Method for separating and determining ethyl gatetate and impurities thereof by using gas chromatography pre-column derivatization

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining ethyl gatetate and impurities thereof through gas chromatography pre-column derivatization. The impurities comprise one or more of an impurity SM1c5, an impurity SM1c8 and an impurity SM1c. The method comprises the following steps: respectively carrying out pre-column derivatization treatment on each component by taking ethanol as a derivatization agent to obtain an impurity SM1c5 derivative, an impurity SM1c8 derivative and / or an impurity SM1c derivative; separating the derivatized compound by adopting gas chromatography; in the gas chromatography, a capillary column taking 6% cyanopropyl phenyl-94% dimethyl polysiloxane as a stationary liquid is adopted as a chromatographic column, a heating program is utilized for separation, and a product enters a detector for detection; and determining the nature of each component according to the chromatogram, and judging whether the impurity content in the detected product is qualified or not by adopting a limit method. The method has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for separating and determining ethyl gatate and its impurities by using gas chromatography pre-column derivatization. Background Art

[0002] Gatinic acid ethyl ester is the key starting material for the synthesis of moxifloxacin hydrochloride. Its scientific name is 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl ester, its English chemical name is Maxifloxacin Difluoro Methoxy Ethyl Ester, and its molecular formula is C 16 H 15 F2NO4, molecular weight is 323.29, CAS number is 112811-71-9, and the structural formula is shown in Formula Ⅰ.

[0003]

[0004] The study found that three impurities are easily introduced into ethyl gatate: SM 1c5 , SM 1c8 and SM 1c The three impurities all have acyl chloride warning structures and are very unstable. Controlling the three acyl chloride impurities in ethyl gatrate is of great significance to improving the quality of moxifloxacin hydrochloride.

[0005] Some prior arts have reported methods for analyzing impurities related to ethyl gatmecarboxylate. For example, patent CN114113353A discloses a method for separating and detecting ethyl gatmecarboxylate and / or related impurities. The method is a high performance liquid chromatography method. The chromatographic column is filled with phenyl bonded silica gel. A buffered salt solution and an organic solvent are used for gradient elution. The flow rate is 0.9 to 1.1 ml / min, and the column temperature is 23 to 27°C. Patent CN114113355B discloses a method for analyzing ethyl gatmecarboxylate and related impurities using high performance liquid chromatography. The chromatographic column is filled with octadecyl bonded silica gel. A buffered salt solution and an organic solvent are used for gradient elution. The flow rate is 0.9 to 1.1 ml / min, and the column temperature is 18 to 22°C; the detection wavelength is 220 nm or 250 nm. These prior arts all use high performance liquid chromatography for analysis, which cannot achieve the detection of SM in ethyl gatmecarboxylate. 1c5 , SM 1c8 and SM 1c Determination of. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a method for separating ethyl gatanoate and its acyl chloride impurities based on gas chromatography, which can complete the separation of multiple substances in a shorter time.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] A method for separating ethyl gatate and its acyl chloride impurities based on gas chromatography, wherein the ethyl gatate and the acyl chloride impurities together constitute a composition, and the acyl chloride impurities include impurities SM 1c5 、Impurity SM 1c8 、Impurity SM 1c Any one or more of the above; the method comprises: using ethanol as a derivatizing agent, performing pre-column derivatization treatment on each component, respectively, to obtain impurity SM 1c5 Derivatives, impurities SM 1c8 Derivatives and / or impurities SM 1c Derivatives; the derivatized compounds are separated by gas chromatography; in the gas chromatography, a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid is used as a chromatographic column, and separation is performed using a temperature program.

[0009] The structural formula of each component and each impurity derivative in the composition is as follows:

[0010]

[0011] After separation and detection, ethyl gatinic acid can be used in the next step of production.

[0012] The above impurities can be arranged and combined in various ways. For example, combination 1: impurity SM 1c5 、Impurity SM 1c8 For example, combination 2: impurity SM 1c5 、Impurity SM 1c8 、Impurity SM 1c For example, combination 3: impurity SM 1c8 、Impurity SM 1c .

[0013] Theoretically, when the upper limit of the substances to be separated, identified and / or detected by the method is n (number), 1-n substances can be detected naturally.

[0014] Furthermore, the composition further comprises any one or more of methanol, triethylamine, glacial acetic acid, ethyl acetate and toluene.

[0015] Further, the heating program is set as follows:

[0016] The first stage: starting temperature 120-150°C, maintained for 2-5 minutes;

[0017] The second stage: heating to 215-225℃;

[0018] The third stage: heating to 235-245℃ and maintaining for 10 minutes;

[0019] The heating rate from the first stage to the second stage is 3-7°C / min; the heating rate from the second stage to the third stage is 8-12°C / min.

[0020] As a preferred solution, the heating program is set as follows:

[0021] Stage 1: Initial temperature 128-132°C, maintained for 3 minutes;

[0022] The second stage: heating to 220℃;

[0023] The third stage: heating to 240℃ and maintaining for 10 minutes;

[0024] The heating rate from the first stage to the second stage is 4-6°C / min; the heating rate from the second stage to the third stage is 10°C / min.

[0025] As the most preferred solution, the heating program is set as follows:

[0026] Stage 1: Initial temperature 130°C, maintained for 3 minutes;

[0027] The second stage: heating to 220℃;

[0028] The third stage: heating to 240℃ and maintaining for 10 minutes;

[0029] The heating rate from the first stage to the second stage is 5°C / min; the heating rate from the second stage to the third stage is 10°C / min.

[0030] Furthermore, the injection port temperature is 180-220° C., the flow rate is 1.0-2.0 ml / min, the split ratio is 18-22:1, and the carrier gas is nitrogen.

[0031] Preferably, the injection port temperature is 200°C.

[0032] Preferably, the split ratio is 20:1.

[0033] Preferably, the flow rate is 1.4-1.6 ml / min, most preferably 1.5 ml / min.

[0034] Preferably, the injection volume is 2 μl.

[0035] Preferably, the chromatographic column specification is DB-62460m×0.32mm, 1.8μm.

[0036] Furthermore, the sample preparation solvent is a mixture of dichloromethane and ethanol.

[0037] Among them, ethanol was used as a derivatizing agent to carry out pre-column derivatization of various impurities.

[0038] Preferably, in the solvent, the volume ratio of dichloromethane to ethanol is 2:1.

[0039] This method also involves a method for processing the sample to be detected, specifically:

[0040] Sample solution: Take an appropriate amount of this product (plus ethyl acetate), weigh accurately, dissolve in solvent and dilute to make a solution containing about 60 mg per 1 ml.

[0041] Reference solution: Accurately weigh impurity SM 1c5 、Impurity SM 1c8 、Impurity SM 1c Appropriate amount, quantitatively diluted with solvent to make each 1ml contain about impurity SM 1c5 2.4μg, impurity SM 1c8 2.4μg with impurity SM 1c 2.4 μg of solution.

[0042] Furthermore, after the sample solution was injected and collected, the high temperature was set to 240°C and balanced for 60 minutes before injecting other solutions.

[0043] The second object of the present invention is to provide a method for identifying acyl chloride impurities in ethyl gatanoate, which can complete the identification of multiple substances in a shorter time.

[0044] To achieve the above object, the technical solution of the present invention is:

[0045] The method for identifying acyl chloride impurities in ethyl gatrate comprises separating the composition by the aforementioned separation method, and entering a detector for detection to obtain a chromatogram; and determining whether the test sample contains the acyl chloride impurity by comparing the chromatogram characteristics of the test sample with those of the reference sample.

[0046] Preferably, the detector is a FID detector, and the detector temperature is 220-280°C.

[0047] Preferably, the components in the composition can be identified in the order of retention time, and the components of the composition are in ascending order: impurity SM 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 derivative.

[0048] As a more preferred embodiment, the retention time is 14.6±0.5min, which is determined to be impurity SM 1c5 Derivative; retention time is 21.4±0.5min, determined to be impurity SM 1cDerivative; retention time is 22.7±0.5min, determined to be impurity SM 1c8 derivative.

[0049] Each component can be identified based on retention time. Retention time refers to the time required for a sample to enter the chromatographic column and be detected by the detector. This time is calculated based on the movement speed of the component on the chromatographic column, that is, the time interval from the start of injection to the chromatographic apex (maximum concentration) of a component. It is mainly used to determine the peak order and position of each component in the sample, and is one of the basic data in chromatographic analysis.

[0050] The third object of the present invention is to provide a method for detecting whether the acyl chloride impurity content in ethyl gatanoate is qualified, and the method can determine whether the acyl chloride impurity content in ethyl gatanoate is qualified within a relatively short time.

[0051] To achieve the above object, the technical solution of the present invention is:

[0052] The method for detecting whether the acyl chloride impurity content in ethyl gatate is qualified is to separate and identify the acyl chloride impurities in ethyl gatate by the above-mentioned identification method to obtain a chromatogram; based on the obtained chromatogram, a limit method is used to determine whether the content of each impurity is qualified.

[0053] Furthermore, if the impurity SM in the test product 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 If the peak area of ​​any one or more impurities in the derivative is greater than the peak area of ​​the corresponding impurity derivative in the chromatogram of the reference solution, it means that the impurity content is unqualified; conversely, if the impurity SM in the test product is 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 The peak area of ​​any one or more impurities in the derivative is not greater than the peak area of ​​the corresponding impurity derivative in the chromatogram of the reference solution, indicating that the impurity content is qualified.

[0054] Furthermore, each impurity limit is 0.004%.

[0055] The above determination method can be used as a drug quality determination model, and further as an indispensable key module in the intelligent production process. The model can achieve accurate separation and quantitative analysis of active ingredients, impurities and degradation products in drugs by accurately controlling parameters such as column flow rate, injection port temperature, heating program, split ratio, etc., providing a scientific basis for the comprehensive evaluation of drug quality. In the intelligent production system, this model is seamlessly integrated, able to receive raw data from the production line in real time, automatically perform analysis tasks, and quickly feedback the determination results based on preset quality standards.

[0056] The beneficial effects of the present invention are:

[0057] 1. The present invention provides a method for separating and determining ethyl gatate and its impurities by gas chromatography column pre-derivation. The impurities include impurity SM 1c5 、Impurity SM 1c8 、Impurity SM 1c Any one or more of the above. Since the three impurities all have acyl chloride warning structures and are extremely unstable, the present invention uses ethanol as a derivatizing agent to carry out pre-column derivatization on the above impurities, and then uses GC direct injection method, selects a capillary column with medium polarity and a programmed temperature rise method for separation, and uses an FID detector for detection. At the same time, the method is verified for its specificity, detection limit, accuracy (standard addition test) and tolerance. The experimental results show that the method is simple, rapid, sensitive, accurate, durable and effective.

[0058] 2. The gas chromatography pre-column derivatization method established by the present invention is used to separate and detect ethyl gatate and its acyl chloride impurities. The separation degree between each impurity can meet the requirements, and is not interfered by the main component ethyl gatate and other impurities (such as methanol, triethylamine, glacial acetic acid, ethyl acetate, toluene, etc.), and has the characteristics of strong specificity.

[0059] 3. This method has the characteristics of high sensitivity and good durability. Impurity SM 1c5 The detection limit concentration was 0.6921 μg / ml, and the impurity SM 1c The detection limit concentration was 0.6756 μg / ml, and the impurity SM 1c8 The detection limit concentration is 0.6792μg / ml; and when the column flow rate, initial column temperature, and heating rate have slight fluctuations, the impurity SM 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 The separation between the derivatives and their adjacent peaks was greater than 1.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is the chromatogram of the blank solution;

[0061] Figure 2 is the chromatogram of the reference solution;

[0062] Figure 3 Chromatograms of solutions mixed with other impurities;

[0063] Figure 4 is the chromatogram of the mixed solution;

[0064] Figure 5 is the chromatogram of the test solution;

[0065] Figure 6 is the chromatogram of the detection limit solution;

[0066] Figure 7 This is the chromatogram of the mixed solution under normal conditions in the durability experiment;

[0067] Figure 8 This is the chromatogram of the mixed solution at a flow rate of 1.4 ml / min in the durability experiment;

[0068] Fig. 9 This is the chromatogram of the mixed solution at a flow rate of 1.6 ml / min in the durability experiment;

[0069] Fig.10 This is the chromatogram of the mixed solution in the durability experiment at an initial column temperature of 128°C;

[0070] Fig.11 This is the chromatogram of the mixed solution in the durability experiment at an initial column temperature of 132°C;

[0071] Fig.12 This is the chromatogram of the mixed solution in the durability experiment at a heating rate of 4°C / min;

[0072] Fig.13 This is the chromatogram of the mixed solution in the durability experiment at a heating rate of 6℃ / min. DETAILED DESCRIPTION

[0073] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0074] Supplementary table to the drawings in the specification. Figure 1-Figure 13 Visual aids are provided for the purpose of understanding and interpretation. In case of any ambiguity, the user should refer to the corresponding numbered table ( Figure 1-Figure 13 The integration results are shown in Tables 1 to 13, respectively, to obtain more detailed information. On the contrary, if any information that may cause misunderstanding or ambiguity is found in the process of reviewing Tables 1 to 13, the content in the corresponding numbered figure shall prevail. The above guidelines are intended to ensure the correct interpretation of this document and the consistency of its information. Although some of the characters in the spectrum of this application document overlap, they can still be viewed clearly, and the integration results of each figure are recorded in detail in the specification. In addition, the numbers in the chromatogram have no effect on the scope of protection of the claims and the full disclosure of the technical solution in the specification.

[0075] Table 1

[0076]

[0077] Table 2

[0078]

[0079] Table 3

[0080]

[0081] Table 4

[0082]

[0083] Table 5

[0084]

[0085] Table 6

[0086]

[0087] Table 7

[0088]

[0089] Table 8

[0090]

[0091] Table 9

[0092]

[0093] Table 10

[0094]

[0095] Table 11

[0096]

[0097] Table 12

[0098]

[0099] Table 13

[0100]

[0101] In the above tables and figures, MOXF-SM1C5 is the impurity SM 1c5 Derivative; MOXF-SM1C8 is impurity SM 1c8 Derivative; MOXF-SM1C is impurity SM 1c derivative.

[0102] In order to enhance the understanding of the present invention, some key technical and scientific terms are clearly defined below. Unless otherwise specifically defined herein, all other technical and scientific terms follow the meanings generally accepted and understood in the field to which the present invention belongs. At the same time, please understand that the terms used in this article are intended to illustrate specific embodiments and are not to be interpreted restrictively.

[0103] In addition, all literature cited herein, including but not limited to patents, patent applications, academic papers, textbooks and further citations therein, are deemed to be incorporated herein by reference as a whole to the extent not directly cited. If there is any inconsistency or conflict between the contents of these cited documents or similar materials and this application, especially in terms of term definitions, term usage, technical descriptions, etc., the contents of this application shall prevail.

[0104] the term

[0105] Limit of Quantitation (LOQ) refers to the lowest amount of the substance to be measured in the sample, and the measurement result should have a certain degree of accuracy and precision. In other words, the limit of quantitation is the lowest level at which the analytical method can accurately and reliably measure the concentration of the substance to be measured in the sample. In gas chromatography, the determination of the limit of quantitation usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the substance to be measured corresponding to the signal-to-noise ratio reaching a certain level is used as the limit of quantitation. The determination of the limit of quantitation is crucial to ensure the accuracy and reliability of the analysis results.

[0106] The limit of detection (LOD) refers to the lowest concentration or amount of the substance in the sample that can be detected. It reflects the sensitivity and noise of the analytical method and instrument, and also indicates the level of the blank (background) value after the sample is processed. The detection limit is an important indicator to measure the sensitivity of the instrument and analytical method.

[0107] Durability refers to the ability of a chromatographic analysis system to maintain stable analytical performance and not significantly affect the results when there is a slight change in the measurement conditions. This durability is crucial to ensure the reliability, repeatability and stability of the analysis results.

[0108] The signal-to-noise ratio (S / N) in gas chromatography refers to the ratio of the peak height of the main component peak in the gas chromatogram to the baseline noise. It is an important indicator for measuring the quality of the measuring electrical system, the detection sensitivity of the instrument, and the reliability of the analytical method. The main component peak height refers to the signal value output by the detector when the component to be measured elutes from the column at the maximum concentration, which is usually used to indicate the relative content or concentration of the component. The noise refers to the fluctuation of the baseline signal, that is, the signal value output by the detector when no sample passes through the chromatographic column. The higher the signal-to-noise ratio, the more accurately the instrument can separate and identify the target components when detecting the sample, while reducing the interference of background noise.

[0109] In the embodiments of the present invention, the structural information of ethyl gatanoate and its impurities is shown in Table A.

[0110] Table A. Compound information table

[0111]

[0112]

[0113] In the embodiments of the present invention, the test conditions are shown in Table B.

[0114] Table B. Test conditions:

[0115]

[0116] If there are any unmentioned conditions in the chromatographic conditions of the present invention, they can be determined by referring to gas chromatography (General Rules 0521 of Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0117] In the examples of the present invention, the test solution is prepared by taking an appropriate amount of the product (plus ethyl acetate), accurately weighing it, adding a solvent to dissolve it and diluting it to prepare a solution containing about 60 mg per 1 ml.

[0118] In the present embodiment, the reference solution is prepared by accurately weighing the impurity SM 1c5 、Impurity SM 1c8 、Impurity SM 1c Appropriate amount, quantitatively diluted with solvent to make each 1ml contain about impurity SM 1c5 2.4μg, impurity SM 1c8 2.4μg with impurity SM 1c 2.4 μg of solution.

[0119] In the embodiment of the present invention, the testing method is as follows:

[0120] Accurately measure 2 μl of the reference solution, inject it into the gas chromatograph, record the chromatogram, and calculate the impurity SM 1c5 Derivatives, SM 1cDerivatives, impurities SM 1c8 The derivatives elute sequentially.

[0121] Accurately measure 2 μl of the test solution, inject it into the gas chromatograph, and record the chromatogram (set the high temperature to 240°C after collection and balance for 60 minutes). 1c5 Derivatives, impurities SM 1c Derivatives and ImpuritiesSM 1c8 The peak area of ​​the derivative shall not be greater than the peak area of ​​the corresponding impurity derivative peak in the chromatogram of the reference solution (0.004%).

[0122] Example 1. Specificity

[0123] (1) Prepare the test solution

[0124] Solvent: dichloromethane:ethanol=2:1.

[0125] Impurity SM 1c5 Stock solution: Accurately weigh the impurity SM 1c5 About 10 mg, put in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0126] Impurity SM 1c8 Stock solution: Accurately weigh the impurity SM 1c8 About 10 mg, put in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0127] Impurity SM 1c Stock solution: Accurately weigh the impurity SM 1c About 10 mg, put in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0128] Mixed stock solutions of other solvents: Take 150 μl each of methanol, triethylamine, glacial acetic acid, ethyl acetate, and toluene, place them in a 50 ml volumetric flask containing an appropriate amount of solvent, add solvent to dilute to the scale, shake well, and the solution is ready.

[0129] Reference stock solution: Precisely pipette impurities SM 1c5 Stock solution, impurities SM 1c8 Stock solution, impurities SM 1c Place 2.4 ml of each stock solution in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, and the solution is ready.

[0130] Reference substance solution: Accurately pipette 1 ml of reference substance stock solution into a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, and the solution is ready.

[0131] Impurity SM 1c5 Positioning solution: Precise removal of impuritiesSM 1c5Place 2.4 ml of the stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0132] Impurity SM 1c8 Positioning solution: Precise removal of impuritiesSM 1c8 Place 2.4 ml of the stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0133] Impurity SM 1c Positioning solution: Precise removal of impuritiesSM 1c Place 2.4 ml of the stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0134] Other solvent mixed solutions: Pipette 2 ml of other solvent mixed stock solution into a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and the solution is ready.

[0135] Test solution: Take 0.6 g of the test sample, weigh accurately, place in a 10 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, and obtain.

[0136] Mixed solution: Accurately weigh 0.6 g of the test sample, transfer 1 ml of the reference sample stock solution, transfer 2 ml of other impurity mixed stock solution, place them in the same 10 ml volumetric flask, dissolve them with solvent and dilute to the scale, shake well, and obtain.

[0137] (2) Detection

[0138] Take 2 μl of the blank solvent, each positioning solution, other solvent mixed solution and mixed solution, respectively, inject them into the liquid chromatograph, test according to the test conditions in Table B, and record the chromatogram.

[0139] The test results are shown in Table C. Figure 1 to Figure 5 , Table 1 to Table 5. Effect of test sample, blank solution and other solvents on impurity SM 1c5 、Impurity SM 1c 、Impurity SM 1c8 No interference in detection; impurities SM 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 The separation between the derivative and the adjacent impurity peaks was greater than 1.5. This result shows that the method of the present invention has good specificity.

[0140] Table C. Method specificity test results

[0141]

[0142] Example 2. Detection Limit

[0143] Reference solution: Prepare the reference solution in the same manner as in “Example 1. Specificity”.

[0144] Detection limit solution: Accurately pipette 3 ml of the reference solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain

[0145] Accurately measure 2 μl of the detection limit solution, inject it three times continuously, perform the test according to the test conditions in Table B, record the chromatogram, and calculate the signal-to-noise ratio of each peak.

[0146] The results are shown in Table D. Figure 6 , Table 6. The data show that impurity SM 1c5 The detection limit concentration is 0.6921μg / ml, and the average S / N is 14.0; the impurity SM 1c The detection limit concentration is 0.6756μg / ml, and the average S / N is 10.7; the impurity SM 1c8 The detection limit concentration was 0.6792 μg / ml, and the average S / N was 11.8.

[0147] Table D. Detection limit test results

[0148]

[0149]

[0150] Example 3. Durability

[0151] Take the mixed solution under "Example 1. Specificity", and conduct experiments according to the normal chromatographic conditions shown in Table B, and fine-tune each chromatographic condition to examine the separation between each peak.

[0152] The test results are shown in Table E. Figure 7 to Figure 13 , Table 7 to Table 13. When the column flow rate, initial column temperature, and first stage heating rate have slight fluctuations, the impurity SM 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 The separation between the derivative peak and its adjacent peaks was greater than 1.5, and the durability of the chromatographic conditions met the verification requirements.

[0153] Table E. Chromatographic system durability test results

[0154]

[0155] Example 4. Standard addition test

[0156] (1) Prepare the test solution

[0157] Reference stock solution: Accurately weigh the impurity SM 1c5 10mg, impurity SM 1c8 10mg, impurity SM 1cPlace 10 mg in a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, then accurately transfer 2.4 ml, place in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and you have the product.

[0158] Reference substance solution: Accurately pipette 1 ml of reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and the solution is ready.

[0159] Test solution: Accurately weigh about 0.6g of the test sample, place it in a 10ml volumetric flask, dissolve it in solvent and dilute it to the mark, shake well, and you have it. (60mg / ml)

[0160] Standard addition solution: accurately weigh 0.6g of the test sample, accurately transfer 1ml of the reference stock solution, place in the same 10ml volumetric flask, dissolve with solvent and dilute to the scale, shake well, and the solution is ready. (Prepare 2 portions in parallel)

[0161] (2) Determination

[0162] Take the reference solution, test solution, and standard spike solution respectively, inject directly according to the chromatographic conditions in Table B, and record the chromatogram. Take the test solution as the unspiked solution and calculate the impurity SM 1c5 、Impurity SM 1c 、Impurity SM 1c8 The spike recovery rate.

[0163] The results are shown in Table F. In the two spiked samples, impurity SM 1c5 The average recovery rate of spiked samples was 106.4%; the impurity SM 1c The average recovery rate of spiked was 107.3%; the impurity SM 1c8 The average recovery rate of spiked products was 102.2%; the recoveries were all in the range of 80% to 115%.

[0164] Table F. Standard addition test results

[0165]

Claims

1. A method for separating ethyl gatanoate and its acyl chloride impurities based on gas chromatography, characterized in that: The ethyl gatrate and the acyl chloride impurities together form a composition, and the acyl chloride impurities include impurities SM 1c5 、Impurity SM 1c8 、Impurity SM 1c Any one or more of the above; the method comprises: using ethanol as a derivatizing agent, performing pre-column derivatization treatment on each component, respectively, to obtain impurity SM 1c5 Derivatives, impurities SM 1c8 Derivatives and / or impurities SM 1c Derivatives; the derivatized compounds are separated by gas chromatography; in the gas chromatography, a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid is used as a chromatographic column, and separation is performed using a temperature program; The structural formula of each component and each impurity derivative in the composition is as follows:

2. The method according to claim 1, characterized in that The heating program is set as follows: The first stage: starting temperature 120-150°C, maintained for 2-5 minutes; The second stage: heating to 215-225℃; The third stage: heating to 235-245℃ and maintaining for 10 minutes; The heating rate from the first stage to the second stage is 3-7°C / min; the heating rate from the second stage to the third stage is 8-12°C / min.

3. The method according to claim 1, characterized in that The injection port temperature is 180-220°C, the flow rate is 1.0-2.0 ml / min, the split ratio is 18-22:1, and the carrier gas is nitrogen.

4. The method according to claim 1, characterized in that: The sample preparation solvent was a mixture of dichloromethane and ethanol.

5. A method for identifying acyl chloride impurities in ethyl gatanoate, characterized in that: The composition is separated by the method described in any one of claims 1 to 4, and is detected in a detector to obtain a chromatogram; and whether the test sample contains the acyl chloride impurity is determined by comparing the chromatogram characteristics of the test sample with those of the reference sample.

6. The method according to claim 5, characterized in that The detector is a FID detector, and the detector temperature is 220-280°C.

7. The method according to claim 5, characterized in that According to the order of retention time, the components in the composition can be identified. The components of the composition are in ascending order: impurity SM 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 derivative.

8. The method according to claim 7, characterized in that The retention time was 14.6±0.5min, which was determined to be impurity SM 1c5 Derivative; retention time is 21.4±0.5min, determined to be impurity SM 1c Derivative; retention time is 22.7±0.5min, determined to be impurity SM 1c8 derivative.

9. A method for detecting whether the acyl chloride impurity content in ethyl gadoxetine is qualified, characterized in that: The method described in any one of claims 5 to 8 is used to separate and identify acyl chloride impurities in ethyl gatrate to obtain a chromatogram; based on the obtained chromatogram, a limit method is used to determine whether the content of each impurity is qualified.

10. The method according to claim 9, characterized in that If the impurity SM in the test product 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 If the peak area of ​​any one or more impurities in the derivative is greater than the peak area of ​​the corresponding impurity derivative in the chromatogram of the reference solution, it means that the impurity content is unqualified; conversely, if the impurity SM in the test product is 1c5 Derivatives, impurities SM 1c Derivatives, impurities SM 1c8 The peak area of ​​any one or more impurities in the derivative is not greater than the peak area of ​​the corresponding impurity derivative in the chromatogram of the reference solution, indicating that the impurity content is qualified.

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