Method for detecting N-fluorenylmethoxycarbonyl protected histidine and enantiomer thereof

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting N-fluorenylmethoxycarbonyl protected histidine and enantiomers thereof, which comprises the following steps: (1) preparing a system applicability solution; (2) injecting the system applicable solution into a high performance liquid chromatograph, and separating by adopting a chiral chromatographic column which takes amylose-tri (3, 5-dimethyl phenyl carbamate) covalently bonded porous silica gel microspheres as a stationary phase; (3) carrying out gradient elution by adopting a binary mobile phase system containing a mobile phase A and a mobile phase B; (4) obtaining a chromatogram map; and (5) calculating the contents of the N-fluorenylmethoxycarbonyl protected histidine and the enantiomer according to the chromatogram. The invention discloses a method for detecting Fmoc-His (Trt)-OH and an enantiomer thereof by an HPLC (High Performance Liquid Chromatography) method. The method has the advantages of very high sensitivity and separation degree, good repeatability and durability, simplicity in operation and stable and reliable result.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for detecting N-fluorenemethyloxycarbonyl-protected histidine and its enantiomers. Background Technology

[0002] Fmoc-His(Trt)-OH (N-fluorenylmethoxycarbonyl protected histidine) is the protected amino acid of histidine and an important amino acid for the synthesis of polypeptide drugs. The Fmoc-His(Trt)-OH molecule contains one chiral carbon atom, with the L configuration being the desired target, while the enantiomer D configuration is not applicable. Therefore, strict quality control of the optical isomer is necessary in drug synthesis.

[0003] The corresponding Fmoc-His(Trt)-OH and its enantiomers are shown below: , .

[0004] No methods for separating and detecting Fmoc-His(Trt)-OH and its enantiomers have been reported. The content of Fmoc-His(Trt)-OH enantiomers directly affects the quality of the finished peptide product. Therefore, developing an efficient and convenient analytical method to detect the content of Fmoc-His(Trt)-OH enantiomers is crucial for controlling the quality of leuprorelin finished products. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the present invention provides a method for detecting N-fluorenemethoxycarbonyl protected histidine and its enantiomers.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for detecting N-fluorenylmethoxycarbonyl-protected histidine and its enantiomers includes the following steps:

[0008] (1) Prepare a system suitability solution, wherein the system suitability solution uses N,N-dimethylformamide as a diluent;

[0009] (2) The system suitability solution was injected into a high performance liquid chromatograph and separated using a chiral column with linear starch-tris(3,5-dimethylphenylcarbamate) covalently bonded porous silica microspheres as the stationary phase;

[0010] (3) Gradient elution is performed using a binary mobile phase system containing mobile phase A and mobile phase B; mobile phase A includes trifluoroacetic acid and water, and mobile phase B is one of acetonitrile, methanol, ethanol, isopropanol or n-propanol;

[0011] (4) Detection was performed at a detection wavelength of 264 nm, a flow rate of 0.54~0.66 mL / min, and a column temperature of 20~25℃ to obtain a chromatogram;

[0012] (5) Calculate the content of N-fluorenemethoxycarbonyl protected histidine and enantiomer based on the retention time and peak area of ​​the main peak and enantiomer peak in the chromatogram.

[0013] The specific preparation method of the system suitability solution is as follows: Take an appropriate amount of Fmoc-His(Trt)-OH and its enantiomers, place them in the same volumetric flask, add diluent to prepare a solution containing 3 mg of Fmoc-His(Trt)-OH and 3 μg of its enantiomers per 1 mL, which is used as the system suitability solution.

[0014] The diluent N,N-dimethylformamide is filtered and degassed using a 0.22 μm microporous membrane before use.

[0015] The chiral chromatographic column used is a CHIRALPAK IB column with a specification of 4.6×250mm and a packing particle size of 5μm.

[0016] The volume ratio of trifluoroacetic acid to water is 1:1000.

[0017] The mobile phase B is n-propanol.

[0018] The mobile phase flow rate was 0.6 mL / min.

[0019] The gradient elution procedure is as follows:

[0020] Gradient 1: 0 to 40 minutes: Mobile phase A 55%, Mobile phase B 45%;

[0021] Gradient 2: 40.1 to 45 minutes: Mobile phase A 10%, Mobile phase B 90%;

[0022] Gradient 3: 45.1 to 58 minutes: mobile phase A 55%, mobile phase B 45%.

[0023] The retention time of the enantiomer in the chromatogram is 19.0-20.0 minutes, the retention time of the main component is 25.0-26.0 minutes, and the resolution between the two is not less than 2.5.

[0024] The beneficial effects of this invention are as follows:

[0025] (1) This application discloses an HPLC method for detecting Fmoc-His(Trt)-OH and its enantiomers. The method has high sensitivity and resolution, good repeatability and robustness, simple operation and stable and reliable results.

[0026] (2) The analytical method provided in this application for detecting the content of Fmoc-His(Trt)-OH and its enantiomers is of great significance for achieving quality control of Fmoc-His(Trt)-OH. Since excessive amino acid isomer impurities will affect the optical purity of the product, for drug molecules, this means a decrease in the proportion of active ingredients. Therefore, strictly controlling the content of isomers plays a crucial role in controlling the quality of peptide products. Attached Figure Description

[0027] Figure 1 This is a liquid chromatogram of Fmoc-His(Trt)-OH and its enantiomers in this invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application are described clearly and completely. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0029] A method for detecting N-fluorenylmethoxycarbonyl-protected histidine and its enantiomers includes the following steps:

[0030] (1) Prepare a system suitability solution, wherein the system suitability solution uses N,N-dimethylformamide as a diluent;

[0031] (2) Inject the system suitability solution into the high performance liquid chromatograph and use a chiral column with linear starch-tris(3,5-dimethylphenylcarbamate) covalently bonded porous silica microspheres as the stationary phase (packing agent) for separation;

[0032] (3) Gradient elution was performed using a binary mobile phase system containing mobile phase A (aqueous phase) and mobile phase B (organic phase);

[0033] (4) Detection was performed at a detection wavelength of 264 nm, a flow rate of 0.54~0.66 mL / min, and a column temperature of 20~25℃ to obtain a chromatogram;

[0034] (5) Calculate the content of N-fluorenemethoxycarbonyl protected histidine and enantiomer based on the retention time and peak area of ​​the main peak and enantiomer peak in the chromatogram.

[0035] The specific preparation method for the system suitability solution is as follows: Take an appropriate amount of Fmoc-His(Trt)-OH (N-fluorenylmethoxycarbonyl protected histidine) and its enantiomers, place them in the same volumetric flask, and add diluent to prepare a solution containing 3 mg of Fmoc-His(Trt)-OH and 3 μg of its enantiomers per 1 mL. This solution is used as the system suitability solution. The diluent N,N-dimethylformamide is filtered through a 0.22 μm microporous membrane and degassed before use. The chiral chromatographic column used is a CHIRALPAK IB column with dimensions of 4.6 × 250 mm and a packing particle size of 5 μm.

[0036] Mobile phase A comprises trifluoroacetic acid and water, with a volume ratio of trifluoroacetic acid to water of 1:1000. Mobile phase B is one of acetonitrile, methanol, ethanol, isopropanol, or n-propanol; preferably, n-propanol is used as mobile phase B. The preferred flow rate of the mobile phase is 0.6 mL / min.

[0037] The gradient elution procedure is as follows:

[0038] Gradient 1: 0 to 40 minutes: Mobile phase A 55%, Mobile phase B 45%;

[0039] Gradient 2: 40.1 to 45 minutes: Mobile phase A 10%, Mobile phase B 90%;

[0040] Gradient 3: 45.1 to 58 minutes: mobile phase A 55%, mobile phase B 45%.

[0041] The retention time of the enantiomers in the chromatogram was 19.0-20.0 minutes, the retention time of the main component was 25.0-26.0 minutes, and the resolution between the two was not less than 2.5.

[0042] Example 1

[0043] 1. Chromatographic conditions

[0044] Column: CHIRALPAK IB (4.6×250mm, 5μm)

[0045] Detection wavelength: 264 nm

[0046] Column temperature: 23℃

[0047] Flow rate: 0.6 mL / min

[0048] Injection volume: 10 μL

[0049] Diluent: N,N-dimethylformamide

[0050] Mobile phase A: 0.1% TFA

[0051] Mobile phase B: n-Propanol

[0052] Elution should be performed according to the gradient in the table below:

[0053]

[0054] 2. Methods and Results

[0055] 2.1 Solution Preparation

[0056] Preparation of test solution: Take an appropriate amount of Fmoc-His(Trt)-OH sample, dilute it with diluent to prepare a solution containing about 3 mg per 1 mL, which is used as the test solution (Table 1 shows the detection content of isomers in the test solution).

[0057] Table 1: Detection content of isomers in test samples

[0058]

[0059] 2.2. System Applicability

[0060] Take appropriate amounts of Fmoc-His(Trt)-OH and its enantiomers, place them in the same volumetric flask, and dilute with solvent to prepare a solution containing approximately 3 mg of Fmoc-His(Trt)-OH and 3 μg of its enantiomers per 1 mL. This solution is used as the system suitability solution. Accurately inject 10 μL of each solution into the liquid chromatograph and record the chromatograms. In the system suitability solution, Fmoc-His(Trt)-OH and its enantiomers are completely separated, with a resolution of 2.8. The retention time of Fmoc-His(Trt)-OH is 25.344 min, and the retention time of the enantiomer is 19.487 min. See [link to relevant documentation]. Figure 1 .

[0061] 2.3. Precision

[0062] Take 20 mg of the enantiomer impurity of Fmoc-His(Trt)-OH and place it in a 100 mL volumetric flask as the enantiomer impurity stock solution. Take an appropriate amount of Fmoc-His(Trt)-OH and the enantiomer impurity stock solution and place them in the same volumetric flask. Dissolve and dilute to the mark with diluent to prepare solutions with approximately 3 mg / mL of Fmoc-His(Trt)-OH and enantiomer contents of 1.2 μg, 3 μg and 6 μg per 1 mL as spiked test solutions. Calculate the enantiomer content and RSD in the 9 spiked test solutions using the area normalization method. The RSD of the 9 results is 0.8% (see Table 2 for the test results). The detection results are basically consistent and meet the requirements of high performance liquid chromatography for enantiomer detection.

[0063] Table 2: Precision Test Results

[0064]

[0065] 2.4. Limit of Quantitation and Limit of Detection

[0066] Take an appropriate amount of Fmoc-His(Trt)-OH enantiomer impurity and prepare a series of solutions of different concentrations. The limit of quantitation is when the signal-to-noise ratio (S / N) is approximately 10, and the limit of detection is when the signal-to-noise ratio (S / N) is approximately 3. The results of each experiment are shown in Table 3.

[0067] Table 3: Results of Limit of Detection and Limit of Quantitation Tests

[0068]

[0069] According to internationally accepted analytical method validation standards (such as ICH Q2(R1)), the limit of detection is usually determined by S / N ≈ 3, and the limit of quantitation is determined by S / N ≈ 10. The signal-to-noise ratios corresponding to the limits of detection and quantitation measured in this application are significantly higher than the above thresholds, indicating that the method has excellent sensitivity and reliability.

[0070] 3. Conclusion

[0071] Under these chromatographic conditions, Fmoc-His(Trt)-OH and its enantiomers can be completely separated. This method is highly specific, precise, reproducible, and has good system applicability, meeting the technical requirements of drug quality research standards.

[0072] The detection method disclosed in this application is based on the principle of chiral high-performance liquid chromatography (Chiral HPLC), utilizing the difference in the ability of enantiomers to form transient, reversible diastereomeric complexes between the chiral stationary phase and the mobile phase to achieve selective separation. Specifically, although N-fluorenemethoxycarbonyl-protected histidine (L-configuration) and its D-enantiomer have the same molecular formula and physicochemical properties, they are mirror images of each other in three-dimensional space. When the sample solution is injected into a chiral column (such as CHIRALPAK IB) with amylose-tris(3,5-dimethylphenylcarbamate) as the stationary phase, under the action of a gradient elution system consisting of an aqueous solution containing 0.1% trifluoroacetic acid (mobile phase A) and n-propanol (mobile phase B), the two enantiomers exhibit different migration rates within the column due to the different strengths of their stereoselective interactions (such as hydrogen bonding, π-π stacking, dipole interaction, and steric hindrance) with the helical cavities and aromatic groups on the chiral stationary phase: the D-enantiomer interacts weakly with the stationary phase, has a shorter retention time (approximately 19.5 minutes), and is eluted first; the L-configuration principal component interacts strongly, has a longer retention time (approximately 25.3 minutes), and elutes later.

[0073] N,N-Dimethylformamide, used as a diluent, not only fully dissolves the highly hydrophobic Fmoc-His(Trt)-OH and its isomers, preventing precipitation or adsorption losses, but also improves peak shape and reduces tailing. The gradient elution program is designed to balance separation efficiency and system equilibrium: a high proportion of aqueous phase is used for the first 40 minutes to maintain a highly polar environment, ensuring complete separation of the two enantiomers; then, a brief switch to a high proportion of n-propanol is made to rapidly wash away any remaining strong impurities; finally, the initial ratio is quickly restored to ensure the column reaches a stable state before the next injection. At a wavelength of 264 nm, the Fmoc group exhibits characteristic UV absorption, enabling highly sensitive detection. Finally, the purity of the main component and the content of the enantiomers are accurately calculated using the area normalization method, based on the retention time and peak area of ​​the two peaks in the chromatogram, meeting the stringent requirements of pharmaceutical quality control.

[0074] The foregoing has shown and described the basic principles, main features, and advantages of this application. Those skilled in the art should understand that this application is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of this application. Various changes and modifications can be made to this application without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of this application as claimed. The scope of protection of this application is defined by the appended claims and their equivalents.

Claims

1. A method for detecting N-fluorenylmethyloxycarbonyl protected histidine and its enantiomer, characterized in that, Includes the following steps: (1) Prepare a system suitability solution, wherein the system suitability solution uses N,N-dimethylformamide as a diluent; (2) The system suitability solution was injected into a high performance liquid chromatograph and separated using a chiral column with linear starch-tris(3,5-dimethylphenylcarbamate) covalently bonded porous silica microspheres as the stationary phase; (3) Gradient elution is performed using a binary mobile phase system containing mobile phase A and mobile phase B; mobile phase A includes trifluoroacetic acid and water, and mobile phase B is one of acetonitrile, methanol, ethanol, isopropanol or n-propanol; (4) Detection was performed at a detection wavelength of 264 nm, a flow rate of 0.54~0.66 mL / min, and a column temperature of 20~25℃ to obtain a chromatogram; (5) Calculate the content of N-fluorenemethoxycarbonyl protected histidine and enantiomer based on the retention time and peak area of ​​the main peak and enantiomer peak in the chromatogram.

2. A method of detecting N-fluorenylmethyloxy carbonyl protected histidine and its enantiomer as claimed in claim 1 wherein, The specific preparation method of the system suitability solution is as follows: Take an appropriate amount of Fmoc-His(Trt)-OH and its enantiomers, place them in the same volumetric flask, add diluent to prepare a solution containing 3 mg of Fmoc-His(Trt)-OH and 3 μg of its enantiomers per 1 mL, which is used as the system suitability solution.

3. The method for detecting N-fluorenemethoxycarbonyl-protected histidine and its enantiomers as described in claim 1, characterized in that: The diluent N,N-dimethylformamide is filtered and degassed using a 0.22 μm microporous membrane before use.

4. The method for detecting N-fluorenemethoxycarbonyl-protected histidine and its enantiomers as described in claim 1, characterized in that: The chiral chromatographic column used is a CHIRALPAK IB column with a specification of 4.6×250mm and a packing particle size of 5μm.

5. The method for detecting N-fluorenemethoxycarbonyl-protected histidine and its enantiomers as described in claim 1, characterized in that: The volume ratio of trifluoroacetic acid to water is 1:1000.

6. The method for detecting N-fluorenemethoxycarbonyl-protected histidine and its enantiomers as described in claim 1, characterized in that: The mobile phase B is n-propanol.

7. The method for detecting N-fluorenemethoxycarbonyl-protected histidine and its enantiomers as described in claim 1, characterized in that: The mobile phase flow rate was 0.6 mL / min.

8. The method for detecting N-fluorenemethoxycarbonyl-protected histidine and its enantiomers as described in claim 1, characterized in that, The gradient elution procedure is as follows: Gradient 1: 0 to 40 minutes: Mobile phase A 55%, Mobile phase B 45%; Gradient 2: 40.1 to 45 minutes: Mobile phase A 10%, Mobile phase B 90%; Gradient 3: 45.1 to 58 minutes: mobile phase A 55%, mobile phase B 45%.

9. The method for detecting N-fluorenemethoxycarbonyl-protected histidine and its enantiomers as described in claim 1, characterized in that, The retention time of the enantiomer in the chromatogram is 19.0-20.0 minutes, the retention time of the main component is 25.0-26.0 minutes, and the resolution between the two is not less than 2.5.

Images