Method for detecting N-hydroxysuccinimide in Fmoc protected amino acid
By using high performance liquid chromatography (HPLC) with a HILIC Amide column, UV detector, and gradient elution, the problem of suboptimal detection of N-hydroxysuccinimide in Fmoc-protected amino acids was solved, achieving highly sensitive and repeatable detection results.
Patent Information
- Application Number
- CN202511010689.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-22
- Publication Date
- 2025-10-03
AI Technical Summary
Existing technologies cannot effectively detect the residual amount of N-hydroxysuccinimide in Fmoc-protected amino acids, and ordinary reversed-phase liquid chromatography is not applicable, resulting in unsatisfactory detection.
High performance liquid chromatography was used with a HILIC Amide column with an alkylamide as the stationary phase, mobile phase A was 0.1% phosphoric acid aqueous solution, mobile phase B was acetonitrile, the detector was an ultraviolet detector, and detection was performed in a gradient elution mode.
The method can be used to quickly, accurately, and easily detect the residual N-hydroxysuccinimide in Fmoc-protected amino acids. It has strong specificity, high sensitivity, good repeatability, and a low detection limit, and is applicable to a variety of Fmoc-protected amino acids.
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Figure CN120741745A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of analytical chemistry, and in particular to a method for detecting the residual amount of N-hydroxysuccinimide (HOSU or NHS) as an impurity in Fmoc-protected amino acids. Background Art
[0002] Amino acids are organic compounds containing an amino group (-NH2) and a carboxylic acid group (-COOH). They are the building blocks of proteins and participate in nearly all aspects of life, including metabolism, immunity, and cell repair. Chemically, amino acids possess amino and carboxyl groups, which allow them to undergo acid-base neutralization reactions, participate in peptide bond formation, and dissociate in acidic or alkaline environments. However, amino acids are susceptible to damage from factors such as oxidation, acid-base reactions, and light. To protect their integrity and stability, they require the use of various methods and techniques, known as amino acid protection. Furthermore, amino acid protection is crucial in peptide synthesis, preventing unwanted reactions and ensuring accurate and efficient synthesis. Protecting groups are typically used to protect the active side chains of amino acids. These protecting groups must selectively protect specific amino acids without affecting other amino acids, while maintaining their stability. Among these, 9-fluorenylmethyloxycarbonyl (Fmoc) is a common protection method. The 9-fluorenylmethyloxycarbonyl group can bind to amino groups to form stable compounds. When it is necessary to remove the protecting group, alkaline conditions can be used to remove the 9-Fmoc group. By using protecting groups, the direction of the reaction can be precisely controlled to obtain the desired peptide product.
[0003] The reagent 9-fluorenylmethyl-N-succinimidyl carbonate (Fmoc-Osu) is often used in the synthesis of Fmoc-protected amino acids. This compound must be stored away from acid and moisture. However, the use of acidic reagents and water is often unavoidable in the synthesis of Fmoc-protected amino acids. This can lead to the hydrolysis of 9-fluorenylmethyl-N-succinimidyl carbonate to produce the impurity N-hydroxysuccinimide. N-hydroxysuccinimide has been identified as a probable human carcinogen by the International Agency for Research on Cancer (IARC), necessitating strict monitoring of its residual levels to ensure the quality and safety of the corresponding products.
[0004] N-hydroxysuccinimide, CAS number 6066-82-6, molecular formula C4H5NO3, molecular weight 115.09, is soluble in water and easily soluble in acetone, alcohol and ethyl acetate. Its boiling point is 262.4±23.0 °C at 760 mmHg, and its LogP (oil-water distribution coefficient) value is -2.00. It is a typical highly polar small molecule compound.
[0005] Regarding methods for detecting residual N-hydroxysuccinimide, existing techniques disclose methods for detecting protein drugs or peptides using conventional reversed-phase liquid chromatography. However, since N-hydroxysuccinimide is a highly polar compound, conventional reversed-phase liquid chromatography does not yield ideal compound retention. Furthermore, these methods cannot be directly applied to the detection of N-hydroxysuccinimide in Fmoc-protected amino acids.
[0006] Therefore, it is necessary to develop a more suitable and simpler high performance liquid chromatography method to detect N-hydroxysuccinimide in Fmoc-protected amino acids. Summary of the Invention
[0007] The present invention provides a method for detecting N-hydroxysuccinimide in Fmoc-protected amino acids. The method of the present invention has strong specificity, high sensitivity, and good repeatability, can quickly and accurately determine the residual amount of HOSU in Fmoc-protected amino acids, and is simple to operate. The method comprises the following steps: 1) Prepare N-hydroxysuccinimide reference solution using diluent; 2) Prepare the test solution by dissolving the Fmoc-protected amino acid in a diluent. 3) testing the reference solution and the test solution using liquid chromatography; The chromatographic conditions include: The chromatographic column is a HILIC Amide column with alkylamide as the stationary phase; Mobile phase A was 0.1% (v / v) phosphoric acid in water, and mobile phase B was acetonitrile; The detector is a UV detector.
[0008] Preferably, the diluent is acetonitrile.
[0009] Preferably, the ultraviolet detector is a diode array detector.
[0010] Preferably, the chromatographic column is Shim-pack GIST Amide, with a specification of 4.6 mm×250 mm, 5 μm, or InertSustain Amide, with a specification of 4.6 mm×250 mm, 5 μm, or ChromCore HILIC Amide, with a specification of 4.6 mm×250 mm, 5 μm.
[0011] Preferably, the detection wavelength of N-hydroxysuccinimide is 220 nm.
[0012] Preferably, the chromatographic conditions further include an injection volume of 20 μL, a flow rate of 0.7 to 0.9 mL / min, and a column temperature of 28 to 32°C.
[0013] More preferably, the chromatographic conditions further include a flow rate of 0.8 mL / min and a column temperature of 30°C.
[0014] Preferably, a gradient elution method is adopted, and the elution gradient is:
[0015] Preferably, the Fmoc-protected amino acids include Fmoc-glycine, Fmoc-Pbf-L-arginine, Fmoc-L-aspartic acid beta-tert-butyl ester, Fmoc-N-trityl-L-glutamine, Fmoc-L-isoleucine, Fmoc-L-leucine, Fmoc-L-phenylalanine, Fmoc-O-tert-butyl-L-serine, Fmoc-O-tert-butyl-L-threonine, Fmoc-L-tryptophan (Boc), Fmoc-O-tert-butyl-L-tyrosine, Fmoc-L-valine and Fmoc-L-glutamate 1-tert-butyl ester.
[0016] Preferably, the content of N-hydroxysuccinimide in the Fmoc-protected amino acid is calculated using an external standard method.
[0017] The beneficial effects achieved by the detection method of the present invention are: The present invention can use a conventional high-performance liquid chromatograph in conjunction with a conventional UV detector or diode array detector. The instrument features a simple, mature structure, easy operation, and a wide range of applicability, allowing standard chromatography laboratories to meet testing requirements. The mobile phase employed in the present invention is the most common acetonitrile and aqueous phosphoric acid system, eliminating the need for complex solution preparation. The operation is simple, the preparation is easy, and even ordinary laboratory technicians can quickly master the method. The chromatographic column used in the present invention is a hydrophilic liquid chromatography column (HILIC Amide column) with an alkylamide group as the stationary phase. These columns are readily available and have low experimental costs. Furthermore, the method of the present invention achieves a short overall analysis time and high efficiency.
[0018] The detection method of the present invention has strong specificity, high sensitivity and good repeatability, and can quickly and accurately determine the residual amount of N-hydroxysuccinimide in Fmoc-protected amino acids. According to methodological verification, the detection method of the present invention has a linear correlation coefficient R of 0.55μg / mL to 5.5μg / mL in the range of N-hydroxysuccinimide concentration. 2 The linearity was 0.9998, with good sensitivity. The detection limit of N-hydroxysuccinimide was 0.18 μg / mL, with high sensitivity. The 13 Fmoc-protected amino acid spiked solutions were tested six times, and the RSD values of the N-hydroxysuccinimide content were between 0.2% and 0.4%, with excellent repeatability. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 is the chromatogram of the blank solution; Figure 2 is the chromatogram of the reference solution; Figure 3 is the chromatogram overlay of the test solution; Figure 4 is a linear relationship graph; Figure 5 is the chromatogram corresponding to Comparative Example 1; Figure 6 is the chromatogram corresponding to Comparative Example 2; Figure 7 is the chromatogram corresponding to Comparative Example 3; Figure 8 is the chromatogram corresponding to Comparative Example 4; Figure 9 is the chromatogram corresponding to Comparative Example 5; Figure 10 is the chromatogram corresponding to Comparative Example 6; Figure 11 is the chromatogram corresponding to Comparative Example 7; Figure 12 is the chromatogram corresponding to Comparative Example 8; Figure 13 is the chromatogram corresponding to Comparative Example 9; Figure 14 is the chromatogram corresponding to Comparative Example 10; Figure 15 This is the chromatogram corresponding to the Fmoc-glycine test solution; Figure 16 This is the chromatogram corresponding to the Fmoc-L-glutamic acid 1-tert-butyl ester test solution. DETAILED DESCRIPTION
[0020] The technical solutions of the present invention are further described below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. Unless otherwise specified, the methods used in the present invention are all conventional production methods; the raw materials used are all commercially available products. Percentages used in the present invention are by mass unless otherwise specified. The electronic balance used in the following examples is a Sartorius Secura 225D-1CN. Except for the intermediate precision experiments, which used a Waters ARC coupled with a Waters 2489 UV detector, the high-performance liquid chromatograph used in the remaining examples was a Waters W2695 coupled with a Waters 2489 UV detector. The chromatographic column used was a Shimadzu Shim-pack GIST Amide (4.6 × 250 mm, 5 μm) column. In the following tables, "ND" indicates not detected, and "N / A" indicates not applicable.
[0021] Fmoc-protected amino acids refer to amino acid monomers whose α-amino groups are protected by 9-fluorenylmethyloxycarbonyl groups. The method of the present invention is applicable to the detection of various Fmoc-protected amino acids. For example, the Fmoc-protected amino acids shown in Table 1 below can be detected using the detection method of the present invention, wherein Pbf is the abbreviation for 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl and Boc is the abbreviation for tert-butyloxycarbonyl. The amino acids in Table 1 were all purchased from Chengdu Zhengyuan Biochemical Technology Co., Ltd.: Table 1
[0022] The present invention utilizes a high-performance liquid chromatograph coupled with a UV detector to detect N-hydroxysuccinimide in Fmoc-protected amino acids. The chromatographic column utilizes a HILIC Amide column with an alkylamide stationary phase, mobile phase A is 0.1% (v / v) aqueous phosphoric acid, and mobile phase B is acetonitrile. By selecting a suitable chromatographic column, detector, and mobile phase, the present invention enables the detection of N-hydroxysuccinimide in Fmoc-protected amino acids using liquid chromatography. This addresses the prior art issue of poor retention of HOSU in conventional reversed-phase chromatography columns and allows direct detection of N-hydroxysuccinimide in Fmoc-protected amino acids, filling the current gap in N-hydroxysuccinimide detection for Fmoc-protected amino acids. Furthermore, the present detection method has a low detection limit, making it more suitable for the detection of trace substances. In a preferred embodiment of the present invention, the chromatographic column is a Shim-pack GIST Amide (4.6 mm × 250 mm, 5 μm) or an InertSustain Amide (4.6 mm × 250 mm, 5 μm) or a ChromCore HILIC Amide (4.6 mm × 250 mm, 5 μm). More preferably, a Shim-pack GIST Amide (4.6 mm × 250 mm, 5 μm) is used, as this further expands the applicable range of the chromatographic column and reduces costs.
[0023] The liquid chromatograph used in the method of the present invention can be a Waters W2695 or ARC high-performance liquid chromatograph, or other manufacturer's model. The detector can be a Waters 2998 diode array detector or 2489 UV detector, or other manufacturer's model, as long as it covers the detection wavelength of the HOSU. In a preferred embodiment of the present invention, the detector used in the present invention is a diode array detector.
[0024] The mobile phase of the present invention is a common system. The ultraviolet absorption wavelength of HOSU is 220 nm, and the ultraviolet absorption wavelengths of acetonitrile and phosphoric acid are both lower than 200 nm. It can elute the target substance / impurity well without affecting the detection of the target substance / impurity.
[0025] In a preferred embodiment of the present invention, the diluent used is acetonitrile. Fmoc-protected amino acids are hydrophobic compounds that are insoluble in water but soluble in organic solvents. Using acetonitrile as a diluent to prepare reference and test solutions not only effectively dissolves the Fmoc-protected amino acids, but also has a UV cutoff wavelength below 200 nm, which does not affect the detection of target substances / impurities.
[0026] In a preferred embodiment of the present invention, the injection volume of the HPLC is 20 μL, which is applicable to most mainstream ordinary HPLC instruments on the market. At this injection volume, the detection limit of HOSU can reach 0.18 μg / mL. Based on this detection limit, those skilled in the art can appropriately adjust the concentration of the test solution according to actual conditions to meet a wider range of detection needs. The flow rate of the present invention is 0.7 to 0.9 mL / min, and the column temperature is 28 to 32°C. More preferably, the flow rate is 0.8 mL / min and the column temperature is 30°C. By further optimizing the chromatographic conditions, the method of the present invention can have better reproducibility, better accuracy, and lower detection limit.
[0027] In a preferred embodiment of the present invention, gradient elution is performed according to the following Table 2: Table 2
[0028] The entire analysis time is only 15 minutes, which is short and efficient.
[0029] In a preferred embodiment of the present invention, the content of N-hydroxysuccinimide in the Fmoc-protected amino acid is calculated by the external standard method.
[0030] The detection method of the present invention can well separate the target peak from the solvent peak, and the target peak has a good peak shape, strong specificity, high sensitivity, good precision, good stability, strong durability, and good reproducibility. It can quickly and accurately detect the residual amount of N-hydroxysuccinimide in Fmoc-protected amino acids.
[0031] The present invention is described in detail below using the 13 Fmoc-protected amino acids listed in Table 1 as examples.
[0032] Example 1: Preparation of solution Blank solution / diluent: acetonitrile.
[0033] Reference solution: Accurately weigh approximately 25 mg of HOSU (Sichuan Puxi'ao Standard Material Technology Co., Ltd., H2408001) into a 100 mL volumetric flask. Dilute to the mark with diluent and shake well to prepare the reference stock solution. The actual concentration is calculated based on the weight of the HOSU weighed. Transfer 0.2 mL of the reference stock solution to a 20 mL volumetric flask, add diluent to the volume, and mix thoroughly to prepare the reference solution.
[0034] Test solution: Accurately weigh approximately 25 mg of each Fmoc-protected amino acid listed in Table 1 and place into separate 10 mL volumetric flasks. Dilute to the mark with diluent and shake well. The actual concentration is calculated based on the weight of the Fmoc-protected amino acid actually weighed.
[0035] Each linear solution: Prepare each linear solution according to the following Table 3: Table 3
[0036] Quantitation limit solution: The linear solution with a concentration level of 20% was used as the quantitation limit solution.
[0037] Detection limit solution: Take 3.3 mL of quantification limit solution, dilute it with diluent and make up to 10 mL, then shake well.
[0038] 100% accuracy solution (test sample spike solution): Accurately weigh approximately 25 mg of each Fmoc-protected amino acid listed in Table 1 and place them in 10 mL volumetric flasks. Pipette 0.1 mL of the reference stock solution into each of the above volumetric flasks, dilute to the mark with diluent, and shake well. Prepare 6 replicates of each Fmoc-protected amino acid.
[0039] Example 2: Detection conditions A Waters 2695 liquid chromatograph coupled with a Waters 2998 diode array detector was used to scan the N-hydroxysuccinimide reference solution at all wavelengths, and the optimal detection wavelength was determined to be around 200 nm. While ensuring that the sensitivity met the requirements, the final detection wavelength was selected to be 220 nm. Furthermore, after determining the detection wavelength, the detection was performed according to the detection conditions shown in Table 4 below: Table 4
[0040] Example 3: System Applicability The reference solution was injected 5 times according to the detection conditions of Example 2. The HOSU determination results are shown in Table 5: Table 5
[0041] As can be seen from Table 5, after 5 injections of the reference solution, the RSD of the peak area was 0.4%, which was less than 1%, indicating good system applicability.
[0042] Example 4: Specificity According to the detection conditions of Example 2, the blank solution, the reference solution, and 13 portions of the Fmoc-protected amino acid test solution were injected once each. The results are shown in Table 6: Table 6
[0043] Among them, the chromatogram of the blank solution is as follows Figure 1 As shown in Figure 2, the blank solution does not interfere with the determination of impurity peaks. The chromatogram of the reference solution is shown in Figure 2. Figure 2 As shown in Figure 2, the retention time of impurity HOSU can be determined by comparing the reference solution with the blank solution. Figure 3 As shown, the retention time of the main peak of each Fmoc-protected amino acid in the test solution is significantly different from the retention time of HOSU, and the main peak of the amino acid does not interfere with the determination of impurities. Figure 15 is the chromatogram corresponding to the Fmoc-glycine test solution, Figure 16 This is the chromatogram corresponding to the Fmoc-L-glutamic acid 1-tert-butyl ester test solution.
[0044] Example 5: Linear According to the detection conditions of Example 2, each linear solution was injected once, and the results are shown in Table 7: Table 7
[0045] The linear relationship diagram obtained from Table 7 is as follows Figure 4 As shown, the linear correlation coefficient R 2 The linearity is good.
[0046] Example 6: Detection Limit The detection limit solution was injected once according to the detection conditions of Example 2. The results are shown in Table 8: Table 8
[0047] The detection limit of the present invention is 0.18 μg / mL, and the present invention has a lower detection limit.
[0048] Example 7: Limit of Quantitation The quantitative limit solution was injected 6 times according to the detection conditions of Example 2. The results are shown in Table 9: Table 9
[0049] It can be seen from Table 9 that the quantification limit of HOSU meets the requirements.
[0050] Example 8: Accuracy, Precision, and Repeatability According to the detection conditions of Example 2, the six parallel sample-spiked solutions prepared in Example 1 were injected once respectively, and the concentration of HOSU in each test solution (sample solution, sample-spiked solution) and the content of HOSU in the sample-spiked solution were determined using the following formulas (I) and (II): C: concentration of HOSU in the test solution; Cs: concentration of HOSU in the reference solution; Au: peak area of HOSU in the test solution; As: average peak area of HOSU in reference solution; m S : The weight of HOSU when preparing the reference solution (mg); m u : The weight of the test sample (mg); V S : Volume of reference solution (mL); V u : Volume of the test solution (mL); R: HOSU content (%).
[0051] The above tests were performed by the same experimenter using the same equipment on the same day. The HOSU test results are shown in Tables 10.1 to 10.13: Table 10.1 Fmoc-glycine
[0052] Table 10.2 Fmoc-Pbf-L-arginine
[0053] Table 10.3 Fmoc-L-aspartic acid beta-tert-butyl ester
[0054] Table 10.4 Fmoc-N-trityl-L-glutamine
[0055] Table 10.5 Fmoc-L-isoleucine
[0056] Table 10.6 Fmoc-L-leucine
[0057] Table 10.7 Fmoc-L-phenylalanine
[0058] Table 10.8 Fmoc-O-tert-butyl-L-serine
[0059] Table 10.9 Fmoc-O-tert-butyl-L-threonine
[0060] Table 10.10 Fmoc-L-tryptophan (Boc)
[0061] Table 10.11 Fmoc-O-tert-butyl-L-tyrosine
[0062] Table 10.12 Fmoc-L-valine
[0063] Table 10.13 Fmoc-L-glutamic acid 1-tert-butyl ester
[0064] As can be seen from Tables 10.1 to 10.13, the detection method of the present invention has excellent accuracy, precision and repeatability.
[0065] Example 9: Intermediate Precision Different personnel, at different times, used liquid chromatographs of the same manufacturer and different types of columns, and chromatographic columns of the same model and different batches. Test solutions were prepared in the same manner as in Example 1. Each sample-spiked solution was injected once. The results were combined with the detected amounts of the sample-spiked solutions in Example 8 to calculate the RSD. The results are shown in Tables 11.1 to 11.13: Table 11.1 Fmoc-glycine
[0066] Table 11.2 Fmoc-Pbf-L-arginine
[0067] Table 11.3 Fmoc-L-aspartic acid beta-tert-butyl ester
[0068] Table 11.4 Fmoc-N-trityl-L-glutamine
[0069] Table 11.5 Fmoc-L-isoleucine
[0070] Table 11.6 Fmoc-L-leucine
[0071] Table 11.7 Fmoc-L-phenylalanine
[0072] Table 11.8 Fmoc-O-tert-butyl-L-serine
[0073] Table 11.9 Fmoc-O-tert-butyl-L-threonine
[0074] Table 11.10 Fmoc-L-tryptophan (Boc)
[0075] Table 11.11 Fmoc-O-tert-butyl-L-tyrosine
[0076] Table 11.12 Fmoc-L-valine
[0077] Table 11.13 Fmoc-L-glutamic acid 1-tert-butyl ester
[0078] It can be seen from Tables 11.1 to 11.13 that the results of the detection method of the present invention are well reproducible.
[0079] Example 10: Solution Stability Following the test conditions of Example 2, reference solution and test sample spiked solution were prepared and stored under analytical conditions (room temperature). Samples were injected at different time points. The rate of change of the HOSU peak area in the reference solution at different injection time points relative to the HOSU peak area in the reference solution at 0 h, as well as the rate of change of the HOSU content in the test sample spiked solution at different injection time points relative to the HOSU content at 0 h, were calculated. The results are shown in Tables 12.1 to 12.14: Table 12.1 Reference solution
[0080] Table 12.2 HOSU in Fmoc-glycine
[0081] Table 12.3 HOSU in Fmoc-Pbf-L-arginine
[0082] Table 12.4 HOSU in Fmoc-L-aspartic acid beta-tert-butyl ester
[0083] Table 12.5 HOSU in Fmoc-N-trityl-L-glutamine
[0084] Table 12.6 HOSU in Fmoc-L-isoleucine
[0085] Table 12.7 HOSU in Fmoc-L-leucine
[0086] Table 12.8 HOSU in Fmoc-L-phenylalanine
[0087] Table 12.9 HOSU in Fmoc-O-tert-butyl-L-serine
[0088] Table 12.10 HOSU in Fmoc-glycine
[0089] Table 12.11 HOSU in Fmoc-L-tryptophan (Boc)
[0090] Table 12.12 HOSU in Fmoc-O-tert-butyl-L-tyrosine
[0091] Table 12.13 HOSU in Fmoc-L-valine
[0092] Table 12.14 HOSU in Fmoc-L-glutamic acid 1-tert-butyl ester
[0093] As can be seen from Tables 12.1 to 12.14, the stability of the reference solution and the test sample spiked solution is good.
[0094] Example 11: Durability The durability of the method of the present invention was verified mainly from the changes in column temperature and flow rate and different chromatographic columns. The conditions were adjusted according to Table 13 below. Except for the conditions listed in Table 13, the other conditions were consistent with those in Example 2.
[0095] Table 13
[0096] The experiment was conducted under the conditions listed in Table 13 above. For each condition, a blank solution was injected until the system was in equilibrium, the reference solution was injected five times, and the test solution was injected once. The results are shown in Table 14: Table 14
[0097] As can be seen from Table 14, the method of the present invention has good durability.
[0098] Comparative Example 1 Gas chromatography was used to detect HOSU in Fmoc protected amino acids. Solution preparation: dimethyl sulfoxide (DMSO) was used as a blank solution, and a reference solution was prepared using DMSO as a diluent in the same manner as in Example 1. Gas chromatography conditions: a DB-WAX column (Agilent) was used, the injection port temperature was set to 200°C, a hydrogen flame ionization detector (FID) was used as the detector, the detector temperature was set to 280°C, an initial temperature of 100°C was maintained for 1 minute, and the temperature was increased to 240°C at a rate of 10°C / min and maintained for 20 minutes. The blank solution and reference solution were injected, and the results were as follows: Figure 5 As shown in the figure, no obvious peak was observed. It can be seen that gas chromatography is not suitable for the determination of HOSU in Fmoc-protected amino acids.
[0099] Comparative Example 2 The blank solution and reference solution in Example 1 were tested under the same detection conditions as in Example 2, except that an Agilent SB-C8, 4.6×250 mm, 5 μm column was used. The results are shown in FIG. Figure 6 As shown in the figure, HOSU showed double peaks, and this column was not suitable for the determination of HOSU in Fmoc-protected amino acids.
[0100] Comparative Example 3 The blank solution and reference solution in Example 1 were tested under the same detection conditions as in Example 2, except that an Agilent Polaris 3 NH2, 4.6×250 mm, 5 μm column was used. The results showed that the HOSU peak was not only interfered by the blank solution, but also had a strange peak shape, as shown in Figure 2. Figure 7 This column is not suitable for the determination of HOSU in Fmoc-protected amino acids.
[0101] Comparative Example 4 The blank solution and reference solution in Example 1 were tested under the same detection conditions as in Example 2 except that Shiseido CAPCLEE PAK ADME-HR, 4.6×250 mm, 5 μm was used as the chromatographic column. The results are shown in FIG. Figure 8 As shown, the HOSU peak partially overlaps with the solvent peak of the blank solution, and this column is not suitable for the determination of HOSU in Fmoc-protected amino acids.
[0102] Comparative Example 5 The reference solution in Example 1 was measured using the same detection conditions as in Example 2, except that 0.1% (v / v) trifluoroacetic acid aqueous solution was used as mobile phase A. The results are shown in Table 15. Figure 9 As shown in the figure, the target peak in the HOSU reference solution has a poor peak shape and a small bifurcation.
[0103] Table 15
[0104] Comparative Example 6 The reference solution and blank solution in Example 1 were tested under the same detection conditions as in Example 2, except that 20 mM ammonium formate solution was used as mobile phase A. The flow rate was set to 0.8 mL / min and the elution gradient shown in Table 16 was used. The results were as follows: Figure 10 As shown, an inverted peak appeared in the blank solution at about 4 minutes, and the peak of the control solution was at the tail of the inverted peak. Not only was the HOSU peak easily affected by the inverted peak, it was also difficult to integrate it. Compared with the blank solution, the HOSU response was not only low and could not meet the experimental requirements, but the peak also followed the solvent peak closely, resulting in poor separation effect.
[0105] Table 16
[0106] Comparative Example 7 The reference solution was prepared using the same detection conditions as in Example 2, except that DMSO was used as the diluent to dissolve HOSU. The comparison results of the reference solution using DMSO as the diluent and the reference solution using 100% acetonitrile as the diluent are shown in Figure 2. Figure 11As shown in the figure, no HOSU peak was observed after injection of the reference solution using DMSO as the diluent because it was covered by the solvent peak DMSO and the diluent was not available.
[0107] Comparative Example 8 The reference solution was tested under the same conditions as in Example 2, except that 0.04% (v / v) ammonia water was used as a diluent to dissolve HOSU and prepare the reference solution. Figure 12 As shown, the HOSU peak diverged and this diluent was unusable.
[0108] Comparative Example 9 The blank solution and reference solution in Example 1 were measured using the same detection conditions as in Example 2, except that acetonitrile: 0.1% (v / v) phosphoric acid aqueous solution = 75:25 (v / v) was used for isocratic elution. Figure 13 As shown, the HOSU peak is interfered by the blank solvent peak.
[0109] Comparative Example 10 The blank solution and reference solution in Example 1 were measured using the same detection conditions as in Example 2, except that acetonitrile: 0.1% (v / v) trifluoroacetic acid aqueous solution = 95:5 (v / v) was used for isocratic elution. Figure 14 As shown, the HOSU peak is interfered by the blank solvent peak.
[0110] From the above examples and comparative examples, it can be seen that the present invention uses a HILICAmide column with an alkylamide group as the stationary phase, 0.1 (v / v)% phosphoric acid aqueous solution as the mobile phase A, pure acetonitrile as the mobile phase B for gradient elution, and acetonitrile as the diluent to enable the HOSU peak in the Fmoc-protected amino acid to be well separated from the solvent peak. The main peak of the Fmoc-protected amino acid in the test sample does not interfere with the determination of HOSU, and the HOSU peak shape is good and the response is good. The linear correlation coefficient R 2 The linearity is 0.9998, with high sensitivity, good repeatability, and good reproducibility of results. The reference solution and the test sample spiked solution of the present invention have good stability, can extend the shelf life of the sample, reduce analytical errors caused by sample degradation or deterioration, and the good solution stability ensures that the components of the solute and solvent do not change during the analysis process, reducing errors caused by solute decomposition or volatilization, and improving the accuracy of the analysis. The detection method of the present invention has strong specificity, high sensitivity, good precision, good stability, and strong durability, and can accurately detect the HOSU residue in Fmoc-protected amino acids.
[0111] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.
Claims
1. A method for detecting N-hydroxysuccinimide in Fmoc-protected amino acids, characterized in that: The method comprises the following steps: 1) Prepare N-hydroxysuccinimide reference solution using diluent; 2) Prepare the test solution by dissolving the Fmoc-protected amino acid in a diluent. 3) testing the reference solution and the test solution using liquid chromatography; The chromatographic conditions include: The chromatographic column is a HILIC Amide column with alkylamide as the stationary phase; Mobile phase A was 0.1% (v / v) phosphoric acid in water, and mobile phase B was acetonitrile; The detector is a UV detector.
2. The detection method according to claim 1, wherein The diluent is acetonitrile.
3. The detection method according to claim 1, wherein The ultraviolet detector is a diode array detector.
4. The detection method according to claim 1, wherein The chromatographic column is Shim-pack GISTAmide, with a specification of 4.6 mm×250 mm, 5 μm, or InertSustain Amide, with a specification of 4.6 mm×250 mm, 5 μm, or ChromCore HILIC Amide, with a specification of 4.6 mm×250 mm, 5 μm.
5. The detection method according to claim 1, wherein The detection wavelength of the N-hydroxysuccinimide is 220 nm.
6. The detection method according to claim 1, characterized in that The chromatographic conditions also include an injection volume of 20 μL, a flow rate of 0.7 to 0.9 mL / min, and a column temperature of 28 to 32°C.
7. The detection method according to claim 1, wherein The chromatographic conditions also include a flow rate of 0.8 mL / min and a column temperature of 30°C.
8. The detection method according to claim 1, wherein: Adopt gradient elution mode, the elution gradient is: 。 9. The detection method according to claim 1, wherein The Fmoc-protected amino acids include Fmoc-glycine, Fmoc-Pbf-L-arginine, Fmoc-L-aspartic acid beta-tert-butyl ester, Fmoc-N-trityl-L-glutamine, Fmoc-L-isoleucine, Fmoc-L-leucine, Fmoc-L-phenylalanine, Fmoc-O-tert-butyl-L-serine, Fmoc-O-tert-butyl-L-threonine, Fmoc-L-tryptophan (Boc), Fmoc-O-tert-butyl-L-tyrosine, Fmoc-L-valine and Fmoc-L-glutamate 1-tert-butyl ester.
10. The detection method according to any one of claims 1 to 9, characterized in that: The content of N-hydroxysuccinimide in Fmoc-protected amino acids was calculated using the external standard method.