Separation and detection method of N-acetyl protected amino acid and its optical isomers

Abstract

The present invention relates to a separation and detection method of N-acetyl protected amino acids and their optical isomers, and belongs to the field of drug analysis and detection. The present invention provides a separation and detection method of N-acetyl protected amino acids and their optical isomers, using high performance liquid chromatography, the stationary phase is O-9-(tert-butyl ester carbamoyl) quinidine bonded silica gel, and the mobile phase is a volume ratio of 500: (5-15): (0.5-1.5) of methanol: glacial acetic acid: triethylamine mixed solution. The detection method provided by the present invention can effectively separate N-acetyl protected amino acids from their optical isomers, realizes accurate detection of optical isomers in N-acetyl protected amino acids, improves the quality control method of N-acetyl protected amino acids, helps to avoid the safety hazards caused by the presence of optical isomers in drugs using N-acetyl protected amino acids as raw materials, and has practical promotion and application value.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis and detection, and specifically relates to a method for separating and detecting N-acetyl protected amino acids and optical isomers thereof. Background Art

[0002] With the rapid development of the pharmaceutical industry, peptide drugs have the advantages of high biological activity, strong specificity, weak toxicity, not easy to aggregate in the body, less interaction with other drugs, and high affinity with receptors in the body. They are one of the research and development hotspots of domestic and foreign pharmaceutical companies. Amino acids with protective groups at the N-terminus are common starting materials for chemically synthesized peptide drugs. Among them, N-acetyl protected amino acids are a type of amino acids protected by acetyl groups at the N-terminus. The acetyl protecting group is generally introduced by acetic anhydride, which is a key starting material for synthesizing peptides. For example, ganirelix acetate is obtained by solid phase synthesis, and the (R)-N-acetyl-beta-naphthylalanine used in its synthesis process is its key starting material.

[0003] Since protected amino acids contain chiral centers, the starting materials and synthesis processes of polypeptide drugs may cause the final product to produce differential peptide impurities. Such impurities are very similar to the structure and chemical properties of the target product and cannot be removed by extraction and recrystallization, which are commonly used for classical small molecules. Liquid phase purification is time-consuming and labor-intensive and may not be effective. Impurities have a direct impact on the safety and effectiveness of drugs, so it is very important to control the optical isomers of protected amino acids from the process and source.

[0004] For the detection of protected amino acid optical isomers, a liquid normal phase system is conventionally used, with n-hexane and isopropanol mixed in different proportions as elution solvents for separation and detection. However, this system is mostly suitable for detecting protected amino acids such as N-Bz, N-Fmoc, N-DNB, etc. whose N-terminal protecting groups have large steric hindrance or whose protecting groups contain π-acid / π-base aromatic functional groups. For N-acetyl protected amino acids, the inventors of the present invention found in their exploration of detection methods that the above conventional liquid phase system is not applicable. The possible reason is that the steric hindrance of the acetyl group is small and it is not a π-acid / π-base aromatic functional group, resulting in a lack of steric action and π-π interaction between the components to be separated and the chiral stationary phase, so the separation effect is poor. For example, patent application CN109725073A discloses a separation and detection method for acetylcysteine ​​enantiomers. In order to increase the interaction between the stationary phase and the chiral substance to be separated in the chromatographic column and improve the selectivity of chiral separation, the method needs to be pre-column derivatization, and acetylcysteine ​​is reacted with the derivatization reagent (R)-(+)-α-methylbenzyl isocyanate. However, the pre-column derivatization reaction operation steps are complicated and not easy to promote and use. Moreover, the n-hexane used in the above-mentioned conventional liquid phase system is highly volatile, highly toxic, and has great harm to the detection personnel, and the chromatographic grade n-hexane and isopropanol are expensive, resulting in higher detection costs.

[0005] Therefore, there is an urgent need to provide an efficient, convenient, safe and low-cost liquid chromatography method for the separation and detection of N-acetyl-protected amino acids and their optical isomers. Summary of the invention

[0006] To solve the above problems, the present invention provides a method for separating and detecting N-acetyl protected amino acids and their optical isomers, which adopts high performance liquid chromatography, wherein the stationary phase is O-9-(tert-butylcarbamoyl)quinadine bonded silica gel, and the mobile phase is a mixed solution of methanol: glacial acetic acid: triethylamine in a volume ratio of 500: (5-15): (0.5-1.5).

[0007] Furthermore, the stationary phase is CHIRALPAK QD-AX or an equivalent chromatographic column.

[0008] Preferably, the length of CHIRALPAK QD-AX is 150 mm.

[0009] Preferably, the inner diameter of CHIRALPAK QD-AX is 2.1 to 4.6 mm.

[0010] More preferably, the inner diameter of CHIRALPAK QD-AX is 4.6 mm.

[0011] Preferably, the filler particle size of CHIRALPAK QD-AX is 5 μm.

[0012] Furthermore, the mobile phase is a mixed solution of methanol: glacial acetic acid: triethylamine in a volume ratio of 500: (8-12): (0.8-1.2).

[0013] Preferably, the mobile phase is a mixed solution of methanol: glacial acetic acid: triethylamine in a volume ratio of 500:10:1.

[0014] Furthermore, the high performance liquid chromatography method adopts isocratic elution.

[0015] Furthermore, the N-acetyl protected amino acid is (R)-N-acetyl-beta-naphthylalanine.

[0016] Furthermore, the flow rate of the mobile phase is 0.6-1.0 ml per minute.

[0017] Preferably, the flow rate is 0.7-0.9 ml per minute.

[0018] More preferably, the flow rate is 0.8 ml per minute.

[0019] Furthermore, the column temperature of the high performance liquid chromatography is set at 25°C to 40°C.

[0020] Preferably, the column temperature is set at 28°C to 32°C.

[0021] More preferably, the column temperature is set to 30°C.

[0022] Furthermore, the high performance liquid chromatography method sets the detection wavelength to 261 nm to 281 nm.

[0023] Preferably, the detection wavelength is set to 265nm-271nm.

[0024] Further preferably, the detection wavelength is set to 271 nm.

[0025] Furthermore, the concentration of the test solution prepared by the high performance liquid chromatography method is 0.1 to 5 mg / ml.

[0026] Preferably, the concentration of the test solution prepared by the high performance liquid chromatography method is 0.8-1.2 mg / ml.

[0027] Further preferably, the concentration of the test solution prepared by the high performance liquid chromatography method is 1.0 mg / ml.

[0028] In the present invention, the concentration of the test solution is calculated based on the amount of the N-acetyl protected amino acid test sample taken.

[0029] Furthermore, the solvent for preparing the test solution is the mobile phase.

[0030] Furthermore, the injection volume of the test solution is 5 to 15 μl.

[0031] Preferably, the injection volume of the test solution is 8-12 μl.

[0032] More preferably, the injection volume of the test solution is 10 μl.

[0033] Furthermore, the detection is qualitative detection and / or quantitative detection.

[0034] Preferably, the quantitative detection adopts the external standard method to determine the content of the optical isomer.

[0035] According to some specific embodiments of the present invention, the method for separating and detecting N-acetyl protected amino acids and their optical isomers comprises the following steps:

[0036] 1) Preparation of test solution: Take the N-acetyl protected amino acid to be tested, add mobile phase to dissolve it, and obtain the test solution;

[0037] 2) Preparation of reference solution: Take N-acetyl protected amino acid optical isomer reference, add mobile phase to dissolve, and obtain reference solution;

[0038] 3) Separately aspirate the test solution, reference solution and system suitability solution and inject them into the HPLC.

[0039] Furthermore, in step 2), the reference substance solution contains 0.5 to 2.5 μg of optical isomer reference substance per 1 ml.

[0040] The detection method provided by the present invention can effectively separate N-acetyl protected amino acids from their optical isomers, and realizes the accurate detection of optical isomers in N-acetyl protected amino acids. The results of the methodological verification experiment show that the specificity, precision, linearity and range, accuracy, etc. of the detection method of the present invention can meet the requirements for accurately detecting the optical isomers of N-acetyl protected amino acids. Moreover, the mobile phase solution used in the present invention is highly safe, low in price, more environmentally friendly, and conducive to reducing the cost of detection. In addition, the detection method of the present invention does not require the pre-column derivatization treatment of the sample to be tested, and has the advantages of high efficiency and convenience. The present invention improves the quality control method of N-acetyl protected amino acids, helps to avoid the potential safety hazards caused by the presence of optical isomers in drugs using N-acetyl protected amino acids as raw materials, and has practical promotion and application value.

[0041] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0042] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a typical chromatogram of the reference substance solution in the embodiment;

[0044] Figure 2 It is a typical chromatogram of the test solution in the embodiment;

[0045] Figure 3 The typical chromatogram of the spiked test solution in the embodiment is shown;

[0046] Figure 4 This is a typical chromatogram of the test solution at a flow rate of 0.7 ml / min under the durability test in the test example;

[0047] Figure 5 This is a typical chromatogram of the spiked test solution at a flow rate of 0.7 ml / min under the durability test in the test example;

[0048] Figure 6 This is a typical chromatogram of the test solution at a flow rate of 0.9 ml / min under the durability test in the test example;

[0049] Figure 7 This is the chromatogram of the spiked test solution at a flow rate of 0.9 ml / min under the durability test in the test example;

[0050] Figure 8 This is a typical chromatogram of the test solution at a column temperature of 28°C under the durability item in the test example;

[0051] Fig. 9 This is a typical chromatogram of the spiked test solution at a column temperature of 28°C under the durability test in the test example;

[0052] Fig.10 This is a typical chromatogram of the test solution at a column temperature of 32°C under the durability item in the test example;

[0053] Fig.11 This is a typical chromatogram of the spiked test solution at a column temperature of 32°C under the durability test in the test example;

[0054] Fig.12 The typical chromatograms of the test sample solutions of different instruments under the durability item in the test example are shown;

[0055] Fig.13 The typical chromatograms of the test solution spiked with different instruments under the durability test example are shown;

[0056] Fig.14 It is a typical chromatogram of the mixed solution in Comparative Example 1;

[0057] Fig.15 It is a typical chromatogram of the mixed solution in Comparative Example 2;

[0058] Fig.16 It is a typical chromatogram of the mixed solution in Comparative Example 3. DETAILED DESCRIPTION

[0059] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0060] In the following experiment, (R)-N-acetyl-beta-naphthylalanine is used as an example to illustrate the separation and detection effect of the method of the present invention on N-acetyl protected amino acids and their optical isomers. The experimental materials are prepared as follows:

[0061] Shimadzu LC-2030C PLUS liquid chromatography system;

[0062] Chromatographic column CHIRALPAK QD-AX (4.6 mm × 150 mm, 5 μm);

[0063] Chromatographic column CHIRALPAK IC (4.6 mm × 250 mm, 5 μm);

[0064] Chromatographic column CHIRALPAK ZWIX (+) (3.0 mm × 250 mm, 3 μm);

[0065] One hundred thousandth electronic balance (SECURA225D-1CN, Sartorius);

[0066] Ac-Nal-OH reference substance ((S)-N-acetyl-beta-naphthylalanine), batch number: GLS181120-20127, purity: 99.22%

[0067] Ac-D-Nal-OH sample ((R)-N-acetyl-beta-naphthylalanine), batch number: GLS181120-20127, manufacturer: Jier Biochemical

[0068] Methanol, n-hexane, isopropanol, and acetonitrile were of chromatographic grade, and glacial acetic acid, triethylamine, formic acid, and diethylamine were of analytical grade.

[0069] Example: Separation and detection of N-acetyl protected amino acids and their optical isomers using the method of the present invention

[0070] 1. Chromatographic conditions:

[0071] Chromatographic column: A chromatographic column filled with O-9-(tert-butylcarbamoyl)quinadine bonded silica gel is used. In this embodiment, CHIRALPAK QD-AX is specifically selected, and the specific specifications are 4.6 mm×150 mm, 5 μm;

[0072] Mobile phase: methanol: glacial acetic acid: triethylamine in a volume ratio of 500:10:1, with isocratic elution;

[0073] Detection wavelength: 271nm;

[0074] Flow rate: 0.8 ml per minute;

[0075] Column temperature: 30°C;

[0076] Injection volume: 10 μl.

[0077] 2. Sample preparation:

[0078] (1) Test solution: Take (R)-N-acetyl-beta-naphthylalanine, weigh accurately, dissolve in mobile phase and quantitatively dilute to make a solution containing about 1 mg per 1 ml.

[0079] (2) Reference solution: Take the optical isomer (S)-N-acetyl-beta-naphthylalanine reference substance and quantitatively dilute it with the mobile phase to make a solution containing about 1 μg per 1 ml.

[0080] (3) Spiked test solution: Take (R)-N-acetyl-beta-naphthylalanine and its optical isomer reference substance, accurately weigh them, dissolve them in the mobile phase and quantitatively dilute them to make a mixed solution containing 1 mg of (R)-N-acetyl-beta-naphthylalanine and 1 μg of its optical isomer per 1 ml.

[0081] Pipette the reference solution, test solution, and spiked test solution into the HPLC, and record the chromatogram. Figure 1 to Figure 3 The experimental results are shown in Table 1. The chromatogram of the test solution shows a chromatographic peak with the same retention time of the optical isomer in the chromatogram of the reference solution, which confirms that the N-acetyl protected amino acid to be tested contains optical isomers, and the content of optical isomers calculated by external standard method is 0.008%.

[0082] Table 1 Example test results

[0083] Injection solution Isomer peak time (min) Theoretical plate number Separation Reference solution 10.045 6691 / Test solution 10.051 6955 8.5 Spiked test solution 10.046 6823 8.1

[0084] The experimental results show that the isomers and the main peak in the spiked test solution are eluted in sequence, and the separation between the two reaches 8.1, which is a good separation; the solvent does not interfere with the elution of the isomers; other impurities do not interfere with the elution of the isomers; the absolute value of the difference between the retention time of the isomer peak in the spiked test solution and the retention time of the isomer peak in the reference solution is 0.007min, and the retention time is consistent. The experiment proves that the system applicability and specificity of the method of the present invention can meet the requirements for accurate detection of isomers.

[0085] Test Example Validation of the separation and detection method of the present invention

[0086] The chromatographic conditions were the same as in Example 1.

[0087] 1. Limit of detection and limit of quantification

[0088] Quantitation limit solution: Take the Ac-Nal-OH impurity stock solution and dilute it to the required concentration so that the signal-to-noise ratio of the impurity peak is 10-20.

[0089] Detection limit solution: Take the quantification limit solution and dilute it until the signal-to-noise ratio of the impurity peak is 3 to 7.

[0090] The quantitative limit solution was accurately measured and injected 6 times continuously, and the detection limit solution was injected 1 time. The experimental results showed that the quantitative limit concentration of Ac-Nal-OH was 0.1026 μg / ml, the detection limit concentration was 0.0513 μg / ml, and the quantitative limit concentration accounted for 0.0102% of the test sample concentration. The detection method of the present invention has good sensitivity.

[0091] 2. Linearity and range

[0092] Linear stock solution: Accurately pipette 1 ml of Ac-Nal-OH impurity stock solution into a 100 ml volumetric flask, add mobile phase (prepared in the example) to dilute to the scale, and shake well to obtain linear stock solution.

[0093] LOQ linear solution: Accurately pipette 2 ml of the linear stock solution into a 100 ml volumetric flask, add the mobile phase (prepared in the example) to dilute to the scale, shake well, and obtain

[0094] 20% linear solution: Densely pipette 2 ml of the linear stock solution into a 50 ml volumetric flask, add the mobile phase (prepared in the example) to dilute to the mark, and shake well to obtain the 20% linear solution.

[0095] 50% linear solution: accurately pipette 1 ml of the linear stock solution into a 10 ml volumetric flask, add the mobile phase (prepared in the example) to dilute to the mark, shake well, and obtain.

[0096] 100% linear solution: accurately pipette 2 ml of the linear stock solution into a 10 ml volumetric flask, add the mobile phase (prepared in the example) to dilute to the scale, shake well, and obtain.

[0097] 150% linear solution: accurately pipette 3 ml of the linear stock solution into a 10 ml volumetric flask, add the mobile phase (prepared in the example) to dilute to the mark, shake well, and obtain.

[0098] 200% linear solution: accurately pipette 4 ml of the linear stock solution into a 10 ml volumetric flask, add the mobile phase (prepared in the example) to dilute to the scale, shake well, and obtain.

[0099] The LOQ, 20%, 50%, 100%, 150%, and 200% linear solutions were accurately measured and injected with one needle each. The experimental results showed that the correlation coefficient r was 1.000; the ratio of the absolute value of the Y-axis intercept to the 100% linear concentration level response value was 1.2%; and the response value RSD was 7.4%.

[0100] 3. Precision

[0101] Ac-Nal-OH reference substance stock solution: accurately pipette 2 ml of Ac-Nal-OH impurity stock solution into a 100 ml volumetric flask, add mobile phase (prepared in the example) to dilute to the scale, and shake well to obtain.

[0102] Test solution: Accurately weigh about 20 mg of (R)-N-acetyl-beta-naphthylalanine, place in a 20 ml volumetric flask, add mobile phase (prepared in the example) to dissolve and dilute to the scale, shake well, and obtain.

[0103] Spiked test solution: Accurately weigh about 20 mg of (R)-N-acetyl-beta-naphthylalanine and place it in a 20 ml volumetric flask. Transfer 2 ml of Ac-Nal-OH reference stock solution to the same 20 ml volumetric flask, add the mobile phase (prepared in the example) to dissolve and dilute to the mark, shake well, and the solution is ready. (Prepare 6 portions in parallel)

[0104] Accurately measure the test solution and the spiked test solution and inject one injection respectively. Repeat the above operation once by different analysts, different days, and different instruments. The experimental results show that the recovery rate of Ac-Nal-OH of the same batch of spiked test solutions (n=6) measured by the same laboratory is between 100.8% and 104.7%, and the recovery rate RSD is 1.2%; the recovery rate RSD of Ac-Nal-OH of 12 spiked test solutions in different laboratories is 2%.

[0105] 4. Accuracy

[0106] 20% recovery solution: Accurately weigh about 20 mg of (R)-N-acetyl-beta-naphthylalanine and place it in a 20 ml volumetric flask. Transfer 0.4 ml of Ac-Nal-OH reference stock solution to the same 20 ml volumetric flask, add the mobile phase (prepared in the example) to dissolve and dilute to the mark, shake well, and the solution is obtained. (Prepare 3 copies in parallel)

[0107] 50% recovery solution: Accurately weigh about 20 mg of (R)-N-acetyl-beta-naphthylalanine and place it in a 20 ml volumetric flask. Transfer 1 ml of Ac-Nal-OH reference stock solution to the same 20 ml volumetric flask, add the mobile phase (prepared in the example) to dissolve and dilute to the mark, shake well, and the solution is obtained. (Prepare 3 copies in parallel)

[0108] 100% recovery solution: Weigh about 20 mg of (R)-N-acetyl-beta-naphthylalanine and place it in a 20 ml volumetric flask. Transfer 2 ml of Ac-Nal-OH reference stock solution to the same 20 ml volumetric flask. Add the mobile phase (prepared in the example) to dissolve and dilute to the mark. Shake well to obtain. (Prepare 3 copies in parallel)

[0109] 150% recovery solution: Weigh about 20 mg of (R)-N-acetyl-beta-naphthylalanine and place it in a 20 ml volumetric flask. Transfer 3 ml of Ac-Nal-OH reference stock solution to the same 20 ml volumetric flask, add the mobile phase (prepared in the example) to dissolve and dilute to the mark, shake well, and the solution is obtained. (Prepare 3 portions in parallel)

[0110] Accurately measure each recovery solution and inject one injection. The experimental results show that the recovery of Ac-Nal-OH in 9 spiked test solutions is between 97.5% and 105.2%, and the recovery RSD is 2.4%.

[0111] 5. Solution stability

[0112] Reference substance solution: accurately pipette 2 ml of Ac-Nal-OH reference substance stock solution into a 20 ml volumetric flask, add mobile phase (prepared in the example) to dilute to the mark, and shake well to obtain.

[0113] Test solution: Accurately weigh 20.40 mg of (R)-N-acetyl-beta-naphthylalanine, place in a 20 ml volumetric flask, add mobile phase (prepared in the example) to dissolve and dilute to the scale, shake well, and obtain.

[0114] Spiked test solution: Accurately weigh 20.07 mg of (R)-N-acetyl-beta-naphthylalanine, place in a 20 ml volumetric flask, add 2 ml of Ac-Nal-OH reference stock solution, add mobile phase (prepared in the example) to dissolve and dilute to the scale, shake well, and obtain.

[0115] The reference solution, test solution, and spiked test solution were accurately measured and injected at 0h, 5h, 9h, 17h, 20.5h, and 24h. The experimental results showed that the peak area ratio of the reference solution injected at each time point within 24h to the peak area at 0h was between 96.3% and 98.1%; the peak area ratio of the spiked test solution injected at each time point within 24h to the peak area at 0h was between 96.6% and 99.5%; the range of the impurity Ac-Nal-OH content of the test solution injected at each time point within 24h and 0h was between 0.0001% and 0.0011%.

[0116] 6. Durability

[0117] Test solution: Accurately weigh 20.11 mg of (R)-N-acetyl-beta-naphthylalanine, place in a 20 ml volumetric flask, add mobile phase (prepared in the example) to dissolve and dilute to the scale, shake well, and obtain.

[0118] Spiked test solution: Accurately weigh 20.21 mg of (R)-N-acetyl-beta-naphthylalanine and place it in a 20 ml volumetric flask. Transfer 2 ml of Ac-Nal-OH reference stock solution to the same 20 ml volumetric flask, add mobile phase (prepared in the example) to dissolve and dilute to the scale, shake well, and obtain.

[0119] Fine-tune the chromatographic conditions: flow rate 0.8ml / min±0.1ml / min, column temperature 30℃±2℃, take one injection of the test solution and the spiked test solution for each instrument and condition, and record the chromatogram, see Figures 4 to 13 , the experimental results are shown in Table 2.

[0120] Table 2 Verification results

[0121]

[0122]

[0123] The experimental results showed that by fine-tuning the column temperature, flow rate and replacing the chromatographic column, the range of the Ac-Nal-OH content in the test solution and the original conditions was in the range of 0 to 0.003%; the ratio of the Ac-Nal-OH content in the spiked test solution to the original conditions was 1.0, and the method had good durability.

[0124] Based on the above experimental results, the detection method provided by the present invention has good system applicability, specificity, precision, linearity, accuracy, solution stability and durability, and can accurately determine the optical isomers present in N-acetyl protected amino acids.

[0125] Comparative Example 1: Separation and detection of N-acetyl protected amino acids and their optical isomers using an existing conventional liquid normal phase system

[0126] 1. Chromatographic conditions

[0127] Chromatographic column: polysaccharide derivative bonded silica gel chiral chromatographic column CHIRALPAK IC (4.6mm×250mm, 5μm);

[0128] Mobile phase: n-hexane:isopropanol volume ratio of 80:20, isocratic elution;

[0129] Detection wavelength: 265nm;

[0130] Flow rate: 0.8 ml per minute;

[0131] Column temperature: 35 °C;

[0132] Injection volume: 10 μl.

[0133] 2. Solution preparation

[0134] Weigh 10.51 mg of Ac-D-Nal-OH and 10.10 mg of Ac-Nal-OH, place them in a 20 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, mix well, and obtain.

[0135] This experiment used the existing conventional liquid phase normal phase system, the chromatographic column filler was polysaccharide derivative bonded silica gel, and a mixed solution of n-hexane and isopropanol was used as the elution solvent. The mixed solution of N-acetyl protected amino acid and its optical isomers was injected and analyzed. The results are shown in Fig.14 .from Fig.14 It can be seen that Ac-Nal-OH and Ac-D-Nal-OH cannot be separated and effectively detected, indicating that the existing conventional liquid normal phase system is not suitable for the separation and detection of N-acetyl protected amino acids and their optical isomers.

[0136] Comparative Example 2: Adjusting the mobile phase composition based on the existing conventional liquid phase normal phase system

[0137] 1. Chromatographic conditions

[0138] Chromatographic column: polysaccharide derivative bonded silica gel chiral chromatographic column CHIRALPAK IC (4.6mm×250mm, 5μm);

[0139] Mobile phase: 0.1% w / w formic acid in water: methanol in a volume ratio of 30:70, isocratic elution;

[0140] Detection wavelength: 265nm;

[0141] Flow rate: 0.8 ml per minute;

[0142] Column temperature: 30°C;

[0143] Injection volume: 10 μl;

[0144] Diluent: methanol: acetonitrile volume ratio is 2:8.

[0145] 2. Solution preparation

[0146] Solvent: methanol:acetonitrile volume ratio is 20:80;

[0147] Mixed solution: Weigh 11.87 mg of Ac-D-Nal-OH and 5.47 mg of Ac-Nal-OH, place them in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, mix well, and the solution is obtained.

[0148] The mixed solution of N-acetyl protected amino acid and its optical isomers was injected and analyzed. The results are shown in Fig.15 .from Fig.15 It can be seen that Ac-Nal-OH and Ac-D-Nal-OH cannot be separated and effectively detected, indicating that adjusting the mobile phase composition based on the existing conventional liquid normal phase system is not applicable to the separation and detection of N-acetyl protected amino acids and their optical isomers.

[0149] Comparative Example 3: Separation and detection of N-acetyl protected amino acids and their optical isomers using a zwitterionic ion exchange chiral chromatographic column CHIRALPAK ZWIX(+)

[0150] 1. Chromatographic conditions

[0151] Chromatographic column: Zwitterionic ion exchange chiral chromatographic column CHIRALPAK ZWIX (+) (3.0 mm × 250 mm, 3 μm);

[0152] Mobile phase: methanol: acetonitrile: water: formic acid: diethylamine in a volume ratio of 95:395:10:0.95:1.3, with isocratic elution;

[0153] Detection wavelength: 265nm;

[0154] Flow rate: 0.3 ml per minute;

[0155] Column temperature: 30°C;

[0156] Injection volume: 10 μl;

[0157] Diluent: methanol: acetonitrile volume ratio is 2:8.

[0158] 2. Solution preparation

[0159] Solvent: methanol:acetonitrile volume ratio is 20:80;

[0160] Mixed solution: Weigh 11.87 mg of Ac-D-Nal-OH and 5.47 mg of Ac-Nal-OH, place them in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, mix well, and the solution is obtained.

[0161] This experiment used a chromatographic column with a zwitterionic chiral stationary phase that is stereoselective for amino acid chiral separation, and used the conventional mobile phase system recommended by the chromatographic column (a mobile phase that can provide protonation, and acid-base additives to promote the protonation of the mobile phase). The experimental results are shown in Fig.16 . Fig.16 The peaks were tailing and could not be effectively detected, indicating that the chromatographic column CHIRALPAKZWIX(+) was not suitable for the separation and detection of N-acetyl protected amino acids and their optical isomers.

[0162] From the above results, it can be seen that the separation and detection of N-acetyl protected amino acids and their optical isomers require the combination of a specific mobile phase and a chromatographic column.

Claims

1. A method for separating and detecting N-acetyl protected amino acids and their optical isomers, characterized in that: The high performance liquid chromatography method is adopted, the stationary phase is O-9-(tert-butylcarbamoyl)quinadine bonded silica gel, and the mobile phase is a mixed solution of methanol: glacial acetic acid: triethylamine in a volume ratio of 500: (5-15): (0.5-1.5); the high performance liquid chromatography method adopts isocratic elution; The high performance liquid chromatography method is set to detect wavelengths of 261 nm to 281 nm; The N-acetyl protected amino acid is (R)-N-acetyl-beta-naphthylalanine.

2. The separation and detection method according to claim 1, characterized in that: The stationary phase is CHIRALPAK QD-AX.

3. The separation and detection method according to claim 2, characterized in that: The length of the CHIRALPAK QD-AX is 150 mm; and / or, the inner diameter of the CHIRALPAK QD-AX is 2.1-4.6 mm; and / or, the filler particle size of the CHIRALPAK QD-AX is 5 μm.

4. The separation and detection method according to claim 3, characterized in that: The inner diameter of the CHIRALPAK QD-AX is 4.6 mm.

5. The separation and detection method according to claim 1, characterized in that: The mobile phase is a mixed solution of methanol: glacial acetic acid: triethylamine in a volume ratio of 500: (8-12): (0.8-1.2).

6. The separation and detection method according to claim 5, characterized in that: The mobile phase is a mixed solution of methanol: glacial acetic acid: triethylamine in a volume ratio of 500:10:

1.

7. The separation and detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.6-1.0 ml per minute.

8. The separation and detection method according to claim 7, characterized in that: The flow rate is 0.7-0.9 ml per minute.

9. The separation and detection method according to claim 8, characterized in that: The flow rate was 0.8 ml per minute.

10. The separation and detection method according to claim 1, characterized in that: The column temperature of the high performance liquid chromatography method is set at 25°C to 40°C.

11. The separation and detection method according to claim 10, characterized in that: The column temperature of the high performance liquid chromatography method is set at 28°C to 32°C.

12. The separation and detection method according to claim 11, characterized in that: The column temperature of the HPLC was set at 30°C.

13. The separation and detection method according to claim 12, characterized in that: The high performance liquid chromatography method is set to have a detection wavelength of 265 nm to 271 nm.

14. The separation and detection method according to claim 13, characterized in that: The high performance liquid chromatography method is set with a detection wavelength of 271 nm.

15. The separation and detection method according to claim 1, characterized in that: The concentration of the test solution prepared by the high performance liquid chromatography method is 0.1-5 mg / ml; and / or, the solvent used to prepare the test solution is the mobile phase; and / or, the injection volume of the test solution is 5-15 μl.

16. The separation and detection method according to claim 15, characterized in that: The concentration of the test solution prepared by the high performance liquid chromatography method is 0.8-1.2 mg / ml; and / or the injection volume of the test solution is 8-12 μl.

17. The separation and detection method according to claim 16, characterized in that: The concentration of the test solution prepared by the high performance liquid chromatography method is 1.0 mg / ml; and / or the injection volume of the test solution is 10 μl.

18. The separation and detection method according to claim 1, characterized in that: The detection is qualitative detection and / or quantitative detection.

19. The separation and detection method according to claim 18, characterized in that: The quantitative detection adopts the external standard method to determine the content of the optical isomer.

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