CN116359410A - Method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine - Google Patents
Method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 37
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- 238000000926 separation method Methods 0.000 claims abstract description 36
- 239000012488 sample solution Substances 0.000 claims abstract description 30
- 238000010606 normalization Methods 0.000 claims abstract description 14
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- 239000000523 sample Substances 0.000 claims description 62
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- 230000000694 effects Effects 0.000 description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
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- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- -1 N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-phenylalanine Chemical compound 0.000 description 2
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- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 2
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Abstract
The invention provides a method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine, which comprises the following steps: chiral impurities and N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine are dissolved together in a solvent to obtain a clear and transparent solution; taking the clear and transparent solution as a sample solution of HPLC, and separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine by an HPLC method; the detection includes: and (3) using the LUX-1 chiral column as a separation carrier, mixing the mobile phase A and the mobile phase B at different time by different proportions to elute the sample solution, and determining the types of each component in the sample solution and the corresponding content of each component by adopting an area normalization method. The method can effectively separate each component in a short time, the separation degree reaches more than 1.5, and the detection result is accurate and reliable.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine.
Background
Polypeptide products, such as liraglutide, cable Ma Lutai, benraglutide, can be prepared from the short peptide N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine as a reaction material. If the impurity condition in the reaction materials can be detected in a short time, the method is important to control the quality of the final product polypeptide. Therefore, detection of impurities in short peptides is required.
N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine as a reactant for a polypeptide is often accompanied by chiral impurities which are caused by the starting material for the preparation of N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine. The method comprises the following steps: the starting materials L-threonine (H-L-Thr-OH) and L-phenylalanine (H-L-Phe-OH) of N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-phenylalanine often contain chiral impurities D-threonine (H-D-Thr-OH) and D-phenylalanine (H-D-Phe-OH), resulting in the produced product Fmoc-L-Thr (tBu) -L-Phe-OH containing chiral isomer impurities Fmoc-D-Thr (tBu) -L-Phe-OH, fmoc-D-Thr (tBu) -D-Phe-OH, fmoc-L-Thr (tBu) -D-Phe-OH. The difficulty in detecting the N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine is that the 3 chiral impurities coexist, the chemical properties and the physical properties of the 3 chiral impurities are similar, the conventional method is difficult to separate and quantitatively analyze at the same time, and the impurity content is relatively low, so that the detection difficulty is increased, and the accurate content determination is more difficult.
The method for detecting the mixture of the plurality of isomers adopts a mode of separating and detecting the plurality of isomers respectively and independently in the prior art, and the detection method can increase time cost and detection cost and also can influence accuracy. However, the quality requirements of Fmoc-L-Thr (tBu) -L-Phe-OH and other protective amino acid products in the downstream industries such as polypeptide and the like are very high (the purity is more than or equal to 99.5 percent, single impurity is less than or equal to 0.1 percent and the like), so that the related impurities in the products are required to be rapidly and efficiently analyzed, and a corresponding separation detection method is required urgently.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provide a method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine, wherein the method can realize the effective separation and quantitative detection of multiple components in a short time through one sample injection.
A method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine, comprising:
chiral impurities and N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine are dissolved together in a solvent to obtain a clear and transparent solution;
taking the clear and transparent solution as a sample solution of HPLC, and detecting by a certain detection means, so as to separate and detect chiral impurities in the N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine;
the certain detection means comprises: and (3) using a LUX-1 chiral column as a separation carrier, mixing the mobile phase A and the mobile phase B in different proportions at different times by using a high performance liquid chromatograph to elute the sample solution, and determining the types of each component in the sample solution and the corresponding content of each component by using an area normalization method.
Further, the method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine comprises the steps of mixing mobile phase A and mobile phase B at different times and corresponding to different ratios:
taking the flow rate of the mobile phase as a reference, taking the volume ratio of the mobile phase A to the mobile phase B as an initial volume ratio of 40:60, and gradually adjusting the volume ratio of the mobile phase A to the mobile phase B in a first time period to obtain a first volume ratio;
gradually adjusting the mobile phase A and the mobile phase B from the first volume ratio to a second volume ratio in a second time period;
gradually adjusting the second volume ratio to the third volume ratio of the mobile phase A and the mobile phase B in a third time period.
Further, the method for separating and detecting chiral impurities in the N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine comprises the steps of, wherein the first time period is 15 minutes, and the first volume ratio is 20:80; the second time period is 15 minutes, and the second volume ratio is 20:80; the third time period is 10 minutes, and the third volume ratio is 40:60.
Further, a method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine as described above, wherein the mobile phase A is a 0.10% TFA aqueous solution; the mobile phase B is acetonitrile.
Further, the method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine as described above, wherein the certain detection means further comprises: the size of the chromatographic column is: 5 μm4.6mm by 250mm; the column temperature was 30 ℃.
Further, the method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine as described above, wherein the certain detection means further comprises: the detection wavelength is 220nm, and the sample injection volume is 5 mu L.
Further, the method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine is described above, wherein the solvent is acetonitrile.
According to the method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine, provided by the invention, chiral impurities and N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine are dissolved together in a solvent to obtain a clear and transparent solution, and the clear and transparent solution is used as a sample solution of HPLC (high performance liquid chromatography) to be detected by a certain detection means, so that each chiral impurity in the N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine can be effectively separated, and the content of each chiral impurity is detected. The method can effectively separate chiral impurities in the N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine in a short time, has a separation degree of more than 1.5, has good specificity and precision, has accurate and reliable detection results, and meets the high standard requirements of detection.
Drawings
FIG. 1 is a chromatogram corresponding to example 1;
FIG. 2 is a chromatogram corresponding to sample 1;
FIG. 3 is a chromatogram corresponding to sample 2;
FIG. 4 is a chromatogram corresponding to sample 3;
FIG. 5 is a chromatogram corresponding to comparative example 1;
FIG. 6 is a chromatogram corresponding to comparative example 2;
FIG. 7 is a chromatogram corresponding to comparative example 3;
FIG. 8 is a chromatogram corresponding to comparative example 4;
FIG. 9 is a chromatogram corresponding to comparative example 5;
FIG. 10 is a chromatogram corresponding to comparative example 6;
FIG. 11 is a chromatogram corresponding to comparative example 7;
FIG. 12 is a chromatogram corresponding to comparative example 8;
FIG. 13 is a chromatogram corresponding to comparative example 9;
FIG. 14 is a chromatogram corresponding to comparative example 10.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
the embodiment provides a separation and detection method for N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine and chiral impurities thereof, which comprises the following steps:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: LUX-1 5 μm4.6mm×250mm;
mobile phase: aqueous 0.10% tfa as mobile phase a and acetonitrile as mobile phase B;
column temperature is 30 ℃; the flow rate is 1.0mL/min; detection wavelength 220nm, sample injection volume 5. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH 2mg, fmoc-D-Thr (tBu) -D-Phe-OH 2mg, fmoc-L-Thr (tBu) -D-Phe-OH 2mg were weighed out, placed in a 10mL volumetric flask, fixed in volume with pure acetonitrile, and dissolved by shaking to give a clear and transparent solution.
The clear and transparent solution is used as a sample solution of HPLC, a chiral column is used as a separation carrier, 0.10% TFA aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the mobile phase A and the mobile phase B are mixed according to different proportions at different time to be used as a mobile phase, the speed of the mobile phase is controlled to be 1.0mL/min, the sample is eluted, the contents of the main component and chiral impurities are determined by adopting an area normalization method, and the specific forms of the mobile phase A and the mobile phase B which are mixed according to different proportions at different time are shown in the table 1.
TABLE 1 volume ratio of mobile phases over time
Using the detection conditions of table 1, a chromatogram of the sample was obtained, as shown in fig. 1, and the peak table corresponding to fig. 1 is shown in table 2.
TABLE 2 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 9.599 | 2484940 | 178290 | 9.023 | 12.067 | -- |
| 2 | 10.887 | 18477934 | 1147463 | 67.097 | 77.665 | 3.196 |
| 3 | 19.170 | 1853499 | 64009 | 6.730 | 4.332 | 13.896 |
| 4 | 26.312 | 4722829 | 87692 | 17.149 | 5.935 | 6.585 |
| Totals to | 27539202 | 1477453 | 100.000 | 100.000 |
The separation degree of 4 components is 3.196-13.896, which is far higher than 1.5 of the conventional requirement, and the separation detection of chiral molecules is realized to reach the expected requirement.
Further, the retention time of each chiral component was confirmed. First, the following treatments were performed for each sample:
preparation of sample 1: fmoc-L-Thr (tBu) -L-Phe-OH10mg and Fmoc-D-Thr (tBu) -D-Phe-OH 1mg were weighed into a 10mL volumetric flask, and dissolved with pure acetonitrile to a constant volume, with shaking, to give a clear and transparent solution, i.e., sample solution 1.
Preparation of sample 2: dividing the sample solution 1 into 2 parts, and reserving 1 part as the sample solution 1; fmoc-L-Thr (tBu) -D-Phe-OH was added to the 2 nd sample solution 1 at about 1mg, and the mixture was dissolved to obtain a sample solution 2.
Preparation of sample 3: dividing the sample solution 2 into 2 parts, and reserving 1 part as the sample solution 2; fmoc-D-Thr (tBu) -L-Phe-OH was added to the 2 nd sample solution 2 at about 1mg, and the mixture was dissolved to obtain a sample solution 3.
Sample 1, sample 2 and sample 3 were run according to the following chromatographic conditions and in the corresponding manner as shown in table 1, respectively, to obtain corresponding chromatograms.
Equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: LUX-1 5 μm4.6mm×250mm;
mobile phase: aqueous 0.10% tfa as mobile phase a and acetonitrile as mobile phase B;
column temperature is 30 ℃; the flow rate is 1.0mL/min; the detection wavelength is 220nm, and the sample injection volume is 5 mu L.
FIG. 2 is a chromatogram corresponding to sample 1; table 3 is a peak table corresponding to fig. 2.
TABLE 3 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 10.954 | 21933602 | 1283035 | 86.962 | 92.247 | -- |
| 2 | 19.358 | 3288322 | 107832 | 13.038 | 7.753 | 13.398 |
| Totals to | 25221923 | 1390868 | 100.000 | 100.000 |
From the chromatograms 2 and Table 3, the Fmoc-L-Thr (tBu) -L-Phe-OH retention time was confirmed to be about 10.954 minutes, and the Fmoc-D-Thr (tBu) -D-Phe-OH retention time was confirmed to be about 19.358 minutes.
FIG. 3 is a chromatogram corresponding to sample 2; table 4 is a peak table corresponding to fig. 3.
TABLE 4 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 9.583 | 2981954 | 211552 | 12.708 | 15.061 | -- |
| 2 | 10.877 | 17883253 | 1104757 | 76.211 | 78.650 | 3.188 |
| 3 | 19.192 | 2600324 | 88347 | 11.081 | 6.290 | 13.799 |
| Totals to | 23465531 | 1404656 | 100.000 | 100.000 |
From chromatogram 3, it was confirmed that Fmoc-L-Thr (tBu) -D-Phe-OH retention time was about 9.583 minutes.
FIG. 4 is a chromatogram corresponding to sample 3, and Table 5 is a peak table corresponding to FIG. 4.
TABLE 5 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 9.598 | 3221237 | 229192 | 12.496 | 15.880 | -- |
| 2 | 10.892 | 17458739 | 1080954 | 67.726 | 74.895 | 3.199 |
| 3 | 19.200 | 2521451 | 86008 | 9.781 | 5.959 | 13.841 |
| 4 | 26.474 | 2576976 | 47134 | 9.997 | 3.266 | 6.616 |
| Totals to | 25778403 | 1443288 | 100.000 | 100.000 |
From chromatogram 4, it was confirmed that Fmoc-D-Thr (tBu) -L-Phe-OH had a retention time of about 26.474 minutes.
In summary, the optimal chromatographic conditions are adopted, and the separation degree of all components in the sample exceeds 1.5, so that the expected separation standard is achieved. The method provided by the invention can be used for sampling once and separating all chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine in a short time, and detecting the main component and the content corresponding to each chiral impurity.
Comparative example 1:
equipment model: shimadzu high performance liquid chromatograph LC-16
Column selection:
IC 5μm 4.6mm×250mm；
mobile phase selection: n-hexane solution of 0.01% TFA as mobile phase A; isopropyl alcohol solution of 0.01% tfa as mobile phase B;
column temperature is 35 ℃; the flow rate is 1.0mL/min; the detection wavelength is 220nm, and the sample injection volume is 10 mu L.
Sample preparation: weighing 5mg of Fmoc-L-Thr (tBu) -L-Phe-OH, 5mg of Fmoc-D-Thr (tBu) -D-Phe-OH, 5mg of Fmoc-L-Thr (tBu) -D-Phe-OH, adding into a 10mL volumetric flask, fixing the volume by absolute ethyl alcohol, shaking and dissolving to obtain a clear and transparent solution;
taking the clear and transparent solution as a sample solution of HPLC, mixing a mobile phase A and a mobile phase B according to different proportions at different times to obtain a mobile phase, eluting chiral impurities in the sample, and determining the contents of the main component and the chiral impurities by adopting an area normalization method; the specific forms of mixing mobile phase A and mobile phase B in different proportions over different time periods are shown in Table 6.
TABLE 6 volume ratio of mobile phases over time
Using the detection conditions of Table 6, a chromatogram 5 corresponding to comparative example 1 was obtained, and the peak table corresponding to FIG. 5 is shown in Table 7.
TABLE 7 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 11.090 | 1224642 | 57234 | 6.114 | 13.629 | -- |
| 2 | 11.688 | 9179602 | 237855 | 45.827 | 56.638 | 0.095 |
| 3 | 16.447 | 3142385 | 55683 | 15.688 | 13.259 | 3.969 |
| 4 | 26.923 | 6484187 | 69185 | 32.371 | 16.474 | 5.599 |
| Totals to | 20030816 | 419957 | 100.000 | 100.000 |
From fig. 5 and table 7, it was found that peaks of 2 components among 4 components in the sample were not completely separated, and partial coincidence occurred. Therefore, the present comparative example method cannot achieve the intended effect.
Comparative example 2:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column:
IC 5μm 4.6mm×250mm；
mobile phase: n-hexane solution of 0.01% TFA as mobile phase A; isopropyl alcohol solution of 0.01% tfa as mobile phase B;
column temperature is 35 ℃; the flow rate is 0.8mL/min; detection wavelength 220nm, sample injection volume 5. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH5mg, fmoc-D-Thr (tBu) -D-Phe-OH5mg, fmoc-L-Thr (tBu) -D-Phe-OH5mg and a 10mL volumetric flask were weighed, fixed in volume with absolute ethyl alcohol, and dissolved by shaking to obtain a clear and transparent solution;
taking the clear and transparent solution as a sample solution of HPLC, mixing a mobile phase A and a mobile phase B at different time by adopting different proportions to serve as a mobile phase, eluting main components and chiral impurities in the sample, and determining the contents of the main components and the chiral impurities by adopting an area normalization method; the specific forms in which mobile phase a and mobile phase B were mixed at different times in different proportions are shown in table 8.
TABLE 8 volume ratio of mobile phases over time
Using the detection conditions of table 8, chromatogram 6 corresponding to comparative example 2 was obtained, and the peak table corresponding to fig. 6 is shown in table 9.
TABLE 9 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 19.121 | 12922026 | 213878 | 53.243 | 70.768 | -- |
| 2 | 29.151 | 3677664 | 39894 | 15.153 | 13.200 | 5.104 |
| 3 | 51.467 | 7670144 | 48453 | 31.604 | 16.032 | 6.957 |
| Totals to | 24269834 | 302226 | 100.000 | 100.000 |
From fig. 6 and table 9, it was found that there were 4 components in the sample, but only 3 peaks appeared, and it was not effective to completely separate. Therefore, the present comparative example method cannot achieve the intended effect.
Comparative example 3:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column:
IC 5μm 4.6mm×250mm；
mobile phase: 0.01% TFA in n-hexane as mobile phase A;0.01% TFA in isopropanol as mobile phase B;
column temperature is 35 ℃; the flow rate is 1.0mL/min; detection wavelength 220nm, sample injection volume 10. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH5mg, fmoc-D-Thr (tBu) -D-Phe-OH5mg, fmoc-L-Thr (tBu) -D-Phe-OH5mg and a 10mL volumetric flask were weighed, fixed in volume with absolute ethyl alcohol, and dissolved by shaking to obtain a clear and transparent solution;
the clear and transparent solution is used as a sample solution of HPLC, a mobile phase A and a mobile phase B are mixed according to different proportions at different times to be used as a mobile phase, so that the main component and chiral impurities in the sample are eluted, and the content of the main component and chiral impurities is determined by adopting an area normalization method, wherein the specific forms of mixing the mobile phase A and the mobile phase B according to different proportions at different times are shown in a table 10.
TABLE 10 volume ratio of mobile phases over time
Using the detection conditions of Table 10, a chromatogram 7 corresponding to comparative example 3 was obtained, and the peak table corresponding to FIG. 7 is shown in Table 11.
TABLE 11 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 48.367 | 2865541 | 19430 | 49.022 | 35.170 | -- |
| 2 | 59.616 | 2979881 | 35816 | 50.978 | 64.830 | 2.038 |
| Totals to | 5845422 | 55246 | 100.000 | 100.000 |
From fig. 7 and table 11, it was found that 60 minutes of operation, 4 components in the sample but only 2 peaks appeared, failed to effectively completely separate and took longer. Therefore, the present comparative example method is ineffective and cannot achieve the intended effect.
Comparative example 4:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: SOD 5 μm4.6mm×250mm;
mobile phase: n-hexane solution of 0.01% TFA as mobile phase A; an ethanol solution of 0.01% tfa as mobile phase B;
column temperature is 35 ℃; the flow rate is 1.0mL/min; detection wavelength 220nm, sample injection volume 10. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH5mg, fmoc-D-Thr (tBu) -D-Phe-OH5mg, fmoc-L-Thr (tBu) -D-Phe-OH5mg and a 10mL volumetric flask were weighed, fixed in volume with absolute ethyl alcohol, and dissolved by shaking to obtain a clear and transparent solution;
the clear and transparent solution is used as a sample solution of HPLC, a mobile phase A and a mobile phase B are mixed according to different proportions at different times to be used as a mobile phase, so that a main component and chiral impurities in the sample are eluted, and the contents of the main component and the chiral impurities are determined by adopting an area normalization method, wherein the specific forms of mixing the mobile phase A and the mobile phase B according to different proportions at different times are shown in Table 12.
TABLE 12 volume ratio of mobile phases over time
Using the detection conditions of table 12, chromatogram 8 corresponding to comparative example 4 was obtained, and the peak table corresponding to fig. 8 is shown in table 13.
TABLE 13 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 7.306 | 99259030 | 2862061 | 100.000 | 100.000 | -- |
| Totals to | 99259030 | 2862061 | 100.000 | 100.000 |
From fig. 8 and table 13, it was found that 40 minutes of operation, 4 components in the sample but only 1 peak appeared, and no separation at all and a long time was taken. Therefore, the present comparative example method cannot achieve the intended effect.
Comparative example 5:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: SOD 5 μm4.6mm×250mm;
mobile phase: n-hexane solution of 0.10% TFA as mobile phase A, isopropanol solution of 0.10% TFA as mobile phase B;
column temperature is 35 ℃; the flow rate is 1.0mL/min; detection wavelength 220nm, sample injection volume 10. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH5mg, fmoc-D-Thr (tBu) -D-Phe-OH5mg, fmoc-L-Thr (tBu) -D-Phe-OH5mg and a 10mL volumetric flask were weighed, fixed in volume with absolute ethyl alcohol, and dissolved by shaking to obtain a clear and transparent solution;
the clear and transparent solution is used as a sample solution of HPLC, the mobile phase A and the mobile phase B are mixed according to different proportions at different times to be used as the mobile phase, so that the main component and chiral impurities in the sample are eluted, and the contents of the main component and chiral impurities are determined by adopting an area normalization method. The specific forms in which mobile phase a and mobile phase B were mixed at different times in different proportions are shown in table 14.
TABLE 14 volume ratio of mobile phases over time
Using the detection conditions of table 14, chromatogram 9 corresponding to comparative example 5 was obtained, and the peak table corresponding to fig. 9 is shown in table 15.
TABLE 15 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 11.968 | 2706826 | 74627 | 12.256 | 16.858 | -- |
| 2 | 13.849 | 2864253 | 86551 | 12.969 | 19.552 | 2.111 |
| 3 | 15.142 | 9981861 | 180458 | 45.197 | 40.766 | 1.126 |
| 4 | 19.480 | 6532049 | 101033 | 29.577 | 22.824 | 2.766 |
| Totals to | 22084989 | 442670 | 100.000 | 100.000 |
From fig. 9 and table 15, it was found that 4 peaks appeared in 4 components in the sample, but 2 peaks were not completely separated. Therefore, the present comparative example method cannot achieve the intended effect.
Comparative example 6:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: LUX-35 μm4.6mm×250mm;
mobile phase: 0.10% TFA in water as mobile phase a and acetonitrile as mobile phase B;
column temperature is 35 ℃; the flow rate is 1.0mL/min; detection wavelength 220nm, sample injection volume 10. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH5mg, fmoc-D-Thr (tBu) -D-Phe-OH5mg, fmoc-L-Thr (tBu) -D-Phe-OH5mg, were weighed into a 10mL volumetric flask, fixed in volume with pure acetonitrile, and dissolved by shaking to give a clear and transparent solution.
The clear and transparent solution is used as a sample solution of HPLC, the mobile phase A and the mobile phase B are mixed at different time and different proportions to be used as mobile phases, so that the main component and chiral impurities in the sample are eluted, and the content of the main component and chiral impurities is determined by adopting an area normalization method, wherein the specific forms of the mobile phase A and the mobile phase B which are mixed at different time and different proportions are shown in Table 16.
TABLE 16 volume ratio of mobile phases over time
Using the detection conditions of Table 16, a chromatogram 10 corresponding to comparative example 6 was obtained, and the peak table corresponding to FIG. 10 is shown in Table 17.
TABLE 17 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 4.746 | 18209699 | 795901 | 100.000 | 100.000 -- | |
| Totals to | 18209699 | 795901 | 100.000 | 100.000 |
From fig. 10 and table 17, it was found that there were 4 components in the sample but only 1 peak, and no separation at all, for 30 minutes of operation. Therefore, the present comparative example method cannot achieve the intended effect.
Comparative example 7:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: LUX-35 mu m 4.6mm.times.250mm;
mobile phase: 0.10% TFA in water as mobile phase a and acetonitrile as mobile phase B;
column temperature is 35 ℃; the flow rate is 1.0mL/min; detection wavelength 220nm, sample injection volume 10. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH5mg, fmoc-D-Thr (tBu) -D-Phe-OH5mg, fmoc-L-Thr (tBu) -D-Phe-OH5mg, were weighed into a 10mL volumetric flask, fixed in volume with pure acetonitrile, and dissolved by shaking to give a clear and transparent solution.
The clear and transparent solution is used as a sample solution of HPLC, the mobile phase A and the mobile phase B are mixed at different time and different proportions to be used as mobile phases, so that the main component and chiral impurities in the sample are eluted, and the content of the main component and chiral impurities is determined by adopting an area normalization method, wherein the specific forms of the mobile phase A and the mobile phase B which are mixed at different time and different proportions are shown in the table 18.
TABLE 18 volume ratio of mobile phases over time
Using the detection conditions of Table 18, a chromatogram 11 corresponding to comparative example 7 was obtained, and the peak table corresponding to FIG. 11 is shown in Table 19.
TABLE 19 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 2.851 | 16150678 | 1435331 | 100.000 | 100.000 | -- |
| Totals to | 16150678 | 1435331 | 100.000 | 100.000 |
From FIG. 11 and Table 19, it was found that the sample was run for 30 minutes with 4 components but only 1 peak, and no separation at all. Therefore, the present comparative example method cannot achieve the intended effect.
Comparative example 8:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: LUX-1 5 μm4.6mm×250mm;
mobile phase: 0.10% TFA in water as mobile phase a and acetonitrile as mobile phase B;
column temperature is 35 ℃; the flow rate is 1.0mL/min; detection wavelength 220nm, sample injection volume 10. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH5mg, fmoc-D-Thr (tBu) -D-Phe-OH5mg, fmoc-L-Thr (tBu) -D-Phe-OH5mg, were weighed into a 10mL volumetric flask, fixed in volume with pure acetonitrile, and dissolved by shaking to give a clear and transparent solution.
Taking the clear and transparent solution as a sample solution of HPLC, mixing a mobile phase A and a mobile phase B according to different proportions at different times to obtain a mobile phase, eluting main components and chiral impurities in the sample, and determining the contents of the main components and the chiral impurities by adopting an area normalization method; the specific forms in which mobile phase a and mobile phase B were mixed at different times in different proportions are shown in table 20.
TABLE 20 volume ratio of mobile phases over time
Using the detection conditions of table 20, chromatogram 12 corresponding to comparative example 8 was obtained, and the peak table corresponding to fig. 12 is shown in table 21.
Table 21 peak table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 15.874 | 17340326 | 946603 | 100.000 | 100.000 | -- |
| Totals to | 17340326 | 946603 | 100.000 | 100.000 |
From FIG. 12 and Table 21, it was found that the sample was run for 30 minutes with 4 components but only 1 peak, and no separation at all. Therefore, the present comparative example method cannot achieve the intended effect.
Comparative example 9:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: LUX-1 5 mu m 4.6mm.times.250mm;
mobile phase: aqueous solution of 0.10% tfa as mobile phase a and acetonitrile as mobile phase B;
column temperature is 30 ℃; the flow rate is 1.0mL/min; detection wavelength 220nm, sample injection volume 10. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH 2mg, fmoc-D-Thr (tBu) -D-Phe-OH 2mg, fmoc-L-Thr (tBu) -D-Phe-OH 2mg, and were weighed into a 10mL volumetric flask, fixed in volume with pure acetonitrile, and dissolved by shaking to give a clear and transparent solution.
Taking the clear and transparent solution as a sample solution of HPLC, mixing a mobile phase A and a mobile phase B at different time by adopting different proportions to serve as a mobile phase, eluting main components and chiral impurities in the sample, and determining the contents of the main components and the chiral impurities by adopting an area normalization method; the specific forms in which mobile phase a and mobile phase B were mixed at different times in different proportions are shown in table 22.
TABLE 22 volume ratio of mobile phases over time
Using the detection conditions of table 22, chromatogram 13 corresponding to comparative example 9 was obtained, and the peak table corresponding to fig. 13 is shown in table 23.
TABLE 23 Peak Table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 17.580 | l7992273 | 792960 | 100.000 | 100.000 | -- |
| Totals to | 17992273 | 792960 | 100.000 | 100.000 |
From FIG. 13 and Table 23, it was found that the operation was performed for 60 minutes, and that there were 4 components in the sample but only 1 peak was present, and that there was no separation at all and it took a long time. Therefore, the present comparative example method cannot achieve the intended effect.
Comparative example 10:
equipment model: shimadzu high performance liquid chromatograph LC-16
Chromatographic column: LUX-1 5 mu m 4.6mm.times.250mm;
mobile phase: 0.10% TFA in water as mobile phase a and acetonitrile as mobile phase B;
column temperature is 30 ℃; the flow rate is 1.0mL/min; the detection wavelength is 220nm; sample injection volume 5. Mu.L
Sample preparation: fmoc-L-Thr (tBu) -L-Phe-OH10mg, fmoc-D-Thr (tBu) -L-Phe-OH 2mg, fmoc-D-Thr (tBu) -D-Phe-OH 2mg, fmoc-L-Thr (tBu) -D-Phe-OH 2mg, and were weighed into a 10mL volumetric flask, fixed in volume with pure acetonitrile, and dissolved by shaking to give a clear and transparent solution.
Taking the clear and transparent solution as a sample solution of HPLC, mixing a mobile phase A and a mobile phase B at different time by adopting different proportions to serve as a mobile phase, eluting main components and chiral impurities in the sample, and determining the contents of the main components and the chiral impurities by adopting an area normalization method; the specific forms in which mobile phase a and mobile phase B were mixed at different times in different proportions are shown in table 24.
TABLE 24 volume ratio of mobile phases over time
Using the detection conditions of Table 24, a chromatogram 14 corresponding to comparative example 10 was obtained, and the peak table corresponding to FIG. 14 is shown in Table 25.
Table 25 peak table of mobile phase over time
Detector A220 nm
| Peak number | Retention time | Area of | Height | Area percent | Height% | Degree of separation (USP) |
| 1 | 9.831 | 2442825 | 161540 | 8.913 | 11.939 | -- |
| 2 | 11.268 | 18519518 | 1050984 | 67.572 | 77.675 | 3.279 |
| 3 | 21.006 | 1833730 | 54057 | 6.691 | 3.995 | 14.370 |
| 4 | 29.072 | 4610836 | 86471 | 16.824 | 6.391 | 7.084 |
| Totals to | 27406909 | 1353052 | 100.000 | 100.000 |
From fig. 14 and table 25, it was found that 4 peaks appear in 4 components in the sample, the separation is complete, and the degree of separation reaches 1.5 or more, but the chromatographic run time is 30 minutes, the retention time of the last component peak is 29.07 minutes, and the overall effect is inferior to that of example 1.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. A method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine, comprising:
chiral impurities and N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine are dissolved together in a solvent to obtain a clear and transparent solution;
taking the clear and transparent solution as a sample solution, and detecting by a certain detection means, so as to separate and detect chiral impurities in the N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine;
the certain detection means comprises: and (3) using a LUX-1 chiral column as a separation carrier, mixing the mobile phase A and the mobile phase B in different proportions at different times by using a high performance liquid chromatograph to elute the sample solution, and determining the types of each component in the sample solution and the corresponding content of each component by using an area normalization method.
2. The method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine according to claim 1, wherein mixing mobile phase a and mobile phase B at different times and in different proportions comprises:
taking the LUX-1 chiral column as a separation carrier, taking the flow rate of a mobile phase as a reference of 1.0mL/min, taking the volume ratio of a mobile phase A to a mobile phase B as an initial volume ratio of 40:60, and gradually adjusting the volume ratio of the mobile phase A to the mobile phase B in a first time period to obtain a first volume ratio;
gradually adjusting the mobile phase A and the mobile phase B from the first volume ratio to a second volume ratio in a second time period;
gradually adjusting the second volume ratio to a third volume ratio of the mobile phase A and the mobile phase B in a third time period.
3. The method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine according to claim 2, wherein the first time period is 15 minutes and the first volume ratio is 20:80; the second time period is 15 minutes, and the second volume ratio is 20:80; the third time period is 10 minutes, and the third volume ratio is 40:60.
4. A method for the separation and detection of chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine according to any one of claims 1-3, wherein mobile phase a is 0.10% tfa in water; the mobile phase B is acetonitrile.
5. The method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine according to claim 4, wherein the certain detection means further comprises: the size of the chromatographic column is: 5 mu m 4.6mm.times.250mm; the column temperature was 30 ℃.
6. The method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine according to claim 4, wherein the certain detection means further comprises: the detection wavelength is 220nm, and the sample injection volume is 5 mu L.
7. The method for separating and detecting chiral impurities in N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonyl-L-phenylalanine according to claim 4, wherein the solvent is acetonitrile.
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| CN117330659A (en) * | 2023-09-13 | 2024-01-02 | 南京汉欣医药科技有限公司 | High performance liquid chromatography analysis method for detecting tetrapeptide non-activated ester isomer |
| CN117330659B (en) * | 2023-09-13 | 2024-05-31 | 南京汉欣医药科技有限公司 | High performance liquid chromatography analysis method for detecting tetrapeptide non-activated ester isomer |
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