Method for measuring content of uric acid in serum by high-phase liquid chromatography based on dispersive liquid phase microextraction technology

Abstract

The invention discloses a method for measuring the content of uric acid in serum by high-phase liquid chromatography based on a dispersive liquid-phase microextraction technology. The method comprises dispersive liquid-phase microextraction pretreatment and high-performance liquid chromatography analysis, the method comprises the following steps: mixing a serum sample with an extracting agent, carrying out vortex oscillation, carrying out ultrasonic treatment under the condition of 1000-3000W for 5-10 minutes, centrifuging at the speed of 12,000 r / min, taking supernate, adding a dispersing agent, further purifying an extracting solution, reducing matrix interference, centrifuging, and removing an upper layer for sample introduction determination. The method provided by the invention can be used for analyzing the content of uric acid by high performance liquid chromatography with less organic solvent consumption and less serum sample amount.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry detection, and in particular to a method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology. Background Art

[0002] Uric acid (UA), also known as 2,6,8-trihydroxypurine, is one of the important components of non-protein nitrogen in plasma and the end product of purine metabolism in the human body. The changes in the content of uric acid in human body fluids can reflect the status of metabolism and immunity in the human body. When a person suffers from gout, leukemia, uremia, cardiovascular disease and other symptoms, the uric acid content in the human blood will increase, especially in severe kidney damage, the uric acid in the blood will increase significantly. The determination of uric acid concentration in plasma is a sensitive indicator of severe kidney damage. Therefore, accurate determination and analysis of uric acid content in human body fluids is of great significance in drug control and clinical diagnosis.

[0003] Liquid chromatography has certain advantages in detecting the uric acid content in serum, with good separation effect, simple mobile phase and fast analysis speed, but the sample pretreatment process is relatively complicated, time-consuming and costly. The traditional high-performance liquid chromatography has cumbersome extraction methods, consumes a large amount of organic solvents, has a large sampling volume, and the chromatographic peaks are not ideal. The mobile phase mostly uses phosphate, which causes great damage to the chromatographic column. At present, there is no public method for detecting uric acid in serum by high-performance liquid chromatography using dispersed liquid phase microextraction technology. This method solves the problems of complex and time-consuming pretreatment and environmental unfriendliness, and has broad application prospects. Summary of the invention

[0004] The purpose of the present invention is to provide a method for determining the uric acid content in serum by high-phase liquid chromatography using dispersed liquid microextraction technology, so as to solve the problem that the serum uric acid content determination method in the prior art is complicated and environmentally unfriendly.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a method for determining the uric acid content in serum, which is determined by high performance liquid chromatography using dispersed liquid phase microextraction technology.

[0006] The dispersed liquid phase microextraction pretreatment technology performs pretreatment on the serum sample, and the pretreatment includes the following steps: The serum sample was mixed with the extractant, vortexed, and then ultrasonicated at 1000-3000W for 5-10 minutes. The supernatant was collected by centrifugation at 12000r / min, and a dispersant was added to further purify the extract to reduce matrix interference. After centrifugation, the upper layer was discarded and sampled for determination.

[0007] Preferably, the extractant is acetonitrile; the volume (μL) ratio of the serum sample to the extractant is 1:1.2.

[0008] Preferably, the dispersant is chloroform; the volume (μL) ratio of the extractant to the dispersant chloroform is 1:1.

[0009] In the high performance liquid chromatography process, methanol-0.002-0.010% ammonium acetate aqueous solution is used as the mobile phase; The methanol is component A, and the 0.002-0.010% ammonium acetate aqueous solution is component B; Preferably, the pH value of the 0.002-0.010% ammonium acetate aqueous solution in the HPLC process is 4.2-4.5.

[0010] Preferably, the chromatographic column in the high performance liquid chromatography process is a C18 reverse phase chromatographic column (250 mm×4.6 mm, 5 μm).

[0011] Preferably, the column temperature used in the HPLC process is 25-30° C., and the injection volume is 10 μL.

[0012] Preferably, the gradient elution program used in the HPLC process is as shown in Table 1.

[0013] Table 1 Gradient elution program Preferably, the flow rate used in the HPLC process is 0.8 to 1.2 mL / min. Technical effects and advantages of the present invention: The present invention establishes a method for determining uric acid in serum, which is fast and simple, has good accuracy, high reproducibility, improves qualitative ability and sensitivity, has a wide linear range, and can meet the configuration requirements of different laboratories. It can provide a method for analyzing uric acid content by high performance liquid chromatography with a small consumption of organic solvent (100 μL) and a small amount of serum sample (100 μL). BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the chromatogram of uric acid standard solution. DETAILED DESCRIPTION

[0015] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0016] Example 1 1 Experimental part 1.1 Instruments and reagents Liquid chromatography (Waters, USA), C18 chromatographic column (250 mm × 4.6 mm, 5 μm) Waters, USA), diode array detector (Waters, USA), methanol, acetonitrile (chromatographic grade, Merck, Germany, purity> 99.9%). Ammonium acetate (analytical grade), Sinopharm Chemical Reagent Co., Ltd. The samples and polyvinyl pyrrolidone used in the experiment were commercially available. 1.2 Test conditions Sample preparation 80 μL of serum was precisely drawn and placed in a 1.5 mL plastic centrifuge tube with a tip. 100 μL of extractant was added and mixed. After vortexing, ultrasonication was performed at 2500 W for 5 min. The supernatant was centrifuged at 12000 r / min. Dispersant chloroform was added to further purify the extract and reduce matrix interference. After centrifugation, the upper layer was discarded and sampled for determination. The volume (μL) ratio of serum sample to extractant was 1:1.2. The volume (μL) ratio of extractant to dispersant chloroform was 1:1.

[0017] Diode array detector: Detection wavelength: 235nm Chromatographic conditions: The chromatographic column was a C18 solid phase extraction silica gel column (250 mm×4.6 mm, 5 μm). Methanol A-0.002-0.010% ammonium acetate aqueous solution B was used as the mobile phase; the pH of B was 4.2-4.5. The column temperature was 30°C, and the injection volume was 10 μL. The flow rate was 1 mL / min. The gradient elution program was as shown in Table 1.

[0018] Table 1 Gradient elution program 1.3 Preparation of standard stock solutions and samples The preparation process of the uric acid standard solution can be as follows: accurately weigh 2.00 mg of the uric acid standard, place it in a 10 mL volumetric flask, add water to dissolve it and then make up the volume, shake well to prepare the standard solutions containing uric acid mass concentrations of 0.20 μg / mL, 0.5 μg / mL, 1.0 mg / L, 2.0 μg / mL, 5.0 μg / mL, and 10.0 μg / mL.

[0019] 2 Results and analysis 2.1 Selection of chromatographic columns In this experiment, two different brands of conventional C18 columns (150 mm × 4.6 mm, 3.5 μm) (Waters, USA) and Atlantis T3 columns (250 mm × 4.6 mm, 5 μm) were compared. The C18 column was selected as the separation column based on the size of the target peak area and the relative standard deviation.

[0020] 2.2 Selection of mobile phase Four mobile phase systems were investigated, including formic acid-water, methanol-ammonium acetate (20 mmol / L) buffer, formic acid-acetonitrile, and methanol A-0.002-0.010% ammonium acetate aqueous solution B (pH 4.2-4.5) as mobile phases. The results showed that on the C18 chromatographic column, methanol A-0.002-0.010% ammonium acetate aqueous solution B (pH 4.2-4.5) as the mobile phase had the best peak shape and no tailing phenomenon.

[0021] 2.3 Optimization of extraction solution The protein content in serum is high, and how to remove the interference of impurities is the key to the extraction experiment. The extraction effects of methanol, acetonitrile, 0.1% acidified acetonitrile, 0.2% acidified acetonitrile, and acetonitrile as extractants were investigated. The results showed that methanol had more interference peaks than acetonitrile. The extraction efficiencies of the five extracts were different, and acetonitrile had the highest extraction effect, so it was used as the extraction solution.

[0022] 2.4 Detection limit and linear range A series of uric acid blood samples with the same concentration of the analyte were prepared and tested according to the test conditions in 1.2. The linear equation of uric acid was Y=38863X-744.55, the correlation coefficient was r=0.9993, and the detection limit and quantification limit were 0.1μg / mL and 0.3μg / mL, respectively.

[0023] 2.5 Method accuracy and precision Serum samples were taken separately, processed and tested according to the above method, and precision and repeatability tests were performed at the same time: the content of each component was calculated, and the RSD corresponding to the content was calculated respectively; Table 2 Serum sample data test results and precision As can be seen from Table 2, the method provided by the present invention can accurately and quickly determine the uric acid content in serum with good precision.

[0024] 2.7 Actual sample measurement The method established in this experiment can be used to quantitatively detect the content of uric acid in blood samples.

[0025] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology, characterized in that: Including dispersed liquid phase microextraction pretreatment and high performance liquid chromatography analysis.

2. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 1, characterized in that: Dispersed liquid phase microextraction pretreatment technology is used to pretreat serum samples. The pretreatment includes the following steps: The serum sample was mixed with the extractant, vortexed, and then ultrasonicated at 1000-3000W for 5-10 minutes. The supernatant was collected by centrifugation at 12000r / min, and a dispersant was added to further purify the extract. After centrifugation, the upper layer was discarded and sampled for determination.

3. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 2, characterized in that: The volume (μL) ratio of serum sample to extractant was 1:1.

2.

4. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 2, characterized in that: The extractant was acetonitrile.

5. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 2, characterized in that: The volume (μL) ratio of the extractant to the dispersant was 1:

1.

6. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 2, characterized in that: The dispersant is chloroform.

7. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 1, characterized in that: In the HPLC process, methanol-0.002-0.010% ammonium acetate aqueous solution was used as the mobile phase; The methanol is component A, and the 0.002-0.010% ammonium acetate aqueous solution is component B.

8. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 7, characterized in that: During the high performance liquid chromatography process, the pH value of the 0.002-0.010% ammonium acetate aqueous solution is 4.2-4.

5.

9. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 7, characterized in that: During the high performance liquid chromatography process, the chromatographic column was a C18 reverse phase column (250 mm×4.6 mm, 5 μm), the column temperature was 25-30°C, and the injection volume was 10 μL.

10. The method for determining the content of uric acid in serum by high-phase liquid chromatography based on dispersed liquid phase microextraction technology according to claim 7, characterized in that: The gradient elution program used in the HPLC process was: The flow rate used in the HPLC process was 0.8 to 1.2 mL / min.

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