Method for simultaneously determining four purine compounds

Abstract

The invention discloses a method for simultaneously determining four purine compounds, and relates to the technical field of determination, and the method comprises the following steps: sample pretreatment: selecting a mixed acid solution with trifluoroacetic acid, formic acid and water in a volume ratio of (8-10): (8-10): (1-3), and hydrolyzing a sample at 80-95 DEG C for 20-30 minutes, so that purine substances are completely hydrolyzed and are not degraded; after the water bath, immediately performing ice bath to room temperature, performing rotary evaporation at 50-70 DEG C to be nearly dry, dissolving residues by using an acetonitrile aqueous solution, fixing the volume, centrifuging, taking supernate, adding the supernate into a purifying agent which comprises N-propylethylenediamine and octadecylsilane, oscillating and uniformly mixing, centrifuging, passing through an organic microfiltration membrane, and waiting for detection on a machine; and UHPLC detection: combining an ultra-high performance liquid chromatography and an ultraviolet detection method. The use of toxic organic solvents is avoided, and the environment-friendly requirement is met; and impurities in the sample are effectively removed by adopting a purifying agent, so that the analysis accuracy and reliability are improved.

Description

Technical Field

[0001] The present invention relates to the field of determination technology, and in particular to a method for simultaneously determining four purine compounds. Background Art

[0002] Purine (C 5 H 5 N 4 ) is an important component of nucleic acid and an important substance in the metabolism of organisms. The main sources of purine in the human body include synthesis in the body, decomposition of nucleic acids in human tissues, and intake from food. Long-term intake of high-purine foods, such as seafood and meat, may lead to abnormal purine metabolism, and then cause metabolic diseases such as hyperuricemia and gout. Therefore, accurate detection of purine content in food is of great significance for the prevention and treatment of related diseases.

[0003] At present, the detection methods of purine in food mainly include acid hydrolysis extraction, ultrasonic extraction and organic solvent extraction. However, these methods have certain limitations in extraction efficiency and detection accuracy. Among them, although the acid hydrolysis extraction method is commonly used, different types of acids have different degrees of hydrolysis on food and may destroy some purine substances; although the ultrasonic extraction method has high extraction efficiency, the equipment cost is high and the operation is complicated; the organic solvent extraction method is cumbersome and time-consuming, and the organic solvent is volatile, toxic, and not environmentally friendly.

[0004] For the simultaneous detection of four major purine compounds (adenine, guanine, xanthine and hypoxanthine) in food, the existing detection method uses high performance liquid chromatography for detection, and the chromatographic column used is a C18 column. Since purine compounds are highly polar compounds, they require a high aqueous phase for elution, and the common C18 columns on the market cannot withstand a high aqueous mobile phase, which will exceed the tolerance of the chromatographic column, thereby leading to unstable and inaccurate data, increased costs and other problems. CN 112881550A discloses an ultra-high performance liquid chromatography-tandem mass spectrometry analysis method for determining four purines in beverages. In the disclosed method, impurities in the sample are not removed during the sample pretreatment process, and the impurities interfere severely, which will affect the instrument analysis and cause problems such as clogging of the chromatographic column. Summary of the invention

[0005] To address the deficiencies in the prior art, the present invention provides a method for simultaneously determining four purine compounds, thereby reducing sample impurities and improving detection accuracy and reliability.

[0006] In order to achieve the purpose of the present invention, the following scheme is proposed: A method for simultaneously determining four purine compounds comprises the following steps: Sample pretreatment: Use a mixed acid solution with a volume ratio of trifluoroacetic acid: formic acid: water of 8-10:8-10:1-3, and hydrolyze the sample at 80°C-95°C for 20-30 minutes to completely hydrolyze the purine substances without degradation; immediately ice-bath to room temperature after water bath, rotary evaporate to nearly dry at 50°C-70°C, dissolve the residue with acetonitrile aqueous solution and make up the volume, take the supernatant after centrifugation and add it to the purifier, which includes N-propylethylenediamine and octadecylsilane, shake and mix well, centrifuge through an organic microporous filter membrane, and wait for machine detection; UHPLC detection: ultra-high performance liquid chromatography combined with ultraviolet detection.

[0007] Furthermore, in the sample pretreatment step: the volume ratio of trifluoroacetic acid:formic acid:water is 9:9:2.

[0008] Furthermore, in the sample pretreatment step: the mixed acid solution was hydrolyzed at 90° C. for 30 minutes.

[0009] Furthermore, in the sample pretreatment step: immediately ice-bath to room temperature after water bath, rotary evaporate to nearly dryness at 60°C, dissolve the residue with 90% acetonitrile aqueous solution and make up to 10 mL, centrifuge at 8000 r / min for 10 min, take 2 mL of supernatant and add 0.225 g of purifier.

[0010] Furthermore, in the sample pretreatment step: the mass ratio of N-propylethylenediamine to octadecylsilane is 1:1.

[0011] Furthermore, in the sample pretreatment step: the sample was shaken and mixed before centrifugation through a 0.22µm organic microporous filter membrane.

[0012] Furthermore, in the UHPLC detection step, the conditions were: Agilent InfinityLab Poroshell 120HILIC-Z (4.6 mm × 100 mm, 2.7 µm) was selected; the mobile phase was 20 mM ammonium acetate aqueous solution (pH = 9); the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 20 µL; and the UV detection wavelength was 254 nm.

[0013] The beneficial effects of the present invention are: 1. A mixed acid solution of trifluoroacetic acid and formic acid is used as the hydrolysis solvent, and the entire detection process also avoids the use of toxic organic solvents, which meets environmental protection requirements; 2. A purification step is added to the sample pretreatment, using N-propylethylenediamine and octadecylsilane as purifiers to effectively remove impurities in the sample and improve the accuracy and reliability of the analysis; 3. Avoid using mobile phases that contain too high a proportion of salt and water to reduce the risk of instrument blockage and column loss, thereby reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Chromatograms of four purine compounds are shown. DETAILED DESCRIPTION

[0015] This embodiment provides a method for simultaneously determining four purine compounds, comprising the following steps: Sample pretreatment: Use a mixed acid solution of trifluoroacetic acid: formic acid: water with a volume ratio of 9:9:2, and hydrolyze the sample at 90°C for 30 minutes to completely hydrolyze the purine substances without degradation; immediately ice-bath to room temperature after water bath, rotary evaporate to nearly dry at 60°C, dissolve the residue with 90% acetonitrile aqueous solution and make up to 10mL, centrifuge at 8000r / min for 10min, take 2mL of supernatant and add 0.225g of purifier, which includes N-propylethylenediamine and octadecylsilane in a mass ratio of 1:1, shake and mix, then centrifuge through a 0.22µm organic microporous filter membrane, and wait for machine detection; UHPLC detection: ultra-high performance liquid chromatography combined with ultraviolet detection was used, and the conditions were: Agilent InfinityLab Poroshell 120 HILIC-Z (4.6mm×100mm, 2.7µm) was selected; the mobile phase was 20Mm ammonium acetate aqueous solution (pH=9); the flow rate was 1.0mL / min; the column temperature was 30℃; the injection volume was 20µL; and the ultraviolet detection wavelength was 254nm.

[0016] like Figure 1 As shown, there are chromatograms of four purine compounds. The retention times of adenine, hypoxanthine, xanthine and guanine are 2.605 min, 4.047 min, 4.781 min and 7.759 min, respectively, all within 8 min, and the detection time is short.

[0017] Experimental verification Under the best experimental conditions, a variety of food samples were tested. Prepare the purine standard stock solution (200 mg / L): accurately weigh 10 mg of adenine, guanine, xanthine and hypoxanthine standards, dissolve them in water and add them to a 50 mL volumetric flask, mix well (if it does not dissolve before adding to the volume, 10 drops of 1 mol / L sodium hydroxide solution can be used to help dissolve it), and store the solution in a refrigerator at 0~4℃. The validity period is 3 months.

[0018] Prepare standard working solutions: aspirate 0.05, 0.1, 0.25, 0.4, 0.5, 1.0, 2.5, and 4.0 mL respectively for pretreatment. The final mass concentrations of the series of standard working solutions are 1, 2, 5, 8, 10, 20, 50, and 80 mg / L respectively.

[0019] The results showed that the method had good linearity in the range of 1 µg / mL to 80 µg / mL. The standard curve regression equations and correlation coefficients R 2 , precision RSD, and spike recovery are shown in Table 1.

[0020] Table 1 The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be the only or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for simultaneously determining four purine compounds, characterized in that: The following steps are involved: Sample pretreatment: Use a mixed acid solution with a volume ratio of trifluoroacetic acid: formic acid: water of 8-10:8-10:1-3, and hydrolyze the sample at 80°C-95°C for 20-30 minutes to completely hydrolyze the purine substances without degradation; immediately ice-bath to room temperature after water bath, rotary evaporate to nearly dry at 50°C-70°C, dissolve the residue with acetonitrile aqueous solution and make up the volume, take the supernatant after centrifugation and add it to the purifier, which includes N-propylethylenediamine and octadecylsilane, shake and mix well, centrifuge through an organic microporous filter membrane, and wait for machine detection; UHPLC detection: ultra-high performance liquid chromatography combined with ultraviolet detection.

2. The method for simultaneously determining four purine compounds according to claim 1, characterized in that: In the sample pretreatment step: the volume ratio of trifluoroacetic acid:formic acid:water was 9:9:

2.

3. The method for simultaneously determining four purine compounds according to claim 1, characterized in that: In the sample pretreatment step: the mixed acid solution was hydrolyzed at 90°C for 30 minutes.

4. The method for simultaneously determining four purine compounds according to claim 1, characterized in that: In the sample pretreatment steps: ice-bath immediately after water bath to room temperature, rotary evaporate at 60°C until nearly dry, dissolve the residue with 90% acetonitrile aqueous solution and make up to 10 mL, centrifuge at 8000 r / min for 10 min, take 2 mL of supernatant and add 0.225 g of purifier.

5. The method for simultaneously determining four purine compounds according to claim 1, characterized in that: In the sample pretreatment step: the mass ratio of N-propylethylenediamine and octadecylsilane is 1:

1.

6. The method for simultaneously determining four purine compounds according to claim 1, characterized in that: Sample pretreatment steps: shake and mix, then centrifuge through a 0.22µm organic microporous filter membrane.

7. The method for simultaneously determining four purine compounds according to claim 1, characterized in that: In the UHPLC detection step, the conditions were: Agilent InfinityLab Poroshell 120 HILIC-Z (4.6 mm × 100 mm, 2.7 µm) was selected; the mobile phase was 20 Mm ammonium acetate aqueous solution (pH = 9); the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 20 µL; and the UV detection wavelength was 254 nm.

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