Detection method of atozepam intermediate and enantiomer thereof

Abstract

The invention discloses a method for detecting an atozepam intermediate and an enantiomer of the atozepam intermediate, which adopts a high performance liquid chromatography, and the chromatographic conditions are as follows: cellulose derivative bonded silica gel is used as a filler, 0.1% formic acid water-acetonitrile-tetrahydrofuran is used as a mobile phase, and the volume ratio of the 0.1% formic acid water to the acetonitrile to the tetrahydrofuran is (650-750): (175-245): (75-105); the detection wavelength is 283 + / -5nm; the flow velocity is 0.4 ml / min to 0.6 ml / min. By adopting the method, the atozepam intermediate and the enantiomer thereof can be effectively separated and detected, and can be accurately quantified. The method has the advantages of strong specificity, good precision and high accuracy, and can rapidly and accurately determine the contents of the atozepam intermediate and the enantiomer thereof.

Description

Technical Field

[0001] The present invention relates to the field of drug analysis methods, and in particular to a method for detecting an atrogepan intermediate and its enantiomers. Background Art

[0002] Atrogepam intermediates (Formula 1) are important chemical raw materials used in the synthesis of atrogepam and ubagepam. The quality of enantiomeric impurities in these intermediates can affect the final quality of the drugs atrogepam and ubagepam. In recent years, requirements for chiral isomers in pharmaceuticals have become increasingly stringent, requiring not only limits but also accurate determination of their content.

[0003]

[0004] Currently, there is no literature report on the HPLC-UV method for determining atrogepan intermediates and their enantiomers. Therefore, it is necessary to establish a method that can quickly and accurately determine atrogepan intermediates and their enantiomers and their contents. Summary of the Invention

[0005] The present invention aims to establish a method for detecting an atrogepan intermediate and its enantiomers. This method utilizes high-performance liquid chromatography (HPLC) analysis to rapidly and accurately determine the content of the atrogepan intermediate and its enantiomers. This method fills a gap in the prior art.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solution is provided.

[0007] In one embodiment, the present invention provides a method for detecting an atrogepam intermediate and its enantiomers using high-performance liquid chromatography (HPLC). The chromatographic conditions include: a chiral chromatographic column filled with a cellulose derivative bonded silica gel; and a mobile phase of 0.1% formic acid in water, acetonitrile, and tetrahydrofuran. The atrogepam intermediate is represented by Formula 1, and the enantiomers of the atrogepam intermediate are represented by Formula 2.

[0008]

[0009] Preferably, in the detection method of the present invention, the volume ratio of 0.1% formic acid water, acetonitrile, and tetrahydrofuran in the mobile phase is (650-750): (175-245): (75-105). Preferably, the volume ratio of 0.1% formic acid water, acetonitrile, and tetrahydrofuran is 700:210:90.

[0010] In some embodiments, in the detection method of the present invention, the chiral chromatographic column is Daicel CHIRALPAK IG (4.6 mm×250 mm, 5 μm).

[0011] Furthermore, the chromatographic conditions of the detection method of the present invention also include: the detection wavelength is 283±5nm; the flow rate is 0.4ml / min to 0.6ml / min; the injection volume is 1μl to 20μl, preferably 3μl; and the column temperature is 35°C to 40°C.

[0012] In a specific embodiment, the present invention provides a method for detecting an atrogepam intermediate and its enantiomers, wherein the method employs high performance liquid chromatography, and the chromatographic conditions include: Chromatographic column: Chiral chromatographic column with cellulose derivative bonded silica gel as filler, column filler particle size is 5μm, column length is 250mm; Mobile phase: 0.1% formic acid water-acetonitrile-tetrahydrofuran was used as the mobile phase, and the volume ratio of 0.1% formic acid water, acetonitrile and tetrahydrofuran was (650-750): (175-245): (75-105); Flow rate: 0.4ml / min~0.6ml / min; Column temperature: 35℃~40℃; Detection wavelength: 283±5nm; Injection volume: 1μl~20μl.

[0013] Optimal chromatographic conditions were as follows: a chromatographic column filled with a cellulose derivative bonded silica gel (Daicel CHIRALPAKIG (4.6 mm × 250 mm, 5 μm); a mobile phase of 0.1% formic acid in water-acetonitrile-tetrahydrofuran (70:21:9); a flow rate of 0.4–0.6 ml / min; a detection wavelength of 283 nm; and an injection volume of 3 μl.

[0014] Preparation of sample solution: Solvent: acetonitrile aqueous solution, where acetonitrile:water = 400:600 (v / v); Test solution: Take about 20 mg of atrogepan intermediate, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with solvent and dilute it to the scale, shake it well, and you have it. Control solution: Accurately pipette 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0015] Determination method: Take the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the peak area according to the main component self-reference method with the addition of correction factors:

[0016] Where: A std - Peak area of main peak of control solution; A sam —Peak area of target substance in test solution; V—dilution multiple of control solution; F—impurity correction factor.

[0017] Advantages and Effects: The detection method of the present invention utilizes high-performance liquid chromatography (HPLC) analysis. The selected chromatographic conditions achieve effective separation of the atrogepam intermediate and its enantiomers on the chromatographic column. The retention times of the atrogepam intermediate and its enantiomers are approximately 9.542 minutes and 11.172 minutes, respectively, resulting in a short analysis time and excellent peak symmetry. The method of the present invention exhibits high recovery, with an average recovery of 10.05%, good reproducibility, and an RSD of 0.8% across six replicates. It also demonstrates high sensitivity, with a limit of quantification of 0.25 µg / ml. The solution is stable for up to 16 hours. The method of the present invention can rapidly and accurately determine the content of the atrogepam intermediate and its enantiomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the liquid chromatogram of the spiked test sample of Scheme 6 of Example 1; Figure 2 This is a working curve diagram of the atrogepam intermediate reference substance and the atrogepam intermediate enantiomer reference substance in Example 3. DETAILED DESCRIPTION

[0019] The present invention will be further described below by way of examples, but the examples do not limit the scope of protection of the present invention.

[0020] Main instruments and chromatographic conditions: Agilent 1200 series high performance liquid chromatograph; equipped with VWD UV detector, autosampler, and Empower chromatography workstation.

[0021] Chromatographic column: A chromatographic column filled with cellulose derivative bonded silica gel.

[0022] Reagents and drugs: acetonitrile (HPLC grade), tetrahydrofuran (HPLC grade), atrogepam intermediate, atrogepam intermediate and its enantiomer reference substance.

[0023] Example 1 Selection of chromatographic conditions

[0024] 1.1 Wavelength selection Approximately 20 mg of the atrogepan intermediate was accurately weighed and placed in a 20 mL volumetric flask. Dissolved in a solvent (acetonitrile-water solution, acetonitrile:water = 400:600, v / v) and diluted to the mark. Shake well to prepare this blank test solution. Accurately measure 1 mL and place it in a 20 mL volumetric flask. Diluted to the mark with a mobile phase (0.1% formic acid in water:acetonitrile:tetrahydrofuran = 700:210:90) and shake well to prepare the standard solution. Using the mobile phase as the blank calibration solution, scan the sample at 190 nm to 400 nm using UV-Vis spectrophotometry (Chinese Pharmacopoeia 2020 edition). The results showed that the standard solution had a maximum absorption peak at 283 nm. Therefore, the detection wavelength was determined to be 283 nm ± 5 nm.

[0025] 1.2 Screening test of the method This experiment screened the chromatographic method by adjusting the mobile phase ratio, type, flow rate, column temperature, and other factors. A cellulose derivative bonded silica gel column (Daicel CHIRALPAK IG, 4.6 mm × 250 mm, 5 μm) was used; the solvent was acetonitrile-water solution (acetonitrile:water ratio of 400:600 (v / v)). Other screening conditions are shown in Tables 1 and 2.

[0026] Table 1 Screening scheme of the method

[0027] Table 2 Screening scheme of the method

[0028] Preparation of reference stock solution: Take about 20 mg of the enantiomer of the intermediate of atrogepan, accurately weigh it, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 2 ml, place it in a 10 ml volumetric flask, and dilute to the scale with solvent.

[0029] Spiked test solution: Take about 20 mg of atrogepan intermediate, accurately weigh it, place it in a 20 ml volumetric flask, add solvent and shake to dissolve, accurately add 1 ml of reference stock solution, dilute to the scale with solvent, shake well, and obtain.

[0030] Detection: Accurately measure a certain volume (see Tables 1 and 2) of the spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. Investigate the changes in the resolution of the enantiomers of the atrogepan intermediate and the adjacent peaks. The results are shown in Table 3, and the chromatogram obtained by Scheme 6 is shown in Figure 1 .

[0031] Table 3 Results of screening tests of the methods

[0032] The results showed that under the chromatographic conditions of Schemes 1 to 5, the resolution between the enantiomers of the atrogepam intermediate and adjacent peaks was no greater than 1.5, posing a high risk for content determination and therefore should not be used. Schemes 6 to 12 all achieved resolution greater than 1.5, making them suitable for content determination of the enantiomers of the atrogepam intermediate. The retention time of the atrogepam intermediate was 9.524 minutes, while the retention time of the enantiomers of the atrogepam intermediate was 11.172 minutes (relative retention time: 1.17).

[0033] Example 2 Solution Stability

[0034] Solvent: acetonitrile-water solution, acetonitrile:water 400:600 (v / v); Preparation of reference solution: Take about 20 mg of the enantiomer of the intermediate of atrogepan, weigh accurately, place in a 20 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, and obtain; Preparation of reference solution: Accurately measure 1 ml of reference solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0035] Testing: 3 μl of the control solution was accurately measured at 0, 4, 12, and 16 hours, and the test was performed according to the chromatographic conditions of Scheme 6 of Example 1. The chromatogram was recorded. Separately, 3 μl of the reference solution was accurately measured at 0, 4, 12, and 16 hours, and the test was performed according to the chromatographic conditions of Scheme 6 of Example 1. The chromatogram was recorded. The results are shown in Table 4.

[0036] Table 4 Solution stability results

[0037] The results obtained using Scheme 6 showed that the reference solution and the control solution were stable over time within 16 hours.

[0038] Example 3 Preparation of standard curve

[0039] Chromatographic conditions: the same as those described in Scheme 6 of Example 1.

[0040] Take approximately 20 mg of the atrogepam intermediate and approximately 20 mg of the atrogepam intermediate enantiomer, accurately weigh them, place them in the same 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain linear stock solutions (both concentrations are approximately 0.2 mg / ml).

[0041] Prepare linearity solutions according to the table below.

[0042] Table 5 Preparation of linear solution

[0043] Note: * refers to 1 mg / ml; ** refers to measuring 1 ml of sample LN3.

[0044] Take the above linear solution and inject it, and plot the peak area and concentration ( Figure 2 ).

[0045] The atrogepant intermediate was linear in the range of 0.5 μg / ml to 19.8 μg / ml, with the linear regression equation being: y = 16574x – 15.0 (y is the peak area, x is the concentration), and the correlation coefficient r = 0.9954.

[0046] The enantiomers of the intermediate atrogepam were linear in the range of 0.5 μg / ml to 20.3 μg / ml, and the linear regression equation was: y = 16589x + 164.8 (y is the peak area, x is the concentration), correlation coefficient r = 0.9951, correction factor 1.00.

[0047] When the signal-to-noise ratio was 10:1, the limit of quantification of (R)-1-tert-butyloxycarbonyl-3-hydroxypiperidine was 0.25 μg / ml.

[0048] The results showed that under the chromatographic conditions of Scheme 6, the linearity was good and the limit of quantification fully met the requirements for the accurate determination of the enantiomers of the atrogepan intermediate.

[0049] Example 4 Recovery Test

[0050] Chromatographic conditions: the same as those described in Scheme 6 in Example 1.

[0051] Solution preparation: Atrogepan intermediate enantiomer stock solution: the same as the reference substance stock solution in Example 1.

[0052] Blank test solution: Take about 20 mg of atrogepan intermediate, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with solvent and dilute it to the scale, and shake it well (prepare 3 copies in parallel).

[0053] 100% limit concentration level solution: Take about 20 mg of atrogepam intermediate, weigh accurately, and place it in a 20 ml volumetric flask. Accurately add 1 ml of the atrogepam intermediate enantiomer stock solution, dissolve and dilute to the scale with solvent, and shake well (prepare 6 copies in parallel).

[0054] Control solution: Accurately measure 1 ml of the three blank test solution and 1 ml of the six 100% limit concentration aqueous solutions, place them in 100 ml volumetric flasks respectively, dilute to the scale with solvent, and shake well to obtain 9 control solutions.

[0055] Detection: Accurately measure 3 μl of each of the three blank test solutions, six 100% limit concentration level solutions, and nine control solutions, and inject them into a high performance liquid chromatograph. Record the chromatogram and calculate the recovery of the six 100% limit concentration level solutions by peak area using the principal component self-reference method with a correction factor. The recovery rates were between 98.4% and 101.6%. The results are shown in Table 6.

[0056] Table 6 Sample recovery determination results

[0057] The results showed that the average recovery rate was 100.5% and the RSD was 0.8%, indicating that the method had good recovery rate and high accuracy.

[0058] Example 5 Repeatability Test

[0059] Chromatographic conditions: the same as those described in Scheme 6 in Example 1.

[0060] Solution preparation: Blank test solution: Same as Example 4; Control solution: Accurately measure 1 ml of blank test solution and place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the control solution.

[0061] Inject blank test solution and control solution, and record the chromatograms. Calculate the enantiomer content of the atrogepan intermediate in the sample using the principal component self-reference method with a correction factor. The results are shown in Table 7.

[0062] Table 7 Sample repeatability test results

[0063] The results showed that the RSD of the repeatability test was 1.8%, which was less than 2.0%, and the repeatability of this method was good.

[0064] Example 6 Determination of samples

[0065] Chromatographic conditions: the same as those described in Scheme 6 in Example 1.

[0066] Take about 20 mg of atrogepan intermediate, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with solvent and dilute it to the scale, shake it well, and use it as the test solution; accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with solvent, and use it as the control solution.

[0067] Inject the test solution and the control solution and record the chromatograms. Calculate the enantiomer content of the atrogepan intermediate in the sample using the principal component self-reference method with a correction factor. The results are shown in Table 8.

[0068] Table 8 Determination results of (R)-1-tert-butyloxycarbonyl-3-hydroxypiperidine content in samples

[0069] The results showed that this method has strong specificity, high sensitivity and good precision. The peak shape of the enantiomers of the atrogepam intermediate is well symmetrical, and it can accurately and quickly determine the content of the enantiomers of the atrogepam intermediate.

Claims

1. A method for detecting an atrogepant intermediate and its enantiomers, the method employing high performance liquid chromatography, wherein the chromatographic conditions comprise: a chiral chromatographic column using a cellulose derivative bonded silica gel as a filler; and a mobile phase of 0.1% formic acid in water-acetonitrile-tetrahydrofuran. in, The atrogepam intermediate is shown in Formula 1, and the enantiomer of the atrogepam intermediate is shown in Formula 2.

2. The detection method according to claim 1, wherein the volume ratio of the mobile phase, 0.1% formic acid water, acetonitrile and tetrahydrofuran is (650-750): (175-245): (75-105).

3. The detection method according to claim 2, wherein the volume ratio of the mobile phase, 0.1% formic acid water, acetonitrile and tetrahydrofuran is 700:210:

90.

4. The detection method according to claim 1, wherein the chiral chromatographic column is Daicel CHIRALPAK IG.

5. The detection method according to claim 1, wherein the chromatographic conditions include a detection wavelength of 283±5 nm.

6. The detection method according to claim 1, wherein the chromatographic condition has a flow rate of 0.4 ml / min to 0.6 ml / min.

7. The detection method according to claim 1, wherein the chromatographic conditions are such that the injection volume is 1 μl to 20 μl. The detection method according to claim 7 , wherein the injection volume is 3 μl.

9. The detection method according to claim 1, wherein the column temperature of the chromatography condition is 35°C to 40°C.

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