Method for determining total phosphorus content in bovine urine

Abstract

The invention discloses a method for measuring the total phosphorus content in bovine urine, and relates to the technical field of bovine urine detection.The method comprises the steps that a bovine urine sample is collected; adjusting the pH value of the cattle urine sample, adding a urease solution, inactivating urease after the reaction is completed, and taking supernate as a standard cattle urine sample; preparing a phosphorus standard stock solution, carrying out gradient dilution, and adding an ammonium vanadium molybdate color developing agent to obtain standard working solutions with different concentrations; measuring the absorbance of each standard working solution, and drawing an absorbance-phosphorus concentration standard curve; the method comprises the following steps: putting a standard bovine urine sample into a microwave digestion tank, adding nitric acid and hydrogen peroxide, executing a multi-stage heating program, and performing constant-temperature acid removal treatment to obtain a digestion solution; absorbing the digestion solution, adding an ammonium vanadium molybdate color developing agent, and measuring the absorbance of the digestion solution; and calculating the total phosphorus content in the standard bovine urine sample according to the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution. The method is simple, convenient, rapid, good in accuracy and suitable for measuring the total phosphorus content in large-batch bovine urine.

Description

Technical Field

[0001] This invention relates to the field of bovine urine detection technology, and more specifically, to a method for determining the total phosphorus content in bovine urine. Background Technology

[0002] In recent years, with the continuous improvement of living standards and the increasing demands for the quality of beef cattle, dairy cattle, and dairy products in my country (Tang Wenjie et al., 2023), large-scale intensive cattle farming has become a development trend. The urine produced by cattle farms mainly contains nutrients such as nitrogen, phosphorus, and potassium, as well as organic matter. Total phosphorus in cattle urine mainly consists of orthophosphate, metaphosphate, and organic phosphorus compounds. Improper treatment can cause serious pollution to surface water, groundwater, soil, and ambient air, directly harming human health and the ecological environment. Regular monitoring of the total phosphorus content in cattle urine from large-scale cattle farms can not only reflect the phosphorus intake and metabolism in cattle diets, thereby adjusting the diet formula and improving farming efficiency, but also allow for the selection of harmless treatment methods for cattle manure, reducing the total phosphorus content of cattle manure organic fertilizer, improving fertilizer utilization efficiency, and avoiding environmental problems such as eutrophication. Therefore, establishing a simple, accurate, and reliable method for determining the total phosphorus content in cattle urine is particularly important.

[0003] Currently, there are few reports on methods for detecting total phosphorus content in bovine urine and related national or industry standards. Only one 1989 paper reported the use of far-infrared digestion combined with ultraviolet spectrophotometry to detect total phosphorus content in human urine. This method has a long digestion time, a complex color development process, and low efficiency. Reported methods for detecting total phosphorus content mainly include inductively coupled plasma mass spectrometry (Wang Fei et al., 2025), ion chromatography (Zhu Hongxia et al., 2022), polarography (Wang Zhonghui et al., 2000), ELISA (Liu Yan et al., 2023), and ultraviolet spectrophotometry (Zou Jing, 2018). Among these, the first four methods require expensive instruments or reagent kits, have high experimental costs, and demanding requirements on operators. In contrast, conventional ultraviolet spectrophotometry remains the most classic and widely used method due to its low experimental cost, mature technology, and stable color development.

[0004] No effective solutions have yet been proposed to address the problems in the relevant technologies. Summary of the Invention

[0005] In view of the problems in related technologies, the present invention proposes a method for determining the total phosphorus content in bovine urine, so as to overcome the above-mentioned technical problems existing in the existing related technologies.

[0006] Therefore, the specific technical solution adopted by the present invention is as follows:

[0007] A method for determining the total phosphorus content in bovine urine, the method comprising:

[0008] S1. Collect all urine from each cow, mix them thoroughly, and then take parallel samples to obtain cow urine samples. Seal the cow urine samples and store them at -20℃ for later use.

[0009] S2. Adjust the pH value of the thawed bovine urine sample and add urease solution to react until no bubbles are produced; after the reaction is completed, deactivate the urease and take the supernatant as the standard bovine urine sample.

[0010] S3. Prepare a phosphorus standard stock solution, add ammonium vanadate colorimetric reagent after gradient dilution, make up to volume and let stand for color development to obtain standard working solutions of different concentrations; based on the preset test conditions, measure the absorbance of each standard working solution and plot the absorbance-phosphorus concentration standard curve.

[0011] S4. Place a standard bovine urine sample in a microwave digestion vessel, add nitric acid and hydrogen peroxide according to the preset ratio, execute a multi-stage heating program for initial digestion, perform constant temperature acid removal treatment after initial digestion, cool and make up to volume to obtain digestion solution.

[0012] S5. Remove the digestion solution, add ammonium vanadate colorimetric reagent, make up to volume and let stand for color development, and measure the absorbance of the digestion solution based on the preset measurement conditions.

[0013] S6. Calculate the total phosphorus content in the standard bovine urine sample based on the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution.

[0014] Further, the pH of the thawed bovine urine sample was adjusted, and urease solution was added to react until no more bubbles were produced; after the reaction was completed, the urease was inactivated, and the supernatant was taken as a standard bovine urine sample, including the following steps:

[0015] S21. Adjust the pH of the thawed cow urine sample to 6.5-7.5 using dilute hydrochloric acid or dilute sodium hydroxide solution.

[0016] S22. Add 5-10 U of urease activity to each milliliter of bovine urine sample after pH adjustment, and react at 35℃-40℃ for 20-30 min until no bubbles are produced to obtain the reacted sample.

[0017] S23. Place the reacted bovine urine sample in a boiling water bath at 95℃-100℃ and heat for 5-10 minutes until no bubbles are generated to obtain a bovine urine sample inactivated by urease.

[0018] S24. Centrifuge the urease-inactivated bovine urine sample at 4000-6000 r / min for 5-10 min, and take the supernatant as the standard bovine urine sample.

[0019] Further, a phosphorus standard stock solution was prepared, serially diluted, and then ammonium vanadate colorimetric reagent was added. The solution was brought to volume and allowed to stand for color development to obtain standard working solutions of different concentrations. Based on preset measurement conditions, the absorbance of each standard working solution was measured, and an absorbance-phosphorus concentration standard curve was plotted, including the following steps:

[0020] S31. Transfer the phosphorus single-element standard solution and dilute it with water to prepare a 50 μg / mL phosphorus standard stock solution;

[0021] S32. The phosphorus standard stock solution was serially diluted, and 10 mL of ammonium vanadate colorimetric reagent was added to each solution. The solution was then brought to a final volume and allowed to stand for color development to obtain standard working solutions of different concentrations.

[0022] S33. Under the preset measurement conditions, measure the absorbance of each standard working solution, and plot the absorbance-phosphorus concentration standard curve with phosphorus mass concentration as the abscissa and absorbance as the ordinate.

[0023] Further, the phosphorus standard stock solution was serially diluted, and 10 mL of ammonium vanadate colorimetric reagent was added to each solution. After making up to volume and allowing to stand for color development, standard working solutions of different concentrations were obtained, including the following steps:

[0024] S321. According to the preset gradient, transfer different volumes of phosphorus standard stock solution into several 50mL volumetric flasks respectively;

[0025] S322. Add 10 mL of ammonium vanadate colorimetric reagent to each 50 mL volumetric flask, dilute to the mark with water, shake well, and let stand for more than 10 minutes to obtain standard working solutions of different concentrations.

[0026] Furthermore, the preset measurement conditions are as follows:

[0027] The absorbance was measured using a 1 cm cuvette and a UV spectrophotometer at a wavelength of 400 nm.

[0028] Further, a standard bovine urine sample was placed in a microwave digestion vessel, and nitric acid and hydrogen peroxide were added according to a preset ratio. A multi-stage heating program was then executed for initial digestion. After initial digestion, a constant-temperature acid removal treatment was performed, followed by cooling and volume adjustment to obtain the digestion solution, which included the following steps:

[0029] S41. Transfer a standard bovine urine sample into a microwave digestion vessel, add nitric acid and hydrogen peroxide according to the preset ratio, shake well, and then seal the microwave digestion vessel.

[0030] S42. Place the sealed microwave digestion vessel into the microwave digester and execute a multi-stage heating program to complete the initial digestion and obtain the initial digested sample.

[0031] S43. At a temperature of 140℃-160℃, the initial digested sample is subjected to isothermal acid removal treatment until about 1 mL of the initial digested sample remains.

[0032] S44. Transfer the initial digestion sample after acid removal to a 25mL volumetric flask, wash the inner wall of the microwave digestion vessel 2-3 times with a small amount of water, combine the washing solution to the 25mL volumetric flask, dilute to the mark with water and shake well to obtain the digestion solution.

[0033] Furthermore, the preset ratio is:

[0034] Add 5 mL of nitric acid and 2 mL of hydrogen peroxide to each 1 mL standard bovine urine sample.

[0035] Furthermore, the multi-stage warming process includes a first stage, a second stage, and a third stage;

[0036] In the first stage, the power control raises the temperature to 80°C within 5-8 minutes and maintains it for 5 minutes.

[0037] The second stage controls the power to raise the temperature to 120℃ within 5-8 minutes and maintain it for 10 minutes;

[0038] The third stage controls the power to raise the temperature to 160℃ within 5-8 minutes and maintain it for 20 minutes.

[0039] Further, the digestion solution is removed, ammonium vanadate colorimetric reagent is added, the volume is adjusted, and the solution is allowed to stand for color development. The absorbance of the digestion solution is then measured based on preset measurement conditions, including the following steps:

[0040] S51. Accurately transfer 5-10 mL of the digestion solution into a 50 mL volumetric flask;

[0041] S52. Add 10 mL of ammonium vanadate colorimetric reagent to a 50 mL volumetric flask, dilute to the mark with water, shake well and let stand for more than 10 minutes.

[0042] S53. Using a 1cm cuvette, the absorbance of the digestion solution was measured at a wavelength of 400nm using an ultraviolet spectrophotometer.

[0043] Furthermore, based on the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution, the total phosphorus content in the bovine urine sample is calculated using the following steps:

[0044] S61. Obtain the phosphorus concentration of the digestion solution based on the absorbance-phosphorus concentration standard curve;

[0045] S62. Multiply the phosphorus concentration by the final volume of the digestion solution and the dilution factor during the determination, and then divide by the original volume of the standard cow urine sample that was digested to calculate the total phosphorus content in the standard cow urine sample.

[0046] The beneficial effects of this invention are as follows: This invention establishes a method for determining the total phosphorus content in bovine urine using microwave digestion pretreatment technology (short time consumption, low pollution) combined with ultraviolet spectrophotometry, while simultaneously validating the methodology. This invention has the advantages of being simple, rapid, low-cost, and accurate, and is suitable for determining the total phosphorus content in large quantities of bovine urine, providing technical support for the formulation of relevant national or industry standards and for improving cattle feed utilization and the breeding environment. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0048] Figure 1 This is a flowchart of a method for determining the total phosphorus content in bovine urine according to an embodiment of the present invention;

[0049] Figure 2 This is an absorbance-phosphorus concentration standard curve in a method for determining the total phosphorus content in bovine urine according to an embodiment of the present invention. Detailed Implementation

[0050] To further illustrate the various embodiments, the present invention provides accompanying drawings, which are part of the disclosure of the present invention. These drawings are mainly used to illustrate the embodiments and can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. With reference to these drawings, those skilled in the art should be able to understand other possible implementation methods and the advantages of the present invention. The components in the drawings are not drawn to scale, and similar component symbols are generally used to represent similar components.

[0051] According to an embodiment of the present invention, a method for determining the total phosphorus content in bovine urine is provided.

[0052] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments, such as... Figure 1 As shown, according to an embodiment of the present invention, a method for determining the total phosphorus content in bovine urine includes:

[0053] S1. Collect all urine from each cow, mix them thoroughly, and then take parallel samples to obtain cow urine samples. Seal the cow urine samples and store them at -20℃ for later use.

[0054] It should be explained that, for each cow as the research subject, all urine from each cow was collected in a clean plastic bucket within 24 hours, mixed thoroughly, and then two parallel samples were taken into clean plastic bottles, approximately 200 mL / sample. One sample was used for testing, and the other was retained as a sample. The sample bottles were sealed and stored in a -20°C refrigerator in a timely manner.

[0055] S2. Adjust the pH value of the thawed bovine urine sample and add urease solution to react until no bubbles are produced; after the reaction is completed, deactivate the urease and take the supernatant as the standard bovine urine sample.

[0056] It should be explained that, considering the matrix characteristics of bovine urine samples, this invention utilizes the catalytic properties of urease to pre-decompose and remove high concentrations of urea in bovine urine, thereby eliminating its interference with subsequent microwave digestion and phosphorus colorimetric determination; at the same time, this pretreatment process does not cause the loss of phosphorus forms in bovine urine, thus improving the accuracy of total phosphorus detection results while ensuring the safety of experimental operations.

[0057] In this optional embodiment, adjusting the pH of the thawed bovine urine sample and adding urease solution until no more bubbles are generated; after the reaction is complete, inactivating the urease and taking the supernatant as a standard bovine urine sample includes the following steps:

[0058] S21. Adjust the pH of the thawed cow urine sample to 6.5-7.5 using dilute hydrochloric acid or dilute sodium hydroxide solution.

[0059] Specifically, a pH of 6.5-7.5 is the optimal neutral environment for urease to exert its catalytic activity, which can avoid the inactivation of urease due to acidic or alkaline conditions and ensure that the subsequent urea enzymatic hydrolysis reaction is efficient and complete. Using dilute acid-base regulators instead of concentrated acid-bases can prevent excessively high local acid-base concentrations from causing the adsorption or speciation of phosphorus in bovine urine samples, thus ensuring the recovery rate of phosphorus.

[0060] S22. Add 5-10 U of urease activity to each milliliter of bovine urine sample after pH adjustment, and react at 35℃-40℃ for 20-30 min until no bubbles are produced to obtain the reacted sample.

[0061] Specifically, under these conditions, urease can rapidly decompose urea into ammonia and carbon dioxide. The urea enzymatic hydrolysis reaction is considered complete when no more bubbles are produced. This eliminates the safety hazard of urea reacting with acid reagents in subsequent microwave digestion, which could generate a large number of bubbles and cause a sudden increase in the pressure of the digestion tank. At the same time, it avoids the complexation interference of urea decomposition products on the phosphorus colorimetric reaction.

[0062] S23. Place the reacted bovine urine sample in a boiling water bath at 95℃-100℃ and heat for 5-10 minutes until no bubbles are generated to obtain a bovine urine sample inactivated by urease.

[0063] Specifically, high temperature completely inactivates urease, preventing residual urease from continuing to undergo non-specific reactions and introducing additional impurities during subsequent sample processing; heating until no bubbles are generated ensures that residual gases produced by the enzymatic hydrolysis reaction are completely released, avoiding the impact of residual gases on subsequent digestion and volume adjustment operations.

[0064] S24. Centrifuge the urease-inactivated bovine urine sample at 4000-6000 r / min for 5-10 min, and take the supernatant as the standard bovine urine sample.

[0065] Specifically, centrifugation at 4000-6000 r / min for 5-10 min can quickly separate the trace flocculent precipitate and inactivated urease protein and other solid impurities produced after enzymatic digestion in bovine urine samples. The supernatant is then used as a standard bovine urine sample for subsequent digestion. This avoids the solid impurities from encapsulating phosphorus or generating additional impurities during microwave digestion, further ensuring the thoroughness of digestion and the accuracy of the test results. This centrifugation speed is a mild centrifugation condition, which can prevent the adsorption loss of phosphorus caused by high-speed centrifugation.

[0066] S3. Prepare a phosphorus standard stock solution, add ammonium vanadate colorimetric reagent after gradient dilution, make up to volume and let stand for color development to obtain standard working solutions of different concentrations; based on the preset test conditions, measure the absorbance of each standard working solution and plot the absorbance-phosphorus concentration standard curve.

[0067] It should be noted that the phosphorus single-element standard solution was selected at 1000 μg / mL from the National Nonferrous Metals and Electronic Materials Analysis and Testing Center. Ammonium metavanadate and ammonium molybdate were selected from analytical grade reagents from Sinopharm Chemical Reagent Co., Ltd.

[0068] The preparation process of ammonium vanadate colorimetric reagent is as follows: Weigh 1.25g of ammonium metavanadate, add 200mL of water and heat to dissolve. After cooling, add 250mL of nitric acid. Separately, take 25g of ammonium molybdate, add 400mL of water and heat to dissolve. Under cooling conditions, mix the two solutions, dilute with water to 1000mL, and store in the dark. If a precipitate is formed, it cannot be used.

[0069] In this optional embodiment, a phosphorus standard stock solution is prepared, serially diluted, and then ammonium vanadate colorimetric reagent is added. The solution is then brought to a final volume and allowed to stand for color development to obtain standard working solutions of different concentrations. Based on preset measurement conditions, the absorbance of each standard working solution is measured, and an absorbance-phosphorus concentration standard curve is plotted, including the following steps:

[0070] S31. Transfer the phosphorus single-element standard solution and dilute it with water to prepare a 50 μg / mL phosphorus standard stock solution;

[0071] S32. The phosphorus standard stock solution was serially diluted, and 10 mL of ammonium vanadate colorimetric reagent was added to each solution. The solution was then brought to a final volume and allowed to stand for color development to obtain standard working solutions of different concentrations.

[0072] S33. Under the preset measurement conditions, measure the absorbance of each standard working solution, and plot the absorbance-phosphorus concentration standard curve with phosphorus mass concentration as the abscissa and absorbance as the ordinate.

[0073] In this optional embodiment, the phosphorus standard stock solution is serially diluted, and 10 mL of ammonium vanadate colorimetric reagent is added to each solution. After making up to volume and allowing to stand for color development, standard working solutions of different concentrations are obtained by the following steps:

[0074] S321. According to the preset gradient, transfer different volumes of phosphorus standard stock solution into several 50mL volumetric flasks respectively;

[0075] S322. Add 10 mL of ammonium vanadate colorimetric reagent to each 50 mL volumetric flask, dilute to the mark with water, shake well, and let stand for more than 10 minutes to obtain standard working solutions of different concentrations.

[0076] In this optional embodiment, the preset measurement conditions are:

[0077] The absorbance was measured using a 1 cm cuvette and a UV spectrophotometer at a wavelength of 400 nm.

[0078] It should be explained that the preparation process of the standard working solution is as follows: Accurately transfer 5.00 mL of phosphorus single element standard solution into a 100 mL volumetric flask, dilute to the mark with water, and shake well to obtain a 50 μg / mL standard stock solution; then accurately transfer 0, 0.50, 1.00, 2.00, 5.00, 10.00 mL and 15.00 mL of the standard stock solution into 50 mL volumetric flasks (equivalent to mass concentrations of 0, 0.50, 1.00, 2.00, 5.00, 10.00 μg / mL and 15.00 μg / mL, respectively), add 10 mL of ammonium vanadate colorimetric reagent to each volumetric flask, dilute to the mark with water, shake well, and let stand at room temperature for more than 10 minutes. Measure the absorbance of each working solution at a wavelength of 400 nm using a 1 cm cuvette and an ultraviolet spectrophotometer.

[0079] Under optimized instrument conditions, the standard working solution was injected, and a working curve was plotted with phosphorus mass concentration (X) on the x-axis and absorbance (Y) on the y-axis. The results are as follows. Figure 2 As shown.

[0080] from Figure 2 As can be seen (Abs in the figure represents absorbance), phosphorus exhibits a good linear relationship in the range of 0.00 μg / mL to 15.00 μg / mL, with a correlation coefficient (R0).2 The value is 1.0000, and the regression equation obtained through linear fitting is Y=0.0890X+0.0018.

[0081] S4. Place a standard bovine urine sample in a microwave digestion vessel, add nitric acid and hydrogen peroxide according to the preset ratio, execute a multi-stage heating program for initial digestion, perform constant temperature acid removal treatment after initial digestion, cool and make up to volume to obtain digestion solution.

[0082] In this optional embodiment, a standard bovine urine sample is placed in a microwave digestion vessel, and nitric acid and hydrogen peroxide are added in a preset ratio. A multi-stage heating program is executed for initial digestion. After the initial digestion is completed, a constant-temperature acid removal treatment is performed, followed by cooling and volume adjustment to obtain the digestion solution, which includes the following steps:

[0083] S41. Transfer a standard bovine urine sample into a microwave digestion vessel, add nitric acid and hydrogen peroxide according to the preset ratio, shake well, and then seal the microwave digestion vessel.

[0084] S42. Place the sealed microwave digestion vessel into the microwave digester and execute a multi-stage heating program to complete the initial digestion and obtain the initial digested sample.

[0085] S43. At a temperature of 140℃-160℃, the initial digested sample is subjected to isothermal acid removal treatment until about 1 mL of the initial digested sample remains.

[0086] S44. Transfer the initial digestion sample after acid removal to a 25mL volumetric flask, wash the inner wall of the microwave digestion vessel 2-3 times with a small amount of water, combine the washing solution to the 25mL volumetric flask, dilute to the mark with water and shake well to obtain the digestion solution.

[0087] In this optional embodiment, the preset ratio is:

[0088] Add 5 mL of nitric acid and 2 mL of hydrogen peroxide to each 1 mL standard bovine urine sample.

[0089] In this optional embodiment, the multi-stage heating process includes a first stage, a second stage, and a third stage;

[0090] In the first stage, the power control raises the temperature to 80°C within 5-8 minutes and maintains it for 5 minutes.

[0091] The second stage controls the power to raise the temperature to 120℃ within 5-8 minutes and maintain it for 10 minutes;

[0092] The third stage controls the power to raise the temperature to 160℃ within 5-8 minutes and maintain it for 20 minutes.

[0093] It should be explained that 1.00 mL (W) of bovine urine sample should be accurately transferred into a microwave digestion vessel, and 5 mL of nitric acid and 2 mL of hydrogen peroxide should be added. Digestion should be carried out according to the microwave digestion procedure, and the digestion conditions are shown in Table 1. After the digested sample is cooled, the acid should be removed to approximately 1 mL at 140℃~160℃. After the digestion vessel has cooled, the digestion solution should be transferred to a 25 mL (V) volumetric flask. The digestion vessel should be washed 2 to 3 times with a small amount of water. The washings should be combined in the volumetric flask and diluted to the mark with water. A blank test should be performed simultaneously. Nitric acid and hydrogen peroxide should be analytical grade, both from Sinopharm Chemical Reagent Co., Ltd.

[0094] According to the method for determining the limit of detection and limit of quantitation in the "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods" (GB / T 27417-2017), the lowest acceptable concentration of phosphorus standard solution was added to the blank sample. After microwave digestion pretreatment according to a multi-stage temperature program, the results were measured by ultraviolet spectrophotometer, and the standard deviation (s) of the detection results was calculated. The limit of detection is expressed as 3s and the limit of quantitation is expressed as 10s. The limit of detection of this method is 0.8 μg / mL and the limit of quantitation is 2.6 μg / mL.

[0095] As shown in Table 1, the process involves a multi-stage heating phase for microwave digestion.

[0096]

[0097] S5. Remove the digestion solution, add ammonium vanadate colorimetric reagent, make up to volume and let stand for color development, and measure the absorbance of the digestion solution based on the preset measurement conditions.

[0098] In this optional embodiment, the digestion solution is aspirated, ammonium vanadate colorimetric reagent is added, the volume is adjusted, and the solution is allowed to stand for color development. The absorbance of the digestion solution is then measured based on preset measurement conditions, including the following steps:

[0099] S51. Accurately transfer 5-10 mL of the digestion solution into a 50 mL volumetric flask;

[0100] S52. Add 10 mL of ammonium vanadate colorimetric reagent to a 50 mL volumetric flask, dilute to the mark with water, shake well and let stand for more than 10 minutes.

[0101] S53. Using a 1cm cuvette, the absorbance of the digestion solution was measured at a wavelength of 400nm using an ultraviolet spectrophotometer.

[0102] The following explanation should be provided: Accurately transfer 5.00–10.00 mL (V1) of the sample solution into a 50 mL (V2) volumetric flask. Add 10 mL of ammonium vanadate colorimetric reagent, dilute to the mark with water, shake well, and let stand at room temperature for at least 10 minutes. Using a 1 cm cuvette, measure the absorbance of the sample solution at a wavelength of 400 nm using a UV spectrophotometer. Calculate the phosphorus content of the sample solution using the working curve. If the phosphorus content of the sample solution exceeds the range of the phosphorus standard working curve, the sample solution should be diluted.

[0103] S6. Calculate the total phosphorus content in the standard bovine urine sample based on the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution.

[0104] It should be explained that the formula for calculating the phosphorus content X of the sample is as follows (unit: μg / mL):

[0105] ;

[0106] In the formula, C is the concentration of phosphorus in the sample obtained from the working curve, in μg / mL.

[0107] In this optional embodiment, calculating the total phosphorus content in the bovine urine sample based on the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution includes the following steps:

[0108] S61. Obtain the phosphorus concentration of the digestion solution based on the absorbance-phosphorus concentration standard curve;

[0109] S62. Multiply the phosphorus concentration by the final volume of the digestion solution and the dilution factor during the determination, and then divide by the original volume of the standard cow urine sample that was digested to calculate the total phosphorus content in the standard cow urine sample.

[0110] It should be noted that the main instruments of this invention include a dual-beam UV-Vis spectrophotometer (T9S type, Beijing Purkinje General Instrument Co., Ltd.); a microwave digester (MD8H type, Opler Instruments Co., Ltd.); a hot plate (IKAC-MAGHP10 type, Suzhou Science Instruments Co., Ltd.); and an electronic balance (sensitivity 0.01g, Me2002t type, Mettler Toledo Instruments (Shanghai) Co., Ltd.).

[0111] The specific experimental data are as follows: Three cow urine samples with different mass concentrations were selected (cow urine 1, cow ... # 2 cow urines # Wagyu urine 3 # Precision tests were conducted, with each sample measured in parallel five times. The results are shown in Table 2.

[0112]

[0113] As shown in Table 2, the average total phosphorus content in the three bovine urine samples was 40.1, 114.7 and 396.8 μg / mL, respectively, with relative standard deviations (RSD) of 2.6% to 2.8%, which is a satisfactory result.

[0114] Two different concentrations of phosphorus standard solution were added to three cow urine samples, with five replicates for each addition. The recovery rate and relative standard deviation were calculated, and the specific values ​​are shown in Table 3.

[0115] Table 3: Recovery rate determination results (n=5)

[0116] As can be seen from Table 3, cow urine 1 # The spiked recoveries were 85.0%–99.6%, with relative standard deviations (RSDs) of 4.0%–6.1%; bovine urine 2 # The spiked recoveries ranged from 92.2% to 102.2%, with relative standard deviations (RSDs) of 2.8% to 3.4%; bovine urine 3 # The spiked recoveries ranged from 99.2% to 107.8%, and the relative standard deviations (RSDs) ranged from 2.2% to 3.7%, indicating that the method has good accuracy and precision and can meet the experimental requirements.

[0117] In summary, by utilizing the above-mentioned technical solution of this invention, a method for determining the total phosphorus content in bovine urine using microwave digestion pretreatment technology (short time consumption, low pollution) combined with ultraviolet spectrophotometry is established, and the methodological validation is performed simultaneously. This invention has the advantages of being simple, rapid, low-cost, and accurate, and is suitable for determining the total phosphorus content in large quantities of bovine urine, providing technical support for the formulation of relevant national or industry standards and for improving cattle feed utilization and the breeding environment.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the total phosphorus content in bovine urine, characterized in that, The method includes: S1. Collect all urine from each cow, mix them thoroughly, and then take parallel samples to obtain cow urine samples. Seal the cow urine samples and store them at -20℃ for later use. S2. Adjust the pH value of the thawed bovine urine sample and add urease solution to react until no bubbles are produced; after the reaction is completed, deactivate the urease and take the supernatant as the standard bovine urine sample. S3. Prepare a phosphorus standard stock solution, add ammonium vanadate colorimetric reagent after gradient dilution, make up to volume and let stand for color development to obtain standard working solutions of different concentrations; based on the preset test conditions, measure the absorbance of each standard working solution and plot the absorbance-phosphorus concentration standard curve. S4. Place a standard bovine urine sample in a microwave digestion vessel, add nitric acid and hydrogen peroxide according to the preset ratio, execute a multi-stage heating program for initial digestion, perform constant temperature acid removal treatment after initial digestion, cool and make up to volume to obtain digestion solution. S5. Remove the digestion solution, add ammonium vanadate colorimetric reagent, make up to volume and let stand for color development, and measure the absorbance of the digestion solution based on the preset measurement conditions. S6. Calculate the total phosphorus content in the standard bovine urine sample based on the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution.

2. The method for determining the total phosphorus content in bovine urine according to claim 1, characterized in that, The process of adjusting the pH of the thawed bovine urine sample and adding urease solution until no more bubbles are produced, followed by inactivating the urease and taking the supernatant as a standard bovine urine sample, includes the following steps: S21. Adjust the pH of the thawed cow urine sample to 6.5-7.5 using dilute hydrochloric acid or dilute sodium hydroxide solution. S22. Add 5-10 U of urease activity to each milliliter of bovine urine sample after pH adjustment, and react at 35℃-40℃ for 20-30 min until no bubbles are produced to obtain the reacted sample. S23. Place the reacted bovine urine sample in a boiling water bath at 95℃-100℃ and heat for 5-10 minutes until no bubbles are generated to obtain a bovine urine sample inactivated by urease. S24. Centrifuge the urease-inactivated bovine urine sample at 4000-6000 r / min for 5-10 min, and take the supernatant as the standard bovine urine sample.

3. The method for determining the total phosphorus content in bovine urine according to claim 1, characterized in that, The prepared phosphorus standard stock solution was serially diluted, then ammonium vanadate colorimetric reagent was added, the volume was adjusted, and the solution was allowed to stand for color development to obtain standard working solutions of different concentrations. Based on preset measurement conditions, the absorbance of each standard working solution was measured, and an absorbance-phosphorus concentration standard curve was plotted, including the following steps: S31. Transfer the phosphorus single-element standard solution and dilute it with water to prepare a 50 μg / mL phosphorus standard stock solution; S32. The phosphorus standard stock solution was serially diluted, and 10 mL of ammonium vanadate colorimetric reagent was added to each solution. The solution was then brought to a final volume and allowed to stand for color development to obtain standard working solutions of different concentrations. S33. Under the preset measurement conditions, measure the absorbance of each standard working solution, and plot the absorbance-phosphorus concentration standard curve with phosphorus mass concentration as the abscissa and absorbance as the ordinate.

4. The method for determining the total phosphorus content in bovine urine according to claim 3, characterized in that, The step of serially diluting the phosphorus standard stock solution, adding 10 mL of ammonium vanadate colorimetric reagent, making up to volume, and allowing it to stand for color development to obtain standard working solutions of different concentrations includes the following steps: S321. According to the preset gradient, transfer different volumes of phosphorus standard stock solution into several 50mL volumetric flasks respectively; S322. Add 10 mL of ammonium vanadate colorimetric reagent to each 50 mL volumetric flask, dilute to the mark with water, shake well, and let stand for more than 10 minutes to obtain standard working solutions of different concentrations.

5. The method for determining the total phosphorus content in bovine urine according to claim 3, characterized in that, The preset measurement conditions are as follows: The absorbance was measured using a 1 cm cuvette and a UV spectrophotometer at a wavelength of 400 nm.

6. The method for determining the total phosphorus content in bovine urine according to claim 1, characterized in that, The process involves placing a standard bovine urine sample in a microwave digestion vessel, adding nitric acid and hydrogen peroxide in a preset ratio, and performing a multi-stage heating program for initial digestion. After initial digestion, a constant-temperature acid removal treatment is performed, followed by cooling and volume adjustment to obtain the digest solution. The steps include: S41. Transfer a standard bovine urine sample into a microwave digestion vessel, add nitric acid and hydrogen peroxide according to the preset ratio, shake well, and then seal the microwave digestion vessel. S42. Place the sealed microwave digestion vessel into the microwave digester and execute a multi-stage heating program to complete the initial digestion and obtain the initial digested sample. S43. At a temperature of 140℃-160℃, the initial digested sample is subjected to isothermal acid removal treatment until about 1 mL of the initial digested sample remains. S44. Transfer the initial digestion sample after acid removal to a 25mL volumetric flask, wash the inner wall of the microwave digestion vessel 2-3 times with a small amount of water, combine the washing solution to the 25mL volumetric flask, dilute to the mark with water and shake well to obtain the digestion solution.

7. The method for determining the total phosphorus content in bovine urine according to claim 6, characterized in that, The preset ratio is: Add 5 mL of nitric acid and 2 mL of hydrogen peroxide to each 1 mL standard bovine urine sample.

8. The method for determining the total phosphorus content in bovine urine according to claim 6, characterized in that, The multi-stage heating process includes a first stage, a second stage, and a third stage; In the first stage, the power control raises the temperature to 80°C within 5-8 minutes and maintains it for 5 minutes. The second stage controls the power to raise the temperature to 120℃ within 5-8 minutes and maintain it for 10 minutes; The third stage controls the power to raise the temperature to 160℃ within 5-8 minutes and maintain it for 20 minutes.

9. The method for determining the total phosphorus content in bovine urine according to claim 1, characterized in that, The digestion solution is prepared by adding ammonium vanadate colorimetric reagent, adjusting the volume, allowing it to stand for color development, and measuring the absorbance of the digestion solution based on preset measurement conditions, including the following steps: S51. Accurately transfer 5-10 mL of the digestion solution into a 50 mL volumetric flask; S52. Add 10 mL of ammonium vanadate colorimetric reagent to a 50 mL volumetric flask, dilute to the mark with water, shake well and let stand for more than 10 minutes. S53. Using a 1cm cuvette, the absorbance of the digestion solution was measured at a wavelength of 400nm using an ultraviolet spectrophotometer.

10. The method for determining the total phosphorus content in bovine urine according to claim 1, characterized in that, The calculation of the total phosphorus content in the bovine urine sample based on the absorbance-phosphorus concentration standard curve and the absorbance of the digestion solution includes the following steps: S61. Obtain the phosphorus concentration of the digestion solution based on the absorbance-phosphorus concentration standard curve; S62. Multiply the phosphorus concentration by the final volume of the digestion solution and the dilution factor during the determination, and then divide by the original volume of the standard cow urine sample that was digested to calculate the total phosphorus content in the standard cow urine sample.

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