Method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector

Abstract

The invention discloses a method for quantitatively detecting GDP-L-fucose based on a high performance liquid chromatography-diode array detector, the method adopts high performance liquid chromatography for analysis, and the chromatographic conditions are as follows: an Agilent C18 chromatographic column; the method comprises the following steps: carrying out gradient elution by taking a 2.5-3.0 g / L 2.72 g / L monopotassium phosphate aqueous solution (containing 0.2-0.3% of tetrabutylammonium bromide and adjusting the pH value to 3.5-4.5 by phosphoric acid) as a mobile phase A and acetonitrile as a mobile phase B, and adopting a diode array detector as a detector and a detection wavelength of 254 nm. The method is low in detection limit and good in precision and accuracy, spectral information can be provided for qualitative and quantitative analysis of GDP-L fucose, the reaction condition in the GDP-L-fucose synthesis process can be monitored in real time, and an analysis method is provided for improvement of the purity and yield of GDP-L-fucose and screening of multifunctional FKP enzyme activity.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and is a method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detection (HPLC-DAD). Background Art

[0002] GDP-L-fucose is a key substrate for glycosylation modification and is widely used in the fields of medicine, food, and health products, with huge market development potential. Since GDP-L-fucose plays an important role in related biological reactions and human treatment of related diseases and is expensive, its purity improvement and quantitative analysis are crucial to drug development. At present, its detection methods mostly rely on thin layer chromatography, gas chromatography, ordinary liquid chromatography, and liquid chromatography-mass spectrometry. GDP-L-fucose has a very large polarity and is not easy to volatilize. Derivatization is required before gas chromatography detection, and the operation is cumbersome. Thin layer chromatography is a normal phase chromatography method, which has low separation of sugar compounds, and it is difficult to accurately identify and quantify substances with high polarity. The laboratory environment and operator level have a great influence on the experimental results. Liquid chromatography-mass spectrometry has high sensitivity and selectivity, but the samples analyzed need to be purified and the reaction situation cannot be monitored in real time. For example, Wang Hongchao et al. (Study on the production of GDP-L-fucose by Mortierella alpina fermentation [J]. Food Science and Technology, 2016, 41(9):3; Production of GDP-L-fucose by Mortierella alpina fermentation [J]. Food Science And Technology, 2016, 41(9):3.) used UV-liquid chromatography and mass spectrometry to detect the content and purity of GDP-L-fucose produced by fermentation. This method requires the configuration of a higher ion exchange column to improve the separation degree, which is cumbersome to operate. In addition, the use of liquid chromatography-mass spectrometry requires the sample to be purified in advance, resulting in high costs. Wei Wang (Chemoenzymatic synthesis of GDP-L-fucose and the Lewis Xglycan derivatives [J]. Journal of PNAS, 2009, 106(38):16097.) et al. used thin layer chromatography to analyze the crude reaction mixture of GDP-L-fucose. This method requires the preparation of cumbersome developing agents and colorants, resulting in severe tailing of the chromatogram. Therefore, it is urgent to develop a simple, sensitive, accurate, rapid and economical analytical method to achieve the quantitative detection of GDP-L-fucose.

[0003] Purpose of the Invention

[0004] The purpose of the present invention is to provide a simple, sensitive, accurate and easy-to-operate GDP-L-fucose detection method, which adopts a high performance liquid chromatography-diode array detector for detection, and can not only provide spectral information for qualitative and quantitative analysis of GDP-L-fucose, but also can monitor the reaction conditions in the GDP-L-fucose synthesis process in real time, and provide an analysis method for improving the purity and yield of GDP-L-fucose and screening the multifunctional FKP enzyme activity.

[0005] The method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector provided by the present invention comprises the following steps:

[0006] Step 1: Prepare GDP-L-fucose test solution.

[0007] Step 2: Prepare GDP-L-fucose standard working solutions of different mass concentrations.

[0008] Step 3: High performance liquid chromatography was used to detect GDP-L-fucose standard working solutions of different mass concentrations. The chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; 0.2% to 0.3% of tetrabutylammonium bromide by mass was added to a 2.5 to 3.0 g / L potassium dihydrogen phosphate aqueous solution and the pH value was adjusted to 3.5 to 4.5 with phosphoric acid as mobile phase A, acetonitrile was used as mobile phase B, gradient elution was performed, the detector was a diode array detector, and the elution program was: 0 to 5 minutes: The volume percentage of mobile phase B is 10%; 5 to 25 minutes: the volume percentage of mobile phase B increases from 10% to 35%; 25 to 28 minutes: the volume percentage of mobile phase B decreases from 35% to 10%; 28 to 30 minutes: the volume percentage of mobile phase B is 10%; the chromatographic peak area is recorded, and a linear regression is performed with the mass concentration of the GDP-L-fucose standard working solution as the abscissa and the corresponding chromatographic peak area as the ordinate, a standard working curve of GDP-L-fucose is established, and a linear regression equation is calculated.

[0009] Step 4: Detect the GDP-L-fucose test solution of step 1 according to the chromatographic conditions of step 3, and calculate the content of GDP-L-fucose in the test solution in combination with the linear regression equation of step 3.

[0010] In the above step 1, when the reaction status during the synthesis of GDP-L-fucose is monitored in real time, the preparation method of the test solution is: the GDP-L-fucose reaction solution obtained by the reaction of adenine nucleoside triphosphate, guanosine triphosphate and L-fucose is allowed to stand at room temperature for 5 to 10 minutes and then centrifuged, the supernatant is diluted with water, filtered with a 0.22 μm filter, and the filtrate is taken to obtain the test solution.

[0011] In the above step 1, when the test sample is a biological sample (such as blood, tissue, etc.), the method for preparing the test solution is: after centrifuging the biological sample, taking the supernatant and using methanol or acetonitrile to precipitate the protein to release GDP-L-fucose from biological macromolecules such as proteins, and then centrifuging to take the supernatant to obtain the test solution.

[0012] In the above step 1, when the test sample is a food or agricultural product sample, the method for preparing the test solution is: after the food or agricultural product sample is crushed, GDP-L-fucose is extracted by ultrasonic extraction or heating reflux extraction with ethanol or water to obtain the test solution.

[0013] Furthermore, in the above step 3, the elution flow rate is preferably 0.8 to 1.2 mL / min.

[0014] Furthermore, in the above step 3, the column temperature is preferably detected to be 25-32°C.

[0015] Furthermore, in the above step 3, the detection wavelength is preferably 254±2 nm.

[0016] The beneficial effects of the present invention are as follows:

[0017] 1. Optimize sample pretreatment process: The present invention has developed a unique extraction and purification step, which can effectively remove various impurities in complex sample matrices and significantly reduce the interference of impurities on GDP-L-fucose detection. Compared with traditional methods, the purity of the target is greatly improved, thereby improving the detection accuracy, providing high-quality samples for subsequent high-performance liquid chromatography analysis, and ensuring the reliability of the test results from the source.

[0018] 2. Systematic optimization of chromatographic conditions: The present invention comprehensively and deeply optimizes a variety of chromatographic conditions. By screening chromatographic columns of different types and specifications, accurately adjusting the composition and proportion of the mobile phase, and precisely controlling the flow rate, the baseline separation of GDP-L-fucose and other coexisting substances is achieved. The obtained chromatographic peaks are symmetrical and sharp, which greatly improves the detection resolution, makes the detection results clearer and more accurate, and solves the problem of poor separation effect in traditional methods.

[0019] 3. Accurately determine the detection wavelength: The present invention innovatively determines the optimal detection wavelength by means of in-depth research on the spectral characteristics of GDP-L-fucose. This measure significantly enhances the detection sensitivity and can detect lower concentrations of targets compared to detection methods without optimized wavelengths, thus broadening the scope of application of this method, especially for the detection of trace amounts of GDP-L-fucose.

[0020] 4. Wide linear range and high sensitivity: The analytical method established by the present invention exhibits an extremely wide linear range, covering a concentration range from as low as 1.005 μg / mL to as high as 21.100 μg / mL, with a linear correlation coefficient of more than 0.9999, and good linear correlation. At the same time, the detection limit is as low as 0.3 μg / mL, which not only ensures high sensitivity but also takes into account the detection needs of samples of different concentrations, and has significant advantages among similar detection methods.

[0021] 5. Excellent precision and accuracy: The relative standard deviation (RSD) of the repeatability experiment of the method of the present invention is less than 0.05%, and the average recovery rate of spiked is 99.6%. These data show that the method has good precision, reliable and stable test results, high accuracy, and can provide reliable test data for scientific research, production and other fields.

[0022] 6. Efficient and rapid detection: This invention successfully shortens the single detection time to less than 30 minutes, greatly improving the detection efficiency compared to traditional detection methods. This not only saves time costs, but also processes more samples per unit time, meeting the needs of modern scientific research and production for high-throughput detection, and has significant practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the UV absorption spectrum of ADP, GDP-L-fucose, ATP, and GTP.

[0024] Figure 2 It is the isobsorbance line diagram of ADP, GDP-L-fucose, ATP, and GTP.

[0025] Figure 3 It is the chromatogram of blank solution, GDP-L-fucose reference solution and sample solution.

[0026] Figure 4 It is a linear graph of GDP-L-fucose. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0028] Example 1

[0029] 1.1 Preparation of test solution

[0030] 320 μL of Mn with a final concentration of 1.95 mg / mL 2+ , 1.875 mg / mL adenine nucleoside triphosphate (ATP), 1.875 mg / mL guanosine triphosphate (GTP), 7.5 mg / mL Tris-HCl buffer at pH 7.5, 25.625 mg / mL L-fucose and 9.06 mg / mL L-fucokinase / GDP-fucose pyrophosphorylase (FKP enzyme) reaction system was placed in a 37°C constant temperature incubator shaker at 225 rpm for 12 hours. After ATP and GTP reacted completely, the enzyme reaction was terminated by boiling at 100°C for 10 minutes to obtain GDP-L-fucose reaction solution. The GDP-L-fucose reaction solution was taken and allowed to stand at room temperature for 5 minutes, and then the reaction solution was centrifuged at 10000 r / min for 2 minutes, 5 μL of the supernatant was taken and injected into a sample injection vial, the sample was diluted 1000 times with water using a pipette, filtered with a 0.22 μm syringe filter, and the filtrate was taken to obtain a test solution (hereinafter referred to as sample solution).

[0031] 1.2 Preparation of solution

[0032] L-fucose reference solution: Accurately weigh 600.3 mg of L-fucose reference, place in a 5 mL volumetric flask, dissolve with water and make up to the mark.

[0033] GDP-L-fucose reference solution: Accurately weigh 63.30 mg of GDP-L-fucose reference, place in a 5 mL volumetric flask, dissolve with water and make up to the mark.

[0034] ADP reference solution: Accurately weigh 0.05 mg of adenosine diphosphate (ADP) reference, place it in a 5 mL volumetric flask, dissolve it in water and make up to the mark.

[0035] ATP reference solution: Accurately weigh 0.6010 g of ATP reference, place in a 5 mL volumetric flask, dissolve with water and make up to the mark.

[0036] GTP reference solution: Accurately weigh 0.6003 g of GTP reference, place in a 5 mL volumetric flask, dissolve with water and make up to the mark.

[0037] GDP-L-fucose series standard working solutions: accurately measure 0.1μL, 0.2μL, 0.5μL, 1.0μL, 1.5uL, and 2μL of the above GDP-L-fucose reference solution, respectively, and dilute with water using a pipette to prepare gradients containing 0.001055mg / mL, 0.00211mg / mL, 0.005275mg / mL, 0.01055mg / mL, 0.015825mg / mL, and 0.0211mg / mL of GDP-L-fucose, respectively, as GDP-L-fucose series standard working solutions.

[0038] 1.3 Determination of detection wavelength

[0039] A diode array detector was used to scan the ADP reference solution, ATP reference solution, GTP reference solution, L-fucose reference solution, and GDP-L-fucose reference solution in the wavelength range of 200 to 400 nm. The spectra are shown in FIG. Figure 1 As shown. Figure 1 It can be seen that the maximum absorption wavelengths of ADP, ATP and GTP are 258.58nm, 259.02nm and 254.64nm respectively, the maximum absorption wavelength of GDP-L-fucose is 254.16nm, L-fucose has no ultraviolet absorption in this wavelength range, the spectra of GTP and GDP-L-fucose overlap, and the spectra of ADP and ATP overlap.

[0040] A diode array detector was used to detect the isobsorptive lines of ADP, GDP-L-fucose, ATP, and GTP, such as Figure 2 As shown in the isobsorption line diagram, it can be seen that the baseline of GDP-L-fucose, ADP, ATP, and GTP is stable at 254nm, and the response of each component is high, and all have strong ultraviolet absorption, which is the best detection wavelength.

[0041] 1.4 Liquid phase analysis

[0042] GDP-L-fucose was detected by high performance liquid chromatography, and the chromatographic conditions were as follows: the chromatographic column was Agilent C18 (250 mm×4.6 mm, 5 μm, octadecylsilane bonded silica gel was used as the filler); 0.2% by weight of tetrabutylammonium bromide was added to a 2.72 g / L potassium dihydrogen phosphate aqueous solution and the pH value was adjusted to 4.0 with phosphoric acid as the mobile phase A, acetonitrile was used as the mobile phase B, gradient elution was performed, the flow rate was 1.0 mL / min, the column temperature was 30° C., the detector was a diode array detector, the detection wavelength was 254 nm, and the injection volume was 20 μL; the elution method was gradient elution, and the elution program is shown in Table 1.

[0043] Table 1 Gradient elution program

[0044]

[0045] 1.5 Establishment of standard curve and equation

[0046] (1) Exclusivity

[0047] According to the chromatographic conditions in 1.4, the blank solution (ultrapure water), GDP-L-fucose reference solution and sample solution are measured. The superimposed chromatogram results are shown in Figure 3 The results showed that there were no impurity peaks in the blank solution, no interfering peaks at the elution position of GDP-L-fucose, and the elution time of the sample and the reference substance was consistent, indicating that the method had good specificity.

[0048] (2) Linearity and detection limit

[0049] Take the GDP-L-fucose series standard working solution and inject it according to the chromatographic conditions of 1.4 for analysis, record the chromatographic peak area, and the results are shown in Table 2. Use the mass concentration of the GDP-L-fucose series standard working solution as the abscissa and the corresponding chromatographic peak area as the ordinate for linear regression to establish the GDP-L-fucose standard working curve, and calculate the linear regression equation and correlation coefficient.

[0050] Table 2 Determination results of GDP-L-fucose series standard working solutions

[0051]

[0052] Depend on Figure 4 It can be seen that the linear regression equation of GDP-L-fucose is y=1920.9x+1.2828, and the linear correlation coefficient is 0.9999, indicating that the mass concentration of GDP-L-fucose has a good linear relationship in the range of 1.005-21.100 μg / mL, and the detection limit is 0.3 μg / mL.

[0053] 1.6 Sample determination

[0054] (1) Precision

[0055] Take 6 portions of the sample solution in 1.1, measure according to the chromatographic conditions in 1.4 and calculate the content of GDP-L-fucose and relative standard deviation in the sample solution using the linear regression equation in 1.5. The results are shown in Table 3.

[0056] Table 3 Repeatability test results

[0057]

[0058]

[0059] It can be seen from Table 3 that the average content of GDP-L-fucose is 1.20 mg / mL, and the relative standard deviation of the determination result is 0.04% (n=6), indicating that the precision of this method is good.

[0060] (2) Sample spike recovery

[0061] Take 9 portions of the sample solution in 1.1, add GDP-L-fucose reference solution respectively, use the chromatographic conditions in 1.4 for determination and use the linear regression equation in 1.5 to calculate the spike recovery rate. The results are shown in Table 4.

[0062] Table 4 Spike recovery test results

[0063]

[0064] As shown in Table 4, the average recovery rate of GDP-L-fucose was 99.6%, indicating that the method had good accuracy.

Claims

1. A method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector, characterized in that: Step 1: Prepare GDP-L-fucose test solution; Step 2: Prepare GDP-L-fucose standard working solutions of different mass concentrations; Step 3: High performance liquid chromatography was used to detect GDP-L-fucose standard working solutions of different mass concentrations. The chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; 0.2% to 0.3% of tetrabutylammonium bromide by mass was added to a 2.5 to 3.0 g / L potassium dihydrogen phosphate aqueous solution and the pH value was adjusted to 3.5 to 4.5 with phosphoric acid as mobile phase A, acetonitrile was used as mobile phase B, gradient elution was performed, the detector was a diode array detector, and the elution program was: 0 to 5 minutes: The volume percentage of mobile phase B is 10%; 5 to 25 minutes: the volume percentage of mobile phase B increases from 10% to 35%; 25 to 28 minutes: the volume percentage of mobile phase B decreases from 35% to 10%; 28 to 30 minutes: the volume percentage of mobile phase B is 10%; the chromatographic peak area is recorded, and a linear regression is performed with the mass concentration of the GDP-L-fucose standard working solution as the abscissa and the corresponding chromatographic peak area as the ordinate, a standard working curve of GDP-L-fucose is established, and a linear regression equation is calculated; Step 4: Detect the GDP-L-fucose test solution of step 1 according to the chromatographic conditions of step 3, and calculate the content of GDP-L-fucose in the test solution in combination with the linear regression equation of step 3.

2. The method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector according to claim 1, characterized in that: In step 1, the preparation method of the test solution is: the GDP-L-fucose reaction solution obtained by the reaction of adenine nucleoside triphosphate, guanosine triphosphate and L-fucose is allowed to stand at room temperature for 5 to 10 minutes and then centrifuged, the supernatant is diluted with water, filtered with a 0.22 μm filter, and the filtrate is taken to obtain the test solution.

3. The method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector according to claim 1, characterized in that: In step 1, the method for preparing the test solution is: after centrifuging the biological sample, taking the supernatant and using methanol or acetonitrile to precipitate the protein to release GDP-L-fucose, and then centrifuging to take the supernatant to obtain the test solution.

4. The method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector according to claim 1, characterized in that: In step 1, the method for preparing the test solution is: after crushing the food or agricultural product sample, ultrasonically extracting GDP-L-fucose with ethanol or water or heating and refluxing to extract GDP-L-fucose to obtain the test solution.

5. The method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector according to claim 1, characterized in that: In step 3, the elution flow rate is 0.8-1.2 mL / min.

6. The method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector according to claim 1, characterized in that: In step 3, the column temperature is detected to be 25-32°C.

7. The method for quantitatively detecting GDP-L-fucose based on high performance liquid chromatography-diode array detector according to claim 1, characterized in that: In step 3, the detection wavelength is 254 nm.

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