A kit and method for simultaneously detecting nine taste nucleotides in livestock and poultry products

Abstract

This invention provides a kit for the simultaneous detection of nine flavor nucleotides in livestock and poultry products. The flavor nucleotides are cytidine monophosphate, uridine monophosphate, guanylic acid, inosine monophosphate, adenosine monophosphate, hypoxanthine, inosine, adenosine diphosphate, and adenosine triphosphate. The kit includes: a high-concentration extraction reagent, a standard curve solution, a high-concentration phosphate buffer solution, pH adjustment solution A, and pH adjustment solution B. The high-concentration extraction reagent is perchloric acid; the standard curve solution is a mixed solution of the nine flavor nucleotides; the high-concentration phosphate buffer solution is a solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate; pH adjustment solution A is sodium hydroxide; and pH adjustment solution B is phosphate. This invention also provides a method for the simultaneous detection of the nine flavor nucleotides using the above kit. This invention enables the simultaneous determination of the content of nine flavor nucleotides in livestock and poultry products. The method is simple, rapid, accurate, and sensitive, and can be used for batch determination, showing good prospects for practical application.

Description

Technical Field

[0001] This invention belongs to the field of food testing and relates to a reagent kit, specifically a reagent kit and method for simultaneously detecting nine flavor nucleotides in livestock and poultry products. Background Technology

[0002] Nucleotides are important low-molecular-weight compounds in living organisms, possessing many unique physiological functions. Cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) all exhibit strong umami-enhancing effects and are known important compounds that produce delicious flavors, particularly enhancing the umami taste of meat. Inosine monophosphate (IMP) is a mononucleotide, a product of ATP metabolism, naturally occurring in muscle. In food, it is also a flavor compound that enhances the umami taste of meat, with its umami-enhancing ability being 40 times stronger than monosodium glutamate (MSG). The umami effect of inosine monophosphate is primarily due to the strong positive correlation between it and MSG. A mixture of inosine monophosphate and MSG in a ratio of 1:5 to 1:20 can increase the umami taste of MSG several times over, and it also has a controlling effect on sour and bitter tastes, i.e., it acts as a taste buffer. Its mechanism of action is that nucleotides remove metal ions from the umami sensory sites, thereby enabling monosodium glutamate to have an effective effect on the taste nerves.

[0003] The inosinic acid content in livestock and poultry products is influenced not only by intrinsic factors such as breed, age, and cut of meat, but also by external conditions such as slaughter age, slaughter method, and muscle storage time. Furthermore, factors such as stocking density and light exposure also affect inosinic acid content to varying degrees. Currently, as an important indicator for meat quality assessment, the determination of inosinic acid content in muscle tissue is widely used in the quality evaluation of various animal meat products and has become a crucial indicator for measuring meat quality. Therefore, establishing an accurate, rapid, and efficient method for determining the inosinic acid content in livestock and poultry muscle is of great significance for the quality evaluation of livestock and poultry products.

[0004] As one of the important umami substances in livestock and poultry meat, there are literature reports on the determination of inosinic acid (IMP) content in livestock and poultry products both domestically and internationally. Currently, the main methods used for IMP content detection include thin-layer chromatography, high-performance electrophoresis combined with ultraviolet spectrophotometry, and high-performance liquid chromatography (HPLC). When determining IMP content in meat products using ultraviolet spectrophotometry, the process is easily affected by interference from other substances due to the complex composition, leading to inaccurate results. High-performance liquid chromatography (HPLC), with its advantages of high separation efficiency, fast analysis speed, and high detection sensitivity, has been widely used in food analysis, biological and pharmaceutical testing, and chemical engineering.

[0005] Currently, the main domestic and international standards for the determination of nucleotide content in livestock and poultry meat include JECFA standard INS No. 630 "5'-Inosinic Acid", Appendix A of GB 1886.97—2015 "National Food Safety Standard Food Additive 5'-Inosinic Acid Disodium" and QB / T 4261—2011 "Food Additive 5′-Inosinic Acid Disodium". All three standards use ultraviolet spectrophotometry. In the past two years, the Shandong Provincial Market Supervision Administration issued the local standard DB 37 / T 3816—2019 "Determination of Inosinic Acid Content in Livestock and Poultry Muscle by High Performance Liquid Chromatography" and the Ningxia Chemical Analysis and Testing Association issued T / NAIA 003—2020 "Determination of Inosinic Acid in Muscle by High Performance Liquid Chromatography". Meanwhile, the national standard GB 5413.40—2016 "National Food Safety Standard Determination of Nucleotides in Infant Food and Dairy Products" provides determination of five nucleotides for infant food and dairy products, but does not establish relevant standards for livestock and poultry meat products. Summary of the Invention

[0006] To address the aforementioned technical problems in the prior art, this invention provides a kit and method for simultaneously detecting nine flavor nucleotides in livestock and poultry products. This kit and method for simultaneously detecting nine flavor nucleotides in livestock and poultry products solves the complex technical problems of existing methods for detecting the content of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) in livestock and poultry products.

[0007] This invention provides a kit for the simultaneous detection of nine flavor nucleotides in livestock and poultry products. The flavor nucleotides are cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP). The kit includes: a high-concentration extraction reagent, a standard curve solution, a high-concentration phosphate buffer solution, pH adjustment solution A, and pH adjustment solution B.

[0008] The high-concentration extraction reagent is perchloric acid; the standard curve solution is a mixed solution of nine flavor nucleotides; the high-concentration phosphate buffer solution is a solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate; the pH adjustment solution A is sodium hydroxide with a concentration of 0.8 mol / L to 1 mol / L; the pH adjustment solution B is phosphoric acid with a concentration of 4 mol / L to 5 mol / L.

[0009] Furthermore, the concentration of perchloric acid in the high-concentration extraction reagent is 5%~6% (V / V); the concentrations of potassium dihydrogen phosphate and dipotassium hydrogen phosphate in the high-concentration phosphate buffer solution are 8 mol / L, respectively.

[0010] Furthermore, the aforementioned nine flavor nucleotide mixture solution was prepared in eight batches. The concentration of any one flavor nucleotide in the first batch was 0.2 μg / mL, the concentration of any one flavor nucleotide in the second batch was 0.5 μg / mL, the concentration of any one flavor nucleotide in the third batch was 1.0 μg / mL, the concentration of any one flavor nucleotide in the fourth batch was 5.0 μg / mL, the concentration of any one flavor nucleotide in the fifth batch was 10.0 μg / mL, the concentration of any one flavor nucleotide in the sixth batch was 20.0 μg / mL, the concentration of any one flavor nucleotide in the seventh batch was 50.0 μg / mL, and the concentration of any one flavor nucleotide in the eighth batch was 100.0 μg / mL.

[0011] This invention discloses a method for simultaneously detecting the content of nine flavor nucleotides in livestock and poultry products, used for simultaneously detecting the content of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP), comprising the following steps:

[0012] (1) Add high-concentration extraction reagent to livestock and poultry products, mix with a homogenizer to obtain the first extract; clean the homogenizer blade with high-concentration extraction reagent to obtain the second extract; mix the first extract and the second extract, centrifuge, take the supernatant and pass it through a membrane to obtain the sample solution, and then perform high-performance liquid chromatography (equipped with a UV detector) for detection.

[0013] (2) Accurately pipette 5 mL of high-concentration phosphate buffer solution, add 900 mL of ultrapure water, adjust the pH to 4.6 with pH adjustment solution A and pH adjustment solution B, and make up to 1000 mL to prepare a 0.04 mol / L phosphate buffer solution as the mobile phase;

[0014] (3) The mixed standard working solution of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) was detected by high performance liquid chromatography. A standard working curve was plotted with mass concentration as the abscissa and peak area as the ordinate. The chromatographic peak areas of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) in the sample solution were substituted into the standard working curve to calculate the content of each substance.

[0015] The preferred embodiment of the above preparation method is as follows:

[0016] The high-concentration extraction reagent in step (1) is a 5%~6% (V / V) perchloric acid solution.

[0017] In step (2), the concentrations of potassium dihydrogen phosphate and dipotassium hydrogen phosphate in the high-concentration phosphate buffer solution are 8 mol / L.

[0018] The parameters for high-performance liquid chromatography (equipped with an ultraviolet detector) in steps (1) and (3) are as follows:

[0019] Column: C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm) or equivalent; injection volume: 5 μL ~ 10 μL; column temperature: 30 ℃;

[0020] Mobile phase: Accurately pipette 5 mL of a mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate with concentrations of 8 mol / L, add 900 mL of ultrapure water, adjust the pH to 4.6 with pH adjustment solution A and pH adjustment solution B, and bring the volume to 1000 mL to prepare a 0.04 mol / L phosphate buffer solution as the mobile phase;

[0021] Detection wavelength: 254 nm; Flow rate: 0.8 mL / min;

[0022] The mixed standard working solution in step (3) is specifically prepared by dissolving cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) standard solutions in water to prepare mixed standard working solutions of 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL, and 100.0 μg / mL.

[0023] Furthermore, in step (1), the mass-to-volume ratio of livestock and poultry products to high-concentration extraction reagent is 5 g: 10 mL.

[0024] Furthermore, in step (1), the centrifugation rate is 8000 r / min and the centrifugation time is 5 min.

[0025] The kit of the present invention can simultaneously detect the levels of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP).

[0026] The standard working curve for the cytidine monophosphate (CMP) is: y = 11.516x - 0.9346, R0 2 =1;

[0027] The standard working curve for uridine monophosphate (UMP) is: y = 14.196x + 12.237, R0 2 =0.9999;

[0028] The standard working curve for the guanylic acid (GMP) is: y = 18.565x + 0.1313, R0 2 =1;

[0029] The standard working curve for inosinic acid (IMP) is: y = 12.403x + 0.2704, R0 2 =1;

[0030] The standard working curve for adenosine monophosphate (AMP) is: y = 24.041x + 1.2446, R0 2 =1;

[0031] The standard working curve for hypoxanthine (HX) is: y = 41.09x - 0.5925, R0 2 =1;

[0032] The standard working curve for inosine (HXR) is: y = 24.191x - 2.6361, R 2 =1;

[0033] The standard working curve for adenosine diphosphate (ADP) is: y = 14.106x + 2.0176, R0 2 =1;

[0034] The standard working curve for adenosine triphosphate (ATP) is: y = 17.514x + 2.4349, R 2 =1.

[0035] This invention utilizes perchloric acid solution and pH adjustment to extract and purify flavor nucleotides from livestock and poultry samples. A liquid chromatography method is then used to simultaneously determine the content of nine flavor nucleotides in the samples, with a sample spike recovery rate ranging from 80.0% to 97.6% and a coefficient of variation of less than 9.3%. This invention employs perchloric acid extraction to extract nucleotides from the product, and high-performance liquid chromatography (HPLC) to detect the nine flavor nucleotide compounds. Qualitative confirmation is achieved through peak time analysis, demonstrating high specificity and significantly reducing the probability of false positives.

[0036] Compared with existing technologies, the technical effects of this invention are positive and significant. This invention breaks away from the traditional single-method ultraviolet spectrophotometry, enabling simultaneous processing of nine flavor nucleotides in livestock and poultry products. The instrument is simple to use, greatly increasing batch processing capacity. The method of this invention is simple to operate, rapid, accurate, and sensitive, and can achieve batch testing, showing promising practical application prospects. This invention fills the gap in flavor nucleotide detection methods for livestock and poultry products, demonstrating excellent application potential. Attached Figure Description

[0037] Figure 1 The chromatograms show the separation of three nucleotides using two different chromatographic columns. The left column is a C18 column, and the right column is an amino column. a and e are mixed standards, b and f are adenosine monophosphate (AMP), c and g are guanosine monophosphate (GMP), and d and h are inosine monophosphate (IMP).

[0038] Figure 2 The chromatograms show the separation of three nucleotides using two mobile phases. The left chromatogram is for 100% 0.05 mol / L phosphate buffer (pH=4.5), and the right chromatogram is for 5% methanol-95% 0.05 mol / L phosphate buffer (pH=4.5), both with isocratic elution. a and e are mixed standards, b and f are adenosine monophosphate (AMP), c and g are guanosine monophosphate (GMP), and d and h are inosine monophosphate (IMP).

[0039] Figure 3 The effect of column temperature on the retention time and peak shape of the analyte is shown, with the upper value at 30℃ and the lower value at 35℃.

[0040] Figure 4 The effect of different concentrations of perchloric acid on the extraction efficiency.

[0041] Figure 5 This is the standard working curve for cytidine monophosphate (CMP).

[0042] Figure 6 This is the standard working curve for uridine monophosphate (UMP).

[0043] Figure 7 This is the standard working curve for inosinic acid (IMP).

[0044] Figure 8 This is the standard working curve for guanylic acid (GMP).

[0045] Figure 9 This is the standard working curve for adenosine monophosphate (AMP).

[0046] Figure 10 This is the standard working curve for hypoxanthine (HX).

[0047] Figure 11 This is the standard working curve for inosine (HXR).

[0048] Figure 12 This is the standard working curve for adenosine diphosphate (ADP).

[0049] Figure 13 This is the standard working curve for adenosine triphosphate (ATP).

[0050] Figure 14 The chromatograms of nine flavor nucleotides are shown, with the peaks from left to right being CMP, UMP, GMP, IMP, ADP, ATP, HX, AMP, and HXR. Detailed Implementation

[0051] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0052] I. Instruments, Reagents and Materials

[0053] Ultra-high performance liquid chromatography (UHPLC) system: Agilent 1260 (Agilent Technologies, USA); refrigerated centrifuge: Thermo Fisher Scientific, USA; Milli-Q ultrapure water system: Millipore, USA; high-speed centrifuge: Thermo Fisher Scientific, USA; vortex mixer; homogenizer.

[0054] Ultrapure water; dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and sodium hydroxide (analytical grade, Merck, Germany); perchloric acid and phosphoric acid (analytical grade, Sigma, USA).

[0055] Standards: Cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP), with a purity of not less than 95%, purchased from Dr. Ehrenstorfer GmbH, Germany.

[0056] Example 1:

[0057] Based on previous literature review, the focus of this invention is the separation degree of inosinic acid (IMP) and guanosine monophosphate (GMP).

[0058] (1) Selection of chromatographic column

[0059] To ensure the separation of flavor nucleotides, this invention investigated C 18 The separation effects of chromatographic columns and amino columns on three nucleotides, such as Figure 1 As shown. It is clear that the amino column has difficulty separating guanosine monophosphate (GMP) and inosine monophosphate (IMP), while C... 18 The chromatographic column provides good resolution and peak shape, therefore C10 was chosen. 18 The chromatographic column was further optimized.

[0060] (2) Selection of mobile phase ratio

[0061] Due to the complexity of livestock and poultry samples, this invention focuses on the mobile phase ratio that provides better separation, taking into account relevant standards and literature. This invention examines the effect of mobile phase ratio on the separation of GMP and IMP. The invention investigates the ratio of methanol-phosphate buffer as the mobile phase and compares two mobile phases: 100% 0.05 mol / L phosphate buffer and 5% methanol-95% 0.05 mol / L phosphate buffer.

[0062] from Figure 2 It was found that while adding methanol (the organic phase) to the mobile phase advanced the elution time, it was difficult to separate GMP and IMP. Subsequent gradient elution showed that GMP and IMP were difficult to separate under elution conditions containing methanol. The results indicated that a better separation effect could be obtained in a 100% aqueous phase system; therefore, 100% phosphate buffer was selected as the mobile phase.

[0063] The applicant also discovered that column temperature affects the retention time and peak shape of the analytes. This invention compared the effects of column temperatures of 30℃ and 35℃ on the retention and peak shape of the analytes. The results showed that at a column temperature of 35℃, the peak elution was faster, the retention time was shorter, and the peak shape was narrower. Simultaneously, the peak separation of guanylic acid and inosinic acid met the requirements. Figure 3 ).

[0064] (3) Selection of mobile phase concentration

[0065] To obtain better resolution, the separation strength of 0.01, 0.02, 0.03, 0.04, and 0.05 mol / L phosphate buffer solutions for nine nucleotides was investigated (see Table 3), with a focus on the resolution of GMP and IMP. The results showed that higher concentrations resulted in later peak elution times, possibly due to higher concentrations leading to higher column pressures and peak shift. Furthermore, the peak resolution initially increased and then decreased. Therefore, a 0.04 mol / L phosphate buffer solution was selected as the mobile phase.

[0066] Table 3. Effect of phosphate buffer concentration on the separation degree of GMP and IMP

[0067] .

[0068] (4) Selection of pretreatment conditions

[0069] This invention uses different pretreatment conditions to extract nucleotides from pork. The samples were extracted with perchloric acid at concentrations of 2%, 5%, and 10% (v / v), respectively. The results showed that ( Figure 4 The recovery rates of methods with volume percentage concentrations of 5% and 10% both meet the requirements of 85%~115%. In accordance with the principle of environmental friendliness, this invention selects a 5% perchloric acid concentration for nucleotide extraction.

[0070] Example 2:

[0071] (1) Pretreatment method

[0072] Extraction: Accurately weigh 5.00 g (accurate to 0.01 g) of livestock and poultry sample into a 50 mL plastic centrifuge tube, accurately add 10 mL of 5% perchloric acid solution, homogenize for 5 min, then wash the blade with another 50 mL centrifuge tube containing 10 mL of 5% perchloric acid solution, combine the solutions, cap, and shake vigorously for about 30 s. Adjust the pH to 4.6 with phosphoric acid solution and sodium hydroxide solution (100 g / L), bring the volume to 50 mL with water, vortex for 1 min, and centrifuge at 8000 rpm for 10 min. Filter the supernatant through a 0.45 µm aqueous microporous membrane for analysis using ultra-high performance liquid chromatography (UHPLC).

[0073] (2) Preparation of standard solutions

[0074] (a) Standard stock solution (1 mg / mL): Accurately transfer 10 mg of the standard into a 10 mL volumetric flask, dilute to the mark with water, mix well, and store at 4 ℃. Shelf life is 3 months.

[0075] (b) Mixed standard intermediate solution (500 mg / L): Accurately transfer 0.50 mL each of the standard stock solutions of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) into a 10 mL volumetric flask, dilute to the mark with water, mix well, and store at 4 ℃. Shelf life is 1 month.

[0076] (c) Standard working curve: Dilute the mixed standard intermediate solution with water to prepare a series of matrix standard solutions with mass concentrations of 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL and 100.0 μg / mL. The standard working curve should be prepared and used immediately.

[0077] (3) Detection by ultra-high performance liquid chromatography

[0078] Liquid chromatography conditions:

[0079] (a) Chromatographic column: C 18 Column (1.8 μm * 3.0 mm * 150 mm); Column temperature: 35℃

[0080] (b) Injection volume: 5 µL.

[0081] (c) Flow rate: 1 mL / min

[0082] (d) Mobile phase: 0.04 mol / L dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer solution (accurately pipette 5 mL of a mixed solution of 8 mol / L potassium dihydrogen phosphate and dipotassium hydrogen phosphate, add 900 mL of ultrapure water, adjust the pH to 4.6 with pH adjustment solutions A and B, and bring the volume to 1000 mL to prepare a 0.04 mol / L phosphate buffer solution as the mobile phase); pH 4.6. Isocratic elution.

[0083] (4) Development and quantitative analysis of standard working curves

[0084] A mixed standard working solution of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) was detected under the above-mentioned liquid chromatography conditions. The detection chromatogram is shown below. Figure 12 As shown, regression analysis was performed on the peak areas of the target analytes and their corresponding concentrations to obtain the target analyte standard working curves and their regression equations, as shown below. Figures 5 to 14 As shown.

[0085] A standard curve was plotted with mass concentration (x) on the x-axis and peak area (y) on the y-axis to obtain the linear regression equation and correlation coefficient (R²) for each target compound. 2 The results showed that the target compound exhibited good linearity in the concentration range of 0.2 μg / mL to 100 μg / mL, and the correlation coefficients R between cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) were [value missing]. 2 All are greater than 0.999.

[0086] In this embodiment, the detection limit for cytidine monophosphate (CMP) and uridine monophosphate (UMP) is 1 mg / kg, and the quantitation limit is 2 mg / g; the detection limit for guanylic acid (GMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) is 3 mg / kg, and the quantitation limit is 10 mg / kg; and the detection limit for inosine monophosphate (IMP) is 6 mg / kg, and the quantitation limit is 20 mg / kg, demonstrating high sensitivity.

[0087] In this embodiment, when the sample addition concentration was 2 mg / kg to 100 mg / kg, the recovery rate was 80.0% to 97.6%, which can achieve a high recovery rate and meet the detection requirements. For details, please refer to Table 3.

[0088] Table 3. Accuracy of spiked recovery of nine nucleotides (n=2)

[0089] .

[0090] In this embodiment, the samples were subjected to repeatability tests with the following concentrations: 2 mg / kg cytidine monophosphate (CMP), uridine monophosphate (UMP), 10 mg / kg guanylic acid (GMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), and 20 mg / kg inosine monophosphate (IMP). The coefficients of variation were all less than 9.3%, indicating good repeatability and stability. For details, please refer to Table 4.

[0091] Table 4. Precision of the content of nine flavor nucleotides (n=6)

[0092] .

[0093] Example 3:

[0094] (1) Pretreatment method

[0095] Extraction: Accurately weigh 5.00 g each of pork, chicken, and beef samples (accurate to 0.01 g) into 50 mL plastic centrifuge tubes. Accurately add 10 mL of 5% perchloric acid solution, homogenize for 5 min, then wash the blade with another 50 mL centrifuge tube containing 10 mL of 5% perchloric acid solution. Combine the solutions, cap, and shake vigorously for about 30 s. Adjust the pH to 4.6 with phosphoric acid solution and sodium hydroxide solution (100 g / L), and bring the volume to 50 mL with water. Vortex for 1 min, and centrifuge at 8000 rpm for 10 min. Filter the supernatant through a 0.45 µm aqueous microporous membrane for analysis using ultra-high performance liquid chromatography (UHPLC). Each sample was analyzed in duplicate.

[0096] (3) Preparation of standard solutions

[0097] Same as Example 2.

[0098] (3) Detection by ultra-high performance liquid chromatography

[0099] Same as Example 2.

[0100] (4) Development and quantitative analysis of standard working curves

[0101] Same as Example 2.

[0102] A standard curve was plotted with mass concentration (x) on the x-axis and peak area (y) on the y-axis to obtain the linear regression equation and correlation coefficient (R²) for each target compound. 2The results showed that the target compound exhibited good linearity in the concentration range of 0.2 μg / mL to 100 μg / mL, and the correlation coefficients R between cytidine monophosphate (CMP), uridine monophosphate (UMP), guanylic acid (GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP), hypoxanthine (HX), inosine (HXR), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) were [value missing]. 2 All are greater than 0.999.

[0103] In this embodiment, the concentrations of nine nucleotides in the three samples ranged from 12.53 mg / kg to 3400.17 mg / kg, with relative differences of less than 10%, which enables the detection of flavor nucleotides in different livestock and poultry products.

[0104] Table 5. Content of nine nucleotides in pork, chicken, and beef (n=2)

[0105] .

Claims

1. A method for simultaneously detecting the content of nine flavor nucleotides in livestock and poultry products, wherein the nine flavor nucleotides are cytidine monophosphate, uridine monophosphate, guanylic acid, inosine monophosphate, adenosine monophosphate, hypoxanthine, inosine, adenosine diphosphate, and adenosine triphosphate, characterized in that... Includes the following steps: (1) Add high-concentration extraction reagent to livestock and poultry products, mix with a homogenizer to obtain the first extract; clean the homogenizer blade with high-concentration extraction reagent to obtain the second extract; mix the first extract and the second extract, centrifuge, take the supernatant and pass it through a membrane to obtain the sample solution, and then perform high-performance liquid chromatography detection. (2) Accurately pipette 5 mL of high-concentration phosphate buffer solution, add 900 mL of ultrapure water, adjust the pH to 4.6 with pH adjustment solution A and pH adjustment solution B, and make up to 1000 mL to prepare a 0.04 mol / L phosphate buffer solution as the mobile phase; (3) High-performance liquid chromatography (HPLC) was performed on the mixed standard working solution of cytidine, uridine, guanylic acid, inosine, adenosine, hypoxanthine, inosine, adenosine diphosphate, and adenosine triphosphate. A standard working curve was plotted with mass concentration as the abscissa and peak area as the ordinate. The chromatographic peak areas of cytidine, uridine, guanylic acid, inosine, adenosine, hypoxanthine, inosine, adenosine diphosphate, and adenosine triphosphate in the sample solution were substituted into the standard working curve to calculate the content of each substance. The high-concentration extraction reagent in step (1) was a 5% (V / V) perchloric acid solution. The concentrations of potassium dihydrogen phosphate and dipotassium hydrogen phosphate in the high-concentration phosphate buffer solution in step (2) were 8 mol / L. The parameters for high-performance liquid chromatography in steps (1) and (3) are as follows: Column: C 18 Chromatographic column; injection volume: 5 μL~10 μL; column temperature: 30℃; detection wavelength: 254 nm; flow rate: 0.8 mL / min; mobile phase: accurately pipette 5 mL of a mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate with concentrations of 8 mol / L, add 900 mL of ultrapure water, adjust the pH to 4.6 with pH adjustment solution A and pH adjustment solution B, and make up to 1000 mL to prepare a 0.04 mol / L phosphate buffer solution as the mobile phase; The mixed standard working solution in step (3) is specifically prepared by dissolving the standard solutions of cytidine, uridine, guanylic acid, inosine, adenosine, hypoxanthine, inosine, adenosine diphosphate and adenosine triphosphate in water to prepare mixed standard working solutions of 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL and 100.0 μg / mL.

2. The method of claim 1, wherein: In step (1), the mass-to-volume ratio of livestock and poultry products to high-concentration extraction reagent is 5 g: 10 mL.

3. The method of claim 1, wherein: In step (1), the centrifugation rate is 8000 r / min and the centrifugation time is 5 min.

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