Method for detecting content of purine in shellfish

Abstract

The invention discloses a shellfish purine content detection method, and belongs to the technical field of purine detection, and the method comprises the following steps: S1, adding mixed acid into a container, uniformly mixing with a shellfish sample, carrying out water bath heating on the container, rapidly cooling after the water bath heating is finished, placing an acid hydrolysate in the container in a nitrogen blowing concentrator to remove volatile matters, s2, respectively preparing adenine, guanine, hypoxanthine and xanthine single standard stock solutions, mixing and diluting the single standard stock solutions into a mixed stock solution, gradually diluting the mixed stock solution into standard series solutions, and filtering the standard series solutions for later use; and S4, carrying out reversed-phase high-performance liquid chromatography separation on the filtrate, and calculating the purine content of the shellfish sample according to the peak area of the filtrate and the standard curve. According to the method disclosed by the invention, a reliable technical support is provided for marine product purine detection by combining optimization of an acid hydrolysis process with optimization of a high performance liquid chromatography technology.

Description

Technical Field

[0001] This invention belongs to the field of purine detection technology, and particularly relates to a method for detecting purine content in shellfish. Background Technology

[0002] Purines, as key components of nucleic acids, are also the basis of many bioactive molecules, mainly including adenine, guanine, hypoxanthine, and xanthine. Under normal circumstances, purines ingested by the human body are converted into uric acid under the catalysis of enzymes, with dietary intake accounting for one-third of this. However, when purine intake is too high, the abnormal increase in purine levels can interfere with uric acid metabolism, leading to elevated serum uric acid concentrations. Persistent hyperuricemia can cause urate crystals to form and deposit in tissues, thereby triggering gout and other health problems, such as severe joint pain and swelling, as well as various diseases associated with diabetes, metabolic syndrome, dyslipidemia, chronic kidney disease, and stroke. Therefore, adopting a low-purine diet strategy is crucial for preventing and alleviating these symptoms, especially for individuals with abnormal purine metabolism; reducing dietary purine intake is of great significance in lowering serum uric acid levels. Given the close link between diet and hyperuricemia and gout, adjusting dietary habits, combined with modern nutrition science and traditional dietary knowledge, can effectively prevent and control the development of these metabolic diseases.

[0003] Given the diversity of marine shellfish species and the impact of their growth environment and seasonal variations on their nutritional composition, particularly purine content, in-depth research into the relationship between marine shellfish and purine content is crucial for assessing the nutritional value of these delicious and nutritious seafoods and their impact on specific health conditions. According to the WS / T 560-2017 guideline, it is recommended to discard the broth after cooking seafood and meat to reduce purine intake and help manage diseases. Therefore, studying the purine content of marine shellfish can help consumers make healthier dietary choices.

[0004] There are various methods for determining purine content in food, with high-performance liquid chromatography (HPLC) being the mainstream method. Current research mainly focuses on beer, soy products, and livestock and poultry meat, while there is relatively little research on the purine content of seafood, and the detection methods and data need to be updated. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting purine content in shellfish, thereby overcoming at least one of the aforementioned defects in the prior art.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] This invention provides a method for detecting purine content in shellfish, comprising the following steps: S1: Sample pretreatment, placing shellfish samples into a container, adding mixed acid to the container and mixing it evenly with the shellfish samples, heating the container in a water bath, rapidly cooling after the water bath heating is completed, placing the acid hydrolysate in the container in a nitrogen blower to remove volatiles, dissolving the residue in water and making up to volume to obtain a sample solution, then centrifuging the sample solution, taking the supernatant and filtering it to obtain the filtrate for later use; S2: Preparation of standard solutions, preparing single standard stock solutions of adenine, guanine, hypoxanthine and xanthine respectively, mixing and diluting them to form a mixed stock solution, and then diluting them stepwise to form a series of standard solutions, filtering them for later use; S3: Plotting a standard curve, plotting a standard curve with the mass concentration of the standard series solutions in step S2 as the abscissa and the corresponding peak area as the ordinate; S4: Calculating purine content, performing reversed-phase high-performance liquid chromatography separation on the filtrate in step S1, and calculating the purine content of the shellfish sample based on the peak area of ​​the filtrate and the standard curve.

[0008] Preferably, step S1 specifically includes the following steps: Take 1.50-2.50g of shellfish sample and put it into a 40-60mL stoppered centrifuge tube. Add mixed acid to the stoppered centrifuge tube and mix it evenly with the shellfish sample. Heat the stoppered centrifuge tube in a water bath at 80-90℃ for 13-17min. After the water bath heating is completed, cool it rapidly in an ice-water bath. Place the acid hydrolysate in the stoppered centrifuge tube in a nitrogen blower at 55-65℃ to remove volatiles. Dissolve the residue with pure water and make up to 8-12mL to obtain a sample solution. Then, centrifuge the obtained sample solution at 5000-7000g for 12-18min. Take the supernatant and filter it through a 0.20-0.24μm organic microporous membrane to obtain the filtrate for later use.

[0009] Preferably, in step S1, the mixed acid consists of trifluoroacetic acid with a mass concentration of 90.00% and formic acid with a mass concentration of 55.00%, and the volume ratio of trifluoroacetic acid to formic acid is 1:1.

[0010] Preferably, in step S1, the water bath heating temperature is 85°C, the water bath heating time is 15 min, and the solid-liquid ratio of the shellfish sample to the mixed acid is 1:5.

[0011] Preferably, step S2 specifically includes the following steps: accurately weigh 55.00-65.00 mg of adenine, guanine, hypoxanthine, and xanthine standards respectively, dissolve them in ultrapure water, and dilute them to 40-60 mL volumetric flasks. Simultaneously, use 0.8-1.2 mol / L sodium hydroxide solution to aid dissolution, and prepare single standard stock solutions of adenine, guanine, hypoxanthine, and xanthine with a mass concentration of 1100-1300 mg / L respectively. Store them in a refrigerator at a constant temperature of 4°C. Take equal volumes of the single standard stock solutions of the above four purines, mix and dilute them to prepare mixed standard stock solutions with a mass concentration of 350-450 mg / L, and then dilute them stepwise with ultrapure water to prepare a series of standard solutions with different mass concentrations. Filter the solutions using a 0.20-0.24 μm microporous membrane for later use.

[0012] Preferably, in step S4, a Waters high-performance liquid chromatography system is used, equipped with a variable wavelength UV 2489 detector and an autosampler, using an InertSustain-C18 column (250mm×4.6mm×5μm, GLscience), with the column temperature maintained at 30℃, the detection wavelength at 254nm, the mobile phase flow rate set at 0.8mL / min, and the injection volume at 10μL.

[0013] Preferably, in step S4, the mobile phase is water, methanol, glacial acetic acid and tetrabutylammonium hydroxide, the volume ratio of the water, methanol, glacial acetic acid and tetrabutylammonium hydroxide solution is 882:100:15:3, and the mass concentration of tetrabutylammonium hydroxide is 40%.

[0014] Preferably, in step S4, the formula for calculating the purine content in the shellfish sample is as follows:

[0015]

[0016] In the formula, X represents the content of adenine, guanine, xanthine, or hypoxanthine in the shellfish sample, in milligrams per 100 grams; c represents the mass concentration of each component of adenine, guanine, xanthine, and hypoxanthine in the filtrate as determined by the standard curve, in milligrams per liter; v represents the final volume of the sample solution, in milliliters; m represents the mass of the shellfish sample, in grams; and 100 / 1000 represents the unit conversion factor.

[0017] The beneficial effects of this invention are as follows:

[0018] By optimizing the acid hydrolysis process and combining it with high-performance liquid chromatography (HPLC), a mixed acid hydrolysis method using trifluoroacetic acid (TFA) and formic acid (FA) (85℃, 15 min, solid-liquid ratio 1:5, TFA concentration 80%, FA concentration 55%) was developed. This method efficiently releases bound purines, achieving an extraction rate of 248.53 ± 2.58 mg / 100 g, with a stable recovery rate between 93.04% and 103.07%. The established HPLC method achieved baseline separation of adenine, guanine, hypoxanthine, and xanthine within 7 min, exhibiting a wide linear range (0.2-300 mg / L) and high sensitivity (LOD 0.01-0.10 mg / L), providing reliable technical support for purine detection in seafood. Attached Figure Description

[0019] Figure 1 These are chromatograms of purine standards under different mobile phases according to the present invention.

[0020] Figure 2 This is a chromatogram of purine samples under different mobile phases according to the present invention (Philippine clam as sample).

[0021] Figure 3 This is a chromatogram of adenine, guanine, hypoxanthine and xanthine mixture at 254 nm.

[0022] Figure 4 This is a graph showing the effect of hydrolysis temperature on the extraction of purines using the trifluoroacetic acid / formic acid method of this invention.

[0023] Figure 5 This is a graph showing the effect of hydrolysis time on the extraction of purines using the trifluoroacetic acid / formic acid method of the present invention.

[0024] Figure 6 This is a graph showing the effect of TFA mass concentration on the extraction of purines using the trifluoroacetic acid / formic acid method of this invention.

[0025] Figure 7 This is a graph showing the effect of FA mass concentration on the extraction of purines using the trifluoroacetic acid / formic acid method of this invention.

[0026] Figure 8 This is a graph showing the effect of different solid-liquid ratios of shellfish samples and mixed acids on the extraction of purines using the trifluoroacetic acid / formic acid method.

[0027] Figure 9 This is a 3D response surface and contour plot showing the effect of the interaction between hydrolysis temperature and time on purine content.

[0028] Figure 10 This invention presents a 3D response surface and contour plot showing the effect of the interaction between formic acid concentration and hydrolysis time on purine content.

[0029] Figure 11This invention presents a 3D response surface and contour plot showing the effect of the interaction between formic acid concentration and hydrolysis temperature on purine content. Detailed Implementation

[0030] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments.

[0031] This embodiment provides a method for detecting purine content in shellfish, comprising the following steps:

[0032] S1: Sample pretreatment. Take 2.00g (accurate to 0.01g) of shellfish sample and put it into a 50mL stoppered centrifuge tube. Add mixed acid to the stoppered centrifuge tube and mix it evenly with the shellfish sample. The mixed acid consists of 90.00% trifluoroacetic acid and 55.00% formic acid, with a volume ratio of 1:1. Heat the stoppered centrifuge tube in an 85℃ water bath for 15min. After the water bath heating is completed, cool it rapidly in an ice-water bath. Place the acid hydrolysate in the stoppered centrifuge tube in a nitrogen blower at 60℃ to remove volatiles. Dissolve the residue with pure water and make up to 10mL to obtain the sample solution. Then centrifuge the obtained sample solution at 6000g for 15min. Take the supernatant and filter it through a 0.22μm organic microporous membrane to obtain the filtrate for later use.

[0033] S2: Preparation of standard solutions: Accurately weigh 60.00 mg of adenine, guanine, hypoxanthine, and xanthine standards respectively, dissolve them in ultrapure water, and dilute to 50 mL in volumetric flasks. Mix well, and use 1 mol / L sodium hydroxide solution to aid dissolution. Prepare single standard stock solutions of adenine, guanine, hypoxanthine, and xanthine with a mass concentration of 1200 mg / L. Store them in a refrigerator at a constant temperature of 4℃. Take equal volumes of the single standard stock solutions of the above four purines, mix and dilute them to prepare a mixed standard stock solution with a mass concentration of 400 mg / L. Then, dilute with ultrapure water stepwise to prepare a series of standard solutions with different mass concentrations. Filter the solutions using a 0.22 μm microporous membrane for later use.

[0034] S3: Plot the standard curve. Plot the standard curve with the mass concentration of the standard series solutions from step S2 as the x-axis and the corresponding peak area as the y-axis.

[0035] S4: Purine content calculation. The filtrate from step S1 was separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Specifically, a Waters HPLC system equipped with a variable wavelength UV-2489 detector and an autosampler was used. An InertSustain-C18 column (250 mm × 4.6 mm × 5 μm, GLscience) was employed, with the column temperature maintained at 30 °C. The detection wavelength was 254 nm, the mobile phase flow rate was set to 0.8 mL / min, and the injection volume was 10 μL. The mobile phase consisted of water, methanol, glacial acetic acid, and tetrabutylammonium hydroxide, with a volume ratio of 882:100:15:3. The mass concentration of tetrabutylammonium hydroxide was 40%. The purine content of the shellfish sample was calculated based on the peak area of ​​the filtrate and the standard curve.

[0036] The formula for calculating the purine content in shellfish samples is as follows:

[0037]

[0038] In the formula, X represents the content of adenine, guanine, xanthine, or hypoxanthine in the shellfish sample, in milligrams per 100 grams; c represents the mass concentration of each component of adenine, guanine, xanthine, and hypoxanthine in the filtrate as determined by the standard curve, in milligrams per liter; v represents the final volume of the sample solution, in milliliters; m represents the mass of the shellfish sample, in grams; and 100 / 1000 represents the unit conversion factor.

[0039] To obtain the optimal process parameters for detecting purine content in shellfish, the following experiments were conducted in this embodiment:

[0040] 1.1 Experimental Methods

[0041] 1.1.1 Sampling and Preprocessing

[0042] Twenty-one fresh shellfish species (including jade mussels, Manila clams, spotted whelks, four-cornered clams, razor clams, American clams, red whelks, tube snails, blunt-spotted clams, hard clams, warty whelks, giant otter clams, wrinkled abalone, mud clams, wavy clams, oysters, *Myxocys squarrosa*, purple stone clam, large bamboo clam, noble scallop, and comb-shaped clams) were purchased from the Xiamen Seafood Wholesale Market (April-June). The shellfish meat (including viscera) was dissected in the laboratory, homogenized, stored at -20℃, and analyzed within 2 days.

[0043] 1.1.2 Purine Detection Method

[0044] 1.1.2.1 Liquid Chromatography Separation Method

[0045] A Waters high-performance liquid chromatography (HPLC) system equipped with a variable wavelength UV-2489 detector and an autosampler was used. An InertSustain-C18 column (250 mm × 4.6 mm × 5 μm, GLscience) was employed, maintained at 30 °C, and the detection wavelength was 254 nm. To ensure proper separation of all target analytes, the mobile phase consisted of water, methanol, glacial acetic acid, and 40% tetrabutylammonium hydroxide in a volume ratio of 882:100:15:3 (v / v / v / v). The mobile phase flow rate was set to 0.8 mL / min, and the injection volume was 10 μL.

[0046] 1.1.2.2 Preparation of Standard Solutions

[0047] Prepare 1200 mg / L single standard stock solutions by accurately weighing 60.00 mg of adenine, guanine, hypoxanthine, and xanthine standards, dissolving them in ultrapure water, and diluting each solution to 50 mL in volumetric flasks. Use 1 mol / L sodium hydroxide solution to aid dissolution and store at 4°C. Prepare a mixed standard stock solution with a concentration of 400 mg / L by taking equal volumes of the single standard stock solutions of the four purines. Then dilute with ultrapure water to prepare solutions of different concentrations. Filter the solutions through a 0.22 μm microporous membrane and perform chromatographic analysis.

[0048] 1.1.2.3 Method Validation

[0049] The newly developed detection method of this invention was comprehensively validated in accordance with ISO / IEC 17025:2018, "General requirements for the competence of testing and calibration laboratories". The following key analytical performance indicators were evaluated: linearity of the calibration curve, accuracy, precision, and repeatability of the method. Repeatability was calculated based on test results over a continuous 48-hour period. The linear response relationship was determined by plotting the analyte concentration versus peak area, and the coefficient of determination (R²) was used. 2 The relationship was evaluated. The limits of detection and quantitation were determined by plotting standard curves, corresponding to concentration levels with signal-to-noise ratios of 3 and 10, respectively. To assess the precision of the method and the reproducibility of the purine extraction process, six replicate tests were performed on the mixed purine base standard solution and the samples extracted from Manila clams, and the relative standard deviation (RSD%) was calculated. This step ensured the reliability and consistency of the method in practical applications.

[0050] 1.1.3 Purine Extraction Method

[0051] 1.1.3.1 TFA / FA hydrolysis

[0052] Weigh 2.00 g (accurate to 0.01 g) of the sample into a 50 mL stoppered centrifuge tube, add 10 mL of LTFA / FA (1:1, v / v) into a 50 mL centrifuge tube, mix well, incubate at 85 °C for 15 min, then rapidly cool (ice-water bath). Place the acid hydrolysate in a nitrogen blower at 60 °C to remove volatiles. Dissolve the residue in pure water and bring the volume to 10 mL. Centrifuge at 6000 g for 15 min, collect the supernatant, and filter through a 0.22 μm organic microporous membrane. The filtrate is ready for analysis. Purine content calculation formula:

[0053]

[0054] In the formula, X represents the content of adenine, guanine, xanthine, or hypoxanthine in the shellfish sample, in milligrams per 100 grams; c represents the mass concentration of each component of adenine, guanine, xanthine, and hypoxanthine in the filtrate as determined by the standard curve, in milligrams per liter; v represents the final volume of the sample solution, in milliliters; m represents the mass of the shellfish sample, in grams; and 100 / 1000 represents the unit conversion factor.

[0055] 1.1.3.2 One-way test

[0056] Several methods are known for extracting purines from samples. Among them, acid hydrolysis is the most commonly used pretreatment method because it can efficiently hydrolyze purine compounds such as nucleic acids, nucleotides, and nucleosides in food into free purine bases while preventing further degradation of purine bases. Under acidic conditions, the protonation degree of purines increases, leading to the breakage of the C1-N9 glycosidic bond, thereby releasing purine bases. This experiment selected a mixed acid method using trifluoroacetic acid and formic acid. Hydrolysis conditions were optimized using five factors at different levels: hydrolysis temperature (40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃); hydrolysis time (5, 10, 15, 20, 25, 30, 35, 40 min); trifluoroacetic acid (TFA) and formic acid (FA) concentrations (20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%); and shellfish meat-TFA / FA (1:1, v / v) solid-liquid ratio (1:1, 1:3, 1:5, 1:7, 1:10, 1:13, 1:15, 1:20).

[0057] 1.1.3.3 Response Surface Design

[0058] This experiment used response surface methodology to optimize purine extraction process parameters. Based on single-factor experiments to determine the influence of each parameter, the Box-Behnken experimental design principle was applied. With trifluoroacetic acid concentration and solid-liquid ratio fixed, three key factors significantly affecting the hydrolysis effect were selected as independent variables: hydrolysis temperature (A), hydrolysis time (B), and formic acid concentration (C). A three-factor, three-level response surface methodology was used, with purine extraction rate as the response value. A mathematical model was constructed using Design-Expert 10.0 statistical software to establish a quantitative relationship between process parameters and response values, aiming to determine the optimal combination of extraction conditions. The experimental design included 17 experimental points, including 5 centroids for evaluating experimental error. The level design of the response surface experimental factors is shown in Table 1.

[0059] Table 1. Box-Behnken Experimental Factor Level Design

[0060]

[0061] 1.1.3.4 Analyte spike recovery rate

[0062] The recoveries of adenine, guanine, hypoxanthine, and xanthine were determined using samples with previously tested purine content. In each case, the known number of single purine base standards were 0.5, 1, and 2 times, respectively. The formulas are as follows:

[0063]

[0064] In the formula, %Recovery is the spiked recovery rate, α is the purine content in the sample with added purine base standard, β is the purine content in the sample without added purine base standard, and C is the known content of added purine base standard in the sample.

[0065] 1.1.4 Storage Experiment

[0066] Manila clams, abalone, whelks, razor clams, and oysters were selected and stored at 25℃ (3, 6, 9, 12, 15, 18, 21, 24h), 4℃ (3, 6, 12, 18, 24, 36, 48, 60, 72h), and -20℃ (0, 2, 4, 6, 8, 10, 12, 15, 30, 60, 90, 120, 180d) for storage experiments. Fresh shellfish were used as blank controls. The changes in purine content under different storage temperatures were analyzed by HPLC.

[0067] 1.1.5 Determination of volatile basic nitrogen

[0068] Apply water-soluble adhesive to the edge of the diffusion dish, then add 1.0 mL of boric acid solution to the center of the dish, along with one drop of mixed indicator. After completing these steps, add 1.0 mL of filtrate to the outer chamber. Cover with a ground glass lid, leaving a small gap, and carefully check the airtightness of the apparatus. Once the airtightness check is confirmed, quickly add 1.0 mL of saturated potassium carbonate solution to the gap, seal the lid, and mix thoroughly by circular motion. Incubate at 37°C for 2 hours, then titrate with a 0.01 mol / L standard titration solution of hydrochloric acid or sulfuric acid, using a mixed indicator of 1 part methyl red ethanol solution and 5 parts bromocresol green ethanol solution. The endpoint should be purple-red. Prepare a reagent blank simultaneously.

[0069] 1.2 Results and Discussion

[0070] 1.2.1 Establishment of a purine detection method

[0071] This experiment investigated the mobile phase, column temperature, flow rate, and other conditions for purine processing in order to establish a suitable method.

[0072] Different mobile phases: such as Figure 1-2 As shown, mobile phase A: 0.01M ammonium formate / methanol; mobile phase B: 0.02M KH2PO4 / methanol; mobile phase C: water / methanol / glacial acetic acid / 40% tetrabutylammonium hydroxide. The results show that all three mobile phases can effectively separate the chromatographic peaks. Compared to KH2PO4 and ammonium formate as mobile phases, using a mobile phase prepared with water, methanol, glacial acetic acid, and 40% tetrabutylammonium hydroxide in a ratio of 882:100:15:3 (v / v / v / v) significantly shortens the time required for purine separation, thus achieving a more efficient separation effect.

[0073] In terms of column selection, the InertSustain-C18 column and the Ultimate-AQ-C18 column were compared. It was found that the InertSustain-C18 column provided highly stable analytical chromatograms for the quantification of all compounds.

[0074] To achieve ideal peak shape and separation, different column temperatures and flow rates were subsequently evaluated. Column temperature and flow rate had little impact on the spectra; the optimal resolution was obtained when the temperature was set at 30℃ and the flow rate at 0.8 mL / min.

[0075] This experiment optimized the HPLC detection method to simultaneously and quantitatively determine the contents of adenine, guanine, hypoxanthine, and xanthine in marine shellfish. Figure 3 As shown, the four purine bases were separated within 7 minutes. The purine compounds had short elution times and good separation effects. There were no excessive interference peaks around the retention times of the target compounds in the sample, and the chromatographic separation was more stable.

[0076] 1.2.1.2 Validation of Analytical Methods

[0077] Strict use of standard solutions eliminated the potential influence of sample matrix effects on the slope of the calibration curve, ensuring the accuracy and reliability of the validation results. Table 2 lists the detailed linear regression equations. Within a specific concentration range, guanine was 0.2–300 mg / L, while adenine, hypoxanthine, and xanthine were 0.2–250 mg / L. The calibration curves for all targets showed excellent linearity, with correlation coefficients (R²) of approximately 100%. 2 The values ​​all exceeded 0.9997, which further verified the stability and reliability of the method.

[0078] Table 2. Results of linearity, precision, repeatability, and stability (n=6)

[0079]

[0080]

[0081] Analysis of the calibration curve slope revealed that this method exhibits the highest detection sensitivity for adenine, followed by hypoxanthine, while its sensitivity for guanine and xanthine is relatively low. The limits of detection (LOD) and quantitation (LOQ), as well as the precision, accuracy, and reproducibility of the method, were evaluated and tested according to the method described in 1.1.2.3. Table 2 also provides the LOD and LOQ values ​​for each compound, along with their precision (expressed as CV%, ranging from 0.99% to 1.03%), stability (ranging from 0.02% to 0.13%), and repeatability (ranging from 0.01% to 0.10%). These data confirm the high accuracy of the developed detection method.

[0082] 1.2.2 Optimization of TFA / FA hydrolyzed purines

[0083] 1.2.2.1 Single-factor experiment

[0084] Hydrolysis conditions were optimized through single-factor experiments to completely convert bound purines in marine shellfish into free purines, which were then detected using high-performance liquid chromatography (HPLC). The study showed that the type and amount of acid significantly affected the purine extraction efficiency. Compared to sulfuric acid, phosphoric acid, and hydrochloric acid, a mixture of trifluoroacetic acid and formic acid, as well as perchloric acid, were more effective at extracting purines. Although perchloric acid showed good extraction results, its long hydrolysis time, low extraction rate, and the potential for purine degradation and the generation of toxic chlorine gas at high concentrations led this experiment to choose a mixed acid method using trifluoroacetic acid and formic acid. The results indicated that hydrolysis time, hydrolysis temperature, solid-liquid ratio, and the concentrations of trifluoroacetic acid (TFA) and formic acid (FA) solutions all significantly affected the purine extraction efficiency.

[0085] like Figure 4 As shown, the hydrolysis temperature significantly affects the purine hydrolysis efficiency (P < 0.05). The hydrolysis rate increases rapidly with rising temperature, reaching a maximum of 241.68 mg / 100g at 80℃. However, the overall extraction efficiency decreases slightly when the temperature exceeds 80℃. This may be because at lower temperatures, the average kinetic energy of the reactant molecules is lower, weakening the acid's catalytic effect and leading to a slower reaction rate and incomplete hydrolysis. Furthermore, when the temperature exceeds 80℃, purine bases may undergo thermal degradation, destroying their structure and making them undetectable or unusable.

[0086] according to Figure 5 The results show that the solid-liquid ratio significantly affects the acid hydrolysis rate of purines by influencing factors such as reactant concentration, diffusion rate, and reaction surface area. The optimal extraction efficiency of purines is achieved when the solid-liquid ratio is 1:5. Figure 6 The results showed that different purines exist in different forms and have varying stability in aquatic products, and the degree of purine hydrolysis is determined by the hydrolysis time. In a shorter time, only a portion of the purines are hydrolyzed; however, as time increases, more purines are released, leading to an increase in the free purine content in the food. The purine content reaches its highest value of 245.49 mg / 100g at 15 min, and then decreases and tends to stabilize.

[0087] like Figure 7 and Figure 8 As shown, acid concentration has a significant impact on purine extraction efficiency. Too low an acid concentration leads to insufficient hydrolysis efficiency, while excessively high concentrations of formic acid or trifluoroacetic acid can disrupt the stability of purine molecules, causing purine degradation or conversion into other compounds, thus reducing the actual measured purine content. Therefore, the optimal purine extraction efficiency is achieved when the formic acid concentration is 60% and the trifluoroacetic acid concentration is 80%. Figure 8 In the diagram, the left vertical axis corresponds to the purine content of each analyte: adenine, guanine, hypoxanthine, and xanthine, while the right vertical axis corresponds to the total purine content of the five analytes.

[0088] 1.2.2.2 Response Surface Experiment of Purine Hydrolysis Process

[0089] 1.2.2.2.1 Box-Behnken Experimental Design and Results

[0090] Based on the results of previous single-factor experimental analysis, this study used response surface methodology to optimize the purine extraction process. The solid-liquid ratio (1:5, g / mL) and trifluoroacetic acid concentration (80%, v / v) were set as fixed parameters. Three key influencing factors—hydrolysis temperature (A), hydrolysis time (B), and formic acid concentration (C)—were selected as independent variables, with purine extraction yield as the response index. Following the Box-Behnken central composite design principle, a three-factor, three-level experimental scheme was constructed, resulting in 17 experimental sites (including 5 central sites for error analysis). Specific experimental parameter combinations and corresponding response values ​​are detailed in Table 3.

[0091] Table 3. Box-Behnken design and purine content determination results (n=3)

[0092]

[0093]

[0094] The data in the table are the mean ± standard deviation.

[0095] 1.2.2.2.2 Model Establishment and Analysis of Variance

[0096] The experimental data were analyzed using Design-Expert 8.06 statistical software, and the following quadratic polynomial regression equation was established:

[0097] Y=251.98+3.03A+4.95B-1.79C+5.01AB+12.74AC-4.72BC-13.44A 2 -9.78B 2 -4.67C 2 . square

[0098] The difference analysis results (Table 4) show that the model is highly significant (F = 40.96, P < 0.0001), and the lack-of-fit term is not significant (P = 0.4605 > 0.05), indicating that the model fits well and the prediction results are reliable. The model's coefficient of determination R0 2 =0.9723, indicating that the model can explain 97.23% of the response value variation; the corrected coefficient of determination R0 2 adj = 0.9366 and the predictive determination coefficient R 2 The difference between pred = 0.7797 and pred = 0.1569 is less than 0.2, further validating the model's applicability. Furthermore, the coefficient of variation (CV%) is 1.34% (<10%), confirming the high accuracy of the experimental data. Significance analysis shows that the substrate concentration B and its quadratic term A... 2 B 2 C 2The effects on the response values ​​were all highly significant (P < 0.001). By comparing the F values ​​of each factor, the influence of the experimental parameters on purine content was as follows: hydrolysis temperature (A) > hydrolysis time (B) > formic acid concentration (C).

[0099] Table 4. Analysis of Variance for Response Surface Regression Model

[0100]

[0101]

[0102] * indicates a statistically significant difference (P < 0.05); ** indicates a highly significant difference (P < 0.01); - indicates no significant difference. Analysis of variance (ANOVA) and Dunnett's multiple comparison test were used to determine the significance of the differences.

[0103] 1.2.2.2.3 Interaction Analysis

[0104] Based on the established model and fitted regression formula, this study, through analysis of the three-dimensional response surface plot and its contour plot, explored in depth the influence of the interaction between two other factors on purine hydrolysis content, assuming one factor is fixed and its central value remains constant. By observing the slope of the three-dimensional response surface and the shape of the contour lines, the strength of the interaction between the two factors on the response value can be more intuitively reflected. Specifically, the steeper the three-dimensional response surface and the denser and more elliptical the contour lines, the more significant the interaction on the response value, and the stronger the interaction between the two factors. Figures 9-11 show the 3D response surface and contour plot of the influence of the pairwise interactions of hydrolysis temperature (A), hydrolysis time (B), and formic acid concentration (C) on the purine content response value. Figure 9 Partially, when the formic acid concentration is within the ideal range, the response value shows a trend of first increasing and then rapidly decreasing with the simultaneous increase of hydrolysis temperature and time, and the steepness of the reaction surface is observed to be relatively consistent. This means that the hydrolysis effect of purines is not ideal under relatively low temperature and time conditions.

[0105] according to Figure 10 The results showed that when the hydrolysis temperature reached the ideal state, the effect of hydrolysis time on purine content was not significant if the formic acid concentration was high. However, with the extension of hydrolysis time, the response value showed a trend of first increasing and then decreasing when the formic acid concentration was high; while when the formic acid concentration was kept constant, the response value first increased and then decreased significantly with the extension of hydrolysis time. Furthermore, the influence of hydrolysis time on the reaction surface was significantly greater than that of formic acid concentration, indicating that hydrolysis time had a more significant effect on the response value.

[0106] according to Figure 11 Analysis showed that at lower temperatures, when the hydrolysis time reached its optimal state, the formic acid concentration had no significant effect on the response value; however, at higher temperatures, the response value first increased and then decreased. Keeping the formic acid concentration constant, with increasing hydrolysis time, the response value first rose rapidly and then slowly decreased or stabilized. Analysis of the ellipticity of the contour lines and the F-values ​​of the AB, AC, and BC interaction terms in Table 4 revealed that the interaction effect between formic acid concentration and hydrolysis time significantly outweighed the influence of the other two interactions on purine content.

[0107] After processing and analysis using Design-Expert 10.0 software, the optimal preparation process parameters for purine hydrolysis were determined: hydrolysis temperature of 83.40℃, hydrolysis time of 15.06 min, and formic acid concentration of 56.52%. Under these conditions, the model predicted a total purine hydrolysis value of 253.15 mg / 100g. To better adapt to the controllability of the experimental operation, the optimal hydrolysis process parameters were appropriately adjusted, and the final determined process conditions were: hydrolysis temperature of 85℃, hydrolysis time of 15 min, formic acid concentration of 55%, combined with a trifluoroacetic acid concentration of 80% and a solid-liquid ratio of 1:5 for experimental operation. Based on these conditions, three repeated experiments were conducted for verification. The purine content was 248.53±2.58 mg / 100g. The relative error of the effectiveness of the prediction model was 1.83%, and the difference between the predicted and actual values ​​was less than 5% (Table 5). The purine content extracted by mixed acid in this experiment was 3.94%. The purine hydrolysis regression model extracted by trifluoroacetic acid and formic acid has high practicality and high fitting degree, and can be used for the preparation of purine hydrolysis.

[0108] Table 5. Optimal conditions, adjustment conditions, predicted response values ​​under these conditions, and experimental values ​​(n=6)

[0109]

[0110] 1.2.2.3 Spike Recovery Analysis

[0111] All spiked samples were treated using the optimized sample pretreatment method. The recoveries of each concentration were evaluated, and three samples at each concentration were measured to estimate the recoveries and RSD% of each purine. As shown in Table 6, the recoveries of the four purines ranged from 93.04% to 103.07%. The results indicate that the established sample preparation method is suitable for the determination of purine content in marine shellfish. Based on the purine extraction rate of 83.75% using perchloric acid, the recovery results in this experiment are superior.

[0112] Table 6 Results of the spiked recovery test (n=3)

[0113]

[0114] The purine content of edible parts of 21 marine shellfish was systematically studied by optimizing acid hydrolysis combined with high-performance liquid chromatography (HPLC). The results showed that mixed acid hydrolysis with trifluoroacetic acid (TFA) and formic acid (FA) (85℃, 15 min, solid-liquid ratio 1:5, TFA concentration 80%, FA concentration 55%) efficiently released bound purines, achieving an extraction rate of 248.53 ± 2.58 mg / 100 g, with a stable recovery rate between 93.04% and 103.07%. The established HPLC method achieved baseline separation of adenine, guanine, hypoxanthine, and xanthine within 7 min, exhibiting a wide linear range (0.2-300 mg / L) and high sensitivity (LOD 0.01-0.10 mg / L), providing reliable technical support for purine detection in seafood.

[0115] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting purine content in shellfish, characterized in that, Includes the following steps: S1: Sample pretreatment: Take shellfish samples and put them into a container. Add mixed acid to the container and mix it evenly with the shellfish samples. Heat the container in a water bath. After the water bath heating is completed, cool it quickly. Place the acid hydrolysate in the container in a nitrogen blower to remove volatiles. Dissolve the residue in water and make up to volume to obtain the sample solution. Then centrifuge the sample solution, take the supernatant and filter it to obtain the filtrate for later use. S2: Preparation of standard solutions: Prepare single standard stock solutions of adenine, guanine, hypoxanthine and xanthine respectively, mix and dilute them to form a mixed stock solution, and then dilute them stepwise to form a series of standard solutions. Filter and use them for later use. S3: Plotting the standard curve, using the mass concentration of the standard series solutions from step S2 as the x-axis and the corresponding peak area as the y-axis; S4: Purine content calculation. The filtrate from step S1 is separated by reversed-phase high-performance liquid chromatography. The purine content of the shellfish sample is calculated based on the peak area of ​​the filtrate and the standard curve.

2. The method for detecting purine content in shellfish according to claim 1, characterized in that, Step S1 specifically includes the following steps: Take 1.50-2.50g of shellfish sample and place it into a 40-60mL stoppered centrifuge tube. Add mixed acid to the stoppered centrifuge tube and mix it evenly with the shellfish sample. Heat the stoppered centrifuge tube in a water bath at 80-90℃ for 13-17 minutes. After the water bath heating is completed, cool it rapidly in an ice-water bath. Place the acid hydrolysate in the stoppered centrifuge tube in a nitrogen blower at 55-65℃ to remove volatiles. Dissolve the residue with pure water and make up to 8-12mL to obtain the sample solution. Then centrifuge the sample solution at 5000-7000g for 12-18 minutes. Take the supernatant and filter it through a 0.20-0.24μm organic microporous membrane to obtain the filtrate for later use.

3. The method for detecting purine content in shellfish according to claim 1, characterized in that: In step S1, the mixed acid consists of trifluoroacetic acid with a mass concentration of 90.00% and formic acid with a mass concentration of 55.00%. The volume ratio of trifluoroacetic acid to formic acid is 1:

1.

4. The method for detecting purine content in shellfish according to claim 1, characterized in that: In step S1, the water bath heating temperature is 85℃, the water bath heating time is 15min, and the solid-liquid ratio of the shellfish sample to the mixed acid is 1:

5.

5. The method for detecting purine content in shellfish according to claim 1, characterized in that, Step S2 specifically includes the following steps: Accurately weigh 55.00-65.00 mg of adenine, guanine, hypoxanthine, and xanthine standards respectively, dissolve them in ultrapure water, and dilute to 40-60 mL in volumetric flasks. Mix well, and use 0.8-1.2 mol / L sodium hydroxide solution to aid dissolution. Prepare single standard stock solutions of adenine, guanine, hypoxanthine, and xanthine with a mass concentration of 1100-1300 mg / L respectively, and store them in a refrigerator at a constant temperature of 4℃. Take equal volumes of the single standard stock solutions of the above four purines, mix and dilute them to prepare mixed standard stock solutions with a mass concentration of 350-450 mg / L, and then add ultrapure water stepwise to dilute them to prepare a series of standard solutions with different mass concentrations. Filter the solutions using a 0.20-0.24 μm microporous membrane for later use.

6. The method for detecting purine content in shellfish according to claim 1, characterized in that: In step S4, a Waters high-performance liquid chromatography system was used, equipped with a variable wavelength UV 2489 detector and an autosampler. An InertSustain-C18 column (250 mm × 4.6 mm × 5 μm, GLscience) was used, with the column temperature maintained at 30 °C, the detection wavelength at 254 nm, the mobile phase flow rate set at 0.8 mL / min, and the injection volume at 10 μL.

7. The method for detecting purine content in shellfish according to claim 1, characterized in that: In step S4, the mobile phase is water, methanol, glacial acetic acid and tetrabutylammonium hydroxide, and the volume ratio of the water, methanol, glacial acetic acid and tetrabutylammonium hydroxide solution is 882:100:15:3, and the mass concentration of tetrabutylammonium hydroxide is 40%.

8. The method for detecting purine content in shellfish according to claim 1, characterized in that: In step S4, the formula for calculating the purine content in the shellfish sample is as follows: In the formula, X represents the content of adenine, guanine, xanthine, or hypoxanthine in the shellfish sample, in milligrams per 100 grams; c represents the mass concentration of each component of adenine, guanine, xanthine, and hypoxanthine in the filtrate as determined by the standard curve, in milligrams per liter; v represents the final volume of the sample solution, in milliliters; m represents the mass of the shellfish sample, in grams; and 100 / 1000 represents the unit conversion factor.

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