CN112986445A - Detection method of Fmoc-Pbf-arginine related substances - Google Patents
Detection method of Fmoc-Pbf-arginine related substances Download PDFInfo
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- 239000000126 substance Substances 0.000 title claims abstract description 31
- 239000004475 Arginine Substances 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 39
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 3
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract 2
- 238000010828 elution Methods 0.000 claims description 4
- 238000010606 normalization Methods 0.000 claims description 4
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
- 239000012088 reference solution Substances 0.000 claims 1
- 239000012535 impurity Substances 0.000 abstract description 24
- 238000000926 separation method Methods 0.000 abstract description 7
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 24
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 16
- 239000000243 solution Substances 0.000 description 15
- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 description 12
- 239000011259 mixed solution Substances 0.000 description 11
- LINBWYYLPWJQHE-UHFFFAOYSA-N 3-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)NCCC(=O)O)C3=CC=CC=C3C2=C1 LINBWYYLPWJQHE-UHFFFAOYSA-N 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000012490 blank solution Substances 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 7
- 239000013558 reference substance Substances 0.000 description 7
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- -1 Fmoc amino acids Chemical class 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical group [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to a method for detecting Fmoc-Pbf-arginine related substances, which comprises the following parameter conditions: the detection wavelength is 220nm, the mobile phase is acetonitrile and water, acetic acid is added, the acetonitrile volume content in the gradient change is 50-85-50%, and the water volume content is 50-15-50%; the stationary phase of the chromatographic column used in the detection is octadecylsilane. The Fmoc-Pbf-arginine related substance detection method provided by the invention has the advantages of high detection speed, accurate result, good impurity separation degree, simple method steps, convenience in operation and wide application prospect.
Description
Technical Field
The invention relates to the field of polypeptides, in particular to a detection method of Fmoc-Pbf-arginine related substances.
Background
Fmoc-Arg (PBF), Fmoc-Arg (PBF) -OH, also known as Fmoc-Pbf-arginine, N-fluorenylmethoxycarbonyl-2, 2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, N α -FMOC-N ω -PBF-L-arginine or Fmoc-protecting group arginine, which has the following structure:
arginine is essential ammonia for human bodyOne of the amino acids is an important raw material for synthesizing polypeptides. Arginine is basic and contains a plurality of functional groups, so that the arginine cannot be directly used for researching the property and synthesizing polypeptide, the amino group and the side chain guanidyl group of the arginine must be effectively protected, and pbf is most sensitive to acid and is in TFA/H2The O system can be conveniently removed at normal temperature, so Pbf is the most widely used arginine protecting group, and the development of the Fmoc-Pbf-arginine detection method has important industrial value.
Chinese patent publication No. CN109115899A discloses a method for analyzing Fmoc amino acids, but it does not mention any related substances, and the applicant has repeated the method used and found that the separation degree of the main impurities is not ideal.
Acetic acid is less acidic than trifluoroacetic acid, and trifluoroacetic acid is generally not used for mass spectrometry because the trifluoroacetate signal is strong, which can mask almost all negative ions and even contaminate mass analyzers. The existing method is to detect related substances of Fmoc-Arg (pbf) -OH by using a trifluoroacetic acid system, TFA plays a role of similar ion pairs, the concentration is generally 0.05-0.1%, and the solution is acidic due to overhigh concentration, so the service life of a chromatographic column can be influenced after long-time use. The invention also makes a comparison between an acetic acid system and a trifluoroacetic acid system, and the conclusion that the acetic acid system has more advantages in the aspects of detecting the separation degree of Fmoc-Arg (pbf) -OH related substances and protecting a chromatographic column.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a method for detecting Fmoc-Pbf-arginine related substances, which has convenient and quick operation and accurate result.
The technical scheme for solving the technical problems is as follows:
a method for detecting Fmoc-Pbf-arginine related substances comprises the following parameter conditions:
the detection wavelength is 220nm, the mobile phase is acetonitrile and water, acetic acid is added, the acetonitrile volume content is 50-85-50% in gradient change, and the water volume content is 50-15-50%.
Preferably, the chromatographic column in the related substance detection method can be selected from octadecylsilane as stationary phaseThe chromatography column of (1); specifically, it can be selected from ZORBAX ODS 4.6mmx250mm 5 μ Kromasil 100A C185 u 250 4.6mm, Sinochrom ODS-BP 5 μ 4.6mmx250mm, Symmetry Shield RP 185 um 4.6.6 mm 250mm, and Synergi MAX-RP 4.6mmx250mm 5 μ C18A column chromatography column.
Preferably, the column temperature in the related substance detection method is 20-30 ℃, and the flow rate of the mobile phase is 0.8-1.2 ml/min.
Furthermore, in the related substance detection method, the column temperature is 25 ℃, and the flow rate of the mobile phase is 1 ml/min.
Preferably, the method for detecting the related substances directly adopts a mobile phase dissolved sample, and the sample injection concentration is 0.25-0.75 mg/ml.
Preferably, the related substance detection method does not use a reference substance solution, and the purity of Fmoc-Pbf-arginine is directly calculated by an area normalization method.
Preferably, the amount of acetic acid added to the mobile phase in the method for detecting a substance of interest is 0.1% (by volume).
Preferably, the elution gradient in the method for detecting a substance is changed to:
TABLE 1
| Time (min) | Mobile phase A% | Mobile phase B% |
| 0 | 50 | 50 |
| 20 | 15 | 85 |
| 25 | 15 | 85 |
| 26 | 50 | 50 |
| 32 | 50 | 50 |
The Chinese naming of the compound of the invention conflicts with the structural formula, and the structural formula is taken as the standard; except for obvious errors in the formula.
The Fmoc-Pbf-arginine related substance detection method provided by the invention has the advantages of high detection speed, accurate result, good impurity separation degree, simple method steps, convenience in operation and wide application prospect.
Drawings
FIG. 1 is a high performance liquid chromatogram of Fmoc-Pbf-arginine according to an embodiment of the present invention;
FIG. 2 is a high performance liquid chromatogram of a blank solution provided by an embodiment of the present invention;
FIG. 3 is a high performance liquid chromatogram of the impurity Fmoc-Arg-OH provided by an embodiment of the invention;
FIG. 4 is a high performance liquid chromatogram of the impurities Fmoc-Arg (pbf) -OH provided by an embodiment of the present invention;
FIG. 5 is a high performance liquid chromatogram of the impurity Fmoc-beta-Ala-Arg (pbf) -OH provided by an embodiment of the invention;
FIG. 6 is a high performance liquid chromatogram of the impurity Fmoc-beta-Ala-OH provided by an embodiment of the present invention;
FIG. 7 is a high performance liquid chromatogram (trifluoroacetic acid system) of a mixed solution of all mixtures provided by an embodiment of the present invention;
fig. 8 is a high performance liquid chromatogram (acetic acid system) of a mixed solution of all mixtures provided by an embodiment of the present invention.
Detailed Description
The invention is illustrated but not limited by the following examples. The technical solutions protected by the present invention are all the simple replacements or modifications made by the skilled person in the art.
Example 1
The chromatographic conditions adopted are as follows:
detection wavelength: 220nm, column temperature: 25 ℃, sample introduction: 10 μ l
Mobile phase: a100% H2O plus 0.1% TFA B100% ACN plus 0.1% TFA
The gradient B phase change is performed for 0min 50-20 min 100-25 min 100-26 min 50-32 min 50-32 min Stop
The method comprises the following steps: respectively dissolving Fmoc-Arg (pbf) -OH and impurity reference substances Fmoc-Arg-OH, Fmoc-beta-Ala-Arg (pbf) -OH and Fmoc-Arg (pbf) -OH in acetonitrile to obtain a sample solution and an impurity reference substance solution with the concentrations of 0.5mg/ml and taking the acetonitrile as a blank solution.
Step two: 1ml of Fmoc-Arg (pbf) -OH solution and 1ml of each impurity control solution were aspirated to prepare a mixed solution.
Step three: gradient elution was performed using ZORBAX ODS 4.6mmx250mm 5 μ, column temperature 35 ℃ and wavelength 220nm, as shown in Table 2:
TABLE 2
Step four: sampling sequence and sampling quantity, namely firstly, 10ul of the mixed solution in the step two, and then, respectively, 10ul of each single impurity reference substance solution so as to determine the corresponding impurity peak-off time in the mixed solution peak-off process;
step five: acetonitrile is used as a blank solution for 1 needle, and then Fmoc-Arg (pbf) -OH is added, and the purity is obtained by integrating through an area normalization method.
Fmoc-Arg (PBF), i.e., Fmoc-Arg (PBF) -OH solution, blank solution (mobile phase), impurity Fmoc-Arg-OH solution, impurity Fmoc-Arg (PBF) -OH solution, impurity Fmoc- β -Ala-OH solution, and mixed solution of all the mixtures were injected, respectively, the retention time of Fmoc-Arg (PBF) -OH was about 10.5min (see FIG. 1), the blank solution (see FIG. 2), the retention time of impurity Fmoc-Arg-OH was about 5.7min (see FIG. 3), the retention time of impurity Fmoc-Arg (PBF) -OH was about 13.3min (see FIG. 4), the retention time of impurity Fmoc- β -Ala-Arg (PBF) -OH was about 9.7min (see FIG. 5), the retention time of the impurity Fmoc-beta-Ala-OH was about 6.7min (see FIG. 6). The peak condition of the mixed solution of all the mixtures (as shown in figure 7) can be well separated from the fluorenylmethoxycarbonyl-arginine (PBF), namely Fmoc-Arg (PBF) -OH and related substances thereof, so that the quality of the Fmoc-Arg (PBF) -OH can be effectively controlled.
TABLE 3 degree of separation
| Fmoc-Arg-OH | Fmoc-β-Ala-OH | Fmoc-β-Ala-Arg(pbf)-OH | Fmoc-Arg(pbf)-OH |
| 3.77 | 12.31 | 3.41 | 10.16 |
TABLE 4 relative Retention time
| Fmoc-Arg-OH | Fmoc-β-Ala-OH | Fmoc-β-Ala-Arg(pbf)-OH | Fmoc-Arg(pbf)-Arg(pbf)-OH |
| 54％ | 64％ | 92％ | 126％ |
Example 2
The chromatographic conditions adopted are as follows:
detection wavelength: 220nm, column temperature: 35 ℃, sample introduction: 10 μ l
Mobile phase: a is 100% H2O with 0.1% acetic acid, B with 100% acetonitrile with 0.1% acetic acid
The gradient B phase change is 0min 50-20 min 85-22 min 85-24 min 50-26 min 50% Stop
The method comprises the following steps: respectively dissolving Fmoc-Arg (pbf) -OH and impurity reference substances Fmoc-Arg-OH, Fmoc-beta-Ala-Arg (pbf) -OH and Fmoc-Arg (pbf) -OH in acetonitrile to obtain a sample solution and an impurity reference substance solution with the concentrations of 0.5mg/ml and taking the acetonitrile as a blank solution.
Step two: 1ml of Fmoc-Arg (pbf) -OH solution and 1ml of each impurity control solution were aspirated to prepare a mixed solution.
Step three: c of Synergi MAX-RP 4.6mmx250mm 5 mu is adopted18The column temperature was set at 35 deg.C, the wavelength was set at 220nm, and the gradient elution process is as follows
TABLE 5
| Time min | Mobile phase A% | Mobile |
| 0 | 50 | 50 |
| 20 | 15 | 85 |
| 25 | 15 | 85 |
| 26 | 50 | 50 |
| 32 | 50 | 50 |
Step four: and (3) sampling sequence and sampling quantity, namely firstly, 10ul of the mixed solution obtained in the step two, and then, respectively, 10ul of each single impurity reference substance solution so as to determine the corresponding impurity peak-off time in the mixed solution peak-off.
Step five: acetonitrile is used as a blank solution for 1 needle, and then Fmoc-Arg (pbf) -OH is added, and the purity is obtained by integrating through an area normalization method.
When the mixed solution is added (as shown in figure 8), the separation degree of an acetic acid system is higher than that of a trifluoroacetic acid system, and the peak type is not influenced. Fmoc-Arg-OH (about 2.0 min), Fmoc-beta-Ala-OH (about 6.8 min), Fmoc-beta-Ala-Arg (pbf) -OH (about 12.1 min), Fmoc-Arg (pbf) -OH (about 13.5 min), Fmoc-Arg (pbf) -Arg (pbf) -OH (about 18.9 min)
TABLE 6 degree of separation
| Fmoc-Arg-OH | Fmoc-β-Ala-OH | Fmoc-β-Ala-Arg(pbf)-OH | Fmoc-Arg(pbf)-OH |
| 28.06 | 23.17 | 4.95 | 20.48 |
TABLE 7 relative Retention time
| Fmoc-Arg-OH | Fmoc-β-Ala-OH | Fmoc-β-Ala-Arg(pbf)-OH | Fmoc-Arg(pbf)-Arg(pbf)-OH |
| 15％ | 50％ | 90％ | 140％ |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (8)
1. The Fmoc-Pbf-arginine related substance detection method is characterized by comprising the following parameter conditions:
the detection wavelength is 220nm, the mobile phase is acetonitrile and water, acetic acid is added, the acetonitrile volume content is 50-85-50% in gradient change, and the water volume content is 50-15-50%.
2. The method for detecting Fmoc-Pbf-arginine-related substance according to claim 1, wherein the chromatography column used in the method for detecting related substance is selected from the group consisting of chromatography columns having octadecylsilane as a stationary phase.
3. The Fmoc-Pbf-arginine-related substance detection method according to claim 1, wherein the column temperature is 20 to 30 ℃ and the flow rate of the mobile phase is 0.8 to 1.2 ml/min.
4. The Fmoc-Pbf-arginine-related substance assay method according to claim 1, wherein the column temperature is 25 ℃ and the mobile phase flow rate is 1 ml/min.
5. The Fmoc-Pbf-arginine-related substance detection method according to claim 1, wherein the related substance detection method directly employs a mobile phase dissolution sample, and the injection concentration is 0.25-0.75 mg/ml.
6. The method for detecting Fmoc-Pbf-arginine-related substance according to claim 1, wherein the purity of Fmoc-Pbf-arginine is calculated by directly using an area normalization method without using a reference solution.
7. The method for detecting Fmoc-Pbf-arginine-related substance according to claim 1, wherein the amount of acetic acid added to the mobile phase in the method for detecting related substance is 0.1% (by volume).
8. The Fmoc-Pbf-arginine-related substance detection method of claim 1, wherein the elution gradient in the related substance detection method is changed to:
。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080287650A1 (en) * | 2007-03-01 | 2008-11-20 | Avi Tovi | High purity peptides |
| CN109115899A (en) * | 2018-06-19 | 2019-01-01 | 南京肽业生物科技有限公司 | A kind of analysis method of Fmoc amino acid |
| CN111505161A (en) * | 2020-05-11 | 2020-08-07 | 成都市科隆化学品有限公司 | Method for detecting enantiomer of protected amino acid |
| WO2020190757A1 (en) * | 2019-03-15 | 2020-09-24 | Novetide Ltd. | Improved processes for the preparation of semaglutide |
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- 2021-03-26 CN CN202110327485.5A patent/CN112986445A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080287650A1 (en) * | 2007-03-01 | 2008-11-20 | Avi Tovi | High purity peptides |
| CN109115899A (en) * | 2018-06-19 | 2019-01-01 | 南京肽业生物科技有限公司 | A kind of analysis method of Fmoc amino acid |
| WO2020190757A1 (en) * | 2019-03-15 | 2020-09-24 | Novetide Ltd. | Improved processes for the preparation of semaglutide |
| CN111505161A (en) * | 2020-05-11 | 2020-08-07 | 成都市科隆化学品有限公司 | Method for detecting enantiomer of protected amino acid |
Non-Patent Citations (3)
| Title |
|---|
| Y.TANG 等: "Investigation on enantiomeric separations of fluorenylmethoxycarbonyl amino acids and peptides by high-performance liquid chromatography using native cyclodextrins as chiral stationary phases", 《JOURNAL OF CHROMATOGRAPHY A》, vol. 743, no. 2, 6 September 1996 (1996-09-06), pages 261 - 271, XP004020289, DOI: 10.1016/0021-9673(96)00302-0 * |
| 张梦悦 等: "柱前衍生化RP-HPLC法测定L-鸟氨酸盐酸盐的有关物质", 《药学与临床研究》, vol. 24, no. 04, 15 August 2016 (2016-08-15), pages 284 - 288 * |
| 陈蓉等: "柱前衍生-HPLC法同时测定不同产地茯苓中18种氨基酸含量", 《药物分析杂志》, vol. 37, no. 02, 28 February 2017 (2017-02-28), pages 297 - 303 * |
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