CN115656380A - HPLC-UV detection method of MTV enantiomer - Google Patents
HPLC-UV detection method of MTV enantiomer Download PDFInfo
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- CN115656380A CN115656380A CN202211399699.4A CN202211399699A CN115656380A CN 115656380 A CN115656380 A CN 115656380A CN 202211399699 A CN202211399699 A CN 202211399699A CN 115656380 A CN115656380 A CN 115656380A
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Abstract
The invention discloses an HPLC-UV detection method of MTV enantiomer, which comprises the steps of dissolving an MTV sample by using a mobile phase diluent, dissolving an enantiomer by using the mobile phase diluent, preparing a system applicability solution containing 2mg and 0.003mg of enantiomer in every 1mL of diluent, dissolving the MTV sample by using the diluent, preparing a sample solution containing 2mg of MTV sample in every 1mL of diluent, respectively injecting 10 mu L of the solution into a liquid chromatograph, recording a chromatogram, and detecting the enantiomer percentage area of the MTV sample; the method adopts a polysaccharide derivative normal phase coating type chiral chromatographic column and a normal hexane-isopropanol-trifluoroacetic acid (90.
Description
Technical Field
The 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) enantiomer by using HPLC.
Background
N- [ N-methyl-N- [ [ (2-isopropyl-4-thiazolyl) methyl ] methyl]Amino group]Carbonyl radical]The name of L-valine is N- [ N-methyl-N- [ [ (2-isoproyl-4-thiazolyl) methyl] amino] carbonyl]-L-valine, abbreviated as MTV, of formula
Molecular formula is C 14 H 23 N 3 O 3 S can be used as an organic synthesis intermediate and a pharmaceutical intermediate, is used for the synthesis of laboratory research and development processes and pharmaceutical chemicals, and is also a key starting material for synthesizing ritonavir serving as a raw material medicament. It is desirable to control the content of the MTV enantiomer during the production of the target compound ritonavir.
MTV is used as a key starting material of ritonavir, and the content of the enantiomer plays a key role in quality control of the content of the enantiomer of the ritonavir serving as a raw material drug, so that the quality control of the content of the enantiomer of MTV is significant in quality control in the production process of the ritonavir.
Disclosure of Invention
The invention aims to provide an HPLC-UV method for detecting the content of MTV enantiomer serving as a key starting material of ritonavir.
The technical scheme adopted by the invention for solving the technical problems is as follows: an HPLC-UV detection method of MTV enantiomer comprises the following detection conditions: a chromatographic column: DAICEL CHIRALPAK AD-H,250mm 4.6mm,5 μm, column temperature of 30 ℃; mobile phase: n-hexane: isopropyl alcohol: trifluoroacetic acid =90:10:0.1 (v/v/v) of a mixed solution; the flow rate is 0.8 mL/min; elution procedure: gradient elution and isocratic elution, wherein the running time is 30min; detection wavelength: 240nm is the maximum absorption wavelength and is used for carrying out quantitative detection on the enantiomer; comprises the following steps
(1) Taking the structural formula as
The MTV sample is dissolved by using a mobile phase mixed solution as a diluent to prepare a sample solution containing 2mg of the MTV sample per 1mL of the diluent;
(2) Respectively taking the MTV sample and the MTV enantiomer to dissolve by using a mobile phase mixed solution as a diluent to prepare a system applicability solution containing 2mg of the MTV sample and 0.003mg of the enantiomer in every 1mL of the diluent;
(3) And (3) respectively injecting 10 mu L of the solution obtained in the step (1) and the step (2) into a liquid chromatograph, recording a chromatogram, and detecting the enantiomer percentage area of the N- [ N-methyl-N- [ [ (2-isopropyl-4-thiazolyl) methyl ] amino ] carbonyl ] -L-valine.
Further, the liquid chromatograph was a Thermo Ultimate 3000/VC, DAD detector.
The invention has the beneficial effects that: the method selects the mobile phase n-hexane-isopropanol-trifluoroacetic acid (90, 10, v/v/v) to dissolve the MTV sample, avoids solvent effect, reduces the possibility that the diluent is not matched with the mobile phase and the MTV sample is separated out in a system, and has certain stability; the method selects DAICEL CHIRALPAK AD-H (250mm x 4.6mm,5 μm), a conventional chromatographic column with a column temperature of 30 ℃ is suitable for all HPLC instruments, and under a normal hexane-isopropanol-trifluoroacetic acid (90: 10:0.1, v/v/v), an MTV sample is completely separated from an enantiomer thereof at a baseline, and does not interfere with the detection of the enantiomer with other known impurities in the MTV sample, so that the method has good specificity and high selectivity; the method can simply and rapidly measure the purity, the content of related substances and the size of residual solvent of the MTV sample, and solves the problem of reaction monitoring of the MTV sample in the production process.
Drawings
Fig. 1 is an HPLC diagram of comparative example 1 of the present invention with n-hexane-ethanol-trifluoroacetic acid (90;
fig. 2 is a mobile phase n-hexane-ethanol-trifluoroacetic acid (95;
fig. 3 is a graph of the mobile phase n-hexane-isopropanol-trifluoroacetic acid (90;
FIG. 4 is a chart of a specific HPLC according to an embodiment of the present invention.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
The applicant finds that by using DAICEL CHIRALPAK AD-H (250mm x 4.6mm,5 μm), N-hexane-isopropanol-trifluoroacetic acid (90.
The key initiator N- [ N-methyl-N- [ [ (2-isoproyl-4-thiazolyl) methyl ] of the invention
The HPLC-UV detection method of amino ] carbonyl ] -L-valine enantiomer can be realized according to the following method:
(1) An appropriate amount of the MTV sample was taken, and the sample was dissolved with a diluent (n-hexane-isopropyl alcohol-trifluoroacetic acid (90.
(2) MTV sample and MTV enantiomer are dissolved by diluent to prepare a system applicability solution containing 2mg of MTV sample and 0.003mg of MTV enantiomer in 1mL of diluent.
(3) And (3) injecting the solutions obtained in the steps (1) and (2) into a liquid chromatograph according to the sample injection volume of 10 mu L, completing sample detection, and recording a chromatogram so as to detect the percentage area of the MTV enantiomer.
Wherein the detection conditions are as follows:
high performance liquid chromatograph: thermo Ultimate 3000, dad detector.
A chromatographic column: DAICEL CHIRALPAK AD-H (250 mm. Times.4.6 mm,5 μm).
Mobile phase: n-hexane-isopropanol-trifluoroacetic acid (90.
Elution time: 30min
Detection wavelength: 240nm.
Flow rate: 0.8mL/min.
Column temperature: 35 ℃ is carried out.
Sample introduction volume: 10 μ L.
The experimental conditions are used for carrying out the pre-verification investigation, and the specific implementation is as follows:
(1) Specificity study is shown in the following table.
The attached figure can be seen in figure 4, which is a blank solution, a separation degree solution, a reference substance solution, a sample solution, a labeled test substance solution and a positioning solution from bottom to top in sequence. Peak No. 2 in the figure is an enantiomeric peak, separated from the adjacent peak by a complete baseline.
As a result, blanks and other known impurities were not perturbed at MTV and enantiomeric peak positions; MTV and enantiomer peak positions in the reference solution, the sample solution and the added standard sample solution are consistent with the resolution solution; the separation degree of MTV and enantiomer in the solution is more than 1.5, and the purity of the main peak is more than 990. The results all meet the specificity acceptance criteria.
(2) The LOQ survey is shown in the table below.
As a result, the quantitative limit and the detection limit concentration level of the main component MTV (with the number of BL 1024-S03) and the enantiomer (with the number of BL1024-S03-IM 04) are lower than the limit level (0.15%), the signal-to-noise ratio of the detection limit solution of the two components is not less than 3, and the signal-to-noise ratio of the quantitative limit solution is not less than 10; 6 portions of the quantitative limiting solution are repeated, the RSD of the retention time of the two components is less than or equal to 2.0 percent, and the RSD of the peak area is less than or equal to 10.0 percent. The results all meet the acceptance criteria of detection limit and quantification limit.
(3) And (5) linear and range investigation.
The linear results (limit levels) of the major component MTV (No. BL 1024-S03) are shown in the following table.
The results (content levels) of the linearity of the major component MTV (BL 1024-S03) are shown in the following table.
The results (limit levels) for the linearity of the enantiomer (code No. BL1024-S03-IM 04) are shown in the following table.
As a result, at the limiting level, the principal component and the enantiomer are quantitatively limited to within 200% of the limiting concentration, the linear correlation coefficient is greater than 0.990, the response factor RSD is less than 10.0%, and the ratio of the y-axis intercept to the peak area at 100% level is less than 25%; for the content level, the main component is in the range of 50% to 150%, the linear correlation coefficient is larger than 0.990, the response factor RSD is smaller than 10.0%, the ratio of the y-axis intercept to the peak area of 100% level is smaller than 2.0%, and the linear slope ratio to the limit level is in the range of 0.9 to 1.1; in conclusion, the main component has a good linear relationship with the enantiomer.
(4) And (3) repeatability inspection: preparing 6 standard sample solutions (same-system applicability solutions) in parallel, wherein the content RSD of the detected main component is less than 2.0%, and the content RSD of the enantiomer is less than 10.0%; the repeatability is good.
(5) Investigation of solution stability: and (3) carrying out solution stability investigation on the added standard sample solution, and standing the added standard sample solution at room temperature for 20 hours to show that the added standard sample solution is stable within 20 hours at room temperature, and the RSD of the main component and the enantiomer content is less than 2.0%.
Comparative example 1
Instruments and conditions:
high performance liquid chromatograph: thermo Ultimate 3000, dad detector.
A chromatographic column: DAICEL CHIRALPAK AD-H (250 mm. Times.4.6 mm,5 μm).
Mobile phase: n-hexane-ethanol-trifluoroacetic acid (90.
Elution time: and (3) 30min.
Detection wavelength: and (4) full sweeping.
Flow rate: 1.0mL/min.
Column temperature: at 30 ℃.
Sample injection volume: 10 μ L.
The experimental steps are as follows: taking 40mg of MTV solid, placing the MTV solid in a 20mL volumetric flask, taking a proper amount of enantiomer and other known impurities to be controlled of MTV, adding mobile phase for dissolving, diluting to a scale, shaking up, and preparing 1mL of mixed solution containing 10 mu g of the sample 2mg, the enantiomer and the other known impurities as the resolution solution for developing the method.
And taking the sample solution, carrying out high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The results are shown in FIG. 1, where peak No. 2 is the enantiomer peak, peak No. 1 is its nearest neighbor, and peak No. 3 is the sample peak, under which condition the enantiomer does not reach baseline separation from its neighbor.
Comparative example 2
Instruments and conditions:
high performance liquid chromatograph: thermo Ultimate 3000, dad detector.
A chromatographic column: DAICEL CHIRALPAK AD-H (250 mm. Times.4.6 mm,5 μm).
Mobile phase: n-hexane-ethanol-trifluoroacetic acid (95.
Elution time: and (3) 30min.
Detection wavelength: and (4) full sweeping.
Flow rate: 1.0mL/min.
Column temperature: at 30 ℃.
Sample introduction volume: 10 μ L.
The experimental steps are as follows: taking 40mg of MTV solid, placing the MTV solid in a 20mL volumetric flask, taking a proper amount of enantiomer and other known impurities to be controlled of MTV, adding a mobile phase for dissolving, diluting to a scale, shaking up, and preparing 1mL of mixed solution containing 10 mu g of sample 2mg, isomer and other known impurities as resolution solution for developing the method.
And taking the sample solution, carrying out high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The results are shown in figure 2, wherein peak No. 2 is an enantiomer peak, peak No. 1 is the nearest adjacent peak, and peak No. 3 is a sample peak, and it can be seen from the figure that under the condition, the enantiomer and the adjacent peak thereof still do not achieve the baseline separation, and the separation degree is poorer than that of example 1.
Comparative example 3
Instruments and conditions:
high performance liquid chromatograph: thermo Ultimate 3000, dad detector.
A chromatographic column: DAICEL CHIRALPAK AD-H (250 mm. Times.4.6 mm,5 μm).
Mobile phase: n-hexane-isopropanol-trifluoroacetic acid (90.
Elution time: and (3) 30min.
Detection wavelength: and (5) full sweeping.
Flow rate: 1.0mL/min.
Column temperature: 35 ℃ is carried out.
Sample introduction volume: 10 μ L.
The experimental steps are as follows: taking 40mg of MTV solid, placing the MTV solid in a 20mL volumetric flask, taking a proper amount of enantiomer and other known impurities to be controlled of MTV, adding mobile phase for dissolving, diluting to a scale, shaking up, and preparing 1mL of mixed solution containing 10 mu g of the sample 2mg, the enantiomer and the other known impurities as the resolution solution for developing the method.
And taking the sample solution, carrying out high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The result is shown in figure 3, in figure 1, the peak 1 is the sample peak, the peak 2 is the nearest adjacent peak of the enantiomer, and the peak 3 is the enantiomer peak, and it can be seen from the figure that, under the condition, after the mobile phase is changed from ethanol to isopropanol, the peak-out sequence is changed, and the enantiomer and the adjacent peak thereof are in proportion 1 and example 2, the effect is good, but still needs to be further optimized.
The above-described embodiments are merely illustrative of the principles and effects of the present invention, and some embodiments may be applied, and it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the inventive concept of the present invention, and these embodiments are within the scope of the present invention.
Claims (2)
1. An HPLC-UV detection method of MTV enantiomer, which is characterized in that: the detection conditions are
A chromatographic column: DAICEL CHIRALPAK AD-H,250mm 4.6mm,5 μm, column temperature 30 ℃;
mobile phase: n-hexane: isopropyl alcohol: trifluoroacetic acid =90:10:0.1 (v/v/v) of a mixed solution; the flow rate is 0.8 mL/min;
elution procedure: isocratic elution for 30min;
detection wavelength: 240nm;
comprises the following steps
(1) Taking an MTV sample, dissolving the MTV sample by using a mobile phase mixed solution as a diluent to prepare a sample solution containing 2mg of the MTV sample in every 1mL of the diluent;
(2) Respectively taking the MTV sample and the MTV enantiomer to dissolve by using a mobile phase mixed solution as a diluent to prepare a system applicability solution containing 2mg of the MTV sample and 0.003mg of the enantiomer in every 1mL of the diluent;
(3) And (3) respectively injecting 10 mu L of the solution obtained in the step (1) and the step (2) into a liquid chromatograph, recording a chromatogram, and detecting the percentage area of the MTV enantiomer.
2. An HPLC-UV detection method of MTV enantiomer according to claim 1, wherein the liquid chromatograph is a Thermo Ultimate 3000/VC, DAD detector.
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