A method for the determination of the content of iodixanol

Abstract

The present invention discloses a method for determining the iodixanol content in iodixanol injection, which utilizes high-performance liquid chromatography for detection and specifically comprises the following steps: a. preparing a test solution; b. preparing a reference solution; and c. respectively injecting the reference solution and the test solution into a liquid chromatograph, and recording a chromatogram. The method for determining the iodixanol content of the present invention can also be used to detect the iodixanol content in iodixanol injection and other iodixanol preparations, and has greater versatility, good specificity, good linearity, high precision, high accuracy, and good durability.

Description

Technical Field

[0001] The present invention relates to the field of drug detection, and particularly to a method for determining the content of iodixanol. Background Art

[0002] Iodinated contrast agents are increasingly used in clinical diagnosis, and several non-ionic iodinated contrast agents have been marketed. Iodixanol injection is a non-ionic, dimeric, hexaiodinated, water-soluble X-ray contrast agent. The biggest feature that differentiates it from other non-ionic iodinated contrast agents as a third-generation non-ionic iodinated contrast agent is that it is isotonic with plasma osmotic pressure. It not only has good imaging effect but also low toxicity and few side effects, and has good clinical tolerance for patients. However, iodixanol injection has a high concentration and high viscosity, which is not conducive to its quality control such as content determination. The current legal standards for iodixanol injection at home and abroad, such as the content determination methods in the United States Pharmacopoeia (USP40-NF35) and the import registration standards, are all potentiometric titration methods. The main principle is to add an alkali solution and zinc powder to remove iodine, and then titrate with silver nitrate titrant. The main operation process includes: sampling and dilution → adding an alkali solution and zinc powder for heating reaction, refluxing for 30 min (the United States Pharmacopoeia requires heating reflux for 1 h) → cooling, washing the condenser → filtering, combining the filtrate and washing solution → adding glacial acetic acid for titration (it can be known from actual operation that a single determination takes about 2 h, and a sample is determined in parallel twice, totaling about 4 h). The main disadvantages of this method are: (1) The determination operation involves chemical reactions for deiodination, filtration, and washing. Whether the reaction is complete is not easily controlled, and whether the reaction is complete and the washing is complete directly affect the content determination result, with large human errors; (2) Since there are iodide ions and chloride ions in the solution (introduced by the pH regulator hydrochloric acid and the excipients sodium chloride and calcium chloride), there will be two inflection points during the titration process, resulting in difficulty in judging the titration end point; (3) The determination process is cumbersome and time-consuming, and is not suitable for content detection during intermediate control in commercial large-scale production.

[0003] Chinese Patent CN111272608 discloses a method for detecting the density of iodinated contrast agents using a densitometer and obtaining the density of the sample according to the mathematical relationship between the content and the density. This method requires specific density measurement instruments, which are expensive and have a low usage frequency, and are only applicable to special varieties with high density, so the generality is not strong. This patent compares the density method with high performance liquid chromatography, and mentions that high performance liquid chromatography has the disadvantage of too long detection time. In Example 4, it determines the content of iopamidol injection by high performance liquid chromatography and concludes that a single determination of the liquid medicine takes about 2.5 h, and a sample is repeatedly determined twice for about 5 h. Therefore, this patent believes that high performance liquid chromatography has the disadvantage of long determination time and is not suitable for drug content detection.

[0004] Sun Min et al., Determination of the Content of Iodixanol in Iodixanol Injection by High Performance Liquid Chromatography [J]. Shanxi Medical Journal, 2012, 41(11), which discloses a method for detecting the content of iodixanol. This method uses gradient elution for 80 minutes and has two main peaks. Therefore, this method still has the problem of too long detection time.

[0005] When detecting the content during the commercial large-scale production of intermediates, it is necessary to be rapid and accurate. If the titration method and high performance liquid chromatography method in the current existing technologies are used, the time is too long. Once the measurement result is abnormal, the retest takes even longer, which will seriously affect the production progress. At the same time, due to the fact that the liquid preparation process time limit exceeds the specified requirements, the risk of microbial contamination is increased, making the quality of the final product uncontrollable. Therefore, there is an urgent need to develop a content determination method with simple operation, short determination time, high accuracy, and strong universality. Summary of the Invention

[0006] To solve the above problems, the present invention provides a method for determining the content of iodixanol, which is characterized in that it uses high performance liquid chromatography for detection, and specifically includes the following steps:

[0007] a. Preparation of the reference solution: Take the iodixanol reference substance and dissolve it with a diluent to obtain it.

[0008] b. Preparation of the test solution: Take the test sample and dilute it with a diluent to obtain it.

[0009] c. Respectively aspirate the reference solution and the test solution and inject them into the liquid chromatograph. The chromatographic conditions are as follows:

[0010] Chromatographic column: Packed with ethyl-bridged hybrid particles; Mobile phase: water - acetonitrile; Elution program: Water - acetonitrile is isocratic eluted at a volume ratio of 20 - 40:60 - 80; or from 0 to 25 minutes, acetonitrile is gradient eluted from 70 - 75% to 65 - 70% by volume percentage.

[0011] d. Calculate the content of iodixanol by the external standard method.

[0012] The diluent is water, acetonitrile, methanol or a mixture of water - acetonitrile.

[0013] Further, each 1 ml of the reference solution contains a solution of iodixanol at 0.2 - 1.2 mg.

[0014] Further, each 1 ml of the test solution contains a solution of iodixanol at 0.2 - 1.2 mg.

[0015] Further, the test sample is an iodixanol solid preparation, an iodixanol raw material drug or an iodixanol solution preparation; the iodixanol solution preparation includes an iodixanol injection.

[0016] Further, the diluent is water, acetonitrile, methanol or a mixture of water and acetonitrile.

[0017] Furthermore, the diluent is a mixture of water and acetonitrile, wherein the volume ratio of water to acetonitrile is 20 - 40:60 - 80.

[0018] Further, the volume of the solution injected into the liquid chromatograph in step c is 5 - 10 μl.

[0019] Furthermore, the volume of the solution injected into the liquid chromatograph in step c is 5 μl.

[0020] Further, the chromatographic column in step c is Waters HILIC, with specifications including: 4.6 mm × 150 mm, 3.5 μm; 4.6 mm × 250 mm, 5 μm; 4.6 mm × 100 mm, 5 μm; 4.6 mm × 100 mm, 3.5 μm or 4.6 mm × 150 mm, 5 μm.

[0021] Further, the wavelength of the chromatographic conditions in step b is 245 nm, the column temperature is 25°C - 35°C, and the flow rate is 0.8 ml / min - 1.1 ml / min.

[0022] Further, the column temperature in the chromatographic conditions is 30°C and the flow rate is 1.0 ml / min.

[0023] Further, the elution program: water - acetonitrile is isocratic eluted at a volume ratio of 25 - 35:65 - 75; or acetonitrile is gradient eluted from 70% - 75% to 65% - 70% by volume percentage in 0 - 15 min.

[0024] Further, the elution program: water - acetonitrile is isocratic eluted at a volume ratio of 35:65; or acetonitrile is gradient eluted from 70% to 65% by volume percentage in 0 - 15 min.

[0025] The present invention provides a method for determining the content of iodixanol in an iodixanol injection. The time for one determination is within 10 min, and it has high accuracy. It can overcome the disadvantages of the potentiometric titration method, such as the chemical reaction being not easily controlled, the operation being cumbersome, the personnel error being large, it being not easy to judge the titration end point, and the detection time being long; it can also overcome the disadvantages of poor accuracy and long time consumption in the existing liquid chromatography detection. Verified by actual production, the method for determining the content of iodixanol in the present invention has better universality, and has the characteristics of good specificity, good linear relationship, high precision, high accuracy and good durability.

[0026] Obviously, based on the above content of the present invention, according to the common technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0027] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Ultraviolet absorption spectrum of iodixanol

[0029] Figure 2 Column 1(C 18 Screening chromatogram of chromatographic column

[0030] Figure 3 Screening chromatogram of column 2 (gel column)

[0031] Figure 4 Screening chromatogram of column 3 (chiral column)

[0032] Figure 5 Screening chromatogram of column 4 (reversed-phase column)

[0033] Figure 6 Screening chromatogram of elution program

[0034] Figure 7 Screening chromatograms of test sample concentration and injection volume DETAILED DESCRIPTION

[0035] Example 1: Solution preparation:

[0036] Preparation of reference solution: Take an appropriate amount of iodixanol reference substance, accurately weigh it, place it in a volumetric flask, add diluent water, acetonitrile, or a water-acetonitrile mixture with a volume ratio of 20-40:60-80 to dissolve it and dilute it to a solution of about 0.2-1.2 mg of iodixanol per 1 ml. Shake well and use this as the reference solution.

[0037] Preparation of test solution: Accurately measure iodixanol injection and dilute with water, acetonitrile, or a water-acetonitrile mixture with a volume ratio of 20-40:60-80 to make a solution containing 0.2-1.2 mg of iodixanol per 1 ml.

[0038] Example 2 Determination of iodixanol content of the present invention

[0039] Prepare the solution according to the solution preparation method of Example 1, and take 5 μl of the reference solution and the test solution and inject them into the liquid chromatograph respectively. The chromatographic conditions are as follows:

[0040] Chromatographic column: Waters HILIC, 4.6 mm × 150 mm, 5 μm; Mobile phase: water - acetonitrile with a volume ratio of 35:65 as the mobile phase; Wavelength 245 nm, column temperature 30 °C, flow rate 1.0 ml / min;

[0041] The content of iodixanol is calculated by the external standard method.

[0042] Example 3 Determination method of the content of iodixanol in the present invention

[0043] Prepare the solution according to the solution preparation method of Example 1. Respectively draw 5 μl of the reference solution and the test solution and inject them into the liquid chromatograph. The chromatographic conditions are as follows:

[0044] Chromatographic column: Waters HILIC, 4.6 mm × 250 mm, 5 μm; Mobile phase: water - acetonitrile with a volume ratio of 20:80 as the mobile phase; Wavelength 245 nm, column temperature 28 °C, flow rate 1.1 ml / min;

[0045] The content of iodixanol is calculated by the external standard method.

[0046] Example 4 Determination method of the content of iodixanol in the present invention

[0047] Prepare the solution according to the solution preparation method of Example 1. Respectively draw 5 μl of the reference solution and the test solution and inject them into the liquid chromatograph. The chromatographic conditions are as follows:

[0048] Chromatographic column: Waters HILIC, 4.6 mm × 150 mm, 3.5 μm; Mobile phase: water - acetonitrile with a volume ratio of 40:60 as the mobile phase; Wavelength 245 nm, column temperature 28 °C, flow rate 0.9 ml / min;

[0049] The content of iodixanol is calculated by the external standard method.

[0050] Example 5 Determination method of the content of iodixanol in the present invention

[0051] Prepare the solution according to the solution preparation method of Example 1. Respectively draw 5 μl of the reference solution and the test solution and inject them into the liquid chromatograph. The chromatographic conditions are as follows:

[0052] Chromatographic column: Waters HILIC, 4.6 mm × 150 mm, 5 μm; Mobile phase: water - acetonitrile with a volume ratio of 35:65 as the mobile phase; Wavelength 245 nm, column temperature 32 °C, flow rate 0.9 ml / min,

[0053] The content of iodixanol is calculated by the external standard method.

[0054] Example 6 Determination method of the content of iodixanol in the present invention

[0055] Prepare the solution according to the solution preparation method of Example 1, and separately aspirate 5 μl of the reference solution and the test solution and inject them into the liquid chromatograph. The chromatographic conditions are as follows:

[0056] Chromatographic column: Waters HILIC, 4.6 mm × 150 mm, 5 μm; Mobile phase: water - acetonitrile; Wavelength 245 nm, column temperature 30 °C, flow rate 1.0 ml / min, gradient elution program is

[0057] Time (min) 0 min 15 min 16 min 25 min Mobile phase A 30 35 30 30 Mobile phase B 70 65 70 70

[0058] Calculate the content of iodixanol by the external standard method.

[0059] The beneficial effects of the present invention are further illustrated by the following test examples

[0060] Test Example 1 Research on the analysis method of iodixanol

[0061] 1. Selection of detection wavelength

[0062] The maximum ultraviolet absorption wavelength of iodixanol is 245 nm, as shown specifically in Figure 1 , so 245 nm is selected as the detection wavelength.

[0063] 2. Selection of chromatographic column

[0064] Both iodixanol and known impurities show the phenomenon of multiple peaks on a common C18 column, and multiple known impurities elute at the peak position of the main peak, interfering with the detection of iodixanol. For example, in the analysis method of related substance 1, the main peak is two incompletely separated peaks, and impurities C, E, F, and G all elute at the peak position of the main peak, interfering with the detection of iodixanol. As shown specifically in Figure 2 . Therefore, the chromatographic column was re - screened, mainly considering some chromatographic columns with separation modes different from C18, in order to improve the peak shape of iodixanol. The screening results of the chromatographic column are shown in Tables 1 - 3, and the chromatograms are shown in Figures 3 - 5 .

[0065] Table 1 Screening results of chromatographic column 2 (gel column)

[0066]

[0067]

[0068] Table 2 Screening results of chromatographic column 3 (chiral column)

[0069]

[0070] Table 3 Screening results of chromatographic column 4 (reversed - phase chromatographic column)

[0071]

[0072] 3. Selection of elution program

[0073] Based on the "Waters HILIC column", the elution program was screened. The screening process is shown in Table 4, and the chromatogram is shown in<T Figure 6 .

[0074] Table 4 Screening results of elution program

[0075]

[0076] 4. Selection of diluent

[0077] Considering the solubility of iodixanol, the diluent was investigated. Water, acetonitrile, methanol or water-acetonitrile were used as diluents in the experiments. It was found that when acetonitrile or methanol was used, the sample could not be completely dissolved; when water was used, the peak shape of the main peak was poor; when water-acetonitrile with a ratio of 20-40:60-80 was used, the solubility and peak shape of iodixanol were better.

[0078] 5. Determination of the concentration of test sample and injection volume

[0079] To balance the peak shape of the main peak and the requirements of method detection sensitivity, under the above determined chromatographic conditions, the concentration of test sample and injection volume of the method were confirmed. The screening process is shown in Table 5, and the chromatogram is shown in Figure 7 .

[0080] Table 5 Determination of the concentration of test sample and injection volume

[0081]

[0082] 6. Screening of the preparation method of test sample solution and needle washing solution

[0083] Considering the solubility of iodixanol and the particularity of the HILIC chromatographic column, the preparation methods of reference solution and test sample solution and the needle washing solution were screened, and the stability of reference solution and test sample solution was investigated. The screening process is shown in Table 6.

[0084] Table 6 Determination of the preparation method of test sample solution and needle washing solution

[0085]

[0086] 7. Finally determined analytical method

[0087] Diluent: water-acetonitrile (20-40:60-80).

[0088] Chromatographic conditions: Use an ethylene-bridged hybrid particle as the filler (Waters XBridge HILIC, 4.6 mm × 150 mm, 5 μm or a chromatographic column of equivalent efficiency); use water-acetonitrile (35:65) as the mobile phase; isocratic elution; flow rate is 1.0 ml per minute; column temperature is 30 °C; detection wavelength is 245 nm; injection volume is 5 μl; the needle washing solution is water-acetonitrile (50:50).

[0089] System suitability requirements: In the chromatogram of the reference solution, the number of theoretical plates calculated based on the iodixanol peak should be not less than 5000.

[0090] Assay method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate by the external standard method based on the peak area.

[0091] Test Example 2 Comparison of Determination Methods

[0092] 1. Test group - Method for determining the content of iodixanol injection by high performance liquid chromatography

[0093] a. Use a chromatographic column with an ethylene-bridged hybrid particle as the filler (Waters HILIC, 4.6 mm × 150 mm, 5 μm or a chromatographic column of equivalent efficiency), use water-acetonitrile (35:65) as the mobile phase, isocratic elution; detection wavelength is 245 nm; column temperature is 30 °C; flow rate is 1.0 ml per minute; the needle washing solution is water-acetonitrile (50:50).

[0094] b. Take an appropriate amount of iodixanol reference substance, accurately weigh it, place it in a volumetric flask, dissolve and dilute it with a diluent to prepare a solution containing about 0.5 mg of iodixanol per 1 ml, shake well, and use it as the reference solution.

[0095] Accurately measure 5 μl of the reference solution, inject it into the liquid chromatograph, and the number of theoretical plates calculated based on the iodixanol peak should be not less than 5000.

[0096] c. Accurately measure an appropriate amount of iodixanol injection, dilute it with a diluent to prepare a solution containing about 0.5 mg per 1 ml, and use it as the test solution.

[0097] Accurately measure 5 μl of the test solution and inject it into the liquid chromatograph, record the chromatogram. Take an appropriate amount of iodixanol reference substance, accurately weigh it, dissolve it with a diluent and quantitatively dilute it to prepare a solution containing about 0.5 mg per 1 ml, and determine it in the same way. Calculate by the external standard method based on the peak area to obtain the result.

[0098] 2. Control group - Method for determining the content of iodixanol injection by potentiometric titration

[0099] a. Pipette accurately 5 ml of the reference preparation with a volumetric pipette, transfer it to a 100-ml volumetric flask, dilute it to the mark with water, mix well, pipette accurately 15 ml, transfer it to a saponification flask, add 25 ml of 5% sodium hydroxide solution and 0.5 g of zinc powder, and reflux for 30 minutes.

[0100] b. Cool, wash the condenser with a small amount of water, transfer the washing liquid into the saponification flask, filter, wash the saponification flask and the filter with water, combine the filtrate and the washing liquid, and add 5 ml of glacial acetic acid.

[0101] c. According to the potentiometric titration method (General Principles 0701, Volume IV, Chinese Pharmacopoeia 2020 Edition), titrate with silver nitrate titrant (0.1 mol / L). Each 1 ml of silver nitrate titrant (0.1 mol / L) is equivalent to 12.69 mg of I. Each 1 ml of silver nitrate titrant (0.1 mol / L) is equivalent to 25.84 mg of C 35 H 44 I6N6O 15 。

[0102] 3. Comparison of the operation time of the method

[0103] High performance liquid chromatography has the advantages of simple operation and short detection time compared with the potentiometric titration method. Detailed analysis can be carried out according to the specific operation steps of the two methods. The specific time used for the determination of the content by the two methods is shown in Table 7 and Table 8.

[0104] Table 7. Statistical table of the time used for the determination of the content by the potentiometric titration method

[0105]

[0106] Table 8. Statistical table of the time used for the determination of the content by high performance liquid chromatography

[0107]

[0108] As can be seen from Table 7 and Table 8 above, when using high performance liquid chromatography to determine the content, the determination of one sample is completed in less than 10 minutes. After receiving the sample to be detected, the time required to detect the sample is much less than that of the potentiometric titration method and the liquid chromatography method reported in the literature. It is especially suitable for the process control during large-scale production.

[0109] Test Example 3 Repeatability and intermediate precision tests for the determination of the content of iodixanol injection by high performance liquid chromatography

[0110] 1. Repeatability

[0111] Diluent, mobile phase, reference solution 1 and reference solution 2: Prepared according to the method of Test Example 1; Blank solution: Diluent is used as the blank solution.

[0112] Test solution: Dilute the iodixanol injection according to the method in Test Example 1. Prepare 6 portions in parallel.

[0113] Then, measure the prepared blank solution, reference solution 1 and 2, and test solution 6 times for the same batch of samples by the same person according to the chromatographic conditions and determination method in Example 1, calculate the content of the samples, and the test results are shown in Table 10:

[0114] Table 10 Results of repeatability of high performance liquid chromatography

[0115]

[0116] The results in Table 10 show that the RSD of the content measured 6 times for the same batch of samples by the same person is < 2%, indicating that the repeatability of the high performance liquid chromatography method is good.

[0117] 2. Intermediate precision:

[0118] The preparation methods of the diluent, mobile phase, blank solution, reference solution 1, reference solution 2, and test solution are prepared according to Test Example 1. Prepare 6 portions of the test solution in parallel.

[0119] It is required that different personnel measure the content of 6 samples by high performance liquid chromatography on different dates respectively, and count the results of the 6 samples measured on the same day and the results of the 12 samples measured under "Repeatability". The detailed results are shown in Table 11:

[0120] Table 11 Summary of repeatability and intermediate precision results

[0121]

[0122]

[0123] The results in Table 11 show that 6 samples of the same batch are measured by different personnel, on different dates, using different instruments and different numbered chromatographic columns respectively. The content of iodixanol in the 6 samples calculated by the external standard method meets the requirements, and the RSD of the content of iodixanol in the 6 samples measured is < 2.0%. The RSD of the content of iodixanol in the 12 samples under "Repeatability" is < 2.0%, and the intermediate precision of the content determined by the high performance liquid chromatography method is good.

[0124] 3. Accuracy

[0125] At the measured levels of 80%, 100%, and 120%, the recoveries are all between 98% and 102%, and the RSD of the recovery at each concentration level and the RSD of the recoveries at all concentration levels are less than 2%. Therefore, the accuracy of this method meets the requirements. See Table 12 for details.

[0126] Table 12 Table of accuracy results

[0127]

[0128] In summary, for the method for determining the content of iodixanol in the iodixanol injection of the present invention, the time for one determination is within 10 minutes, and it has high accuracy. It can overcome the disadvantages of the potentiometric titration method, such as the chemical reaction being not easily controlled, the operation being cumbersome, the human error being large, it being not easy to judge the titration end point, and the detection time being long. It can also overcome the disadvantages of poor accuracy and long time consumption in the liquid chromatography detection in the prior art. Verified by actual production, the method for determining the content of iodixanol of the present invention has better generality, and has the characteristics of good specificity, good linear relationship, high precision, high accuracy and good durability.

Claims

1. A method for determining iodixanol content, characterized in that: It is detected by high performance liquid chromatography, which includes the following steps: a. Preparation of reference solution: Take iodixanol reference solution, dissolve and dilute with diluent to obtain; b. Preparation of test solution: Take the sample to be tested and dilute it with diluent to a concentration of 0.5 mg / ml; c. Pipette the reference solution and the test solution into the liquid chromatograph respectively. The chromatographic conditions are as follows: Chromatographic column: Waters HILIC; mobile phase: water-acetonitrile; column temperature 30°C, flow rate 1.0 ml / min, wavelength 254 nm; injection volume of the solution into the liquid chromatograph was 5 μl; elution procedure: isocratic elution with water:acetonitrile at a volume ratio of 35:65, or gradient elution with acetonitrile at a volume ratio of 70% to 65% from 0 to 15 min; needle wash solution was acetonitrile:water at a volume ratio of 50:

50. d. Calculate the iodixanol content using the external standard method; The diluent is a mixture of water and acetonitrile, wherein the volume ratio of water to acetonitrile is 20-40:60-80.

2. The assay method according to claim 1, wherein: In step a, each 1 ml of the reference solution contains 0.2 to 1.2 mg of iodixanol; and in step b, each 1 ml of the test solution contains 0.2 to 1.2 mg of iodixanol.

3. The assay method according to claim 1 or 2, wherein: The sample to be tested is an iodixanol solid preparation, an iodixanol bulk drug or an iodixanol solution; the iodixanol solution includes an iodixanol injection.

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