Method for separating and detecting ethyl gatetate starting material SM1c and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and detecting an ethyl gatetate starting material SM1c and impurities thereof. The initial material SM1c of ethyl tetracate is 2, 4, 5-trifluoro-3-methoxybenzoyl chloride, and the initial material SM1c of ethyl tetracate is 2, 4, 5-trifluoro-3-methoxybenzoyl chloride. The impurities comprise one or more of an impurity SM1c10, an impurity SM1c3, an impurity SM1c13 and an impurity SM1c4. According to the method, a gas chromatographic method is adopted for analysis, a capillary column with (5%-phenyl)-methylpolysiloxane as a stationary liquid is adopted as a chromatographic column, 2, 4, 5-trifluoro-3-methoxybenzoyl chloride and related substances are separated by utilizing a heating procedure, and the 2, 4, 5-trifluoro-3-methoxybenzoyl chloride and the related substances enter a detector for detection; and calculating the content of each impurity by adopting a main component self-contrast method according to the chromatogram. The method has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and particularly relates to a method for separating and detecting a starting material SM of ethyl gatrate. 1c and its impurities. Background Art

[0002] Gatinic acid ethyl ester, also known as gaticarboxylic acid ethyl ester or gatifloxacin cyclized ester, is an important intermediate for the synthesis of moxifloxacin hydrochloride. The structural formula of gatinic acid ethyl ester is shown in Formula XV.

[0003]

[0004] SM 1c It is the key starting material for the synthesis of ethyl gatidine. Its scientific name is 2,4,5-trifluoro-3-methoxybenzoyl chloride, its English chemical name is 2,4,5-Trifluoro-3-methoxybenzoyl chloride, its molecular formula is C8H4ClF3O2, its molecular weight is 224.56, its CAS number is 112811-66-2, and its structural formula is shown in Formula I.

[0005]

[0006] The study found that SM 1c Easy to introduce SM 1c3 , SM 1c4 , SM 1c10 , SM 1c13 These four impurities are all mutagenic impurities with warning structures, and the detection control SM 1c The four impurities in SM are helpful for the quality control of ethyl gatrate, which is of great significance for the subsequent synthesis of moxifloxacin hydrochloride. 1c Medium impurities SM 1c3 , SM 1c4 , SM 1c10 and SM 1c13 The analysis method is reported. 1c It is also easy to introduce SM 1e , SM 1c1 , SM 1c2 , SM 1c11 , SM 1c5 , SM 1c6 , SM 1c7 , SM 1c8 , SM 1c9 The presence of other impurities further increases the difficulty of determining the four mutagenic impurities.

[0007] In the prior art, patent CN110794046B discloses a method for testing 2,4,5-trifluoro-3-methoxybenzoyl chloride in moxifloxacin intermediates. This patent uses LC-MS / MS to determine the content of 2,4,5-trifluoro-3-methoxybenzoyl chloride in moxifloxacin intermediates, and the chromatographic conditions used are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, the flow rate is 0.5±0.1mL / min, the column temperature is 30℃±5℃, the injection volume is 0.1-0.3μl, formic acid: water system is used as mobile phase A, methanol is used as mobile phase B, and gradient elution is used. Although this method can determine 2,4,5-trifluoro-3-methoxybenzoyl chloride in moxifloxacin intermediates, it cannot effectively separate SM in 2,4,5-trifluoro-3-methoxybenzoyl chloride. 1c3 , SM 1c4 , SM 1c10 , SM 1c13 . Summary of the invention

[0008] One of the purposes of the present invention is to provide a method for separating ethyl gatanoate starting material SM based on gas chromatography. 1c The method can separate a variety of substances in a shorter time.

[0009] To achieve the above object, the technical solution of the present invention is:

[0010] Separation of ethyl gatate starting material SM based on gas chromatography 1c and impurity methods thereof, the SM 1c and the impurities together form a composition, wherein the impurities include SM 1c10 , SM 1c3 , SM 1c13 , SM 1c4 Any one or more of the gas chromatography method comprising: a chromatographic column using a capillary column with 5% phenyl-95% methyl polysiloxane as a stationary liquid, using a heating program to SM 1c and its impurities for separation;

[0011] The structural formula of each component in the composition is as follows:

[0012]

[0013] Ethyl gatate starting material SM 1c After separation and testing, it can be used in the next step of production.

[0014] The above impurities can be arranged and combined in many ways. For example, combination 1: SM 1c10 and SM 1c3 . Another example, combination 2: SM 1c10 , SM1c3 and SM 1c13 . For example, combination 3: SM 1c10 , SM 1c3 , SM 1c13 and SM 1c4 .

[0015] 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.

[0016] Further, the heating program is set as follows:

[0017] Stage 1: Starting temperature 80-120°C, maintained for 2-5 minutes;

[0018] The second stage: heating to 180-300℃ and maintaining for 8-15 minutes;

[0019] The heating rate from the first stage to the second stage is 10-20°C / min.

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

[0021] Stage 1: Initial temperature 99-101°C, maintained for 3 minutes;

[0022] The second stage: heating to 200°C and maintaining for 12 minutes;

[0023] The heating rate from the first stage to the second stage is 14-16°C / min.

[0024] As the most preferred solution, the heating program is set as follows:

[0025] Stage 1: Initial temperature 100°C, maintained for 3 minutes;

[0026] The second stage: heating to 200°C and maintaining for 12 minutes;

[0027] The heating rate from the first stage to the second stage was 15°C / min.

[0028] Further, the composition may also contain other substances, including SM 1e , SM 1c1 , SM 1c2 , SM 1c11 , SM 1c5 , SM 1c6 , SM 1c7 , SM 1c8 , SM 1c9 Any one or more of the following, its structural formula is as follows:

[0029]

[0030] Furthermore, the injection port temperature is 150-300° C., the flow rate is 1-2 ml / min, the carrier gas is nitrogen, and the split ratio is 25-35:1.

[0031] As a preferred solution, the injection port temperature is 180°C.

[0032] As a preferred solution, the split ratio is 30:1.

[0033] As a preferred solution, the flow rate is 1.4 to 1.6 ml / min, and most preferably 1.5 ml / min.

[0034] As a preferred solution, the chromatographic column specifications are 30m×0.32mm, 0.25μm.

[0035] As a preferred solution, the injection volume is 1 μl.

[0036] As a preferred solution, the sample preparation solvent is dichloromethane.

[0037] This method also involves a method for processing the sample to be detected, specifically:

[0038] Preparation of sample solution: Take an appropriate amount of the product, weigh accurately, add dichloromethane and dilute to make a solution containing about 50 mg per 1 ml.

[0039] Preparation method of control solution: Accurately pipette sample solution, such as 0.5 ml, into a 100 ml volumetric flask, dilute to the mark with dichloromethane, shake well, and the solution is ready.

[0040] Preparation method of system suitability solution: Take the test sample (containing impurities SM 1c13 ) about 0.5g, add potassium carbonate 0.1g and water 20μl, immediately add dichloromethane 10ml, shake for 30 seconds and filter, take the filtrate, and immediately add an appropriate amount of impurity SM 1c10 , SM 1c3 , SM 1c4 , accurately weighed, dissolved and diluted with dichloromethane to make SM per 1 ml 1c3 75μg, SM 1c4 75μg, SM 1c10 0.4mg of mixed solution.

[0041] The second object of the present invention is to provide a method for identifying the starting material SM of ethyl gatanoate. 1c The method can complete the identification of multiple substances in a shorter time.

[0042] To achieve the above object, the technical solution of the present invention is:

[0043] Identification of ethyl gatate starting material SM 1c The method for separating the composition and its impurities is to separate the composition by the aforementioned separation method, and enter the composition into a detector for detection to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample and the reference sample, it is determined whether the test sample contains SM 1c and its impurities.

[0044] As a preferred embodiment, the detector temperature is 220-300°C.

[0045] As a preferred solution, the components in the composition can be identified according to the order of retention time. The components of the composition are in ascending order: SM 1c , SM 1c10 , SM 1c3 , SM 1c13 , SM 1c4 .

[0046] As a preferred method, the retention time is 4.9±0.5min, which is determined to be the SM 1c ; The retention time was 5.4±0.5min, which was determined to be the SM 1c10 ; The retention time was 6.3±0.5min, which was determined to be the SM 1c3 ; The retention time was 13.4 ± 0.5min, which was determined to be the SM 1c13 ; The retention time was 14.2±0.5min, which was determined to be the SM 1c4 .

[0047] Retention time can be used to determine the components. Retention time refers to the time required for the 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 value) 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.

[0048] The third object of the present invention is to provide a method for detecting the starting material SM of ethyl gatrate 1c The method can complete the identification and content determination of multiple substances in a relatively short time.

[0049] To achieve the above object, the technical solution of the present invention is:

[0050] Detection of ethyl gatrate starting material SM 1c and its impurity content, using the aforementioned identification method to separate and identify SM 1cand its impurities to obtain a chromatogram; based on the obtained chromatogram, the content of each impurity is calculated using the main component self-comparison method.

[0051] Through content determination, we can further determine whether the impurity content in 2,4,5-trifluoro-3-methoxybenzoyl chloride is qualified. 1c10 、Impurity SM 1c3 、Impurity SM 1c13 、Impurity SM 1c4 Any one or more impurity content in (the impurity content is calculated by the principal component self-comparison method: impurity peak area / SM in the control solution 1c If the peak area / dilution multiple of the control solution × 100%) is greater than the specified limit, it indicates that the impurity content is unqualified; otherwise, it indicates that the impurity content is qualified.

[0052] 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. This model can achieve accurate separation and quantitative analysis of active ingredients, impurities and degradation products in drugs by accurately controlling parameters such as the heating program, split ratio, flow rate, and inlet temperature, 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.

[0053] The beneficial effects of the present invention are:

[0054] 1. Considering that the acyl chloride compound is unstable when exposed to water, acid, and alkali, the present invention adopts GC method to separate and determine the starting material SM of ethyl ester 1c and its impurities (SM 1c3 , SM 1c4 , SM 1c10 , SM 1c13 ), and dichloromethane, which is immiscible with water, is used as the solvent to avoid hydrolysis of the acyl chloride and its impurities. A capillary column with a weakly polar 5% phenyl-95% methyl polysiloxane as the stationary liquid is used as the separation chromatographic column, which can directly replace the starting material SM of ethyl ester without derivatization. 1c The separation degree between the four mutagenic impurities can meet the requirements, and it is simple, rapid, sensitive and effective.

[0055] 2. This method has high sensitivity, and the main component SM 1c The limit of quantification was 10.0920 μg / ml, and the limit of detection was 3.0276 μg / ml; SM 1c10 The limit of quantification was 10.6894 μg / ml, and the limit of detection was 3.2068 μg / ml; SM 1c3The limit of quantification was 14.2276 μg / ml, and the limit of detection was 4.2683 μg / ml; SM 1c4 The limit of quantification was 14.3521 μg / ml and the limit of detection was 4.3056 μg / ml.

[0056] 3. Separation and determination of ethyl gatanoate starting material SM by the method of the present invention 1c and its impurities. The determination of each component is not affected by the test sample and other impurities (SM 1e , SM 1c1 , SM 1c2 , SM 1c11 , SM 1c5 , SM 1c6 , SM 1c7 , SM 1c8 and SM 1c9 ) interference, will not cause the degradation of acyl chloride, has strong specificity and good separation effect.

[0057] 4. This method can simultaneously separate the starting material SM of ethyl ester in 22 minutes 1c and its 4 impurities with short separation time, high accuracy and good durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is the GC graph of blank solution;

[0059] Figure 2 is the GC graph of the control solution;

[0060] Figure 3 GC graph of solution I for mixed location of other impurities;

[0061] Figure 4 GC profile of the II solution for the mixed location of other impurities;

[0062] Figure 5 is the GC graph of the mixed solution;

[0063] Figure 6 It is the GC graph of the test solution;

[0064] Figure 7 GC graph of the quantitative limit solution;

[0065] Figure 8 is the GC graph of the detection limit solution;

[0066] Fig. 9 This is the GC graph of the mixed solution at a flow rate of 1.4 ml / min in the durability experiment;

[0067] Fig.10This is the GC graph of the mixed solution at a flow rate of 1.6 ml / min in the durability experiment;

[0068] Fig.11 This is the GC graph of the mixed solution in the durability experiment at an initial column temperature of 99°C;

[0069] Fig.12 This is the GC graph of the mixed solution in the durability experiment at an initial column temperature of 101°C;

[0070] Fig.13 This is the GC graph of the mixed solution in the durability experiment at a heating rate of 14℃ / min;

[0071] Fig.14 This is the GC graph of the mixed solution in the durability experiment at a heating rate of 16℃ / min. DETAILED DESCRIPTION

[0072] 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.

[0073] Supplementary table to the drawings in the specification. Figure 1 to Figure 14 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 to Figure 14 Correspond to Table 1 to Table 14) respectively, and obtain more detailed information. Fig.14 If any information that may cause misunderstanding or ambiguity is found during the process, the content of 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.

[0074] Table 1

[0075] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates total

[0076] Table 2

[0077] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 4.768 552645 286612 100.00 1.13 -- 156374 total 552645 286612 100.00

[0078] In Table 2, the peak name of peak number 1 is the main component SM 1c .

[0079] Table 3

[0080] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 4.240 91865 22694 0.73 2.49 -- 35804 2 6.766 1 2369855 1924340 98.15 0.59 20.76 30975 3 7.602 141495 16809 1.12 3.89 5.47 40007 total 12603215 1963842 100.00

[0081] Table 4

[0082] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.344 47384 26341 27.35 1.06 -- 40930 2 5.344 35861 18103 20.70 1.33 63.66 199156 3 5.481 50783 25516 29.31 1.12 2.86 214199 4 7.649 39234 23517 22.64 1.18 48.96 551067 total 173262 93477 100.00

[0083] Table 5

[0084] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.859 117066 43314 0.10 1.02 -- 27982 2 4.260 113567 31393 0.09 1.21 17.40 33821 3 4.910 SM1C 118461414 21924458 97.74 0.55 4.82 12338 4 5.370 SM1C10 503207 277505 0.42 1.04 4.16 230409 5 6.190 36653 20961 0.03 1.13 18.47 317095 6 6.291 SM1C3 135830 70279 0.11 1.32 2.29 321177 7 6.502 40062 21921 0.03 1.19 4.75 338824 8 13.410 1689428 466105 1.39 0.92 99.58 325262 9 14.181 SM1C4 103483 24678 0.09 1.07 7.63 274591 total 121200711 22880613 100.00

[0085] In Table 5, the peak number 8 is the impurity SM 1c13 .

[0086] Table 6

[0087] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.859 114791 45554 0.09 0.98 -- 29127 2 4.914 120863168 22073740 98.97 0.54 17.43 13563 3 5.370 228262 110021 0.19 1.05 4.19 194517 4 6.190 38082 21868 0.03 1.13 17.40 295454 5 6.503 42190 21579 0.03 1.16 7.07 365620 6 13.395 832289 228825 0.68 0.99 101.90 337321 total 122118782 22504586 100.00

[0088] In Table 6, the peak name of peak number 2 is the main component SM 1c ; The peak number 3 is the impurity SM 1c10 ; The peak number 6 is the impurity SM 1c13 .

[0089] Table 7

[0090] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 4.772 SM1c 17040 8125 15.54 1.32 -- 158934 2 5.362 SM1C10 31525 15100 28.75 1.26 12.16 190699 3 6.289 SM1C3 34753 16722 31.70 1.59 18.90 264093 4 14.173 SM1C4 26324 6044 24.01 1.16 94.11 228490 total 109642 45991 100.00

[0091] Table 8

[0092] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 4.774 SM1c 8618 4121 23.70 1.29 -- 146225 2 5.363 SM1C10 9774 4525 26.88 1.34 12.13 205983 3 6.292 SM1C3 10457 4631 28.75 1.66 19.01 249164 4 14.176 SM1C4 7517 1744 20.67 1.04 113.31 408237 total 36365 15022 100.00

[0093] Table 9

[0094] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.988 126982 41000 0.09 1.06 -- 20968 2 4.404 164237 35528 0.12 1.00 13.47 19031 3 5.068 SM1c 133872411 23200415 97.19 0.53 4.07 10406 4 5.513 SM1C10 577693 323890 0.42 1.05 3.67 251984 5 6.332 42381 24749 0.03 1.11 18.85 349327 6 6.432 SM1C3 148070 82048 0.11 1.29 2.28 323740 7 6.643 44808 26289 0.03 1.16 4.81 386690 8 6.742 31570 9083 0.02 3.08 1.63 119626 9 13.704 2613912 667872 1.90 0.81 76.80 281403 10 14.511 SM1C4 113863 27230 0.08 1.04 7.48 265977 total 137735927 24438104 100.00

[0095] In Table 9, the peak number 9 is the impurity SM 1c13 .

[0096] Table 10

[0097] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.747 126695 43183 0.09 1.02 -- 21026 2 4.131 164522 36108 0.12 1.00 14.13 18906 3 4.794 SM1c 133552495 23335786 97.08 0.53 4.46 11698 4 5.243 SM1C10 576876 317107 0.42 1.03 4.02 204558 5 6.062 42114 23755 0.03 1.10 17.84 284029 6 6.163 SM1C3 148560 84105 0.11 1.28 2.26 314171 7 6.3375 44843 25978 0.03 1.15 4.77 324941 8 6.484 38636 10944 0.03 3.10 1.68 91490 9 13.159 2753653 741671 2.00 0.82 72.63 288264 10 13.878 SM1C4 115289 29101 0.08 1.05 7.15 289514 total 137563682 24647739 100.00

[0098] In Table 10, the peak name of peak number 9 is impurity SM 1c13 .

[0099] Table 11

[0100] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.917 128097 47199 0.09 0.99 -- 23575 2 4.344 165368 36545 0.12 0.98 14.43 19358 3 5.008 SM1c 134710904 23299437 97.48 0.53 4.73 16522 4 5.456 SM1C10 580561 319335 0.42 1.04 4.46 232973 5 6.271 42063 24321 0.03 1.11 17.94 302187 6 6.372 SM1C3 148703 83341 0.11 1.28 2.26 336617 7 6.583 44794 25797 0.03 1.16 4.84 376131 8 6.691 31165 9095 0.02 3.05 1.59 83086 9 13.478 2222046 607687 1.61 0.88 70.88 298476 10 14.246 SM1C4 115445 27534 0.08 1.00 7.56 298028 total 138189146 24480289 100.00

[0101] In Table 11, the peak name of peak number 9 is impurity SM 1c13 .

[0102] Table 12

[0103] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.804 126724 46167 0.09 1.00 -- 22046 2 4.178 164263 36539 0.12 1.01 14.23 20244 3 4839 SM1c 131964014 22942760 97.22 0.53 5.09 18392 4 5.287 SM1C10 592184 312233 0.44 1.04 4.76 216192 5 6.108 42946 24104 0.03 1.14 18.27 303234 6 6.210 SM1C3 151670 81304 0.11 1.32 2.31 320861 7 6.422 46403 25147 0.03 1.16 4.80 335685 8 6.526 36852 9049 0.03 3.50 1.31 50600 9 13.346 2501006 645320 1.84 0.83 63.88 300078 10 14.107 SM1C4 114375 27756 0.08 1.06 7.46 279345 total 135740437 24150378 100.00

[0104] In Table 12, the peak name of peak number 9 is impurity SM 1c13 .

[0105] Table 13

[0106] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.861 125653 37728 0.09 1.04 -- 18265 2 4.280 161904 34025 0.12 0.96 13.50 18584 3 4.961 SM1c 1 32534743 22605261 97.05 0.53 4.86 16503 4 5.426 8M1C10 567138 302110 0.42 1.03 4.63 218021 5 6.281 SM1C3 41831 22535 0.03 1.09 18.07 272992 6 6.386 147364 78568 0.11 1.30 2.19 280884 7 6.608 44637 24341 0.03 1.15 4.70 330628 8 13.770 2832154 725281 2.07 0.81 97.60 298578 9 14.520 SM1C4 113534 27681 0.08 1.05 7.41 327315 total 136568958 23857531 100.00

[0107] In Table 13, the peak name of peak number 8 is impurity SM 1c13 .

[0108] Table 14

[0109] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation Number of plates 1 2.863 126696 39859 0.09 0.96 -- 19905 2 4.248 164295 36128 0.12 0.95 13.58 19196 3 4.891 SM1c 134092811 24215728 97.06 0.53 4.15 10915 4 5.322 SM1C10 579742 328276 0.42 1.04 3.73 235884 5 6.108 42470 25635 0.03 1.10 18.23 332082 6 6.206 SM1C3 152057 83999 0.11 1.28 2.25 319467 7 6.407 46728 27531 0.03 1.12 4.69 373820 8 6.502 40158 12308 0.03 3.17 1.58 111176 9 13.104 2800073 728608 2.03 0.79 73.35 263979 10 13.870 SM1C4 115559 28234 0.08 1.06 7.34 271110 total 138160588 25526305 100.00

[0110] In Table 14, the peak name of peak number 9 is impurity SM 1c13 .

[0111] 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.

[0112] 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.

[0113] the term

[0114] 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.

[0115] 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.

[0116] 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.

[0117] Accuracy refers to the closeness between the measured value and the true value. In gas chromatography, the main purpose of the accuracy experiment is to verify the accuracy of the gas chromatography analysis method. By comparing the measured value with the known true value (or standard value), the error size of the analysis method can be evaluated to determine whether it meets the analysis requirements. Accuracy experiments are very important to ensure the reliability and accuracy of gas chromatography analysis. It reflects the combined influence of systematic errors and random errors in the measurement results.

[0118] 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.

[0119] In the present invention, the structural formula information of 2,4,5-trifluoro-3-methoxybenzoyl chloride and its related impurities is shown in A.

[0120] Table A. Structural information of 2,4,5-trifluoro-3-methoxybenzoyl chloride and its related impurities

[0121]

[0122]

[0123] In the present invention, the structural formula information of other related substances in 2,4,5-trifluoro-3-methoxybenzoyl chloride is shown in B.

[0124] Table B. Structural information of other related substances in 2,4,5-trifluoro-3-methoxybenzoyl chloride

[0125]

[0126] In the embodiments of the present invention, the main test conditions are shown in Table C.

[0127] Table C. Test conditions list

[0128]

[0129]

[0130] If there are any unmentioned places in the chromatographic conditions of the present invention, reference can be made to the gas chromatography method (General Rules 0521 of Part IV of the Chinese Pharmacopoeia 2020 Edition) test.

[0131] In the examples of the present invention, the test solution is prepared as follows: take an appropriate amount of the product (2,4,5-trifluoro-3-methoxybenzoyl chloride), accurately weigh it, add dichloromethane to dissolve it and dilute it to make a solution containing about 50 mg per 1 ml.

[0132] In the examples of the present invention, the control solution is prepared by accurately pipetting 0.5 ml of the test solution into a 100 ml volumetric flask, diluting to the mark with dichloromethane, and shaking to obtain the control solution.

[0133] In the embodiment of the present invention, the system suitability solution is prepared by taking the test sample (containing impurities SM 1c13 ) about 0.5g, add potassium carbonate 0.1g and water 20μl, immediately add dichloromethane 10ml, shake for 30 seconds and filter, take the filtrate, and immediately add an appropriate amount of impurity SM 1c10 , SM 1c3 , SM 1c4 , accurately weighed, dissolved and diluted with dichloromethane to make SM per 1 ml 1c3 75μg, SM 1c4 75μg, SM 1c10 0.4mg of mixed solution.

[0134] In the embodiment of the present invention, the testing method is as follows:

[0135] Accurately measure 1 μl of system suitability solution, test solution, and control solution, inject them into the gas chromatograph, and record the chromatogram. The order of peaks in the chromatographic column of the system suitability solution is the main component SM 1c 、Impurity SM 1c10 、Impurity SM 1c3 、Impurity SM 1c13 、Impurity SM 1c4 , the separation between the peaks of each component should meet the requirements. In the chromatogram of the test solution, except for the solvent peak, the content of the impurity SM 1c10、Impurity SM 1c3 、Impurity SM 1c4 Not more than 0.15%, impurity SM 1c13 The GC chromatogram of the control solution is as follows: Figure 2 The integration results are shown in Table 2.

[0136] Example 1. Specificity

[0137] (1) Prepare the test solution

[0138] Solvent: dichloromethane.

[0139] Impurity SM 1c3 Stock solution: Accurately weigh the impurity SM 1c3 About 375mg, put in a 50ml volumetric bottle, add solvent to dissolve and dilute to scale, shake well, and you have it. (about 7.5mg / ml)

[0140] Impurity SM 1c4 Stock solution: Accurately weigh the impurity SM 1c4 About 37.5 mg, put in a 20 ml volumetric bottle, add dichloromethane to dissolve and dilute to the scale, shake well, and you have it. (about 1.88 mg / ml)

[0141] Impurity SM 1c10 Stock solution: Accurately weigh the impurity SM 1c10 About 50mg, put in a 10ml volumetric bottle, add dichloromethane to dissolve and dilute to the scale, shake well, and you have it. (about 5mg / ml)

[0142] Impurity SM 1c3 With SM 1c4 Mixed stock solution: Accurately weigh the impurity SM 1c3 Stock solution 2.5ml, impurity SM 1c4 Place 10 ml of stock solution in a 25 ml volumetric flask, dilute to scale with solvent, shake well, and you have it. (0.75 mg / ml)

[0143] Other impurity location solutions I: Take the impurity SM 1e About 50mg, impurity SM 1c1 、Impurity SM 1c2 、Impurity SM 1c11 About 7.5 mg of each, put in the same 10 ml volumetric bottle, add solvent to dissolve and dilute to scale, shake well, and you are done. (Impurity SM 1e 5mg / ml, impurity SM 1c1 、Impurity SM 1c2 、Impurity SM 1c11 0.75mg / ml each)

[0144] Other impurity location solution II: Accurately weigh the impurity SM 1c5、Impurity SM 1c6 、Impurity SM 1c7 、Impurity SM 1c8 、Impurity SM 1c9 About 10 mg of each, put in different 10 ml volumetric bottles, add solvent to dissolve and dilute to scale, shake well, then accurately transfer 0.2 ml, put in the same 10 ml volumetric bottle, add solvent to dissolve and dilute to scale, shake well, and you have it. (about 0.02 mg / ml)

[0145] Test solution: Accurately weigh 0.5g of the test sample, place in a 10ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and the solution is ready. (50mg / ml)

[0146] Mixed solution: Accurately weigh 0.25 g of the test sample (containing impurities SM 1c13 ), put it into a 5ml volumetric flask, and accurately transfer the impurity SM 1c3 With SM 1c4 Mixed stock solution 0.5ml, impurity SM 1c10 0.1 ml of stock solution, 0.5 ml of other impurities localization solution I, and 0.5 ml of other impurities localization solution II are placed in the same 5 ml volumetric flask, and solvent is added to dissolve and dilute to the scale, and shake well to obtain.

[0147] (2) Determination

[0148] Take 1 μl of each of the blank solvent, other impurities localization solution I, other impurities localization solution II and the mixed solution, inject them into the liquid chromatograph respectively, perform detection according to the experimental conditions in Table C, and record the chromatogram.

[0149] The test results are shown in Table D. Figure 1 , Figure 3 to Figure 6 , Table 1, Table 3 to Table 6. In the mixed (system suitability) solution spectrum, the main component SM 1c (RT≈4.91min), impurity SM 1c10 (RT≈5.37min), impurity SM 1c3 (RT≈6.29min), SM 1c13 (RT≈13.41min), impurity SM 1c4 (RT≈14.18min) peaks appeared in sequence. Blank solvent, other impurities 1 (SM 1e , SM 1c1 , SM 1c2 , SM 1c11 ) does not interfere with SM 1c10、 SM 1c3、 SM 1c4、 SM 1c13 Detection; Other impurities 2 (SM 1c5 , SM 1c6 , SM1c7 , SM 1c8 , SM 1c9 ) does not interfere with SM 1c10 , SM 1c3 , SM 1c4 , SM 1c13 Detection. SM 1c3 、Impurity SM 1c4 , SM 1c10 , SM 1c13 The separation degree between the peaks and other adjacent peaks is greater than 1.5. This result shows that the method of the present invention has good specificity.

[0150] Table D. Method specificity test results

[0151]

[0152]

[0153] Example 2. Limit of detection and limit of quantification

[0154] (1) Prepare the test solution

[0155] Test sample quantitative limit stock solution: Accurately pipette 1 ml of the test sample solution under "Example 1. Specificity" into a 100 ml volumetric flask, dilute to scale with solvent, and shake well; (about 0.5 mg / ml)

[0156] Impurity SM 1c3 With SM 1c4 Mixed stock solution: Same as impurity SM under "Example 1. Specificity" 1c3 With SM 1c4 Prepare mixed stock solution.

[0157] Impurity SM 1c10 Stock solution: Same as impurity SM under "Example 1. Specificity" 1c10 Preparation of stock solution.

[0158] Quantitative limit solution: Accurately pipette 0.5 ml of the quantitative limit stock solution of the test sample and the impurity SM 1c10 Stock solution 2.5ml, impurity SM 1c3 With SM 1c4 Mix 0.5 ml of the stock solution, place it in a 25 ml volumetric flask, dilute to the mark with solvent, and shake well; 1c :10μg / ml, SM 1c10 :10μg / ml, SM 1c3 :15μg / ml, SM 1c4 :15μg / ml、)

[0159] Detection limit solution: Accurately pipette 3 ml of the quantitative limit solution into a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and the solution is ready.

[0160] (2) Determination

[0161] Accurately measure the quantitative limit solution and inject it continuously for 6 times, and the detection limit solution and inject it continuously for 3 times. Perform the test according to the experimental conditions in Table C and record the chromatogram; calculate the signal-to-noise ratio of each peak.

[0162] The results of the quantitation limit test are shown in Table E. Figure 7 , Table 7, the detection limit experimental results are shown in Table F, Figure 8 , Table 8. The data show that the principal component SM 1c The limit of quantification was 10.0920 μg / ml, and the limit of detection was 3.0276 μg / ml; SM 1c10 The limit of quantification was 10.6894 μg / ml, and the limit of detection was 3.2068 μg / ml; SM 1c3 The limit of quantification was 14.2276 μg / ml, and the limit of detection was 4.2683 μg / ml; SM 1c4 The limit of quantification was 14.3521 μg / ml and the limit of detection was 4.3056 μg / ml, indicating that this method has the characteristics of high sensitivity.

[0163] Table E. Quantitation limit test results

[0164]

[0165]

[0166] Table F. Detection limit determination results

[0167]

[0168] Example 3. Durability

[0169] Take the mixed solution under "Example 1. Specificity" and conduct experiments based on the chromatographic conditions in Table C by fine-tuning each chromatographic condition (including column flow rate of 1.5±0.1 ml / min, initial column temperature of 100±1°C, and heating rate of 15±1°C / min) to examine the separation between the peaks.

[0170] The test results are shown in Table G. Figures 9 to 14 , Table 9 to Table 14. When the column flow rate, initial column temperature, and heating rate fluctuate slightly, the main component SM 1c , SM 1c10 , SM 1c3 , SM 1c4 , SM 1c13The separation degrees with its adjacent peaks were greater than 1.5, and the durability of the chromatographic conditions met the verification requirements.

[0171] Table G. Chromatographic system durability test results

[0172]

[0173] Example 4. Accuracy

[0174] (1) Prepare the test solution

[0175] Impurity SM 1c10 Stock solution, impurities SM 1c3 Stock solution, SM 1c4 Stock solution: Same as impurity SM under "Example 1. Specificity" 1c10 Stock solution, impurities SM 1c3 , SM 1c4 Preparation of stock solution.

[0176] Test sample stock solution: Accurately weigh 12.5g of the test sample, place in a 25ml volumetric flask, add solvent to dissolve and dilute to scale, shake well, and the solution is ready. (0.5g / ml)

[0177] Accurate impurity stock solution: Precise pipetting of impurities SM 1c10 Stock solution 5ml, impurities SM 1c3 Stock solution 2.5ml, SM 1c4 Place 10 ml of the stock solution in a 25 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0178] Reference solution: Accurately pipette 1 ml of the accurate impurity stock solution into the same 10 ml volumetric flask, dilute to the mark with solvent, shake well, and the solution is ready.

[0179] Test sample solution: Accurately pipette 1 ml of the test sample stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0180] Accuracy 80% solution: Accurately pipette 1 ml of the sample stock solution and 0.8 ml of the accuracy impurity stock solution into the same 10 ml volumetric flask, dilute to the mark with solvent, shake well, and you have it. (Prepare 3 portions in parallel)

[0181] Accuracy 100% solution: Accurately pipette 1 ml of the test sample stock solution and 1 ml of the accuracy impurity stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with solvent, shake well, and you have it. (Prepare 3 portions in parallel)

[0182] Accuracy 120% solution: Accurately pipette 1 ml of the sample stock solution and 1.2 ml of the accuracy impurity stock solution into the same 10 ml volumetric flask, dilute to the mark with solvent, shake well, and you have it. (Prepare 3 portions in parallel)

[0183] (2) Measurement method

[0184] Take the reference solution, test solution, 80% accuracy solution, 100% accuracy solution, and 120% accuracy solution respectively, inject directly, test according to the experimental conditions in Table C, and record the chromatogram. Calculate the content of each impurity in the test sample by the external standard method, and calculate the recovery rate.

[0185] Impurity SM 1c10 The test results are shown in Table H. Impurity SM 1c3 The detection results are shown in Table 1. The impurity SM 1c4 The detection results are shown in Table J. The results show that the method of the present invention has high accuracy.

[0186] Table H. Impurity SM 1c10 Accuracy test results table

[0187]

[0188] Table I. Impurity SM 1c3 Accuracy test results table

[0189]

[0190]

[0191] Table J. Impurity SM 1c4 Accuracy test results table

[0192]

Claims

1. Separation of ethyl gatanoate starting material SM based on gas chromatography 1c and impurities thereof, characterized in that, The SM 1c and the impurities together form a composition, wherein the impurities include SM 1c10 , SM 1c3 , SM 1c13 , SM 1c4 Any one or more of the gas chromatography method comprising: a chromatographic column using a capillary column with 5% phenyl-95% methyl polysiloxane as a stationary liquid, using a heating program to SM 1c and its impurities for separation; The structural formula of each component in the composition is as follows:

2. The method according to claim 1, characterized in that The heating program is set as follows: Stage 1: Starting temperature 80-120°C, maintained for 2-5 minutes; The second stage: heating to 180-300℃ and maintaining for 8-15 minutes; The heating rate from the first stage to the second stage is 10-20°C / min.

3. The method according to claim 1, characterized in that The composition may also contain other substances, including SM 1e , SM 1c1 , SM 1c2 , SM 1c11 , SM 1c5 , SM 1c6 , SM 1c7 , SM 1c8 , SM 1c9 Any one or more of the following, its structural formula is as follows:

4. The method according to claim 1, characterized in that The injection port temperature is 150-300°C, the flow rate is 1-2 ml / min, the carrier gas is nitrogen, and the split ratio is 25-35:

1.

5. The method according to claim 1, characterized in that The sample preparation solvent was dichloromethane.

6. Identification of ethyl gatanoate starting material SM 1c and impurities thereof, characterized in that, The composition is separated by the method described in any one of claims 1 to 5, and is detected by a detector to obtain a chromatogram; by comparing the chromatogram characteristics of the test product with those of the reference product, it is determined whether the test product contains SM 1c and its impurities.

7. The method according to claim 6, characterized in that The detector temperature is 220~300℃.

8. The method according to claim 6, characterized in that According to the order of retention time, the components in the composition can be identified, and the components of the composition are in ascending order: SM 1c , SM 1c10 , SM 1c3 , SM 1c13 , SM 1c4 .

9. The method according to claim 8, characterized in that The retention time was 4.9±0.5min, which was determined to be the SM 1c ; The retention time was 5.4±0.5min, which was determined to be the SM 1c10 ; The retention time was 6.3±0.5min, which was determined to be the SM 1c3 ; The retention time was 13.4 ± 0.5min, which was determined to be the SM 1c13 ; The retention time was 14.2±0.5min, which was determined to be the SM 1c4 .

10. Detection of ethyl gatrate starting material SM 1c and its impurity content, characterized in that, Using the method described in any one of claims 6 to 9 to separate and identify SM 1c and its impurities to obtain a chromatogram; based on the obtained chromatogram, the content of each impurity is calculated using the main component self-comparison method.

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