A method for quantitative determination of GDP-L-fucose based on high performance liquid chromatography-diode array detector

Abstract

This invention discloses a method for the quantitative detection of GDP-L-fucose based on high-performance liquid chromatography (HPLC) with a diode array detector. The method employs HPLC analysis under the following chromatographic conditions: an Agilent C18 column; mobile phase A consisting of 2.5–3.0 g / L 2.72 g / L potassium dihydrogen phosphate aqueous solution (containing 0.2%–0.3% tetrabutylammonium bromide, adjusted to pH 3.5–4.5 with phosphoric acid); acetonitrile as mobile phase B; gradient elution; and a diode array detector at a detection wavelength of 254 nm. This method exhibits low detection limits, good precision and accuracy, and not only provides spectral information for qualitative and quantitative analysis of GDP-L-fucose but also enables real-time monitoring of the reaction process during GDP-L-fucose synthesis. This provides an analytical method for improving the purity and yield of GDP-L-fucose and for screening the activity of multifunctional FKP enzymes.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology and is a method for quantitative detection of GDP-L-fucose based on high performance liquid chromatography-diode array detection (HPLC-DAD). Background Technology

[0002] GDP-L-fucose is a key substrate for glycosylation modification and is widely used in pharmaceuticals, food, and health products, possessing enormous market development potential. Due to its important role in related biological reactions and the treatment of related diseases, and its high cost, improving its purity and quantitative analysis are crucial for drug development. Currently, its detection methods largely rely on thin-layer chromatography (TLC), gas chromatography (GC), conventional liquid chromatography (LC), and liquid chromatography-mass spectrometry (LC-MS). GDP-L-fucose is highly polar and non-volatile, requiring derivatization for gas chromatography detection, which is cumbersome. TLC, being a normal-phase method, has low resolution for carbohydrate compounds and struggles with accurate qualitative and quantitative determination of highly polar substances; laboratory environment and operator skill significantly influence experimental results. LC-MS offers high sensitivity and selectivity, but requires sample purification and cannot monitor the reaction process in real time. For example, Wang Hongchao et al. (Study on the production of guanylate-L-fucose by fermentation of Mortierella alpina[J]. Food Science and Technology, 2016, 41(9):3.) used both UV-LC and mass spectrometry to detect the content and purity of GDP-L-fucose produced by fermentation. This method requires a high-performance ion exchange column to improve the separation, which is cumbersome. Furthermore, the liquid chromatography-mass spectrometry analysis requires prior sample purification, resulting in high costs. Wei Wang et al. (Chemoenzymatic synthesis of GDP-L-fucose and the Lewis Xglycan derivatives[J]. Journal of PNAS, 2009, 106(38):16097.) used thin-layer chromatography to analyze the crude reaction mixture of GDP-L-fucose. This method requires the preparation of cumbersome developing and colorimetric reagents, resulting in severe tailing of the chromatogram. Therefore, there is an urgent need to develop a simple, sensitive, accurate, rapid and economical analytical method to achieve the quantitative detection of GDP-L-fucose. Summary of the Invention

[0003] The purpose of this invention is to provide a simple, sensitive, accurate, and easy-to-operate method for detecting GDP-L-fucose. This method uses high-performance liquid chromatography with a diode array detector, which can not only provide spectral information for qualitative and quantitative analysis of GDP-L-fucose, but also monitor the reaction in the GDP-L-fucose synthesis process in real time. This provides an analytical method for improving the purity and yield of GDP-L-fucose and for screening the activity of multifunctional FKP enzymes.

[0004] The method for quantitative detection of GDP-L-fucose based on high-performance liquid chromatography-diode array detector provided by the present invention includes the following steps:

[0005] Step 1: Prepare GDP-L-fucose test solution.

[0006] Step 2: Prepare standard working solutions of GDP-L-fucose at different mass concentrations.

[0007] Step 3: High-performance liquid chromatography (HPLC) was used to detect GDP-L-fucose standard working solutions of different mass concentrations. The chromatographic conditions were as follows: the column was packed with octadecylsilane-bonded silica gel; 0.2%–0.3% (by mass) of tetrabutylammonium bromide was added to a 2.5–3.0 g / L potassium dihydrogen phosphate aqueous solution, and the pH was adjusted to 3.5–4.5 with phosphoric acid as mobile phase A; acetonitrile was used as mobile phase B for gradient elution; a diode array detector was used; and the elution program was 0–5 minutes. The volume percentage of mobile phase B is 10%; from 5 to 25 minutes: the volume percentage of mobile phase B increases from 10% to 35%; from 25 to 28 minutes: the volume percentage of mobile phase B decreases from 35% to 10%; from 28 to 30 minutes: the volume percentage of mobile phase B is 10%; record the chromatographic peak area, and perform linear regression with the mass concentration of GDP-L-fucose standard working solution as the abscissa and the corresponding chromatographic peak area as the ordinate to establish the standard working curve of GDP-L-fucose, and calculate the linear regression equation.

[0008] Step 4: Detect the GDP-L-fucose test solution from Step 1 according to the chromatographic conditions of Step 3, and calculate the content of GDP-L-fucose in the test solution based on the linear regression equation from Step 3.

[0009] In step 1 above, when the reaction situation during the synthesis of GDP-L-fucose is monitored in real time, the preparation method of the test solution is as follows: the GDP-L-fucose reaction solution obtained by reacting 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 taken, diluted with water, filtered through a 0.22 μm filter, and the filtrate is taken to obtain the test solution.

[0010] In step 1 above, when the test sample is a biological sample (such as blood, tissue, etc.), the preparation method of the test solution is as follows: after centrifuging the biological sample, take the supernatant and use methanol or acetonitrile to precipitate the protein, so that GDP-L-fucose is released from the biological macromolecules such as proteins, and then centrifuge to take the supernatant to obtain the test solution.

[0011] In step 1 above, when the test sample is a food or agricultural product sample, the preparation method of the test solution is as follows: after crushing the food or agricultural product sample, extract GDP-L-fucose by ultrasonic extraction with ethanol or water or by heating and reflux extraction to obtain the test solution.

[0012] Furthermore, in step 3 above, the preferred elution flow rate is 0.8–1.2 mL / min.

[0013] Furthermore, in step 3 above, the preferred detection column temperature is 25–32°C.

[0014] Furthermore, in step 3 above, the preferred detection wavelength is 254±2nm.

[0015] The beneficial effects of this invention are as follows:

[0016] 1. Optimized Sample Pretreatment Process: This invention develops a unique extraction and purification procedure that effectively removes various impurities from complex sample matrices, significantly reducing interference from impurities in the detection of GDP-L-fucose. Compared with traditional methods, this greatly improves the purity of the target analyte, thereby enhancing detection accuracy and providing high-quality samples for subsequent high-performance liquid chromatography analysis, ensuring the reliability of detection results from the source.

[0017] 2. System Optimization of Chromatographic Conditions: This invention comprehensively and thoroughly optimizes various chromatographic conditions. By screening different types and specifications of chromatographic columns, precisely adjusting the composition and ratio of the mobile phase, and accurately controlling the flow rate, baseline separation of GDP-L-fucose from other coexisting substances is achieved. The obtained chromatographic peaks are symmetrical and sharp, greatly improving detection resolution and making the detection results clearer and more accurate, solving the problem of poor separation effect in traditional methods.

[0018] 3. Precise Determination of Detection Wavelength: This invention innovatively determines the optimal detection wavelength by leveraging in-depth research on the spectral characteristics of GDP-L-fucose. This significantly enhances detection sensitivity, enabling the detection of lower concentrations of the target analyte compared to detection methods without optimized wavelengths, thus broadening the applicability of this method, especially suitable for the detection of trace amounts of GDP-L-fucose.

[0019] 4. Wide linear range and high sensitivity: The analytical method established in this 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 exceeding 0.9999, demonstrating excellent linear correlation. Simultaneously, the detection limit is as low as 0.3 μg / mL, ensuring high sensitivity while also meeting the detection needs of samples with different concentrations, giving it a significant advantage over similar detection methods.

[0020] 5. Excellent precision and accuracy: The relative standard deviation (RSD) of the repeatability experiment of the method of this invention is less than 0.05%, and the average recovery rate of spiked samples is 99.6%. These data indicate that the method has good precision, reliable and stable detection results, and high accuracy, and can provide reliable detection data for scientific research, production and other fields.

[0021] 6. Highly Efficient and Rapid Detection: This invention successfully reduces the single-test time to less than 30 minutes, significantly improving detection efficiency compared to traditional methods. This not only saves time and costs but also allows for the processing of more samples per unit time, meeting the high-throughput detection needs of modern scientific research and production, and demonstrating significant practical value. Attached Figure Description

[0022] Figure 1 It shows the UV absorption spectra of ADP, GDP-L-fucose, ATP, and GTP.

[0023] Figure 2 It is an isoabsorption curve of ADP, GDP-L-fucose, ATP, and GTP.

[0024] Figure 3 These are chromatograms of the blank solution, GDP-L-fucose reference solution, and sample solution.

[0025] Figure 4 It is a linear graph of GDP-L-fucose. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0027] Example 1

[0028] 1.1 Preparation of the test solution

[0029] 320 μL of Mn was used to achieve a final concentration of 1.95 mg / mL. 2+The reaction system consisting of 1.875 mg / mL adenosine triphosphate (ATP), 1.875 mg / mL guanosine triphosphate (GTP), 7.5 mg / mL Tris-HCl buffer (pH 7.5), 25.625 mg / mL L-fucose, and 9.06 mg / mL L-fucokinase / GDP-fucosylation enzyme (FKP enzyme) was placed in a 37°C incubator with shaking at 225 rpm for 12 h. After the ATP and GTP had completely reacted, the enzyme reaction was terminated by boiling at 100°C for 10 min to obtain the GDP-L-fucose reaction solution. The GDP-L-fucose reaction solution was taken and allowed to stand at room temperature for 5 min. Then, the reaction solution was centrifuged at 10000 rpm for 2 min. 5 μL of the supernatant was injected into a sample vial. The sample was diluted 1000 times with water using a pipette and filtered through a 0.22 μm syringe filter. The filtrate was collected to obtain the test solution (hereinafter referred to as the sample solution).

[0030] 1.2 Preparation of Solution

[0031] L-fucose reference solution: Accurately weigh 600.3 mg of L-fucose reference standard, place it in a 5 mL volumetric flask, dissolve it in water and dilute to the mark.

[0032] GDP-L-fucose reference solution: Accurately weigh 63.30 mg of GDP-L-fucose reference standard, place it in a 5 mL volumetric flask, dissolve it in water and dilute to the mark.

[0033] ADP reference solution: Accurately weigh 0.05 mg of adenosine diphosphate (ADP) reference standard, place it in a 5 mL volumetric flask, dissolve it in water and dilute to the mark.

[0034] ATP standard solution: Accurately weigh 0.6010 g of ATP standard, place it in a 5 mL volumetric flask, dissolve it in water and dilute to the mark.

[0035] GTP reference solution: Accurately weigh 0.6003 g of GTP reference standard, place it in a 5 mL volumetric flask, dissolve it in water and dilute to the mark.

[0036] GDP-L-fucose series standard working solutions: Accurately measure 0.1 μL, 0.2 μL, 0.5 μL, 1.0 μL, 1.5 μL, and 2 μL of the above GDP-L-fucose reference solution, respectively, and dilute with water using a pipette to prepare a gradient containing 0.001055 mg / mL, 0.00211 mg / mL, 0.005275 mg / mL, 0.01055 mg / mL, 0.015825 mg / mL, and 0.0211 mg / mL of GDP-L-fucose as GDP-L-fucose series standard working solutions.

[0037] 1.3 Determination of detection wavelength

[0038] A diode array detector was used to scan ADP, ATP, GTP, L-fucose, and GDP-L-fucose reference solutions in the wavelength range of 200–400 nm. The spectra are shown below. Figure 1 As shown. By Figure 1 It can be seen that the maximum absorption wavelengths of ADP, ATP, and GTP are 258.58 nm, 259.02 nm, and 254.64 nm, respectively, while the maximum absorption wavelength of GDP-L-fucose is 254.16 nm. 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.

[0039] Isosorbite lines for ADP, GDP-L-fucose, ATP, and GTP were detected using a diode array detector. Figure 2 As shown in the isoabsorption plot, GDP-L-fucose, ADP, ATP, and GTP are stable at the baseline of 254 nm, with each component showing high response and strong ultraviolet absorption, which is the optimal detection wavelength.

[0040] 1.4 Liquid Phase Analysis

[0041] GDP-L-fucose was detected by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: an Agilent C18 column (250 mm × 4.6 mm, 5 μm, octadecylsilane-bonded silica gel as the packing material); 0.2% (w / w) of tetrabutylammonium bromide was added to a 2.72 g / L potassium dihydrogen phosphate aqueous solution, and the pH was adjusted to 4.0 with phosphoric acid as mobile phase A; acetonitrile was used as mobile phase B for gradient elution at a flow rate of 1.0 mL / min, a column temperature of 30 °C, a diode array detector at a detection wavelength of 254 nm, and an injection volume of 20 μL; the elution method was gradient elution, and the elution program is shown in Table 1.

[0042] Table 1 Gradient elution program

[0043]

[0044] 1.5 Establishment of Standard Curve and Equation

[0045] (1) Exclusivity

[0046] The blank solution (ultrapure water), GDP-L-fucose reference solution, and sample solution were determined according to the chromatographic conditions in 1.4. The results of the overlay chromatograms are shown in the figure. Figure 3 The results showed that the blank solution had no impurity peaks, the GDP-L-fucose peak position had no interfering peaks, and the peak times of the sample and the control were consistent, indicating that the method has good specificity.

[0047] (2) Linearity and detection limit

[0048] GDP-L-fucose series standard working solutions were injected and analyzed under chromatographic conditions 1.4, and the chromatographic peak areas were recorded. The results are shown in Table 2. A linear regression was performed with the mass concentration of the GDP-L-fucose series standard working solutions as the abscissa and the corresponding chromatographic peak area as the ordinate to establish the standard working curve of GDP-L-fucose. The linear regression equation and correlation coefficient were calculated.

[0049] Table 2. Determination results of GDP-L-fucose series standard working solutions

[0050]

[0051] Depend on Figure 4 As can be seen, the linear regression equation for GDP-L-fucose is y = 1920.9x + 1.2828, with a linear correlation coefficient of 0.9999, indicating that the mass concentration of GDP-L-fucose has a good linear relationship in the range of 1.005 to 21.100 μg / mL, and the detection limit is 0.3 μg / mL.

[0052] 1.6 Sample Determination

[0053] (1) Precision

[0054] Six portions of the sample solution from section 1.1 were taken and the content of GDP-L-fucose and the relative standard deviation in the sample solution were determined according to the chromatographic conditions in section 1.4 and the linear regression equation in section 1.5 was used to calculate the results. The results are shown in Table 3.

[0055] Table 3 Repeatability test results

[0056]

[0057]

[0058] Table 3 shows that the average content of GDP-L-fucose is 1.20 mg / mL, and the relative standard deviation of the determination results is 0.04% (n=6), indicating that the method has good precision.

[0059] (2) Sample spike recovery

[0060] Take 9 portions of the sample solution in 1.1, add GDP-L-fucose reference solution to each, and determine the spiked recovery rate using the chromatographic conditions in 1.4. The results are shown in Table 4.

[0061] Table 4 Results of Spiked Recovery Test

[0062]

[0063] As shown in Table 4, the average recovery rate of GDP-L-fucose spiked to 99.6% indicates that the method has good accuracy.

Claims

1. A method for quantitative detection of GDP-L-fucose based on high-performance liquid chromatography-diode array detector, characterized in that: Step 1: Preparation of GDP-L-fucose test solution: 320 μL of Mn2 with a final concentration of 1.95 mg / mL was added. 2+ The reaction system consisting of 1.875 mg / mL adenosine triphosphate, 1.875 mg / mL guanosine triphosphate, 7.5 mg / mL Tris-HCl buffer (pH 7.5), 25.625 mg / mL L-fucose, and 9.06 mg / mL FKP enzyme was placed in a 37°C incubator and shaken at 225 rpm for 12 h. After the adenosine triphosphate and guanosine triphosphate had completely reacted, the enzyme reaction was terminated by boiling at 100°C for 10 min to obtain the GDP-L-fucose reaction solution. The GDP-L-fucose reaction solution was taken and allowed to stand at room temperature for 5 min. Then, the reaction solution was centrifuged at 10000 r / min for 2 min. 5 μL of the supernatant was injected into a sample vial. The sample was diluted 1000 times with water using a pipette and filtered through a 0.22 μm syringe filter. The filtrate was collected to obtain the test solution. Step 2: Prepare standard working solutions of GDP-L-fucose at different mass concentrations; Step 3: High-performance liquid chromatography (HPLC) was used to detect GDP-L-fucose standard working solutions of different mass concentrations at a wavelength of 254 nm. The chromatographic conditions were as follows: the column was packed with octadecylsilane-bonded silica gel; the pH range was 2.5–3.0 nm. Adding 0.2%–0.3% (by mass) of tetrabutylammonium bromide to a 10 g / L potassium dihydrogen phosphate aqueous solution and adjusting the pH to 3.5–4.5 with phosphoric acid to form mobile phase A, and using acetonitrile as mobile phase B, gradient elution was performed. A diode array detector was used as the detector. The elution program was as follows: 0–5 minutes: mobile phase B volume percentage 10%; 5–25 minutes: mobile phase B volume percentage increased from 10% to 35%; 25–28 minutes: mobile phase B volume percentage decreased from 35% to 10%; 28–30 minutes: mobile phase B volume percentage 10%. The chromatographic peak areas were recorded, and linear regression was performed with the mass concentration of the GDP-L-fucose standard working solution as the x-axis and the corresponding chromatographic peak area as the y-axis to establish a standard working curve for GDP-L-fucose. The linear regression equation was then calculated. Step 4: Detect the GDP-L-fucose test solution from Step 1 according to the chromatographic conditions of Step 3, and calculate the content of GDP-L-fucose in the test solution based on the linear regression equation from Step 3.

2. The method for quantitative detection of 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.

3. The method for quantitative detection of 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 measured at 25–32°C.

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