A HPLC-UV detection method for MTV enantiomers

Abstract

The invention discloses a HPLC-UV detection method for MTV enantiomers. The method comprises the following steps: taking an MTV sample and dissolving it in a mobile phase diluent, and simultaneously taking an enantiomer and dissolving it in the mobile phase diluent to prepare a system suitability solution containing 2 mg of the MTV sample and 0.003 mg of the enantiomer per 1 mL of the diluent; taking an MTV sample and dissolving it in the diluent to prepare a sample solution containing 2 mg of the MTV sample per 1 mL of the diluent; taking 10 μL of the above solution and injecting it into a liquid chromatograph, recording a chromatogram, and detecting the percentage area of ​​the enantiomer of the MTV sample; the method adopts a normal phase coated chiral chromatographic column of a polysaccharide derivative, a n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1, v / v / v) chromatographic system, and isocratic elution, so as to effectively separate and detect the percentage area of ​​the enantiomer, thereby ensuring smooth production in a production workshop.

Description

Technical Field

[0001] The present invention belongs to a high performance liquid chromatography (HPLC) method, in particular to a method for detecting N-[N-methyl-N-[[(2-isopropyl-4-thiazolyl)methyl]amino]carbonyl]-L-valine (MTV) enantiomers by using HPLC. Background Art

[0002] N-[N-methyl-N-[[(2-isopropyl-4-thiazolyl)methyl]amino]carbonyl]-L-valine is called N-[N-methyl-N-[[(2-isopropyl-4-thiazolyl)methyl]amino]carbonyl]-L-valine in English, abbreviated as MTV, and its structural formula is The molecular formula is C 14 H 23 N3O3S can be used as an organic synthesis intermediate and a pharmaceutical intermediate in laboratory research and development and pharmaceutical chemical synthesis. It is also a key starting material for the synthesis of the raw material ritonavir. In the process of producing the target compound ritonavir, the content of the MTV enantiomer needs to be controlled.

[0003] MTV is a key starting material for ritonavir, and its enantiomer content plays a key role in the quality control of the enantiomer content of the raw material ritonavir. Therefore, quality control of the enantiomer content of MTV is of great significance to the quality control in the production process of ritonavir. Summary of the invention

[0004] The object of the present invention is to provide a HPLC-UV method for detecting the enantiomer content of MTV, a key starting material of ritonavir.

[0005] The technical solution adopted by the patent of the present invention to solve its technical problem is: an HPLC-UV detection method for MTV enantiomers, the detection conditions are: chromatographic column: DAICEL CHIRALPAK AD-H, 250mm*4.6mm, 5μm, column temperature is 30°C; mobile phase: a mixed solution of n-hexane: isopropanol: trifluoroacetic acid = 90:10:0.1 (v / v / v); flow rate is 0.8mL / min; elution program: gradient elution and isocratic elution, running time is 30min; detection wavelength: 240nm is the maximum absorption wavelength, which is used for quantitative detection of enantiomers; comprising the following steps

[0006] (1), take the structural formula as The MTV sample was dissolved in the mobile phase mixed solution as a diluent to prepare a sample solution containing 2 mg of MTV sample per 1 mL of diluent;

[0007] (2) Dissolve the MTV sample and the MTV enantiomers separately in a mobile phase mixed solution as a diluent to prepare a system suitability solution containing 2 mg of MTV sample and 0.003 mg of isomers per 1 mL of diluent;

[0008] (3) Take 10 μL of the solution from step (1) and (2) respectively and inject it into a liquid chromatograph, record the chromatogram, and detect the area percentage of the enantiomer of N-[N-methyl-N-[[(2-isopropyl-4-thiazolyl)methyl]amino]carbonyl]-L-valine.

[0009] Furthermore, the liquid chromatograph was Thermo Ultimate 3000 / VC, with a DAD detector.

[0010] The invention has the following beneficial effects: the invention selects a mobile phase of n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1, v / v / v) to dissolve an MTV sample, thereby avoiding a solvent effect, reducing the possibility of the MTV sample being precipitated in the system due to the mismatch between the diluent and the mobile phase, and the sample solution has certain stability; the invention selects DAICEL CHIRALPAK AD-H (250 mm*4.6 mm, 5 μm), a conventional chromatographic column, a column temperature of 30°C, which is suitable for all HPLC instruments; in a n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1, v / v / v) system, the MTV sample and its enantiomer are completely baseline separated, and other known impurities in the MTV sample do not interfere with the detection of the enantiomer, and the specificity is good and the selectivity is high; the method of the invention can simply and quickly determine the purity, the content of related substances and the size of residual solvents of the MTV sample, and solves the problem of reaction monitoring of the MTV sample in the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the HPLC chart of Comparative Example 1 of the present invention, in which the mobile phase is n-hexane-ethanol-trifluoroacetic acid (90:10:0.1);

[0012] Figure 2 This is the HPLC chart of the mobile phase n-hexane-ethanol-trifluoroacetic acid (95:5:0.1) of Comparative Example 2 of the present invention;

[0013] Figure 3 This is a graph of the mobile phase of Comparative Example 3 of the present invention, n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1), column temperature 35°C;

[0014] Figure 4 It is a specific HPLC diagram of the embodiment of the present invention. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0016] The applicant found that using DAICEL CHIRALPAK AD-H (250mm*4.6mm, 5μm), n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1, v / v / v)) as the mobile phase system and as the sample dissolving solvent at the same time, eluting for 30min, the MTV sample and its enantiomeric impurities can be effectively separated, with strong specificity, quantitative limit S / N>10, and good linearity, so that the size of the sample enantiomer can be accurately determined. Therefore, a HPLC-UV detection method for the enantiomers of MTV sample, a key starting material of ritonavir, is provided.

[0017] The HPLC-UV detection method of the key starting material N-[N-methyl-N-[[(2-isopropyl-4-thiazolyl)methyl]amino]carbonyl]-L-valine enantiomer of the present invention can be achieved by the following method:

[0018] (1) Take an appropriate amount of MTV sample and dissolve the sample in a diluent (n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1, v / v / v) to prepare a sample solution containing about 2 mg of MTV sample per 1 mL of diluent.

[0019] (2) Dissolve the MTV sample and the MTV enantiomer in a diluent to prepare a system suitability solution containing 2 mg of the MTV sample and 0.003 mg of the MTV enantiomer per 1 mL of the diluent.

[0020] (3) The solutions of step (1) and (2) were respectively injected into a liquid chromatograph at an injection volume of 10 μL to complete sample detection, record the chromatogram, and thereby detect the percentage area of ​​the MTV enantiomer.

[0021] The detection conditions are:

[0022] High performance liquid chromatograph: Thermo Ultimate 3000, DAD detector.

[0023] Chromatographic column: DAICEL CHIRALPAK AD-H (250 mm × 4.6 mm, 5 μm).

[0024] Mobile phase: n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1) (channel 1).

[0025] Elution time: 30min

[0026] Detection wavelength: 240nm.

[0027] Flow rate: 0.8mL / min.

[0028] Column temperature: 30℃.

[0029] Injection volume: 10 μL.

[0030] The pre-verification investigation was carried out according to the above experimental conditions, and the specific implementation is as follows:

[0031] (1) The specificity test is shown in the following table.

[0032]

[0033] See attached Figure 4 , from bottom to top are blank solution, resolution solution, reference solution, sample solution, spiked test solution, and positioning solution. Peak 2 in the figure is the enantiomer peak, which is completely baseline separated from the adjacent peaks.

[0034] As a result, blank and other known impurities had no interference at the peak positions of MTV and enantiomers; the peak positions of MTV and enantiomers in the reference solution, sample solution, and spiked test solution were consistent with those in the resolution solution; the resolution of MTV and enantiomers in the resolution solution was greater than 1.5, and the purity of the main peak was greater than 990. All results met the specificity acceptance criteria.

[0035] (2) LOQ investigation is shown in the following table.

[0036]

[0037] As a result, the quantitative limit and detection limit concentration levels of the main component MTV (No. BL1024-S03) and the enantiomer (No. BL1024-S03-IM04) were lower than the limit level (0.15%), the signal-to-noise ratio of the detection limit solution of the two components was not less than 3, and the signal-to-noise ratio of the quantitative limit solution was not less than 10; the RSD of the retention time of the two components was ≤2.0%, and the RSD of the peak area was ≤10.0% when the quantitative limit solution was repeated 6 times. The results all met the acceptance criteria of the detection limit and the quantitative limit.

[0038] (3) Linearity and range investigation.

[0039] The linear results (limit levels) of the principal component MTV (No. BL1024-S03) are shown in the following table.

[0040]

[0041] The linear results (content level) of the main component MTV (BL1024-S03) are shown in the following table.

[0042]

[0043] The linearity results (limit levels) of the enantiomers (No. BL1024-S03-IM04) are shown in the table below.

[0044]

[0045] The results showed that at the limit level, the linear correlation coefficient of the main component and the enantiomer was greater than 0.990, the response factor RSD was less than 10.0%, and the ratio of the y-axis intercept to the 100% horizontal peak area was less than 25% in the range of the quantitative limit to 200% of the limit concentration; for the content level, the linear correlation coefficient of the main component was greater than 0.990 in the range of 50% to 150%, the response factor RSD was less than 10.0%, the ratio of the y-axis intercept to the 100% horizontal peak area was less than 2.0%, and the ratio of the linear slope at the limit level was in the range of 0.9 to 1.1; in summary, the main component and the enantiomer had a good linear relationship.

[0046] (4) Repeatability study: Six spiked test solutions (same as system suitability solutions) were prepared in parallel. The RSD of the main component content was less than 2.0%, and the RSD of the enantiomer content was less than 10.0%. The repeatability was good.

[0047] (5) Solution stability study: The spiked test solution was subjected to a solution stability study. The results showed that the RSD of the main component and enantiomer content of the spiked test solution was less than 2.0% after the spiked test solution was placed at room temperature for 20 hours, indicating that the spiked test solution was stable at room temperature for 20 hours.

[0048] Comparative Example 1

[0049] Instruments and conditions:

[0050] High performance liquid chromatograph: Thermo Ultimate 3000, DAD detector.

[0051] Chromatographic column: DAICEL CHIRALPAK AD-H (250 mm × 4.6 mm, 5 μm).

[0052] Mobile phase: n-hexane-ethanol-trifluoroacetic acid (90:10:0.1) (channel 1).

[0053] Elution time: 30min.

[0054] Detection wavelength: full scan.

[0055] Flow rate: 1.0mL / min.

[0056] Column temperature: 30℃.

[0057] Injection volume: 10 μL.

[0058] Experimental steps: Take 40 mg of MTV solid and place it in a 20 mL volumetric flask. Take an appropriate amount of enantiomers and other known impurities to be controlled by MTV, add mobile phase to dissolve and dilute to the scale, shake well, and make a mixed solution containing 2 mg of sample, 10 μg of isomers and other known impurities in 1 ml, which is used as the separation solution for method development.

[0059] Take the above sample solution, perform HPLC analysis under the above conditions, and record the chromatogram. The results are shown in the attached Figure 1 , Figure 1 Peak No. 2 is the enantiomer peak, Peak No. 1 is its closest adjacent peak, and Peak No. 3 is the sample peak. It can be seen from the figure that under this condition, the enantiomer and its adjacent peaks do not achieve baseline separation.

[0060] Comparative Example 2

[0061] Instruments and conditions:

[0062] High performance liquid chromatograph: Thermo Ultimate 3000, DAD detector.

[0063] Chromatographic column: DAICEL CHIRALPAK AD-H (250 mm × 4.6 mm, 5 μm).

[0064] Mobile phase: n-hexane-ethanol-trifluoroacetic acid (95:5:0.1) (channel 1).

[0065] Elution time: 30min.

[0066] Detection wavelength: full scan.

[0067] Flow rate: 1.0mL / min.

[0068] Column temperature: 30℃.

[0069] Injection volume: 10 μL.

[0070] Experimental steps: Take 40 mg of MTV solid and place it in a 20 mL volumetric flask. Take an appropriate amount of enantiomers and other known impurities to be controlled by MTV, add mobile phase to dissolve and dilute to the scale, shake well, and make a mixed solution containing 2 mg of sample, 10 μg of isomers and other known impurities in 1 ml, which is used as the separation solution for method development.

[0071] Take the above sample solution, perform HPLC analysis under the above conditions, and record the chromatogram. The results are shown in the attached Figure 2 , Figure 1Peak No. 2 is the enantiomer peak, Peak No. 1 is its closest adjacent peak, and Peak No. 3 is the sample peak. It can be seen from the figure that under this condition, the enantiomer and its adjacent peaks still cannot achieve baseline separation, and the separation degree is compared with ratio 1, which is worse.

[0072] Comparative Example 3

[0073] Instruments and conditions:

[0074] High performance liquid chromatograph: Thermo Ultimate 3000, DAD detector.

[0075] Chromatographic column: DAICEL CHIRALPAK AD-H (250 mm × 4.6 mm, 5 μm).

[0076] Mobile phase: n-hexane-isopropanol-trifluoroacetic acid (90:10:0.1) (channel 1).

[0077] Elution time: 30min.

[0078] Detection wavelength: full scan.

[0079] Flow rate: 1.0mL / min.

[0080] Column temperature: 35°C.

[0081] Injection volume: 10 μL.

[0082] Experimental steps: Take 40 mg of MTV solid and place it in a 20 mL volumetric flask. Take an appropriate amount of enantiomers and other known impurities to be controlled by MTV, add mobile phase to dissolve and dilute to the scale, shake well, and make a mixed solution containing 2 mg of sample, 10 μg of isomers and other known impurities in 1 ml, which is used as the separation solution for method development.

[0083] Take the above sample solution, perform HPLC analysis under the above conditions, and record the chromatogram. The results are shown in the attached Figure 3 , Figure 1 Peak 1 is the sample peak, peak 2 is the closest adjacent peak of the enantiomer, and peak 3 is the enantiomer peak. It can be seen from the figure that under this condition, after the mobile phase is changed from ethanol to isopropanol, the peak order changes, and the comparison between the enantiomer and its adjacent peak is good in Example 1 and Example 2, but still needs further optimization.

[0084] The above embodiments are only illustrative of the principles and effects of the present invention, as well as some embodiments of its application. For those skilled in the art, several modifications and improvements may be made without departing from the creative concept of the present invention, and all of these belong to the protection scope of the present invention.

Claims

1. A HPLC-UV method for detecting MTV enantiomers, wherein the MTV is N-[N-methyl-N-[[(2-isopropyl-4-thiazolyl)methyl]amino]carbonyl]-L-valine, characterized in that: The detection conditions are Chromatographic column: DAICEL CHIRALPAK AD-H, 250mm*4.6mm, 5μm, column temperature 30℃; Mobile phase: a mixed solution of n-hexane: isopropanol: trifluoroacetic acid in a volume ratio of 90:10:0.1; flow rate: 0.8 mL / min; Elution procedure: isocratic elution for 30 min; Detection wavelength: 240nm; The following steps are included (1) Take an MTV sample and dissolve it in a mobile phase mixed solution as a diluent to prepare a sample solution containing 2 mg of MTV sample per 1 mL of diluent; (2) Take the MTV sample and the MTV enantiomer separately and dissolve them in the mobile phase mixed solution as a diluent to prepare a system suitability solution containing 2 mg of MTV sample and 0.003 mg of isomer per 1 mL of diluent; (3) Take 10 μL of the solution from step (1) and (2) respectively and inject it into a liquid chromatograph, record the chromatogram, and detect the area percentage of the MTV enantiomer.

2. The HPLC-UV detection method for MTV enantiomers according to claim 1, characterized in that: The liquid chromatograph was Thermo Ultimate 3000 / VC with DAD detector.

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