Detection method of succinylcholine chloride intermediate and related impurities

Abstract

The invention relates to a method for detecting an intermediate of succinylcholine chloride and related impurities, which comprises the following steps: detecting a solution to be detected of the intermediate of succinylcholine chloride by high performance liquid chromatography by adopting an octadecyl silane bonded silica gel chromatographic column, and carrying out gradient elution by taking a 0.05-0.5% phosphoric acid aqueous solution as a mobile phase A and methanol as a mobile phase B; the detection wavelength is 200 to 220 nm; the sample size is 5-50 [mu] l; the flow rate is 0.5 to 1.5 ml / min; the temperature of a chromatographic column is 30-60 DEG C; according to the method, succinic acid, succinic acid monomethyl ester, 2, 3-dichloromaleate monomethyl ester and 4-(4-methylphenyl)-4-oxobutyric acid methyl ester can be well separated, and the separation degree is 2.0 or above; the method has the advantages of high impurity recovery rate, good repeatability and high accuracy, and realizes the separation and quality control of the succinyl chloride intermediate succinyl chloride and impurities thereof, thereby ensuring the quality of the final product succinyl chloride.

Description

Technical Field

[0001] The invention belongs to the field of drug intermediate detection, and particularly relates to a method for detecting a succinylcholine chloride intermediate and related impurities. Background Art

[0002] Succinylcholine chloride is an ultra-short-acting depolarizing skeletal muscle relaxant for intravenous administration. Its generic name is succinylcholine and succinylcholine. Its chemical name is 2',2-[(1,4-dioxo-1,4-butylene)bis(oxy)]bis(N,N,N-trimethylethylamine) dichloride dihydrate. Its chemical formula is shown in Formula I. Its molecular formula is C 14 H 30 Cl2N2O4·2H2O, molecular weight is 397.34.

[0003]

[0004] Succinylcholine chloride can replace acetylcholine, bind to the motor end plate of cholinergic receptors to produce depolarization, and has a rapid onset of muscle relaxation (flaccid paralysis) and rapid metabolism. It is widely used in clinical practice. Currently, no non-depolarizing muscle relaxant can fully possess its advantages. Succinylcholine chloride can provide more satisfactory conditions for endotracheal intubation and has become the longest-used muscle relaxant. It is currently the only muscle relaxant selected for difficult intubation and emergency intubation.

[0005] In the mainstream synthesis process of succinylcholine chloride API, succinic acid (i.e. succinic acid) or its anhydride is generally used as the starting material, an acyl chloride reagent such as solid phosgene (BTC) or dichlorothionyl is added, and DMF is used as an initiator to obtain the intermediate succinyl chloride (i.e. succinyl chloride), and then choline chloride is added to the above reaction solution, and succinylcholine chloride is obtained after post-treatment.

[0006] The preparation process of succinylcholine chloride involves the control of the reaction intermediate succinyl chloride. Impurities may exist in the succinyl chloride intermediate: succinic acid, monomethyl succinate, 2,3-dichloromaleic acid monomethyl ester and 4-(4-methylphenyl)-4-oxobutyric acid methyl ester. These impurities may remain in the finished product of succinylcholine chloride, thereby affecting the quality of the finished product. Therefore, they need to be controlled, especially succinic acid, which is also a degradation impurity of succinylcholine chloride and is clearly required to be controlled in the pharmacopoeia.

[0007] Among the current pharmacopoeias of various countries, only the USP pharmacopoeia requires the HPLC method for the detection of related substances of succinylcholine chloride API. Its detection requirements for related substances of succinylcholine chloride API are: succinic acid 0.1%, succinic acid 0.4%, bimodal group 0.4%, others 0.2%, total impurities: 1.5%.

[0008] At present, there is no literature reporting the detection method of succinyl chloride and the above four impurities. The following problems exist in the determination of succinyl chloride, an intermediate of succinylcholine chloride, and its multiple impurities:

[0009] First, the number of impurities is large and their polarity is similar, especially dimethyl succinate and 2,3-dichloromaleic acid monomethyl ester, which are derivatives of succinyl chloride and methanol / water, which are difficult to separate and require high selectivity of the analytical method. Succinic acid has a high polarity and poor retention, and it is easy to produce a secondary retention effect with the residual silanol group in the stationary phase, resulting in poor peak shape.

[0010] Second, the UV absorption of the target analyte is close to the end. The UV absorption of the succinyl chloride intermediate and its impurities is close to the end, which makes it difficult to choose the mobile phase and additive types.

[0011] From the above, it can be seen that there is still great room for exploration in the quality control of succinylcholine chloride intermediate succinyl chloride and its multiple impurities. Summary of the invention

[0012] The object of the present invention is to provide a method for detecting a succinylcholine chloride intermediate and related impurities. The separation degree of succinic acid, monomethyl succinate, 2,3-dichloromaleic acid monomethyl ester and methyl 4-(4-methylphenyl)-4-oxobutyrate is above 2.0, the linearity is good, the repeatability is good, the accuracy is high, the separation and quality control of the succinylcholine chloride intermediate succinyl chloride and its impurities are achieved, thereby ensuring the quality of the final product succinylcholine chloride.

[0013] In order to achieve the above object, the present invention provides the following technical solutions:

[0014] The method for detecting succinylcholine chloride intermediates and related impurities comprises the following steps:

[0015] The test solution of succinylcholine chloride intermediate succinyl chloride is detected by high performance liquid chromatography, using a chromatographic column with octadecylsilane bonded silica gel as a filler, 0.05-0.5% phosphoric acid aqueous solution as mobile phase A, and methanol as mobile phase B for gradient elution; detection wavelength: 200-220nm; injection volume: 5-50μl; flow rate: 0.5-1.5ml / min; chromatographic column temperature: 30-60℃.

[0016] Preferably, the impurities include succinic acid, monomethyl succinate, monomethyl 2,3-dichloromaleate and methyl 4-(4-methylphenyl)-4-oxobutanoate.

[0017] Furthermore, the elution process is as follows: after isocratic elution for 6 to 12 minutes with a mobile phase A of 96 to 100% by volume and a mobile phase B of 0 to 4% by volume as the starting mobile phase, gradient elution for 8 to 15 minutes, after the mobile phase composition reaches 10 to 40% of the mobile phase A and 50 to 90% of the mobile phase B, isocratic elution for 12 to 20 minutes, and then the starting mobile phase is restored for equilibrium, and the equilibrium time is not less than 5 minutes.

[0018] Preferably, the chromatographic column is selected from Waters XBridge C18, Waters Atlantis T3, XtimateC18, Waters ACQUITY HSS T3, and Inertsil ODS-3.

[0019] Furthermore, the chromatographic column filler has a particle size of ≤4.6 μm, a length of ≥150 cm, and an inner diameter of ≤5 mm.

[0020] Furthermore, the concentration of dimethyl succinate in the test solution is 3-8 mg / ml.

[0021] The ultraviolet absorption of the succinyl chloride intermediate and related impurities is close to the end, and there is a problem of terminal absorption. The inventor unexpectedly found that by using a chromatographic column with octadecylsilane bonded silica gel (C18) as a filler and a specific acidic mobile phase, the retention of these compounds can be improved, such as increasing the retention of succinic acid and improving the peak shape of succinic acid. However, since the ultraviolet absorption of the succinyl chloride intermediate and its impurities is close to the end, most mobile phase additives may cause poor baselines and poor target response due to the large ultraviolet cutoff wavelength, so there are many restrictions on the selection of mobile phase types.

[0022] Succinic acid has a large polarity, poor retention, and is prone to secondary retention effects with residual silanol groups in the stationary phase, resulting in poor peak shape. The present invention selects a phosphoric acid ratio of 0.05-0.5%, which further enhances the retention of succinic acid, improves the peak shape, and improves the separation of the target peak. If the phosphoric acid ratio is too low, the effect of enhancing retention and improving peak shape is not obvious, and the separation of dimethyl succinate and 2,3-dichloromaleic acid monomethyl ester is poor; if the phosphoric acid ratio is too high, the chromatographic behavior of the target peak is limitedly improved, and the chromatographic column is irreversibly damaged due to the low pH value of the mobile phase.

[0023] In the present invention, the number of impurities is large and the polarity is similar, especially the derivatives of succinyl chloride and methanol / water, dimethyl succinate and 2,3-dichloromaleic acid monomethyl ester, which are difficult to separate, and an elution gradient is developed according to the retention capacity of each impurity, so that all impurities can be eluted out of the peak and separated better. During elution, the mobile phase A with a volume percentage of 96-100% and the mobile phase B with a volume percentage of 0-4% are used as the starting mobile phase for isocratic elution, and the separation degree is better and the peak shape is better. During isocratic elution, if the proportion of organic solvent is too large, the retention capacity and separation degree of each component will be deteriorated, and if the proportion of organic phase is too small, the chromatographic peak may be broadened, which has a negative impact on the peak shape. After gradient elution for 8-15 minutes, the proportion of mobile phase A in the mobile phase reaches 10-40%, which helps to increase the separation degree of monomethyl succinate, dimethyl succinate and 2,3-dichloromaleic acid monomethyl ester, thereby obtaining good specificity and accurate quantification.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] In the present invention, the blank solvent has no interference on the inspection of each impurity, the separation degree of each impurity is above 2.0, and the specificity is good; the impurity reference substance is used for external standard method quantification, the recovery rate is between 98% and 110%, the accuracy is good, and the accuracy of the detection result is guaranteed; the linear correlation coefficient r is above 0.998, the linearity is good, the recovery rate RSD is between 0.9 and 1.3%, and the repeatability is good.

[0026] The methodological validation of the method of the present invention was carried out through tests such as specificity, linearity, repeatability, and accuracy. The results showed that the method of the present invention has good detection specificity, good repeatability, and high accuracy for the four impurities, and can meet the needs of the determination of impurities in the intermediate succinyl chloride of succinylcholine chloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the reference solution spectrum in Example 1 of the present invention.

[0028] Figure 2 This is a chromatogram of the spiked test solution in Example 1 of the present invention.

[0029] Figure 3 This is the solution spectrum of the system applicability in Example 1 of the present invention.

[0030] Figure 4 It is the chromatogram of the system suitability solution in Comparative Example 1.

[0031] Figure 5 This is the chromatogram of the test sample spiked solution in Comparative Example 2. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the scope of the embodiments.

[0033] The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions; in the chromatograms of the examples and comparative examples, the unit of peak time is minute (min).

[0034] The reference substances used in the examples of the present invention are shown in Table 1.

[0035] Table 1

[0036]

[0037]

[0038] Example 1 Detection method of succinylcholine chloride intermediate succinyl chloride and related impurities, high performance liquid chromatography detection conditions are as follows:

[0039] High performance liquid chromatograph: Waters e2695;

[0040] Column: Waters XBridge C18, 4.6 mm × 250 mm, 5 μm;

[0041] Mobile phase A: 0.2% phosphoric acid aqueous solution, volume percentage;

[0042] Mobile phase B: methanol;

[0043] Elution method: gradient elution. See Table 2 for the elution process.

[0044] Column temperature: 50°C;

[0045] Flow rate: 1.0ml / min;

[0046] Detection wavelength: 210nm;

[0047] Injection volume: 20μl.

[0048] Table 2

[0049] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 98 2 8 98 2 20 30～40 70～60 35 30～40 70～60 36 98 2 45 98 2

[0050] Solution preparation:

[0051] Preparation method of reference solution: Take appropriate amount of succinic acid reference substance, monomethyl succinate reference substance, monomethyl 2,3-dichloromaleate reference substance, and methyl 4-(4-methylphenyl)-4-oxobutyrate reference substance, accurately weigh them, add appropriate amount of methanol to dissolve them, dilute with solvent to make a solution containing approximately 0.05 mg of succinic acid, 0.5 mg of monomethyl succinate, 0.25 mg of monomethyl 2,3-dichloromaleate and 0.01 mg of methyl 4-(4-methylphenyl)-4-oxobutyrate per 1 ml, and shake well.

[0052] Preparation method of spiked test solution: Take 50 mg of intermediate succinyl chloride sample, place it in a 10 ml volumetric flask, add reference solution to dissolve and dilute to the scale, and shake well.

[0053] Since part of the succinyl chloride reacts with the methanol in the mobile phase to form dimethyl succinate, its elution condition needs to be considered.

[0054] Preparation method of system suitability solution: Take an appropriate amount of dimethyl succinate reference substance, accurately weigh it, add an appropriate amount of reference substance solution to dissolve it, dilute it with solvent to make a solution containing approximately 2.5 mg of dimethyl succinate, 0.05 mg of succinic acid, 0.5 mg of monomethyl succinate, 0.25 mg of monomethyl 2,3-dichloromaleate and 0.01 mg of methyl 4-(4-methylphenyl)-4-oxobutyrate per 1 ml, and shake well.

[0055] According to the HPLC method in accordance with the General Rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the reference solution, system suitability solution, and test sample spiked solution were injected into the chromatograph respectively, and the chromatogram was recorded. The results are shown in Figure 1 to Figure 3 And Table 3.

[0056] Table 3

[0057] Peak name Retention time (min) Separation Succinic acid 5.253 / Monomethyl succinate 12.672 39.9 Dimethyl succinate 16.464 25.3 2,3-Dichloromaleic acid monomethyl ester 16.842 2.2 Methyl 4-(4-methylphenyl)-4-oxobutanoate 24.397 57.5

[0058] Depend on Figure 1 to Figure 3 As shown in Table 3, part of succinyl chloride peaks in the form of dimethyl succinate, and succinic acid, monomethyl succinate, dimethyl succinate, 2,3-dichloromaleic acid monomethyl ester, and 4-(4-methylphenyl)-4-oxobutyric acid methyl ester peak in sequence. The separation degrees between the impurities are all greater than 1.5, meeting the separation requirements. Under the detection conditions, the separation degrees of the impurities are above 2.2.

[0059] Embodiments 2 to 9

[0060] In Examples 2 to 9, except for the special conditions in Table 4, the other test conditions are the same as those in Example 1.

[0061] Table 4

[0062]

[0063] The system suitability solution was taken and liquid chromatography was performed on Examples 2 to 9 according to the chromatographic conditions in Table 4. The results are shown in Table 5:

[0064] Table 5 Retention time Unit: min

[0065]

[0066] As can be seen from Table 5, when the mobile phase, wavelength, flow rate and column temperature are changed within the detection range of the present invention, the separation degree between the impurities meets the requirements, the separation degree is above 2.0, and the method has good durability.

[0067] Example 10

[0068] The chromatographic detection conditions and method are the same as those in Example 1.

[0069] 1. The intermediate succinyl chloride was added to the reference solution described in Example 1 according to the theoretical amount of 5 mg / ml as a recovery solution. Six portions were prepared in parallel to test the recovery of succinic acid, succinic acid monomethyl ester, 2,3-dichloromaleic acid monomethyl ester and 4-(4-methylphenyl)-4-oxobutyric acid methyl ester in the present invention. The specific data are shown in Table 6.

[0070] Table 6

[0071]

[0072] It can be seen that the recoveries of succinic acid, monomethyl succinate, monomethyl 2,3-dichloromaleate and methyl 4-(4-methylphenyl)-4-oxobutanoate at a concentration level of 100% are between 99.0 and 101.0%, 100.0 and 103.8%, 98.3 and 102.3%, and 98.7 and 101.6%, respectively, and the recovery RSD (n=6) is between 0.9 and 1.3%, indicating that the method has good accuracy and repeatability.

[0073] 2. Six test solutions L1 to L6 of different concentrations were prepared for the four impurity reference solutions for testing. The chromatographic conditions were the same as those in Example 1. The linear relationship between the concentration of each impurity and the peak area was examined. The test results are shown in Table 7.

[0074] Table 7

[0075]

[0076]

[0077]

[0078] As can be seen from Table 7, under the detection method of the present invention, the correlation coefficient r between the concentration of each impurity and the peak area is between 0.9989 and 1.0000, and the linear relationship is good.

[0079] Comparative Example 1

[0080] The USP pharmacopoeia method was used to detect succinyl chloride, an intermediate of succinylcholine chloride, and its impurities.

[0081] 1. High performance liquid chromatography detection conditions are as follows:

[0082] Chromatographic column: a column filled with octadecylsilane bonded silica gel (Hichrom Alltima C18, 4.6 mm × 250 mm, 5 μm);

[0083] Mobile phase: buffered saline (3.85 g / L anhydrous sodium 1-pentanesulfonate, 2.9 g / L sodium chloride, 1% 1N sulfuric acid aqueous solution)-acetonitrile (95:5);

[0084] Column temperature: 30°C;

[0085] Flow rate: 1.0ml / min;

[0086] Detection wavelength: 214nm;

[0087] Injection volume: 20μl.

[0088] 2. Experimental steps

[0089] Preparation method of reference solution: Take appropriate amount of succinic acid, monomethyl succinate, monomethyl 2,3-dichloromaleate, and methyl 4-(4-methylphenyl)-4-oxobutyrate, accurately weigh, add appropriate amount of methanol to dissolve, dilute with 10% methanol to make a solution containing approximately 0.05 mg of succinic acid, 0.1 mg of monomethyl succinate, 0.05 mg of monomethyl 2,3-dichloromaleate, and 0.005 mg of methyl 4-(4-methylphenyl)-4-oxobutyrate per 1 ml, and shake well.

[0090] Preparation method of system suitability solution: Take an appropriate amount of dimethyl succinate, accurately weigh it, add an appropriate amount of reference solution to dissolve it, dilute it with 10% methanol to make a solution containing approximately 0.5 mg of dimethyl succinate, 0.05 mg of succinic acid, 0.1 mg of monomethyl succinate, 0.05 mg of monomethyl 2,3-dichloromaleate and 0.005 mg of methyl 4-(4-methylphenyl)-4-oxobutyrate per 1 ml, and shake well.

[0091] According to the high performance liquid chromatography method, take the system suitability solution and inject it into the chromatograph, and record the chromatogram. Figure 4 .

[0092] Depend on Figure 4 It can be seen that under this system, succinic acid is poorly retained, the separation from adjacent peaks is poor, and the peak shape is poor. Monomethyl succinate and dimethyl succinate cannot be separated and co-emit peaks.

[0093] Comparative Example 2

[0094] Unless otherwise specified, other detection conditions and methods refer to Example 1.

[0095] 1. Chromatographic conditions

[0096] Chromatographic column: Agilent ZORBAX SB AQ, 4.6 mm × 250 mm, 5 μm;

[0097] Mobile phase A: 0.2% phosphoric acid in water;

[0098] Mobile phase B: methanol;

[0099] Elution method: gradient elution, elution program see Table 7;

[0100] Column temperature: 50°C;

[0101] Flow rate: 1.0ml / min;

[0102] Detection wavelength: 210nm;

[0103] Injection volume: 50μl.

[0104] Table 7

[0105] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 10 95 5 30 30 70 40 30 70 45 95 5 50 95 5

[0106] 2. Experimental steps

[0107] Reference substance mixed solution: Take appropriate amounts of succinic acid, monomethyl succinate, monomethyl 2,3-dichloromaleate, and methyl 4-(4-methylphenyl)-4-oxobutyrate, accurately weigh them, add appropriate amount of methanol to dissolve them, and dilute them with 10% methanol to make a solution containing approximately 0.75 mg of succinic acid, 1 mg of monomethyl succinate, 0.25 mg of monomethyl 2,3-dichloromaleate, and 0.005 mg of methyl 4-(4-methylphenyl)-4-oxobutyrate per 1 ml, and shake well.

[0108] Preparation method of test sample spiked solution: Take 50 mg of intermediate succinyl chloride sample, place it in a 10 ml volumetric flask, add reference sample mixed solution to dissolve and dilute to the scale, and shake well.

[0109] According to the HPLC method in accordance with the General Rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia, inject the spiked solution of the test sample into the chromatograph and record the chromatogram. Figure 5 .

[0110] Depend on Figure 5 It can be seen that under this detection method, monomethyl succinate and monomethyl 2,3-dichloromaleate co-emitted peaks and could not be separated.

Claims

1. A method for detecting succinylcholine chloride intermediates and related impurities, comprising the following steps: The test solution of succinylcholine chloride intermediate succinyl chloride is detected by high performance liquid chromatography, using a chromatographic column with octadecylsilane bonded silica gel as a filler, 0.05-0.5% phosphoric acid aqueous solution as mobile phase A, and methanol as mobile phase B for gradient elution; detection wavelength: 200-220nm; injection volume: 5-50μl; flow rate: 0.5-1.5ml / min; chromatographic column temperature: 30-60℃.

2. The method for detecting the succinylcholine chloride intermediate and related impurities according to claim 1, characterized in that: The impurities include succinic acid, monomethyl succinate, monomethyl 2,3-dichloromaleate and methyl 4-(4-methylphenyl)-4-oxobutanoate.

3. The method for detecting the succinylcholine chloride intermediate and related impurities according to claim 1, characterized in that: The gradient elution process is as follows: after isocratic elution for 6 to 12 minutes with a mobile phase A of 96 to 100% by volume and a mobile phase B of 0 to 4% by volume as the initial mobile phase, gradient elution for 8 to 15 minutes, after the mobile phase composition reaches 10 to 40% of the mobile phase A and 50 to 90% of the mobile phase B, isocratic elution for 12 to 20 minutes, and then the initial mobile phase is restored for equilibrium, and the equilibrium time is not less than 5 minutes.

4. The method for detecting the succinylcholine chloride intermediate and related impurities according to claim 1, characterized in that: The chromatographic column is selected from Waters XBridge C18, Waters Atlantis T3, Waters ACQUITY HSS T3 or InertsilODS-3.

5. The method for detecting the succinylcholine chloride intermediate and related impurities according to claim 1, characterized in that: The chromatographic column filler has a particle size of ≤4.6 μm, a length of ≥150 cm, and an inner diameter of ≤5 mm.

6. The method for detecting the succinylcholine chloride intermediate and related impurities according to claim 1, characterized in that: The concentration of succinyl chloride in the test solution is 3-8 mg / ml.

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