (for example, the column model YMC J' sphere ODS-H18 of example 1, specification 4.6mm. Times.250 mm,4.0 μm or the column model GL Sciences ODS-3 of example 2, specification 4.6mm. Times.250 mm,3 μm), and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in the argatroban injection are separated;

2) On the contrary, when other types of chromatographic columns (for example, methods 2 to 5 of example 2) are used, 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in the argatroban injection solution cannot be separated, and especially, impurity G has poor separation effect.

Example 3: type selection of mobile phase A

System applicability solution: as in example 1;

referring to the test conditions of example 1, the kinds of mobile phases were changed, and other chromatographic conditions were the same as in example 1, and 80. Mu.L of a system-applicable solution was precisely measured for analysis and detection, and the test results are shown in Table 4.

TABLE 4 influence of mobile phase A type on System suitability solution test results

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Conclusion:

when the flow-through system applicable solution of example 1 of the present invention was used for separation (FIG. 1), the degree of separation between the main peak (Argatroban) and the adjacent peak was >1.0, and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) could be separated, with good separation effect.

The separation degree of each peak of the system-applicable solution was <1.0 by the mobile phase type in the methods 1 to 4 of example 3, and 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F), particularly impurity G, could not be separated from the argatroban injection, and the separation effect was poor.

The small knot:

1) The mobile phase type can separate 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in the argatroban injection only when the mobile phase of the present invention (for example, example 1) is used, and the separation effect is good;

2) On the contrary, when other types of mobile phases (for example, methods 1 to 4 of example 3) are used, 12 types of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F), in particular impurity G, in the argatroban injection cannot be separated, and the separation effect is poor.

Example 4: proportion selection of mobile phase A

System applicability solution: as in example 1;

referring to the test conditions of example 1, the ratio of mobile phase A was changed, and other chromatographic conditions were the same as in example 1, and 80. Mu.L of system applicability solution was precisely measured for analysis and detection, and the test results are shown in Table 5.

TABLE 5 influence of the ratio of mobile phase A on the results of the System applicability solution test

Conclusion:

when the system-applicable solution was separated using the mobile phase A in the ratio of 86.5:13.5 (FIG. 1) in example 1 of the present invention, 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) were separated with a degree of separation of >1.0 between the main peak (Argatroban) and the adjacent peaks, and the separation effect was good.

The ratio of mobile phase A in examples 4 methods 1 to 2 was 85:15 and 90:10, the degree of separation between the main peak (Argatroban) and the adjacent peak was >1.0, the degree of separation was good, and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) could be separated, and the separation effect was good.

The fraction of mobile phase A in example 4, method 3, was 82:18,2S, 4R-isomer, and the 2R, 4S-isomer were alternately peaked, and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in Argatroban injection could not be separated, and especially 2S, 4R-isomer, 2R, 4S-isomer did not meet the detection requirements, and the separation effect was poor.

The fraction of mobile phase A in method 4 of example 4 was 92:8, the degree of separation between impurity G and the main peak was less than 1.0, and 12 impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in Argatroban injection could not be separated, and especially impurity G did not meet the detection requirements, and the separation effect was poor.

The small knot:

1) When the ratio of the mobile phase A is (85-90): 15-10) (for example, the method 1-2 of the embodiment 1 and the embodiment 4), 12 impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) in the argatroban injection can be separated, and the separation effect is good;

2) When the ratio of mobile phase A is 86.5:13.5 (for example, example 1), 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in the Argatroban injection can be separated from the Argatroban injection, and the separation effect is the best.

3) On the contrary, when the other ratio of mobile phase A (for example, methods 3 to 4 of example 4) is used, 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in Argatroban injection cannot be separated, and especially 3 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, impurity G) are poor in separation effect.

Example 5: selection of pH of mobile phase A

System applicability solution: as in example 1;

referring to the test conditions of example 1, the pH of mobile phase a was changed:

example 1: the pH of mobile phase A was 6.0;

method 1: the pH of mobile phase A was 5.8;

method 2: the pH of mobile phase A was 6.2;

method 3: the pH of mobile phase A was 5.0;

method 4: the pH of mobile phase A was 5.5;

method 5: the pH of mobile phase A was 6.5;

method 6: the pH of mobile phase A was 7.0;

Other chromatographic conditions were the same as in example 1, 80. Mu.L of the system applicability solution was measured precisely for analysis and detection, and the test results are shown in Table 6.

TABLE 6 pH change and impurity separation of mobile phase A

Conclusion:

when the system-applicable solution was separated using the mobile phase A of example 1 of the present invention at pH 6.0 (FIG. 1), the separation degree between the main peak (Argatroban) and the adjacent peaks and between the peaks of the respective impurities was >1.0, and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) could be separated, with good separation effect.

Example 5 method 1 and method 2 employ mobile phase a having pH of 5.8 and 6.2,2s,4 r-isomer, 2r,4 s-isomer, peak in order, and the chromatographic peak can be separated, and 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) can be separated, and the separation effect is good.

Example 5 method 3 uses mobile phase A having pH of 5.0,2S, 4R-isomer, and 2R, 4S-isomer alternately showing peaks, and the chromatographic peaks cannot be separated, and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) cannot be separated, and particularly 2S, 4R-isomer, 2R, 4S-isomer, and separation effect is poor.

Example 5 method 4 uses mobile phase a having pH of 5.5,2s,4 r-isomer, 2r,4 s-isomer, peak in order, separation degree <1.0, separation effect is poor, 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F), especially 2s,4 r-isomer, 2r,4 s-isomer, separation effect is poor in argatroban injection cannot be separated.

In example 5, method 5 uses mobile phase a having a pH of 6.5, alternately peaks for impurity G and argatroban, and the chromatographic peaks cannot be separated, and 12 impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in argatroban injection cannot be separated, and especially impurity G has poor separation effect.

In example 5, method 6 was carried out by using a mobile phase a having a pH of 7.0 and sequentially peaking for impurity G and argan Qu Banfeng, the degree of separation was <1.0, the separation effect was poor, and 12 impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in argatroban injection, especially impurity G, could not be separated, and the separation effect was poor.

The small knot:

1) When the pH of the mobile phase A is controlled to be 5.8 to 6.2 (for example, methods 1 to 2 in example 1 and example 5), 12 impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) in the argatroban injection can be separated, and the separation effect is good;

2) Wherein the mobile phase A has a pH of 6.0 (e.g., example 1) and is capable of separating 12 impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) with the best separation effect;

3) On the contrary, the other pH of mobile phase a (e.g., methods 3 to 6 in example 5) was not able to separate 12 impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F), especially 3 impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity G) in the argatroban injection, and the separation effect was poor.

Example 6: gradient elution procedure selection of mobile phase

System applicability solution: as in example 1;

referring to the test conditions of example 1, the gradient elution procedure of the mobile phase was changed:

Wherein the gradient elution procedure for the mobile phase of example 1 was:

Method 1: the gradient elution procedure for the mobile phase was:

time/min	Mobile phase a/%	Mobile phase B/%
			0～50	95	5
90～100	95～40	5～60
			100～110	40	60
110～111	40～95	60～5
			111～130	95	5

Method 2: the gradient elution procedure for the mobile phase was:

time/min	Mobile phase a/%	Mobile phase B/%
			0～60	95	5
90～100	95～40	5～60
			100～110	40	60
110～111	40～95	60～5
			111～130	95	5

Method 3: the gradient elution procedure for the mobile phase was:

method 4: the gradient elution procedure for the mobile phase was:

time/min	Mobile phase a/%	Mobile phase B/%
			0～80	95	5
90～100	95～40	5～60
			100～110	40	60
110～111	40～95	60～5
			111～130	95	5

Other chromatographic conditions were the same as in example 1, 80. Mu.L of the system applicability solution was precisely measured for analysis and detection, and the test results are shown in Table 7.

TABLE 7 Effect of mobile phase gradient elution procedure on System applicability solution test results

Conclusion:

when the gradient elution procedure (FIG. 1) of the mobile phase of example 1 of the present invention was employed, the degree of separation between the peaks of the impurities in the system-applicable solution was all greater than 1.0, and 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) could be separated, with good separation effect.

When the gradient elution procedure of the mobile phase of the methods 1 to 4 in example 6 was used, the separation results showed that each impurity could not be eluted completely, and the general standard of the degree of separation (> 1.0) was not satisfied, and the separation effect was poor, and 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F), especially 2r,4 s-isomer, impurity G, in the argatroban injection could not be separated.

The small knot:

1) The mobile phase elution procedure in chromatographic conditions can separate 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in the argatroban injection only by the gradient elution procedure of the present invention (for example, example 1), and the separation effect is good;

2) On the contrary, the 12 kinds of impurities (2S, 4R-isomer, 2R, 4S-isomer, 2S, 4S-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) of the present invention, especially 2R, 4S-isomer and impurity G, cannot be separated by using other mobile phase gradient elution procedures (for example, methods 1 to 4 of example 6), and the separation effect is poor.

Example 7: destructive test

The forced degradation test is to accelerate the damage to the sample under the severe conditions, such as strong light irradiation, high temperature, high humidity, acid-base damage, hydrolysis, oxidation damage and the like, so as to evaluate the effectiveness and applicability of the analysis method by examining the separation condition of the degradation product and main peak of the sample and known impurities. Meanwhile, the detection of the light-emitting diode array is adopted to carry out the detection of the peak purity: in the map obtained by the degradation experiment, when the purity angle of the impurities and the main peak is smaller than the purity threshold value, the determination method is judged to meet the determination requirement.

7-1, preparing a solution:

test solution: as in example 1;

undegraded solution: namely a sample solution;

acid degradation solution: precisely measuring 20mL of a sample solution, placing the sample solution into a 25mL measuring flask, adding 1mL of 1mol/L hydrochloric acid solution, sealing, carrying out water bath at 90 ℃ for 5min, taking out and cooling, adding 1mL of 1mol/L sodium hydroxide solution for neutralization, and shaking uniformly to obtain an acid degradation solution;

alkali degradation solution: precisely measuring 20mL of a sample solution, placing the sample solution into a 25mL measuring flask, adding 1mL of 1mol/L sodium hydroxide solution, sealing, carrying out water bath at 90 ℃ for 20min, taking out, cooling to room temperature, adding 1mL of 1mol/L hydrochloric acid solution for neutralization, and shaking uniformly to obtain an alkali degradation solution;

oxidative degradation solution: precisely measuring 20mL of a sample solution, placing the sample solution into a 25mL measuring flask, adding 1mL of 30% hydrogen peroxide solution, standing at room temperature for 2h, and shaking uniformly to obtain an oxidative degradation solution;

high temperature degradation solution: precisely measuring 20mL of the sample solution, placing the sample solution into a 25mL measuring flask, sealing, placing the sample solution into a water bath at 90 ℃ for 6 hours, taking out the sample solution, and cooling the sample solution to room temperature to obtain a high-temperature degradation solution;

light degradation solution: precisely measuring 20mL of the sample solution, placing in a 25mL measuring flask, placing in an illumination incubator (illumination intensity: 4500 lx+/-500 lx) for 48h, and taking out to serve as an illumination degradation solution.

7-2, test conditions:

Chromatographic conditions were the same as in example 1;

7-3, experimental steps and conclusions:

the sample solution which is not degraded and degraded by acid, alkali, oxidation, high temperature and illumination is taken for analysis and detection, the destructive test chromatograms are shown in fig. 15 (not degraded), fig. 16 (acid degraded), fig. 17 (alkali degraded), fig. 18 (oxidative degraded), fig. 19 (high temperature degraded) and fig. 20 (light degraded), and the test results are shown in tables 8 and 9.

TABLE 8 impurity test results under different degradation conditions

Note that: "ND" means "undetected".

TABLE 9 results of testing the main and adjacent peaks under different degradation conditions

Conclusion:

the samples were tested by high performance liquid chromatography under the conditions of undegraded and degraded (fig. 6-11), and the results showed that:

the degradation percentage of each impurity of the Argatroban injection under the conditions of strong acid, oxidation and high temperature is less than 0.05%, the maximum single impurity content is less than 0.14%, and the total impurity content is less than 0.16%, which indicates that the Argatroban injection is stable under the conditions of strong acid, oxidation and high temperature;

the Argatroban injection has impurity G generated under the strong alkali condition, the impurity G content is 4.33%, the maximum single impurity content is 3.09%, and the total impurity content is 9.20%, which indicates that the Argatroban injection is unstable under the strong alkali condition;

The argatroban injection has impurity K generated under the illumination condition, and the content of the impurity K is 0.47%, which indicates that the argatroban injection is unstable under the illumination condition;

the material conservation proportion of the Argatroban injection is kept within the range of 100% -110% under degradation conditions such as acid, alkali, oxidation, high temperature, illumination and the like, which indicates that the material is basically conserved;

the Argatroban injection achieves good separation between main peak (Argatroban) and adjacent impurities under each degradation condition, and has good separation degree; the purity angles of the main peak 1 and the main peak 2 are obviously smaller than the purity threshold value, which indicates that no co-elution phenomenon exists, and the peak purity can meet the impurity separation requirement;

the results show that the analysis method has good specificity under various degradation conditions.

Example 8: solution stability test

A diluent: mixing glacial acetic acid buffer solution with pH value of 6.0 with methanol, wherein the volume ratio of the glacial acetic acid buffer solution to the methanol is 55:45;

test solution: as in example 1;

control solution: precisely measuring a proper amount of sample solution, and diluting with a diluent to prepare a solution with about 5 mug in each 1mL serving as a control solution;

and (5) placing for 0h, 4h, 9h, 22h and 52h at room temperature, and then performing sample injection detection.

Chromatographic conditions were the same as in example 1;

80. Mu.L of the test solution and the control solution were taken for analysis and detection, and the test results are shown in Table 10.

TABLE 10 test results of stability of test solutions

Note that: "ND" means "undetected".

Table 11 results of stability test of control solutions

Conclusion:

stability test results show that the test solution and the control solution remain stable for 52 hours:

the variation value of the impurity content detected at each time point of the test sample solution is 0 compared with 0h, which is far less than 30.0% of the requirement of the common standard;

the retention time and peak area of the control solution have a maximum value of 3.30% and less than 5.0% compared with 0h, indicating good solution stability in the method of the invention.

Example 9: sensitivity test

Quantitative limiting solution: taking a proper amount of Argatroban reference substance, 2S, 4R-isomer, 2R, 4S-isomer and impurity G reference substance, respectively adding diluents for dissolving and gradually diluting into serial diluted solutions, wherein the signal to noise ratio is 10:1, measuring the quantitative limit, and preparing 6 parts in parallel;

detection limit solution: taking a proper amount of Argatroban reference substance, 2S, 4R-isomer, 2R, 4S-isomer and impurity G reference substance, respectively adding diluents for dissolving and gradually diluting into serial diluted solutions, wherein the signal to noise ratio is 3:1, measuring the detection limit, and preparing 6 parts in parallel;

Chromatographic conditions were the same as in example 1;

80. Mu.L of the above quantitative limit and detection limit solutions were taken for analysis and detection, and the test results are shown in Table 12 below.

Table 12 quantitative limit and detection limit test results

Conclusion:

the quantitative limit of each impurity is as low as 0.5104 mug/mL, which corresponds to 0.1% of the limit concentration, the maximum value of RSD (n=6) of the quantitative limit is 3.50%, which is far smaller than the standard requirement of the quantitative limit of the impurity (< 15.0%);

the detection limit of each impurity is as low as 0.2092 mug/mL, which is equivalent to 0.04% of the limit concentration; the detection limit of each impurity is equal to 0.0418-0.1130% of the concentration ratio of the sample, and is lower than the limit concentration (< 0.2%) of each impurity.

Example 10: linearity and range

Linear studies cover a range of impurity limiting concentrations from quantitative limits to 200%.

A diluent: as in example 1;

200% linear solution: accurately weighing the Argatroban reference substance, 2S, 4R-isomer, 2R, 4S-isomer and impurity G reference substance, adding methanol to dissolve and dilute to obtain solutions containing about 2 μg each of 1mL as 200% linear solution;

50%, 80%, 100%, 120%, 150% linear solution: precisely measuring 2.5mL, 4mL, 5mL, 6mL and 7.5mL of 200% linear solution, respectively placing in a 10mL measuring flask, diluting to scale with a diluent, and shaking uniformly to obtain 50%, 80%, 100%, 120% and 150% linear solution;

Chromatographic conditions were the same as in example 1;

80 mu L of each linear solution is precisely measured for analysis and detection, and is respectively injected into a chromatograph, a chromatogram is recorded, and a linear regression equation is made by taking the concentration as an abscissa and the peak area as an ordinate. Then another experimenter performs a linear test on another instrument, calculates a correction factor, and calculates an average value with the measurement result of experimenter 1 as the correction factor of the impurity. The test results are shown in Table 13.

TABLE 13 Linear and Range detection results

Conclusion:

the linearity of each impurity in the measuring range is good, and the method meets the requirements of methodology:

2s,4 r-isomer in the concentration range of 0.52 to 2.69 μg/mL (corresponding to 50 to 200% of the limit concentration), linear equation y= 74646.4434x-215.6609 and y= 51.4479x-2.5671, peak area being linear with concentration; the linear correlation coefficient (r) =0.9997 is greater than 0.9995, and the peak area of the 2s,4 r-isomer has good linear relation with concentration; the correction factor is 1.0 and is between 0.9 and 1.1, and the measurement result of the 2S, 4R-isomer in the linear range is accurate without correction;

the concentration of the 2R, 4S-isomer is in the range of 0.53-2.13 mug/mL (corresponding to 50-200% of the limit concentration), the linear equation is y= 67312.4041x-1693.4225 and y= 47.0575x-0.9115, and the peak area is linear with the concentration; the linear correlation coefficient (r) =0.9998/0.9997 is greater than 0.9995, and the peak area of the 2r,4 s-isomer has good linear relation with concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement results of the 2R, 4S-isomer in the linear range are accurate without correction;

Impurity G is in the concentration range of 0.53-2.12 μg/mL (corresponding to 50-200% of limit concentration), the linear equation is y=72949.2036x+357.8182 and y= 49.6802x-3.5652, the peak area is linear with concentration; the linear correlation coefficient (r) = 0.9996/0.9999 is larger than 0.9995, and the peak area of the impurity G has good linear relation with the concentration; the correction factor is 1.0 and is between 0.9 and 1.1, and the measurement result of the impurity G in the linear range is accurate without correction;

impurity 5 is in the concentration range of 0.52-2.11 μg/mL (corresponding to 50-200% of limit concentration), the linear equation is y= 58712.4023x-1893.4254 and y= 48.0365x-0.9251, the peak area is linear with the concentration; the linear correlation coefficient (r) = 0.9996/0.9995 is larger than 0.9995, and the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity 5 in the linear range is accurate without correction;

the concentration of impurity 2S, 4S-isomer is in the range of 0.48-2.15 mug/mL (corresponding to 50-200% of limit concentration), the linear equation is y= 68712.4211x-1583.4224 and y= 48.0567x-0.9241, the peak area is linear with the concentration; the linear correlation coefficient (r) =0.9997/0.9996, which is larger than 0.9995, and the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity 2S, 4S-isomer in the linear range is accurate without correction;

Impurity K is in the concentration range of 0.55-2.12 μg/mL (corresponding to 50-200% of limit concentration), the linear equation is y= 58782.4041x-1893.4214 and y= 49.1568x-0.8874, the peak area is linear with concentration; the linear correlation coefficient (r) =0.9997, which is greater than 0.9995, the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity K in the linear range is accurate without correction;

the impurity L is in the concentration range of 0.55-2.12 mug/mL (corresponding to the limit concentration of 50-200%), the linear equation is y= 66812.4041x-1693.4225 and y= 48.2155x-0.9325, and the peak area is linear with the concentration; the linear correlation coefficient (r) =0.9998/0.9996, which is larger than 0.9995, and the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity L in the linear range is accurate without correction;

impurity E is in the concentration range of 0.56-2.18 μg/mL (corresponding to 50-200% of limit concentration), the linear equation is y= 68542.4023x-1704.3282 and y= 48.6274x-0.8578, the peak area is linear with concentration; the linear correlation coefficient (r) =0.9999/0.9995, which is larger than 0.9995, the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity E in the linear range is accurate without correction;

The impurity O is in the concentration range of 0.56-2.18 mug/mL (corresponding to the limit concentration of 50-200%), the linear equation is that y= 70312.4211x-1713.4355 and y= 49.0525x-0.8995, and the peak area is linear with the concentration; the linear correlation coefficient (r) =0.9998, which is larger than 0.9995, the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity O in the linear range is accurate without correction;

impurity C is in the concentration range of 0.52-2.18 μg/mL (corresponding to 50-200% of limit concentration), the linear equation is y= 68355.4338x-1741.3257 and y= 48.0215x-0.9132, the peak area is linear with concentration; the linear correlation coefficient (r) =0.9997/0.9989, which is greater than 0.99, the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity C in the linear range is accurate without correction;

the impurity I is in the concentration range of 0.52-2.18 mug/mL (corresponding to the limit concentration of 50-200%), the linear equation is that y= 68472.4369x-1705.4325 and y= 48.0568x-0.9235, and the peak area is linear with the concentration; the linear correlation coefficient (r) =0.9999/0.9998, which is larger than 0.9995, the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity I in the linear range is accurate without correction;

Impurity F is in the concentration range of 0.54-2.21 μg/mL (corresponding to 50-200% of limit concentration), the linear equation is y= 68572.4035x-1685.4236 and y= 48.0625x-0.9357, the peak area is linear with concentration; the linear correlation coefficient (r) =0.9999/0.9997, which is larger than 0.9995, the peak area of the impurity 5 has good linear relation with the concentration; the correction factor is 1.1 and is between 0.9 and 1.1, and the measurement result of the impurity F in the linear range is accurate without correction;

the above results indicate that: the peak areas and concentrations of the 12 impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity G, 2s,4 s-isomer, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) are linear, the linear relationship is good in the linear range, and the measurement result does not need correction.

Example 11: accuracy test

Accuracy is investigated through a recovery rate test, and the research covers a limit concentration range of 50% -150%.

A diluent: as in example 1;

impurity stock solution: precisely weighing appropriate amounts of 2S, 4R-isomer, 2R, 4S-isomer and impurity G, placing into the same measuring flask, adding methanol for dissolution and quantitatively diluting to prepare a solution containing about 100 mug per 1mL, and taking the solution as an impurity stock solution;

50%, 100%, 150% accuracy solution: taking three parts of 0.5mL, 1.0mL and 1.5mL of impurity stock solution, respectively placing the three parts into 100mL measuring flasks, diluting the three parts to the scale by using Argatroban injection stock solution, and shaking the three parts uniformly to obtain 50%, 100% and 150% accuracy solutions;

Control solution: the impurity stock solution was quantitatively diluted with a diluent to prepare 1mL of a solution containing about 1. Mu.g of each of 2S, 4R-isomer, 2R, 4S-isomer and impurity G as a reference solution.

The chromatographic conditions were the same as in example 1, 80. Mu.L of each of the above solutions was precisely measured, the respective solutions were injected into a chromatograph, the chromatograms were recorded, the peak areas were calculated by the external standard method, and the accuracy test results are shown in Table 14.

Table 14 accuracy test results

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Conclusion:

the average recovery rates of 2S, 4R-isomer, 2R, 4S-isomer, impurity G and other 9 known impurities (2S, 4S-isomer, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) are kept between 96% and 104% under the accurate solutions of 50%, 100% and 150%, and the average recovery rates are respectively 101.3%, 100.8%, 97.8%, 97.2%, 96.9%, 96.8%, 99.1%, 99.3%, 101.8%, 99.5%, 99.7% and 100.1%, the recovery rate distribution ranges are concentrated, and the quantitative determination requirements of each impurity can be accurately met by approaching 100%;

the recovery rates RSD of 2S, 4R-isomer, 2R, 4S-isomer, impurity G and other 9 known impurities (2S, 4S-isomer, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) of the accurate solutions of the respective impurities measured in this example were 1.4%, 1.8%, 0.97%, 1.69%, 2.19%, 2.43%, 1.68%, 1.35%, 1.81%, 0.77%, 1.63%, 1.52%, respectively, and were not more than 2.5%, respectively, and compared with the general standard requirements (recovery rate RSD. Ltoreq.10.0%), the accuracy of the detection results of the detection method of the present invention was significantly improved by 4 times or more, further proving that the accuracy of the detection method for detecting the above-mentioned impurities was good.

Example 12: precision testing

Test solution: as in example 1;

control solution: the sample solution was diluted quantitatively with a mobile phase to prepare a solution containing about 5. Mu.g per 1mL, and 6 parts of the diluted solution were prepared in parallel as a control solution, and the diluted solution was designated as 1 to 6 reproductions.

The chromatographic conditions were the same as in example 1, 80. Mu.L of the above control solution was precisely measured, and the measurement was carried out by injecting into a chromatograph, and the impurity content was calculated, and the results of the precision test are shown in Table 15.

TABLE 15 precision test results

Note that: "ND" means "undetected"

Conclusion: the precision test result shows that all known impurities are not detected, the maximum single impurity content and the total impurity content are 0.02%, and the number of the impurities is consistent, so that the method has good repeatability and good precision.

Example 13: durability test

Durability refers to the degree of tolerance to which the measurement result is not affected when the measurement chromatographic conditions slightly change. The system applicability solution and the sample solution were prepared in the same manner as in example 1, and the measurement results are shown in tables 16 and 17, with the temperature, flow rate, measurement wavelength, mobile phase pH and durability of the measurement method being changed under the conditions of changing different batches of chromatographic columns based on the measurement conditions of example 1.

Table 16 system applicability solution test results

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TABLE 17 test sample solution detection results

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Note that: "ND" means "undetected".

Conclusion:

the durability test results show that on the basis of the embodiment 1, the separation degree of each peak in the system applicability solution is larger than 1.0 when chromatographic conditions such as column temperature change (+ -2 ℃), flow rate change (+ -0.1 mL/min), detection wavelength change (+ -2 nm), pH value change (+ -0.2) of the mobile phase A, and chromatographic column replacement of different batches are changed, and the impurity detection results of the sample solution under the chromatographic detection conditions are not obviously different.

Under different chromatographic conditions, the column temperature is controlled to be 38-42 ℃, the flow rate is controlled to be 0.7-0.9 mL/min, the detection wavelength is 257-261 nm, the pH value of the mobile phase A is 5.8-6.2, and the durability of detecting the impurities in the Argatroban injection is good under different batches of chromatographic column conditions.

Comparative examples 1 to 2

With reference to the detection conditions and methods of the comparative document, 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) in the sample solution and the mixed impurity control solution of the embodiment of the present invention were separated and detected.

Comparative document 1: CN104237397a is a method for detecting related substances of argatroban injection.

Comparison document 2: wang Gang impurity control in Argatroban bulk drug, university of Zhejiang's Shuoshi treatise 2013-12-18.

Table 18 comparison of impurities of the present application with comparative files

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Table 19 comparison of chromatographic conditions of the present application and comparative files

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The conditions and methods for detecting comparative document 1 and comparative document 2 were used to examine 12 kinds of impurities in example 1 of the present invention, and the 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I, impurity F) were analyzed and detected, and the results are shown in table 20.

Table 20 comparison of the test results of the present application with the comparison document

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Conclusion: under the detection conditions of example 1 of the present invention, 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) can be effectively detected: wherein, 3 impurities in the solution chromatogram of the system applicability are separated and quantitatively analyzed: 2S, 4R-isomer, 2R, 4S-isomer, key degradation impurity G, each chromatographic peak separation degree >1.0, accord with impurity detection requirement, separation effect is good.

Under the detection conditions of comparative document 1, 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) cannot be detected effectively, and all 10 kinds of impurities reported in pharmacopoeia and literature cannot be eluted under the present conditions; wherein, 3 kinds of impurities cannot be separated: the chromatographic peaks of the key degradation impurity G and the unreported 2 impurities (2 s,4 r-isomer, 2r,4 s-isomer) alternately appear, the interference of each impurity peak is obvious, the separation degree of each peak is <1.0, the impurity detection requirement is not met, and the separation effect is poor (see fig. 21).

Under the detection conditions of comparative document 2, 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) cannot be effectively detected, and 10 kinds of impurities reported in pharmacopoeia and literature cannot be all eluted under the present conditions; 2s,4 s-isomer, impurity E, impurity O, impurity I cannot be detected; wherein, 3 kinds of impurities cannot be separated: the chromatographic peaks of the key degradation impurity G and the unreported 2 impurities (2 s,4 r-isomer, 2r,4 s-isomer) alternately appear, the interference of each impurity peak is obvious, the separation degree of each peak is <1.0, the separation effect is bad (see fig. 22).

The small knot:

compared with other detection methods, only the detection method of the present invention (e.g., example 1) can realize detection of 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) and separation detection of 3 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity G), with good separation effect.

In contrast, with other detection methods (for example, reference 1 and reference 2), detection of 12 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F) cannot be achieved, and particularly, detection of 3 kinds of impurities (2 s,4 r-isomer, 2r,4 s-isomer, impurity G) cannot be separated and the separation effect is poor.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A detection method of impurities in Argatroban raw materials or preparations adopts a high performance liquid chromatography method for detection;

the chromatographic conditions include:

mobile phase: the mobile phase A is a mixed solution of glacial acetic acid buffer solution and tetrahydrofuran, the volume ratio of the glacial acetic acid buffer solution to the tetrahydrofuran=85-90:15-10, and the pH value of the glacial acetic acid buffer solution is 5.8-6.2;

the mobile phase B is a mixed solution of methanol and acetonitrile, the volume ratio of the methanol to the acetonitrile=58-62:42-38, and the preferable volume ratio of the methanol to the acetonitrile=60:40;

elution mode: gradient elution.

2. The detection method according to claim 1, wherein the column is YMC J' sphere ODS-H18,4.6 mm. Times.250 mm,4.0 μm or GL Sciences ODS-3,4.6 mm. Times.250 mm,3 μm.

3. The detection method according to claim 1 or 2, wherein the volume ratio of the glacial acetic acid buffer and tetrahydrofuran mixed solution in the mobile phase A is: glacial acetic acid buffer, tetrahydrofuran=86.5:13.5.

4. The detection method according to any one of the preceding claims 1 to 3, wherein the pH of the glacial acetic acid buffer in mobile phase a is 6.0.

5. The detection method according to any one of the preceding claims 1 to 4, wherein the gradient elution gradient is:

。

6. The detection method according to any one of the preceding claims 1 to 5, wherein the detection wavelength of the chromatographic conditions is 257 to 261nm, preferably 259nm.

7. The detection method according to any one of the preceding claims 1 to 6, wherein the flow rate of the chromatographic column is 0.7-0.9 mL/min, preferably 0.8mL/min.

8. The detection method according to any one of the preceding claims 1 to 7, wherein the column temperature of the chromatographic column is 38 to 42 ℃, preferably 40 ℃.

9. The assay according to any one of the preceding claims 1-8, wherein the impurities in the argatroban feedstock or formulation comprise one or more of the following:

10. The detection method according to any one of claims 1 to 9, wherein the impurities in the argatroban raw material or preparation comprise impurity 2s,4 r-isomer, impurity 2r,4 s-isomer and impurity G.

11. The detection method according to any one of claims 1 to 10, wherein the impurities in the argatroban starting material or formulation comprise 2r,4 s-isomer and impurity G.

12. The detection method according to any one of claims 1 to 11, wherein the impurities in the argatroban raw material or preparation include 2s,4 r-isomer, 2r,4 s-isomer, impurity 2s,4 s-isomer, impurity G, impurity 5, impurity K, impurity L, impurity E, impurity O, impurity C, impurity I and impurity F.

13. The assay according to any one of the preceding claims 1-12, wherein the formulation is an injection.