Detection method of optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate

Abstract

The invention discloses a method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxyethyl] carbamate, and adopts a high-performance liquid chromatography determination method. The determination method comprises the following steps: using a chromatographic column with amylose-tris(3,5-diphenylcarbamate) as a filler, using n-hexane-ethanol-trifluoroacetic acid with a volume ratio of 700-750:250-300:1 as a mobile phase, preparing a solution for injection, and calculating the optical isomer content by a peak area normalization method. The invention makes up for the current technical gap of lacking high-performance liquid chromatography detection of the substance.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxyethyl]carbamate. Background Art

[0002] Malignant tumors are one of the common causes of death in human diseases. Due to the different pathogenesis of different types of tumors, the diagnosis and treatment of tumors are still difficult. Tumors are composed of tumor cells and tumor stroma. The stroma accounts for the vast majority of tumors, and tumor-associated fibroblasts (CAFs) are an important component of tumor stroma. Fibroblast activation protein (FAP) is overexpressed in CAFs and is closely related to tumor growth, invasion, metastasis, immunosuppression and prognosis. FAP is expressed at low levels in normal organs and highly expressed in tumors, so it is a good diagnostic and therapeutic target.

[0003] Tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a) can be used as a starting material to synthesize FAP targeted drugs. However, the R-configuration optical isomer impurities contained in tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a) will undergo impurity transfer and transformation during the synthesis reaction, resulting in product impurities that lack targeting and may even have other side effects, increasing the difficulty of purifying FAP targeted drugs. Therefore, it is necessary to control the content of R-configuration optical isomers in this material.

[0004] Tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a), CAS No.: 1448440-50-3, molecular formula: C 12 H 17 F2N3O3, molecular weight: 289.28, its structural formula is:

[0005]

[0006] The structural formula of the R-configuration optical isomer impurity of Boc-fapi-a is:

[0007]

[0008] Through literature search and data investigation, it was found that there is currently no public method for detecting the content of optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a). Summary of the invention

[0009] The purpose of the present invention is to solve the above-mentioned problems and to provide a detection method with high sensitivity and good separation effect for detecting the optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a) for the R-configuration optical isomer impurities generated during the synthesis process.

[0010] To achieve the above purpose, the technical solution provided by the present invention is:

[0011] A method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, using high performance liquid chromatography, the determination method is as follows:

[0012] A chromatographic column with amylose-tris (3, 5-xylylcarbamate) as filler was used;

[0013] The mobile phase was hexane-ethanol-trifluoroacetic acid in a volume ratio of 700-750: 250-300: 1;

[0014] Preparation of separation solution: Take tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate and tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate optical isomers, dissolve and dilute with solvent;

[0015] Preparation of test solution: Take tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, weigh accurately, dissolve with solvent and dilute quantitatively;

[0016] After the prepared solution was injected, the content of the optical isomer of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate was calculated by the peak area normalization method.

[0017] In order to optimize the above technical solutions, the specific limitations adopted also include:

[0018] A chromatographic column with a size of 250 mm × 4.6 mm and a size of 5 μm and a filler of amylose-tris (3, 5-xylylcarbamate) was used.

[0019] Preferably, the chromatographic column is CHIRALPAK AD-H, 250 mm×4.6 mm, 5 μm.

[0020] Preferably, the ratio of n-hexane-ethanol-trifluoroacetic acid in the mobile phase is 700:300:1.

[0021] After sample injection, the running time of each HPLC test is 95min-105min.

[0022] The solvent for preparing the separation solution and the test solution is anhydrous ethanol or anhydrous methanol.

[0023] In the separation solution, the concentration ranges of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate and tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate optical isomers were 0.5 mg / ml-1.0 mg / ml, respectively.

[0024] In the test solution, the concentration of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate ranged from 9 mg / ml to 11 mg / ml.

[0025] Detection wavelength: 205nm±5nm; column temperature: 25℃-35℃.

[0026] Flow rate: 0.7-0.9ml / min; injection volume: 5μl-20μl.

[0027] The beneficial technical effects of the present invention are:

[0028] The invention provides a method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a), and adopts high performance liquid chromatography to achieve quality control of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate.

[0029] The R-configuration optical isomer of Boc-fapi-a is similar in structure to Boc-Fapi-a, and is difficult to measure, and suitable high-performance liquid chromatography measurement conditions are difficult to obtain. The present invention fills the current technical gap of the lack of high-performance liquid chromatography detection of the substance, and provides a way to improve the quality of the final product of FAP targeted drugs by controlling the content of optical isomer impurities in tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a).

[0030] The method of the present invention is easy to operate, has good specificity, detection limit and quantification limit, linearity and precision, and the solution is stable, and can be used for the detection of the content of optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the separation solution chromatogram in Comparative Example 1.

[0032] Figure 2 This is the separation solution chromatogram in Comparative Example 2.

[0033] Figure 3 This is the chromatogram of the 2% spiked test solution in Comparative Example 3.

[0034] Figure 4 This is the UV spectrum of the R-configuration optical isomer of Boc-fapi-a in Example 7.

[0035] Figure 5 The chromatograms are of 2% spiked test solution in Example 1 and Example 7.

[0036] Figure 6 This is the blank solvent chromatogram in Example 7.

[0037] Figure 7 This is a comparison chart of the specificity in Example 2.

[0038] Figure 8 It is the detection limit chromatogram in Example 3.

[0039] Fig. 9 It is the quantification limit chromatogram in Example 3.

[0040] Fig.10 It is the linear graph in Example 4.

[0041] Fig.11 This is an overlapping diagram of the precision test in Example 5.

[0042] Fig.12 This is an overlapping diagram of the room temperature light-proof stability test of the test sample solution in Example 6.

[0043] Fig.13 This is an overlapping diagram of the room temperature light stability test of the test solution in Example 6.

[0044] Fig.14 This is the chromatogram of the 2% spiked test solution in Example 8. DETAILED DESCRIPTION

[0045] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0046] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0047] The Boc-Fapi-a isomers and Boc-Fapi-a optical isomers marked in the drawings of the present invention refer to the R-configuration optical isomers of Boc-fapi-a.

[0048] Boc-Fapi-a Application Examples

[0049] With Boc-Fapi-a (S configuration), 7-aminoquinoline-4-carboxylic acid, H7ND-b and HYNIC-NHS as the main raw materials, the S configuration final product is generated through four steps of de-Boc, amidation, dimerization and amidation, with a specific rotation of -103.3°:

[0050]

[0051] The R-configuration optical isomer of Boc-fapi-a, 7-aminoquinoline-4-carboxylic acid, H7ND-b and HYNIC-NHS were used as the main raw materials. The R-configuration final product was generated through four steps of de-Boc, amidation, dimerization and amidation, with a specific rotation of 95.1°:

[0052]

[0053] The above-mentioned S-configuration final product is targeted, while the R-configuration final product is not targeted.

[0054] Example 1 HPLC detection method

[0055] A method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate comprises the following steps: respectively taking a separation solution and a test solution for injection, performing detection according to high performance liquid chromatography conditions, and recording a chromatogram;

[0056] The chromatographic conditions were as follows: a column filled with amylose-tris (3, 5-xylylcarbamate); a mobile phase of n-hexane-ethanol-trifluoroacetic acid (700:300:1); and an isocratic elution.

[0057] Chromatographic conditions also include:

[0058] Detector: UV detector;

[0059] Flow rate: 0.8ml / min;

[0060] Column temperature: 30°C;

[0061] Detection wavelength: 205nm;

[0062] Injection volume: 5 μl;

[0063] Running time: 100 minutes;

[0064] Chromatographic column selection: CHIRALPAK AD-H, 250 mm × 4.6 mm, 5 μm;

[0065] Mobile phase: n-hexane-ethanol-trifluoroacetic acid (700:300:1) (ml / ml);

[0066] The separation solution is prepared by the following process: take appropriate amounts of the R-configuration optical isomer of Boc-fapi-a and Boc-Fapi-a, dissolve and dilute with anhydrous ethanol to make solutions containing 0.5 mg of each in each ml, shake well, and obtain the solution.

[0067] The test solution is prepared by the following process: take an appropriate amount of Boc-Fapi-a, accurately weigh it, dissolve it in anhydrous ethanol and quantitatively dilute it to make a solution containing about 10 mg per 1 ml, shake it well, and the solution is obtained.

[0068] The 2% spiked test solution was prepared by the following process: appropriate amounts of the R-configuration optical isomer of Boc-fapi-a and Boc-Fapi-a were dissolved and diluted with solvent to prepare solutions containing 0.2 mg and 10 mg, respectively, per 1 ml.

[0069] After injecting the 2% spiked test solution, the results are shown in Figure 5 The peak shape of the 2% spiked test solution is better. The theoretical plate number of the R-configuration optical isomer peak of Boc-fapi-a is 18531, and the theoretical plate number of the Boc-Fapi-a peak is 2638. The separation degree between the two is 1.144, achieving baseline separation.

[0070] Example 2 Specificity Experiment

[0071] Blank solvent: anhydrous ethanol.

[0072] Separation solution: prepared as in Example 1.

[0073] Test solution: Prepare the same as in Example 1.

[0074] The determination conditions were the same as those in Example 1. 5 μl of blank solvent, test solution and separation solution were accurately measured and injected into the liquid chromatograph to record the chromatogram.

[0075] Results: The blank solvent had no interference at the retention time of the Boc-Fapi-a peak and the R-configuration optical isomer peak. In the separation solution, the separation degree between the isomer and the main peak was 2.7, which was greater than 2.5, and the specificity met the requirements. The specific results are shown in Table 1. Figure 7 .

[0076] Table 1 Specificity test results

[0077]

[0078] Example 3 Detection limit and quantification limit test

[0079] Preparation of detection limit and quantification limit solutions: Accurately weigh an appropriate amount of Boc-Fapi-a, dissolve it in anhydrous ethanol and inject 5 μl of it after gradual dilution. The quantification limit is established according to the signal-to-noise ratio S / N ≥ 10, and the detection limit is established according to the signal-to-noise ratio S / N ≥ 3.

[0080] The determination conditions were the same as those in Example 1. 5 μl of the detection limit solution and the quantification limit solution were accurately measured and injected into the liquid chromatograph respectively, and the chromatograms were recorded.

[0081] Results: The detection limit of Boc-Fapi-a was 0.02475 mg / ml, equivalent to 0.2% of the test sample concentration, and the S / N value was 6, all greater than 3; the quantification limit was 0.0495 mg / ml, equivalent to 0.5% of the test sample concentration, not greater than 50% of the limit. The S / N value was between 11 and 13, all greater than 10, and the quantitative limit peak area RSD was 3.6% (n=6), less than 10.0%, and both the detection limit and the quantification limit met the requirements. The specific results are shown in Table 2 and Figure 8 , Fig. 9 .

[0082] Table 2 Results of determination of limit of quantification and limit of detection of Boc-Fapi-a

[0083]

[0084] Example 4 Linearity Test

[0085] Preparation of linear solution: Take an appropriate amount of Boc-Fapi-a, add anhydrous ethanol to dissolve and dilute to make a stock solution containing approximately 19.93 mg of Boc-Fapi-a per 1 ml. Accurately pipette an appropriate amount of the stock solution, add solvent to gradually dilute to make solutions containing 0.05 mg, 0.20 mg, 0.50 mg, 4.98 mg, 9.97 mg, and 14.95 mg of Boc-Fapi-a per 1 ml as linear solutions.

[0086] The determination conditions were the same as those in Example 1. 5 μl of each linear solution was accurately measured and injected into the liquid chromatograph to record the chromatogram. The Boc-Fapi-a peak area was recorded, with the injection concentration C (mg / ml) as the abscissa and the peak area A as the ordinate. The regression equation and correlation coefficient were calculated using the least squares method. The specific results are shown in Tables 3 and Fig.10 When the concentration of Boc-Fapi-a was in the range of 0.05~14.95mg / mL (0.5%~1149.5%), the linear regression equation was y=4×10 6 C+875681, correlation coefficient r is 0.998; the ratio of the Y-axis intercept to the 100% limit response value is 2.1%, and the linear relationship is good.

[0087] Table 3 Boc-Fapi-a linear relationship results

[0088]

[0089] Example 5 Precision Test

[0090] 2% spiked test solution: Prepare the same solution as in Example 1. Prepare 6 portions in parallel.

[0091] The determination conditions were the same as in Example 1. 5 μl of each spiked test solution was accurately measured and injected into the liquid chromatograph to record the chromatogram. The content of optical isomers in the 2% spiked test solution was determined by the peak area normalization method. The results are shown in Table 4 and Fig.11 As shown, the precision test results are good.

[0092] Table 4 Precision test results

[0093]

[0094] Example 6 Solution stability test

[0095] After the test solution was placed at room temperature in the dark for 0 hours, 5 hours, 10 hours and 15 hours, and placed under light conditions for 0 hours, 2 hours, 4 hours and 6 hours, the sample was injected and the chromatogram was recorded to investigate the stability of the test solution. Compared with 0 hours, the absolute difference in the isomer content in the test solution was calculated. The results are shown in Table 5 and Fig.12 , Fig.13 The results show that the test solution has good stability when placed at room temperature in the dark for 15 hours, and has good stability when placed under light for 6 hours.

[0096] Table 5 Solution stability results

[0097]

[0098] Comparative Example 1

[0099] The mobile phase was ethanol-trifluoroacetic acid (100:0.1), and the chromatographic column was filled with amylose-tris (3, 5-xylylcarbamate): CHIRALPAK AD-H, 150 mm × 4.6 mm, 5 μm, the detection wavelength was 220 nm, the running time was 30 minutes, the flow rate was 0.8 ml / min, the column temperature was 30 °C, and the injection volume was 5 μl.

[0100] Separation solution: Take appropriate amount of R-configuration optical isomer of Boc-fapi-a and Boc-Fapi-a, dissolve and dilute with anhydrous ethanol to make a solution containing 0.5 mg of each in each ml, shake well, and obtain.

[0101] Test results are shown in Figure 1 , the separation degree of the solution optical isomers completely overlaps with the main peak.

[0102] Comparative Example 2

[0103] Based on Comparative Example 1, the mobile phase was changed to n-hexane-ethanol-trifluoroacetic acid with a volume ratio of 400:600:1, and the running time was 45 minutes.

[0104] Test results are shown in Figure 2 , the resolution of the optical isomers in the solution still cannot be baseline separated.

[0105] After multiple adjustments of the mobile phase, the R-configuration optical isomers of Boc-fapi-a were difficult to separate from Boc-fapi-a.

[0106] Comparative Example 3

[0107] The mobile phase is n-hexane-ethanol-trifluoroacetic acid in a volume ratio of 700:300:1. The chromatographic column is a 250 mm long chromatographic column filled with amylose-tris (3, 5-xylylcarbamate): CHIRALPAK AD-H, 250 mm × 4.6 mm, 5 μm. The detection wavelength is 217 nm. The running time is 60 minutes. Other conditions are the same as in Example 1.

[0108] Test results are shown in Figure 3 , the peak response of the optical isomers of the 2% spiked test solution is large, and the detection sensitivity meets the requirements. There is a bulge at 30 minutes, which is considered to be the residual impurities from the previous injection. Example 7

[0109] On the basis of Comparative Example 3, the running time was extended to 100 min, and 2% spiked test solution and blank solvent were respectively injected. The determination conditions of this example were the same as those of Example 1.

[0110] Test results are shown in Figure 4 , Figure 5 and Figure 6 , there is an impurity peak at about 90 min in the 2% spiked test solution, and the bulge disappears in the blank solvent of the next needle. Example 8

[0111] The mobile phase used was n-hexane-ethanol-trifluoroacetic acid in a volume ratio of 750:250:1. Other conditions were the same as in Example 1. A 2% spiked test solution was injected.

[0112] Test results are shown in Fig.14 The peak shape is better. The theoretical plate number of the R-configuration optical isomer peak of Boc-fapi-a is 18388, and the theoretical plate number of the Boc-Fapi-a peak is 2666. The separation degree between the two is 1.162, achieving baseline separation, and the impurity peak bulge can also be eluted before 100 minutes.

[0113] In summary, there is currently no suitable HPLC method for detecting the content of optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a). The R-configuration optical isomer of Boc-fapi-a will generate non-targeted optical isomer impurities in the subsequent reaction route, affecting the product quality, and the content of the optical isomers in Boc-Fapi-a needs to be controlled. However, the R-configuration optical isomer of Boc-fapi-a is similar in structure to Boc-Fapi-a, making it difficult to determine, and suitable HPLC determination conditions are difficult to obtain.

[0114] The high performance liquid chromatography content determination method proposed in the present invention is easy to operate, has good specificity, detection limit and quantification limit, linearity and precision, and the solution is stable. The peak area normalization method is used to calculate the optical isomer content, and the quality of Boc-Fapi-a can be controlled to meet the detection requirements.

[0115] The above are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention, may make any simple modifications, equivalent substitutions and improvements to the above embodiments, which are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, characterized in that: The high performance liquid chromatography method is as follows: A chromatographic column with amylose-tris (3, 5-xylylcarbamate) as filler was used; The mobile phase was hexane-ethanol-trifluoroacetic acid in a volume ratio of 700-750: 250-300: 1; Preparation of separation solution: Take tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate and tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate optical isomers, dissolve and dilute with solvent; Preparation of test solution: Take tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, weigh accurately, dissolve with solvent and dilute quantitatively; After the prepared solution was injected, the content of the optical isomer of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate was calculated by the peak area normalization method.

2. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: A chromatographic column with a size of 250 mm × 4.6 mm and a size of 5 μm and a filler of amylose-tris (3, 5-xylylcarbamate) was used.

3. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 2, characterized in that: The chromatographic column is CHIRALPAK AD-H, 250 mm×4.6 mm, 5 μm.

4. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: The ratio of n-hexane-ethanol-trifluoroacetic acid in the mobile phase was 700:300:

1.

5. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: After sample injection, the running time of each HPLC test is 95min-105min.

6. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: The solvent for preparing the separation solution and the test solution is anhydrous ethanol or anhydrous methanol.

7. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: In the separation solution, the concentration ranges of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate and tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate optical isomers were 0.5 mg / ml-1.0 mg / ml, respectively.

8. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: In the test solution, the concentration of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate ranged from 9 mg / ml to 11 mg / ml.

9. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: Detection wavelength: 205nm±5nm; column temperature: 25℃-35℃.

10. The method for detecting optical isomers of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: flow rate: 0.7-0.9 ml / min; injection volume: 5 μl-20 μl.

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