Method for separating and determining linezolid intermediate Z4 and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining a linezolid intermediate Z4 and impurities thereof. The impurities comprise any one or more of an impurity Z2, an impurity Z3b, an impurity Z4b, an impurity Z3, an impurity Z4d, an impurity Z3c and an impurity Z4c. The self-built high performance liquid chromatography is used for separating and determining the linezolid intermediate Z4 and impurities thereof. A mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of a buffer salt solution and acetonitrile; the mobile phase B is a mixed solution of methanol and acetonitrile; in a stationary phase, octadecylsilane chemically bonded silica is used as a filler of a chromatographic column, and linear gradient elution is carried out. Then, according to a chromatogram, the content of each impurity is calculated by adopting a main component self-contrast method with a correction factor. The method disclosed by the invention 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 specifically relates to a method for separating and determining the intermediate Z of linezolid. 4 and its impurities. Background Art

[0002] Linezolid is a synthetic oxazolidinone antibiotic that was approved by the U.S. FDA in 2000 for the treatment of infections caused by Gram-positive cocci, including suspected or confirmed hospital-acquired pneumonia (HAP) caused by MRSA, community-acquired pneumonia (CAP), complicated skin or skin and soft tissue infections (SSTI), and vancomycin-resistant enterococci (VRE) infections.

[0003] Linezolid intermediate Z 4 It is a key intermediate in the synthesis of linezolid, and its structural formula is shown in Formula I. 4 The following seven impurities are easily introduced into the 3 、Impurity Z 2 、Impurity Z 3b 、Impurity Z 3c 、Impurity Z 4b 、Impurity Z 4c and impurity Z 4d . Separation and determination of linezolid intermediate Z 4 The seven impurities are of great significance for the quality control of linezolid intermediates and linezolid finished products.

[0004]

[0005] Some documents or patent materials have reported on the analysis methods of linezolid and its related substances. For example, the patent with publication number CN115047117A discloses a detection method for the simultaneous determination of three genotoxic impurities in linezolid, which are 3-fluoro-4-(4-morpholinyl)-aniline, (S)-N-glycidylphthalimide and 2,3-dihydro-phthalazine-1,4-dione; the method is a high-performance liquid chromatography method, in which the chromatographic column uses octadecylsilane bonded silica gel as a filler, the mobile phase A is a phosphate buffer, and the mobile phase B is 10% acetonitrile. For another example, the patent with publication number CN115902018A discloses a method for detecting linezolid isomers, which uses a high-performance liquid chromatography method to detect linezolid isomers, and the stationary phase uses a polysaccharide derivative solvent-resistant chiral chromatographic column, and the mobile phase is a mixture of acetonitrile and ammonium bicarbonate aqueous solution. However, these methods cannot achieve the aforementioned linezolid intermediate Z 4 Separation and determination of its 7 impurities.

[0006] Therefore, it is necessary to establish an efficient and simple method to determine the intermediate Z 4 and its 7 impurities. Summary of the invention

[0007] In view of this, one of the purposes of the present invention is to provide a method for separating linezolid intermediate Z based on high performance liquid chromatography 4 The method can separate a variety of substances in a shorter time.

[0008] To achieve the above object, the technical solution of the present invention is:

[0009] Separation of Linezolid Intermediate Z Based on HPLC 4 and its impurities, the linezolid intermediate Z 4 and the impurities together form a composition, wherein the impurities include impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 、Impurity Z 4d 、Impurity Z 3c 、Impurity Z 4c Any one or more of; the structural formula of each component in the composition is as follows:

[0010]

[0011] In the high performance liquid chromatography method, the mobile phase: mobile phase A is a mixed solution of buffered salt solution and acetonitrile; mobile phase B is a mixed solution of methanol and acetonitrile; the stationary phase: the chromatographic column uses octadecylsilane bonded silica gel as a filler, and linear gradient elution is performed.

[0012] After separation and testing, it is used for the next production step.

[0013] The above impurities can be arranged and combined in many ways. For example, combination 1: impurity Z 2 、Impurity Z 3b For example, combination 2: impurity Z 2 、Impurity Z 3b 、Impurity Z 4b For example, combination 3: impurity Z 3 、Impurity Z 4d 、Impurity Z 4c .

[0014] 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.

[0015] Furthermore, the composition may also contain other related substances: imidazole and / or impurity SM 2 , the impurity SM 2 The structural formula of is shown in Formula IX.

[0016]

[0017] Linezolid intermediate Z 4 The separation and determination of its impurities are not interfered by other related substances.

[0018] Further, the procedure of the linear gradient elution is as follows:

[0019] Time - Minutes Mobile phase A-(volume parts) Mobile phase B-(volume parts) 0 70±10 30±10 2 70±10 30±10 22 30±10 70±10 28 30±10 70±10 28.1 70±10 30±10 35 70±10 30±10

[0020] Preferably, the procedure of the linear gradient elution is as follows:

[0021] Time - Minutes Mobile phase A-(volume parts) Mobile phase B-(volume parts) 0 68-72 32-28 2 68-72 32-28 22 30 70 28 30 70 28.1 68-72 32-28 35 68-72 32-28

[0022] For example, mobile phase A accounts for 70 parts and mobile phase B accounts for 30 parts; or mobile phase A accounts for 72 parts and mobile phase B accounts for 28 parts ( Fig.11 ); or mobile phase A accounts for 71 parts and mobile phase B accounts for 29 parts; or mobile phase A accounts for 69 parts and mobile phase B accounts for 31 parts; or mobile phase A accounts for 68 parts and mobile phase B accounts for 32 parts ( Fig.12 ).

[0023] Most preferably, the procedure of the linear gradient elution is as follows:

[0024] Time - Minutes Mobile phase A-(volume parts) Mobile phase B-(volume parts) 0 70 30 2 70 30 22 30 70 28 30 70 28.1 70 30 35 70 30

[0025] Furthermore, in the mobile phase A, the volume ratio of the buffered saline solution to acetonitrile is 93-97:7-3; in the mobile phase B, the volume ratio of methanol to acetonitrile is 70-80:30-20.

[0026] Preferably, in the mobile phase A, the volume ratio of the buffered saline solution to acetonitrile is 95:5; and in the mobile phase B, the volume ratio of methanol to acetonitrile is 75:25.

[0027] Furthermore, the buffered saline solution in the mobile phase A is a sodium dihydrogen phosphate solution with a concentration of 8-12 mmol / L.

[0028] As a preferred embodiment, the concentration of the buffer salt solution in the mobile phase A is 9-11 mmol / L. For example, the buffer salt concentration is 10 mmol / L; or the buffer salt concentration is 9 mmol / L ( Fig.13 ), or the buffer salt concentration is 11mmol / L ( Fig.14 ).

[0029] As the most preferred solution, the concentration of the buffered salt solution in the mobile phase A is 10 mmol / L.

[0030] Furthermore, the flow rate is 1.3-1.7 ml / min; the column temperature is 25-35°C, and the injector temperature is 4-6°C.

[0031] As a preferred solution, the flow rate is 1.4-1.6 ml / min; the column temperature is 28-32°C. For example, the flow rate is 1.5 ml / min, or the flow rate is 1.4 ml / min ( Figure 7 ), or a flow rate of 1.6 ml / min ( Figure 8 ). For example, the column temperature is 30°C; or the column temperature is 28°C ( Fig. 9 ); or column temperature is 29°C; or column temperature is 31°C; or column temperature is 32°C ( Fig.10 ).

[0032] As the most preferred solution, the flow rate is 1.5 ml / min; the column temperature is 30°C, and the injector temperature is 5°C.

[0033] Furthermore, the injection volume was 5 μl.

[0034] Preferably, the running time is 35 minutes.

[0035] As a preferred solution, the chromatographic column specifications are 4.6 mm×250 mm, 5 μm.

[0036] As the most preferred solution, the chromatographic column selected is Agilent Eclipse XDB-C18, 4.6 mm×250 mm, 5 μm.

[0037] Furthermore, the sample preparation solvent was an acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water was 50:50.

[0038] As a preferred solution, a sample preparation solvent is used to prepare a test solution before separation; the test solution includes a sample solution, a control solution and / or a system suitability solution;

[0039] Preferably, the concentration of the sample solution is 1 mg / ml. The preparation method comprises: taking the linezolid intermediate Z 4 An appropriate amount, accurately weighed, is dissolved in acetonitrile at a volume of 20%, and diluted with solvent to make a solution containing about 1 mg per 1 ml.

[0040] Preferably, the concentration of the control solution is 1.5 μg / ml. The preparation method thereof comprises: accurately measuring an appropriate amount of the sample solution, and diluting it with a solvent to prepare a solution containing about 1.5 μg per 1 ml.

[0041] As a preferred method, the system suitability solution is prepared as follows: 4System suitability reference material 1 (containing Z 4 、Impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 、Impurity Z 4d 、Impurity Z 3c 、Impurity Z 4c ) is accurately weighed, dissolved in acetonitrile at a volume of 20% and diluted with solvent to make a solution containing about 1 mg per 1 ml.

[0042] The second object of the present invention is to provide a method for identifying the intermediate Z of linezolid 4 The method can complete the identification of multiple substances in a shorter time.

[0043] To achieve the above object, the technical solution of the present invention is:

[0044] Identification of Linezolid Intermediate Z 4 The method for separating the composition and its impurities comprises: separating the composition by the aforementioned separation method, and entering the composition into a detector for detection to obtain a chromatogram; and determining whether the test sample contains the linezolid intermediate Z by comparing the chromatogram characteristics of the test sample with those of the reference sample. 4 and its impurities.

[0045] As a preferred solution, the detection wavelength of the detector is 240±10nm. The setting range of ±10nm is a comprehensive consideration based on multiple factors such as error considerations, method superiority, and practical application requirements. This setting range helps to ensure the reliability of the detection results, improve the repeatability and flexibility of the measurement, and meet the requirements of specific experiments.

[0046] Most preferably, the detection wavelength is 240 nm.

[0047] As a preferred solution, the components in the composition can be identified according to the order of relative retention time. The components of the composition are in ascending order: impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 , Linezolid intermediate Z 4 、Impurity Z 4d 、Impurity Z 3c 、Impurity Z 4c .

[0048] Generally, relative retention time is used to describe the relative retention of different components in a mixture on a chromatographic column. It refers to the ratio of the retention time of a component to the retention time of a reference component (usually the main peak or a known component). This ratio reflects the relative retention performance of different components on a chromatographic column and is an important parameter for positioning, qualitative and quantitative analysis in chromatographic analysis. The relative retention time is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0049] As a preferred solution, the linezolid intermediate Z 4 is the reference peak; the relative retention time is 0.25, which is determined to be impurity Z 2 ; The relative retention time is 0.78, which is determined to be impurity Z 3b ; The relative retention time is 0.89, which is determined to be impurity Z 4b ; The relative retention time is 0.92, which is determined to be impurity Z 3 ; The relative retention time is 1.10, which is determined to be impurity Z 4d ; The relative retention time is 1.18, which is determined to be impurity Z 3c ; The relative retention time is 1.26, which is determined to be impurity Z 4c ; The relative retention time of each component fluctuates within 5%.

[0050] In addition to relative retention time, retention time can also be used to determine components. 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 certain 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. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0051] The third object of the present invention is to provide a method for determining the intermediate Z of linezolid 4 The method for determining the content of various substances and their impurities can be completed in a relatively short time.

[0052] To achieve the above object, the technical solution of the present invention is:

[0053] Determination of Linezolid Intermediate Z 4 and its impurity content, using the aforementioned identification method to separate and identify the linezolid intermediate Z 4 and its impurities to obtain a chromatogram; based on the obtained chromatogram, the main component self-comparison method with a correction factor is used to calculate the content of each impurity.

[0054] As a preferred solution, impurity Z2 The correction factor is 0.68; the impurity Z 3b The correction factor is 0.89; the impurity Z 3 The correction factor is 0.80; the impurity Z 4d The correction factor is 2.0; the impurity Z 4c The correction factor is 1.2.

[0055] Through content determination, we can further determine the linezolid intermediate Z 4 And whether its impurity content is qualified. If impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 、Impurity Z 4d 、Impurity Z 3c 、Impurity Z 4c If the peak area of ​​any one or more impurities in the reference solution is greater than the peak area of ​​the corresponding impurities in the reference solution, it indicates that the impurity content is unqualified; conversely, if impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 、Impurity Z 4d 、Impurity Z 3c 、Impurity Z 4c The peak area of ​​any one or more impurities in the sample is not greater than the peak area of ​​the corresponding impurity in the reference solution, indicating that the impurity content is qualified.

[0056] 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 mobile phase composition, flow rate, column temperature, 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.

[0057] The beneficial effects of the present invention are:

[0058] (1) The present invention self-built high performance liquid chromatography method for linezolid intermediate Z 4 The separation and detection of linezolid intermediate Z was successfully achieved. 4 Medium impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 、Impurity Z 4d 、Impurity Z 3c and impurity Z 4cIt can effectively control the system and has the advantages of being simple, fast, accurate and practical.

[0059] (2) The method of the present invention can simultaneously produce linezolid intermediate Z within 35 minutes 4 Medium impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 、Impurity Z 4d 、Impurity Z 3c and impurity Z 4c A total of 7 impurities were effectively separated, and the separation degree between the 7 impurities was greater than 1.5. In addition, other known or unknown impurities in the test sample (such as imidazole, impurity SM 2 etc.) also do not interfere with the detection of the 7 impurities, and meet the requirements of relevant substances.

[0060] (3) The method of the present invention has strong specificity, high sensitivity, good durability, and accurate and reliable detection results, which is of great significance for achieving quality control of linezolid intermediates and linezolid finished products. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 is the chromatogram of the blank solution;

[0062] Figure 2 is the chromatogram of the total mixed solution;

[0063] Figure 3 is the chromatogram of the test solution;

[0064] Figure 4 is the chromatogram of the system suitability solution;

[0065] Figure 5 is the chromatogram of the quantitation limit solution;

[0066] Figure 6 is the chromatogram of the detection limit solution;

[0067] Figure 7 This is a chromatogram for the durability experiment at a flow rate of 1.4 ml / min.

[0068] Figure 8 This is the chromatogram for the durability experiment at a flow rate of 1.6 ml / min.

[0069] Fig. 9 This is the chromatogram of the durability experiment under the condition of column temperature of 28°C;

[0070] Fig.10 This is the chromatogram of the durability experiment under the condition of column temperature of 32°C;

[0071] Fig.11This is the chromatogram under the condition of durability experiment - initial ratio of mobile phase A:B = 72:28;

[0072] Fig.12 This is the chromatogram under the condition of durability experiment - initial ratio of mobile phase A:B = 68:32;

[0073] Fig.13 This is the chromatogram of the durability experiment under the condition of 9 mmol / L buffer salt concentration in mobile phase A;

[0074] Fig.14 This is the chromatogram of the durability experiment under the condition of buffer salt concentration of 11mmol / L in mobile phase A. DETAILED DESCRIPTION

[0075] 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.

[0076] Supplementary table to the drawings in the specification. Figure 1-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-Figure 14 The integral results correspond to Tables 1-14 respectively, and the data can be used to 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 spectrum have no effect on the scope of protection of the claims and the full disclosure of the technical solution in the specification.

[0077] Table 1

[0078]

[0079] Table 2

[0080]

[0081] Table 3

[0082]

[0083] Table 4

[0084]

[0085] Table 5

[0086]

[0087] Table 6

[0088]

[0089] Table 7

[0090]

[0091] Table 8

[0092]

[0093] Table 9

[0094]

[0095] Table 10

[0096]

[0097] Table 11

[0098]

[0099] Table 12

[0100]

[0101] Table 13

[0102]

[0103] Table 14

[0104]

[0105] In order to enhance the understanding of the present invention, some key technologies and scientific terms are clearly defined below. Unless 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. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference substances, and test substances described, but reasonable flexibility and adjustment in these aspects are allowed. At the same time, please understand that the terms used in this article are intended to illustrate specific embodiments rather than to be interpreted restrictively.

[0106] 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.

[0107] In the embodiments of the present invention, if there are any unmentioned conditions in the chromatographic conditions, they can be determined by referring to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part 4, Part 0512).

[0108] the term

[0109] 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 HPLC, 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 a certain level of signal-to-noise ratio 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.

[0110] The durability of chromatographic conditions refers to the ability of the chromatographic analysis system to maintain stable analytical performance and not significantly affect the measurement 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.

[0111] 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.

[0112] The correction factor is a coefficient or parameter used to correct the results during the analysis process. It is intended to improve data accuracy and reliability. In HPLC analysis, since the same detector has different response values ​​for different substances, when different substances of the same mass pass through the detector, the peak areas generated are not necessarily equal. In order to make the peak area accurately reflect the content of the component to be measured, standard substances are used for correction, the correction factor is calculated, and it is applied to the measurement results of the sample to be measured.

[0113] The ratio of peak height to noise (S / N, i.e. signal-to-noise ratio) is used in HPLC to evaluate the detection sensitivity and resolution of the instrument and is an important indicator for measuring instrument performance. Among them: peak height refers to the signal value output by the detector when the component to be tested is eluted from the column; and noise refers to the fluctuation of the baseline signal, that is, the signal value measured by the blank sample. The signal-to-noise ratio is the ratio of the signal measured by a sample of known concentration to the signal measured by a blank sample. The larger the signal-to-noise ratio, the more accurately the instrument can separate and identify the target component when detecting the sample, while also reducing the interference of background noise.

[0114] In the embodiment of the present invention, the linezolid intermediate Z 4 The information on its impurities is shown in Table A.

[0115] Table A. Compound information table

[0116]

[0117]

[0118] In the examples of the present invention, the analytical method validation standards are shown in Table B.

[0119] Table B. Analytical Method Validation Standards

[0120]

[0121]

[0122] Example 1

[0123] (1) Prepare the test solution

[0124] Solvent: water and acetonitrile in a volume ratio of 50:50.

[0125] Test solution: Take this product (linezolid intermediate Z 4 ) is accurately weighed, dissolved in acetonitrile at a volume of 20% and diluted with solvent to make a solution containing about 1 mg per 1 ml.

[0126] Control solution: Accurately measure an appropriate amount of the test sample solution and dilute it with solvent to make a solution containing approximately 1.5 μg per 1 ml.

[0127] System suitability solution: Take Z 4 System suitability reference material 1 (containing Z 4 、Impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 、Impurity Z 4d 、Impurity Z 3c 、Impurity Z4c ) is accurately weighed, dissolved in acetonitrile at a volume of 20% and diluted with solvent to make a solution containing about 1 mg per 1 ml.

[0128] (2) Chromatographic conditions

[0129] Use octadecylsilane bonded silica gel as filler (Agilent Eclipse XDB-C18, 4.6mm×250mm, 5μm or chromatographic column with equivalent performance); use 0.01mol / L sodium dihydrogen phosphate solution-acetonitrile (95:5) as mobile phase A, and methanol-acetonitrile (75:25) as mobile phase B, and perform gradient elution according to Table C; the flow rate is 1.5ml per minute; the column temperature is 30℃; the injector temperature is 5℃; the detection wavelength is 240nm; and the injection volume is 5μl.

[0130] Table C. Gradient elution schedule

[0131] Time - Minutes Mobile phase A-(volume parts) Mobile phase B-(volume parts) 0 70 30 2 70 30 22 30 70 28 30 70 28.1 70 30 35 70 30

[0132] 3. Determination

[0133] Accurately measure the system suitability solution, test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. According to the chromatogram, the main component self-reference method with correction factor is used to quantify each impurity.

[0134] System suitability requirements: In the system suitability solution chromatogram, according to the impurity Z 2 、Impurity Z 3b 、Impurity Z 4b 、Impurity Z 3 , Z 4 、Impurity Z 4d 、Impurity Z 3c and impurity Z 4c The peaks are extracted sequentially, and the separation between the peaks of each component should meet the requirements.

[0135] Limit: If there is an impurity peak in the chromatogram of the test solution, impurity Z 2 , Z 3b , Z 3 , Z 4d , Z 4c The corrected peak areas (multiplied by the correction factors 0.68, 0.89, 0.80, 2.0 and 1.2, respectively) should not be greater than the main peak area of ​​the control solution (0.15%). 4b , Z 3cThe peak area shall not be greater than the main peak area of ​​the control solution (0.15%), the area of ​​other individual impurity peaks shall not be greater than twice the main peak area of ​​the control solution (0.3%), the sum of the impurity peak areas after correction shall not be greater than 6.66 times the main peak area of ​​the control solution (1.0%), and peaks less than 0.33 times the main peak area of ​​the control solution shall be ignored (0.05%).

[0136] Example 2. Specificity

[0137] (1) Prepare the test solution

[0138] Blank solvent (solvent): prepared from water and acetonitrile in a volume ratio of 50:50.

[0139] Impurity Z 3 Stock solution: Accurately weigh impurity Z 3 Place 25.49 mg of the reference substance in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0140] Impurity Z 2 Stock solution: Accurately weigh impurity Z 2 Place 25.79 mg of the reference substance in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0141] Impurity Z 3b Stock solution: Accurately weigh impurity Z 3b Place 10.46 mg of reference substance in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0142] Impurity Z 3c Stock solution: Accurately weigh impurity Z 3c Place 10.35 mg of the reference substance in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0143] Impurity Z 4b Stock solution: Accurately weigh impurity Z 4b Place 25.75 mg of reference substance in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0144] Impurity Z 4c Stock solution: Accurately weigh impurity Z 4c Place 10.43 mg of the reference substance in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0145] Impurity Z 4d Stock solution: Accurately weigh impurity Z 4d Place 9.99 mg of the reference substance in a 10 ml volumetric flask, add 5 ml of acetonitrile and sonicate, then add solvent to dissolve and dilute to the scale, shake well, and the result is obtained.

[0146] Impurity SM 2 Stock solution: Accurately weigh impurities SM 2 Place 50.44 mg of the reference substance in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0147] Imidazole stock solution: Accurately weigh 25.03 mg of imidazole reference substance, place in a 25-volume flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0148] Z 4 Stock solution: Accurately weigh Z 4 Place 25.88 mg of the reference substance in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and the solution is ready.

[0149] Each impurity location solution: accurately measure the impurity Z 3 、Impurity Z 2 、Impurity Z 3b 、Impurity Z 3c 、Impurity Z 4b 、Impurity Z 4c 、Impurity Z 4d 、Impurity SM 2 Place 1.5 ml of each of the stock solutions of imidazole and imidazole in different 100 ml volumetric flasks, dilute to the mark with solvent, shake well, and the solution is ready.

[0150] Test solution: Accurately weigh this product (lot number: LINE-Z 4 -231201) 25.18 mg, put into a 25 ml volumetric flask, add 5 ml of acetonitrile to dissolve, add solvent to dilute to the scale, shake well, and obtain.

[0151] Impurity mixed stock solution: Precise pipetting of impurity Z 3 、Impurity Z 2 、Impurity Z 3b 、Impurity Z 3c 、Impurity Z 4b 、Impurity Z 4c 、Impurity Z 4d Place 1.5 ml of each impurity stock solution in the same 100 ml volumetric flask, dilute to the mark with solvent, shake well, and the product is ready.

[0152] Total mixed solution: accurately weigh 25.36 mg of this product, place it in a 25 ml volumetric flask, add 5 ml of acetonitrile to dissolve, and add impurity mixed stock solution, impurity SM 2 Take 2.5 ml of the positioning solution and imidazole positioning solution respectively, dilute to the scale with solvent, shake well, and you have it.

[0153] (2) Determination

[0154] 5 μl of blank solvent, each impurity location solution, test solution and total mixed solution were injected respectively, and the detection was carried out according to the chromatographic conditions in Example 1, and the chromatogram was recorded to examine the separation between the peaks.

[0155] The test results are shown in Table D. Figure 1 to Figure 3 , Table 1 to Table 3. The results show that the blank solvent does not interfere with the test sample detection; the separation between each impurity peak is greater than 1.5, indicating that the method has good specificity.

[0156] Table D. Specificity test results

[0157]

[0158] Example 3. System Applicability

[0159] (1) Separation

[0160] Separation test: See the total mixed solution spectrum and its test data under "Example 2. Specificity".

[0161] (2) Injection repeatability

[0162] Control solution: Accurately measure 5 ml of the test solution under "Example 2. Specificity", place it in a 100 ml volumetric flask, dilute to the mark with solvent, shake well, accurately measure 3 ml, place it in a 100 ml volumetric flask, dilute to the mark with solvent, shake well, and obtain.

[0163] Accurately measure 5 μl of the control solution, inject it into the liquid chromatograph, measure it continuously for 6 times according to the chromatographic conditions in Example 1, record the chromatogram, and calculate the RSD of the main peak area.

[0164] The test results are shown in Table E. Figure 4 ,Table 4. The results show that in the system suitability solution, the RSD of the main peak area is less than 5%, the tailing factor is less than 2.0, and the number of theoretical plates is greater than 5000, which meets the requirements.

[0165] Table E. System suitability test results

[0166]

[0167] Example 4. Limit of Quantitation

[0168] Quantitative limit solution: Accurately measure the impurity Z under "Example 2. Specificity" 3 , Z 2 , Z 3b , Z 3c , Z 4b , Z 4c , Z 4d , Z 4Place 0.7ml, 0.4ml, 0.9ml, 0.7ml, 0.5ml, 0.8ml, 1.4ml and 0.6ml of stock solution in the same 100ml volumetric flask, add solvent to dilute to the scale, shake well, accurately measure 1ml, place in a 50ml volumetric flask, add solvent to dilute to the scale, shake well.

[0169] The quantitative limit solution was taken and measured 6 times continuously. The chromatographic conditions were the same as those in Example 1. The chromatogram was recorded and the ratio of each peak to noise (signal-to-noise ratio) and the peak area RSD were calculated.

[0170]

[0171] The test results are shown in Table F. Figure 5 , Table 5. The data show that impurity Z 2 The limit of quantification concentration was 0.0818 μg / ml, the RSD value of the peak area was 2.3%, and the mean signal-to-noise ratio was 35.9. 4 The concentration present in is indicated as 0.008%. Impurity Z 3b The limit of quantification concentration was 0.1806 μg / ml, the RSD value of the peak area was 2.4%, and the mean signal-to-noise ratio was 26.1. 4 The concentration present in is indicated as 0.018%. Impurity Z 4b The limit of quantification was 0.1026 μg / ml, the RSD value of the peak area was 7.8%, and the mean signal-to-noise ratio was 21.4. 4 The concentration present in is indicated as 0.010%. Impurity Z 3 The limit of quantification concentration was 0.1413 μg / ml, the RSD value of the peak area was 3.1%, and the mean signal-to-noise ratio was 40.7. 4 The concentration present in is indicated as 0.014%. Impurity Z 4d The limit of quantification concentration was 0.2786 μg / ml, the RSD value of the peak area was 2.2%, and the mean signal-to-noise ratio was 29.9. 4 The concentration present in is indicated as 0.028%. Impurity Z 3c The limit of quantification concentration was 0.1382 μg / ml, the RSD value of the peak area was 3.8%, and the mean signal-to-noise ratio was 31.1. 4 The concentration present in is indicated as 0.014%. Impurity Z 4c The limit of quantification was 0.1609 μg / ml, the RSD value of the peak area was 4.0%, and the mean signal-to-noise ratio was 31.9. 4 The concentration present in is indicated as 0.016%. 4 The limit of quantification was 0.1236 μg / ml, the RSD value of the peak area was 6.7%, and the mean signal-to-noise ratio was 30.4. 4The concentration present in the sample is expressed as 0.012%. At the quantitative limit concentration of each impurity, the signal-to-noise ratio is greater than 10, and the peak area RSD is less than 10%, which meets the detection requirements of the relevant substances, indicating that each impurity can be accurately quantified at this level.

[0172] Table F. Quantitation limit test results

[0173]

[0174]

[0175] Example 5. Detection Limit

[0176] Detection limit solution: Accurately measure 3.5 ml of the quantification limit solution under the "quantification limit" item, place it in a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0177] Take the above detection limit solution and measure it continuously for 3 times, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio).

[0178]

[0179] The test results are shown in Table G. Figure 6 , Table 6. The data show that impurity Z 2 The detection limit concentration was 0.0286 μg / ml, the mean signal-to-noise ratio was 11.7, and the Z 4 The concentration of Z is 0.003%; 3b The detection limit concentration was 0.0632 μg / ml, the mean signal-to-noise ratio was 6.7, and the Z 4 The concentration in Z is 0.006%; 4b The detection limit concentration was 0.0359 μg / ml, the mean signal-to-noise ratio was 6.5, and the Z 4 The concentration in Z is 0.004%; 3 The detection limit concentration was 0.0495 μg / ml, the mean signal-to-noise ratio was 14.2, and the Z 4 The concentration of Z is 0.005%; 4d The detection limit concentration was 0.0975 μg / ml, the mean signal-to-noise ratio was 10.8, and the Z 4 The concentration of Z is 0.010%; 3c The detection limit concentration was 0.0484 μg / ml, the mean signal-to-noise ratio was 10.7, and the Z 4 The concentration of Z is 0.005%; 4c The detection limit concentration was 0.0563 μg / ml, the mean signal-to-noise ratio was 10.6, and the Z 4 The concentration in Z is 0.006%; 4The detection limit concentration was 0.0433 μg / ml, the mean signal-to-noise ratio was 9.6, and the Z 4 The concentration in the sample is 0.004%; at the detection limit concentration of each impurity, the signal-to-noise ratio is greater than 3, which meets the detection requirements of relevant substances, indicating that the impurities can be effectively detected at this level.

[0180] Table G. Detection limit test results

[0181]

[0182] Example 6. Chromatographic System Robustness

[0183] Take the total mixed solution under "Example 2. Specificity", take the chromatographic conditions in Example 1 as normal conditions, adjust the experimental conditions (column flow rate ± 0.1 ml / min, column temperature ± 2 ° C, mobile phase initial ratio ± 2, mobile phase A buffer salt concentration ± 1 mmol / L), and test them after the instrument system is stable to examine the relative retention time and separation between the peaks.

[0184] The test results are shown in Table H, Table I, Figure 7 to Figure 14 , Table 7 to Table 14. When the flow rate, column temperature, initial ratio of mobile phase and buffer salt concentration fluctuate, the peak order of each impurity and main component remains unchanged, and the relative retention time of each impurity under each condition changes slightly; the separation degree of the main peak and each impurity peak is greater than 1.5, and the method is relatively durable.

[0185] Table H. Chromatographic Conditions Durability Test Results (Relative Retention Time)

[0186]

[0187] Table I. Chromatographic conditions robustness test results (resolution)

[0188]

Claims

1. A method for separating linezolid intermediate Z4 and its impurities based on high performance liquid chromatography, characterized in that: The linezolid intermediate Z4 and the impurities together form a composition, and the impurities include impurities Z2, impurities Z 3b 、Impurity Z 4b , impurity Z3, impurity Z 4d 、Impurity Z 3c 、Impurity Z 4c Any one or more of; the structural formula of each component in the composition is as follows: In the high performance liquid chromatography method, the mobile phase: mobile phase A is a mixed solution of buffered saline solution and acetonitrile; mobile phase B is a mixed solution of methanol and acetonitrile; Stationary phase: The chromatographic column uses octadecylsilane bonded silica gel as a filler and performs linear gradient elution.

2. The method according to claim 1, characterized in that The procedure of the linear gradient elution is as follows:

3. The method according to claim 1, characterized in that In the mobile phase A, the volume ratio of the buffered salt solution to acetonitrile is 93-97:7-3; in the mobile phase B, the volume ratio of methanol to acetonitrile is 70-80:30-20.

4. The method according to claim 1, characterized in that: The buffered saline solution in the mobile phase A is a sodium dihydrogen phosphate solution with a concentration of 8-12 mmol / L.

5. The method according to claim 1, characterized in that The flow rate was 1.3-1.7 mL / min; the column temperature was 25-35°C, and the injector temperature was 4-6°C.

6. A method for identifying linezolid intermediate Z4 and its impurities, characterized in that: The composition is separated by the method described in any one of claims 1 to 5, and enters 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 the linezolid intermediate Z4 and its impurities.

7. The method according to claim 6, characterized in that The detection wavelength of the detector is 240±10 nm.

8. The method according to claim 6, characterized in that According to the order of relative retention time, the components in the composition can be identified, and the components of the composition are in ascending order: impurity Z2, impurity Z 3b 、Impurity Z 4b , impurity Z3, linezolid intermediate Z4, impurity Z 4d 、Impurity Z 3c 、Impurity Z 4c .

9. The method according to claim 8, characterized in that The linezolid intermediate Z4 was used as the reference peak; the relative retention time was 0.25, and it was determined to be impurity Z2; The relative retention time is 0.78, which is determined to be impurity Z 3b ; The relative retention time is 0.89, which is determined to be impurity Z 4b ; The relative retention time is 0.92, which is determined to be impurity Z3; The relative retention time is 1.10, which is determined to be impurity Z 4d ; The relative retention time is 1.18, which is determined to be impurity Z 3c ; The relative retention time is 1.26, which is determined to be impurity Z 4c ; The relative retention time of each component fluctuates within 5%.

10. A method for determining the content of linezolid intermediate Z4 and its impurities, characterized in that: The method described in any one of claims 6 to 9 is used to separate and identify the linezolid intermediate Z4 and its impurities to obtain a chromatogram; based on the obtained chromatogram, the content of each impurity is calculated using the principal component self-reference method with a correction factor.

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