High performance liquid chromatography analysis method for N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine
By combining conventional HPLC methods (HPLC-DAD) with silica-bonded diol-based stationary phases and gradient elution, the problem of identifying and determining N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine in existing technologies has been solved, achieving efficient and low-cost separation and quantification.
Patent Information
- Application Number
- CN202511699556.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-19
- Publication Date
- 2026-02-10
AI Technical Summary
Existing technologies cannot efficiently and cost-effectively identify and determine N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine; the operation is complex and the equipment cost is high.
The conventional HPLC method (HPLC-DAD) was used, with silica gel bonded diol groups as the stationary phase and methanol, acetonitrile and water as the mobile phase, gradient elution, and ultraviolet detection.
The method achieves efficient separation and accurate quantification of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine, with simple operation, low cost, good specificity and high accuracy.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of chemical analysis, in particular to a high-performance liquid analysis method for N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine. BACKGROUND
[0002] Glycosaminoglycans (GAGs) are a class of linear polysaccharides existing in the extracellular matrix of animal cells, including heparin, chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronic acid (HA), etc. As a key component of the extracellular matrix, GAGs cover multiple dimensions of physiological functions such as tissue construction, signal regulation, and disease prevention, and are key molecules for maintaining body homeostasis. Based on its multi-dimensional biological activity, GAGs have shown great commercial value, and there is no complete replacement of raw materials in the fields of anticoagulant therapy (heparin), joint repair (CS), and skin care (HA). With the intensification of aging (increase in patients with osteoarthritis) and the upgrading of medical beauty needs (popularization of HA skin care products), the global GAGs market size has exceeded 20 billion US dollars in 2024, and is expected to reach 35 billion US dollars in 2030. Currently, commercialized GAGs raw materials are mainly extracted from animal tissues, such as heparin extracted from pig small intestine, CS extracted from pig cartilage, DS extracted from pig skin, and HA extracted from bovine eye. However, animal-derived GAGs face a high risk of heterologous immunogenicity in clinical use. In recent years, microbial fermentation to obtain GAGs has received widespread attention and promotion. After a large number of research and clinical use verification, GAGS obtained by microbial fermentation has quality comparable to natural extraction sources, and the risk of heterologous immunogenicity is significantly reduced. With the further improvement of technology iteration and product quality requirements, the synthesis of GAGs or GAGs oligosaccharides of specific length and single component through in vitro enzyme method has received widespread attention and research, and based on its more significant biological activity, higher safety, and significantly reduced quality control difficulty, it is expected to become the third generation of GAGs raw materials.
[0003] The in vitro enzyme synthesis process of GAGs or GAGs oligosaccharides involves two important starting materials, N-acetyl-glucosamine (GlcNAc) and N-trifluoroacetyl-glucosamine (GlcNTFA), with the chemical structures as follows Figure 10The quality of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine directly determines the quality and yield of subsequent intermediates and finished products. Therefore, quality control of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine is crucial. Due to the weak ultraviolet absorption characteristics of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine, there is currently no conventional HPLC method for identification and content determination of the two, which can only be realized by combining TLC with qNMR or HPLC with a general detector. The current method has high equipment cost, complex operation and is not intuitive. SUMMARY
[0004] In view of the limitations of the existing N-acetyl-glucosamine (GlcNAc) and N-trifluoroacetyl-glucosamine (GlcNTFA) detection methods, the present application aims to develop a conventional HPLC method (HPLC-DAD) for identification and content determination of the two.
[0005] Firstly, based on the analysis of the structure and physicochemical properties of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine (high polarity, good water solubility and the presence of weakly absorbing chromophores in the structure, acyl group), HILIC retention and ultraviolet detection potential are possessed.
[0006] The technical scheme of the present application is as follows: the test sample is dissolved with high proportion of acetonitrile; silica gel bonded diol group is used as the stationary phase; methanol, acetonitrile and water are used as the mobile phase for gradient elution; and ultraviolet detection is performed.
[0007] Compared with the prior art, the present application provides a high performance liquid analysis method for N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine, which has the following beneficial effects:
[0008] 1. The high performance liquid analysis method of the present application has good retention for N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine, symmetrical peak shape, and baseline separation of N-acetyl-glucosamine and its related substances, and N-trifluoroacetyl-glucosamine and its related substances. Compared with the prior art, the method has good specificity, high accuracy, good precision, simple operation and lower cost. BRIEF DESCRIPTION OF DRAWINGS
[0009] Figure 1 is the high performance liquid chromatogram of Example 1;
[0010] Figure 2 is the high performance liquid chromatogram of Example 2;
[0011] Figure 3 is the high performance liquid chromatogram of Example 3;
[0012] Figure 4 is the high performance liquid chromatogram of Example 4;
[0013] Figure 5 High performance liquid chromatogram of Example 5;
[0014] Figure 6 High performance liquid chromatogram of Example 6;
[0015] Figure 7 High performance liquid chromatogram of Example 7;
[0016] Figure 8 High performance liquid chromatogram of Example 8;
[0017] Figure 9 High resolution mass spectrum of GlcNTFA of Example 10;
[0018] Figure 10 Structures of N-acetyl-glucosamine (GlcNAc) and N-trifluoroacetyl-glucosamine (GlcNTFA). DETAILED DESCRIPTION
[0019] The present application will demonstrate the beneficial effects of the present application through examples. Those skilled in the art will know that these examples are exemplary rather than limiting. These examples will not limit the scope of the present application in any way. The test samples used in the present application are self-made by the applicant, the reference samples are purchased through the market channel, and the mobile phase used is chromatographic grade solvent or ultrapure water.
[0020] Example 1
[0021] This example provides a high performance liquid chromatography method for detecting GlcNAc in GlcNAc synthesis reaction, which is as follows:
[0022] Preparation of test sample solution: dilute the acetonitrile of GlcNAc synthesis reaction solution by 5 times to a final concentration of 1 mg / ml;
[0023] Stationary phase, TriartDiol-HILIC, 4.6mm*250mm, 5μm;
[0024] Injection volume: 5μl;
[0025] Detection wavelength: 214nm;
[0026] Flow rate: 0.5ml / min;
[0027] Mobile phase A, 40%ACN-20%MeOH-40%H2O (v / v); mobile phase B, 80%ACN-20%MeOH (v / v).
[0028] Gradient program:
[0029] Time (min) B% 0 70 5 70 10 60 10.5 0 20 0 20.5 70 25 70
[0030] Results
[0031] The test sample solution was detected by the above method, and the obtained chromatogram is shown in Figure 1 From the figure, it can be seen that the GlcNAc peak type is symmetrical and separated from the key related substance ethyl acetate baseline.
[0032] Among them, Figure 1 The retention time of 5.843 min is the ethyl acetate peak, and the retention time of 8.340 min is the GlcNAc peak.
[0033] Example 2
[0034] Compared with Example 1, the difference lies in that the test sample is the GlcNTFA synthesis reaction solution, and is diluted by acetonitrile to a concentration of 1 mg / ml in GlcNTFA.
[0035] Results
[0036] After the test sample solution was detected, the obtained high performance liquid chromatogram is shown in Figure 2 From the figure, it can be seen that the GlcNTFA peak type is symmetrical and separated from the key related substance ethyl trifluoroacetate baseline.
[0037] Among them, Figure 2 In the above method, the retention time of 5.527 min is the ethyl trifluoroacetate peak, and the retention time of 6.613 min is the GlcNTFA peak.
[0038] Example 3
[0039] Compared with Example 1, the difference lies in that the flow rate is 1 ml / min;
[0040] Results
[0041] After the test sample solution was detected, the obtained chromatogram is shown in Figure 3 From the figure, it can be seen that after the flow rate is increased to 1 ml / min, the retention time of GlcNAc is reduced, but the peak type is symmetrical, and the separation degree from the key related substance ethyl acetate is good.
[0042] Among them, Figure 3 In the above method, the retention time of 2.943 min is the ethyl acetate peak, and the retention time of 4.207 min is the GlcNAc peak.
[0043] Example 4
[0044] Compared with Example 2, the flow rate is 1 ml / min;
[0045] Results
[0046] After the test sample solution was detected, the obtained chromatogram isFigure 4 As shown in the figure, when the flow rate is increased to 1 ml / min, the retention time of GlcNTFA decreases, but the peak shape is symmetrical and the separation from the key related substance ethyl trifluoroacetate is good.
[0047] in, Figure 4 In the sample, the peak at retention time of 2.783 min is ethyl trifluoroacetate, and the peak at retention time of 3.330 min is GlcNTFA.
[0048] Example 5
[0049] The difference from Example 2 is that the detection wavelength is 200 nm; the chromatogram of the test solution is attached. Figure 5 As shown in the figure, compared with the 214nm wavelength, the signal-to-noise ratio of GlcNTF is reduced, but the response is still much higher than the limit of quantitation, and the peak shape is symmetrical, with good separation from the key related substance ethyl trifluoroacetate.
[0050] in, Figure 5 In the sample, the peak at retention time of 5.527 min is ethyl trifluoroacetate, and the peak at retention time of 6.610 min is GlcNTFA.
[0051] Example 6
[0052] The difference from Example 2 is that the injection volume was 20 μl; the chromatogram of the test solution is as follows. Figure 6 As shown in the figure, the response of GlcNTFA is significantly enhanced, the peak shape is symmetrical, and it is baseline separated from the key related substance ethyl trifluoroacetate.
[0053] in, Figure 6 In the sample, the peak at retention time of 5.530 min is ethyl trifluoroacetate, and the peak at retention time of 6.647 min is GlcNTFA.
[0054] Example 7
[0055] The difference from Example 1 is that the flow rate is 1.2 ml / min;
[0056] The chromatogram of the test solution is shown below. Figure 7 As shown in the figure, the retention time of GlcNAc is earlier, but the peak shape is symmetrical and it is separated from the baseline of the key related substance ethyl acetate.
[0057] in, Figure 7 In the sample, the ethyl acetate peak was observed at a retention time of 2.457 min, and the GlcNAc peak was observed at a retention time of 3.503 min.
[0058] Example 8
[0059] The difference from Example 2 is that the flow rate is 1.2 ml / min;
[0060] result
[0061] The chromatogram of the test solution is as follows: Figure 8 As shown in the figure, the retention time of GlcTFA is earlier, but the peak shape is symmetrical and it is baseline separated from the key related substance ethyl trifluoroacetate.
[0062] in, Figure 8 In the sample, the ethyl acetate peak was observed at a retention time of 2.323 min, and the GlcNTFA peak was observed at a retention time of 2.777 min.
[0063] Example 9
[0064] This embodiment verifies the linearity, accuracy, and precision of the method, as detailed below:
[0065] Preparation of reference solution: Weigh an appropriate amount of GlcNTFA reference standard accurately, dissolve and dilute with 85% acetonitrile to a concentration of 1 mg / ml;
[0066] Preparation of precision solution: Dilute the GlcNTFA test solution 5 times with acetonitrile and prepare six parallel solutions;
[0067] Preparation of linear solutions: Accurately weigh an appropriate amount of GlcNTFA reference standard, dissolve it in 85% acetonitrile, and serially dilute it to final concentrations of 2.0 mg / ml, 1.5 mg / ml, 1.0 mg / ml, 0.75 mg / ml, 0.25 mg / ml, and 0.1 mg / ml.
[0068] Preparation of spiked recovery solution: Dilute the GlcNTFA test solution 5 times with acetonitrile, and dissolve the GlcNTFA reference standard in the same solution to make the spiked GlcNTFA concentration approximately 80%, 100% and 120% of the GlcNTFA concentration in the test sample, respectively. Prepare three parallel solutions for each concentration level.
[0069] result
[0070] Accurately measure the above solutions and inject them into the high-performance liquid chromatograph (HPLC). Detect the solutions according to the described method. Calculate the concentration based on peak area using the external standard method. The linearity, system precision, method precision, and spiked recovery results are shown in Appendix Table 1. As can be seen from the table, the method exhibits good linearity, high system and method precision, and high accuracy.
[0071] Appendix 1: Summary Table of Method Validation Results
[0072] Verification item Result Linear Linear equation is y = 46.08x + 1.1028, R² = 0.9996 System precision The GlcNTFA peak area RSD of the same sample solution continuously injected for 5 times was 1.07%, and the retention time RSD was 0.98% Method precision The GlcNTFA concentration RSD of six sample solutions was 1.13% Spiked recovery rate The average spiked recovery rate at the 80% concentration level was 99.1%, the average spiked recovery rate at the 100% concentration level was 99.6%, and the average spiked recovery rate at the 120% concentration level was 98.7%,
[0073] Example 10
[0074] This embodiment mainly illustrates the method and mass spectrometry tandem for mass spectrometry identification of GlcNTFA, and the specific process is as follows;
[0075] The difference from Example 2 is that 0.1% formic acid (v / v) was added to mobile phases A and B, respectively.
[0076] Furthermore, a high-resolution mass spectrometer detector is connected in series after the DAD detector, with positive ion detection mode and a mass range (m / z) of 150–2000 Da;
[0077] result
[0078] The obtained GlcNTFA mass spectrum is as follows Figure 9 As shown, the calculated and measured accurate mass number is 275.0599 Da, which is consistent with the theory.
[0079] This invention illustrates the detailed process equipment and process flow through the above embodiments. However, this invention is not limited to the detailed process equipment and process flow described above, meaning that this invention does not necessarily depend on the detailed process equipment and process flow to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, all fall within the protection scope and disclosure scope of this invention.
Claims
1. A high-performance liquid chromatography (HPLC) method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine, characterized in that, Includes mobile phase A and mobile phase B; The mobile phase A is (35%–45% acetonitrile) – (15%–25% methanol) – 40% water (volume ratio). Mobile phase B is (10%–20% methanol) – (80%–90% acetonitrile).
2. The high-performance liquid chromatography (HPLC) method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, The gradient elution program was as follows: 0–5 min: 30% A; 5–10 min: 30% A–40% A; 10–10.5 min: 40%–100% A; 10.5–20 min: 100% A; 20–20.5 min: 100% A–30% A; 20.5–25 min: 30% A.
3. The high-performance liquid chromatography (HPLC) method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, Fine-tuning based on claim 2 includes shortening or lengthening the gradient time and increasing or decreasing the proportion of A.
4. The high-performance liquid chromatography (HPLC) method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, The flow rate was 0.5–1.2 ml / min, and the detection wavelength was 190–220 nm.
5. The high-performance liquid chromatography (HPLC) method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, The test sample was dissolved in acetonitrile-water solution with a concentration greater than 60%, and the concentration of the test sample was 0.5–1.5 mg / ml.
6. The high-performance liquid chromatography (HPLC) method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, The injection volume is 5–20 μl.
7. The high-performance liquid chromatography (HPLC) method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, Diol groups bonded to silica gel serve as the stationary phase.
8. The high-performance liquid chromatography method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, The mobile phase may or may not contain an acidic regulator, wherein the acidic regulator includes, but is not limited to, formic acid, acetic acid, and phosphoric acid.
9. The high-performance liquid chromatography (HPLC) method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, This method was used to identify and determine the content of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine.
10. The high-performance liquid chromatography method for the analysis of N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine according to claim 1, characterized in that, This method was used to perform mass spectrometry analysis on N-acetyl-glucosamine and N-trifluoroacetyl-glucosamine.