A method for detecting the chelation rate of hydroxymethionine metal chelates

Abstract

This application relates to the field of detection and discloses a method for detecting the chelation rate of hydroxymethionine metal chelates. The steps include: taking two identical hydroxymethionine metal chelate samples to be tested, diluting them with diethyl ether and then ultrasonically extracting them to obtain a first extract and a second extract, and measuring the mass content of free hydroxymethionine in the first extract as w1, and the mass content of metal ions in the second extract as w2, satisfying w2=0; taking the hydroxymethionine raw material sample used to prepare the hydroxymethionine metal chelate sample to be tested, measuring the total hydroxymethionine mass content as w3, and calculating the chelation rate using the formula chelation rate = (w3-w1) / w3×100%. This application uses diethyl ether as the extraction solvent, which improves the detection error caused by the difficulty in separating free and bound hydroxymethionine during the detection process, and improves the accuracy of the detection.

Description

Technical Field

[0001] This invention relates to the field of detection, and more particularly to a method for detecting the chelation rate of hydroxymethionine metal chelates. Background Technology

[0002] Hydroxymethionine metal chelates are an important class of organic trace element feed additives. Hydroxymethionine uses carboxyl oxygen and α-hydroxy oxygen as coordinating atoms to form complexes with metal ions such as iron, copper, zinc, and manganese. Because hydroxymethionine has a bidentate structure, the resulting complexes are five-membered heterocyclic chelates, which are structurally stable. These chelates exist in the animal digestive tract in a molecular state and are not easily dissociated. The metal elements in the molecular state of hydroxymethionine chelates are constrained by chelation steric hindrance and are not easily bound to anti-nutritional substances such as phytic acid, oxalic acid, tannic acid, and soluble cellulose in the digesta, forming large, unabsorbable molecules. On the other hand, the molecular state of metal chelates is absorbed through amino acid absorption channels (carrier-mediated dissimilatory diffusion pathways), avoiding competitive inhibition of metal element absorption channels in the intestinal epithelium, improving the absorption and utilization rate of metal elements, and ensuring the balance of trace elements at the cellular level. Therefore, it improves material and energy metabolism, enhances antioxidant stress function, and protects the integrity of the immune system structure and its responsiveness.

[0003] The chelation rate of hydroxymethionine metal chelates is one of the important indicators for evaluating the quality of hydroxymethionine metal chelate products. However, due to the difficulty in separating free and bound hydroxymethionine, the methods for detecting the chelation rate of hydroxymethionine metal elements have low accuracy. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a method for detecting the chelation rate of hydroxymethionine metal chelates, aiming to solve the problem of low accuracy in related methods for detecting the chelation rate of hydroxymethionine metal elements.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for detecting the chelation rate of hydroxymethionine metal chelates includes the following steps: taking two identical hydroxymethionine metal chelate samples to be tested, respectively diluting with ether and ultrasonically extracting to obtain a first extract and a second extract, and measuring the mass content of free hydroxymethionine in the first extract as w1, and measuring the mass content of metal ions in the second extract as w2, and satisfying w2=0; taking the hydroxymethionine raw material sample used to prepare the hydroxymethionine metal chelate sample to be tested, directly measuring the total hydroxymethionine mass content as w3, and calculating the chelation rate using the formula chelation rate=(w3- w1) / w3×100%.

[0006] This invention uses diethyl ether, which can dissolve free hydroxymethionine but not hydroxymethionine metal chelates, as the extraction solvent. This ensures that after the sample is diluted with ether and extracted ultrasonically, the free and bound states of hydroxymethionine are separated. The resulting extract contains only free hydroxymethionine and zero metal ion content. Therefore, w1 represents the mass content of free hydroxymethionine in the sample, and w2 represents the mass content of metal ions, satisfying w2=0. Hydroxymethionine is one of the raw materials for preparing hydroxymethionine metal chelates. By directly measuring the mass content of hydroxymethionine in the raw material sample, the mass content of total hydroxymethionine in the sample, w3, can be obtained. Finally, the chelation rate can be accurately calculated using the formula: chelation rate = (w3 - w1) / w3 × 100%.

[0007] Further, in the above-mentioned chelation rate detection method, the extraction conditions of the first extract and the second extract are as follows: two identical 0.25g samples of hydroxymethionine metal chelate to be tested are placed in 25mL volumetric flasks respectively, diluted with ether, and extracted by ultrasonication at 40KHz for 20min to obtain the first extract and the second extract. The extraction conditions of the first extract and the second extract are the same.

[0008] Furthermore, the first extract and the second extract need to be filtered and heated before the content determination. The filtration is carried out using a filter membrane with a pore size of 0.45 μm, and the heating is carried out under constant temperature water bath conditions of 80°C to completely evaporate the ether in the filtrate.

[0009] Further, the method for detecting the mass content w1 of free hydroxymethionine in the first extract is as follows: Take the first extract and the hydroxymethionine standard series solution respectively, add 25 mL of 10wt% acetonitrile solution to the first extract and the hydroxymethionine standard series solution after filtration and heating treatment, and extract by ultrasonic extraction at 40KHz for 20min, add 0.5 mL of 50wt% sodium hydroxide solution and shake well, add 1 mL of 50wt% phosphoric acid solution and shake well, filter with a filter membrane with a pore size of 0.45μm to obtain the hydrolysate of the first extract and the hydrolysate of the hydroxymethionine standard series solution respectively, and then determine the mass content of free hydroxymethionine by high performance liquid chromatography.

[0010] Furthermore, the preparation process of the hydroxymethionine standard series solutions is as follows: Take the hydroxymethionine standard sample and prepare a series of standard solutions with concentrations of 0.05, 0.10, 0.20, 0.40, and 1.00 mg / mL using 10wt% acetonitrile solution; The chromatographic conditions for the high performance liquid chromatography method are: flow rate 1.0 mL / min, detection wavelength 210 nm, and injection volume 20 μL~50 μL. The mass content of free hydroxymethionine in the first extract was calculated using the formula w1=(c1×V1) / (m1×1000)×100%, where c1 is the concentration of hydroxymethionine in the hydrolysate of the first extract as determined by high performance liquid chromatography (mg / mL), V1 is the volume of 10wt% acetonitrile solution added to the sample (mL), and m1 is the mass of the sample (g).

[0011] Further, the method for detecting the mass content w2 of metal ions in the second extract is as follows: 5 mL of 1~4 mol / L hydrochloric acid solution is added to the second extract after filtration and heating treatment, and then the solution is heated and dissolved under constant temperature water bath conditions at 80℃. After cooling, the solution is transferred to a 100 mL volumetric flask and diluted with water to obtain the solution of the second extract. Then, the metal ion content in the second extract is determined by atomic absorption spectrophotometry.

[0012] Furthermore, the measurement wavelengths for the atomic absorption spectrophotometry are: 324.8 nm for Cu, 279.5 nm for Mn, 213.8 nm for Zn, and 422.6 nm for Ca. Using the formula w2=[(c2-c20)×50×N] / (m2×10 6 Calculate the metal ion content w2 in the second extract by multiplying c2 by 100%, where c2 is the concentration of metal ions in the solution of the second extract (μg / mL), c20 is the concentration of metal ions in the blank solution (μg / mL), N is the dilution factor, m2 is the mass of the sample to be tested (g), and 50 represents the final volume.

[0013] Furthermore, the total hydroxymethionine content (w3) was determined using the iodometric titration method.

[0014] Furthermore, the iodometric method is as follows: Take 0.13g~0.15g of the hydroxymethionine raw material sample and place it in an iodine flask. Add distilled water to dissolve the sample, then add disodium hydrogen phosphate and sodium dihydrogen phosphate to adjust the pH of the solution to 7. Then add 2g of potassium iodide solid and shake to completely dissolve the sample to obtain a sample solution. Add 50mL of 0.1mol / L iodine standard solution to the iodine flask containing the sample solution, tighten the stopper and shake. After standing for 30min, add 1mL of starch indicator. The solution immediately turns blue. Titrate with 0.1mol / L sodium thiosulfate standard solution until the blue color just disappears, and record the volume of sodium thiosulfate solution consumed. At the same time, replace the hydroxymethionine raw material sample with distilled water and perform the same titration operation, recording the volume of sodium thiosulfate solution consumed in the blank test. The total hydroxymethionine content w3 is calculated using the formula w3=[(V30-V3)×c3×0.0751] / m3×100%, where V30 is the volume of sodium thiosulfate solution consumed in the blank test (mL), V3 is the volume of sodium thiosulfate solution consumed in the sample titration (mL), c3 is the concentration of the sodium thiosulfate standard solution (mol / L), M is the molar mass of hydroxymethionine (g / mol), m3 is the mass of the hydroxymethionine raw material sample (g), and 0.0751 is the mass of hydroxymethionine, expressed in grams, equivalent to 1.00 mL of 1.000 mol / L sodium thiosulfate standard solution.

[0015] Furthermore, the hydroxymethionine metal chelate sample to be tested is any one of hydroxymethionine copper, hydroxymethionine zinc, hydroxymethionine manganese, and hydroxymethionine calcium.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The chelation rate detection method for hydroxymethionine metal chelates provided by this invention measures a metal ion content of 0. This method uses diethyl ether, which can dissolve free hydroxymethionine but not hydroxymethionine metal chelates, as the extraction solvent. This improves the detection accuracy by reducing the detection error caused by the difficulty in separating free and bound hydroxymethionine during the chelation rate detection process. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic flowchart of the method for detecting the chelation rate of hydroxymethionine metal chelates in an embodiment of the present invention. Detailed Implementation

[0019] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0020] In the description of this invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0021] The detection methods involved in this invention are as follows: 1. The specific procedure for detecting the mass content w1 of free hydroxymethionine in the first extract is as follows: (1) Preparation of hydrolysate of the first extract The first extract was filtered through a 0.45 μm pore size filter membrane, and then heated in a constant temperature water bath at 80 °C to completely evaporate the ether in the filtrate. Next, 25 mL of 10 wt% acetonitrile solution was accurately added to the filtered and heated first extract and ultrasonically extracted. 0.5 mL of 50 wt% sodium hydroxide solution was added and shaken well. 1 mL of 50 wt% phosphoric acid solution was added and shaken well. The extract was then filtered through a 0.45 μm nylon filter membrane to obtain the hydrolysate of the first extract.

[0022] (2) Preparation of standard hydrolysate Accurately weigh 0.1136 g of liquid hydroxymethionine (88.00 wt%) standard sample and dilute to 100 mL with 10 wt% acetonitrile solution to obtain a hydroxymethionine stock solution with a concentration of 1.00 mg / mL. Accurately pipette 2.5, 5.0, 10.0, and 20.0 mL of the hydroxymethionine stock solution into 50 mL volumetric flasks, dilute to the mark with 10 wt% acetonitrile solution, and shake well to obtain a series of standard solutions with concentrations of 0.05, 0.10, 0.20, 0.40, and 1.00 mg / mL. Add 25 mL of 10 wt% acetonitrile solution to each solution in the standard series and extract by sonication. Add 0.5 mL of 50 wt% sodium hydroxide solution and shake well. Add 1 mL of 50 wt% phosphoric acid solution and shake well. Filter through a 0.45 μm nylon filter membrane to obtain the standard hydrolysate.

[0023] (3) Determination by high performance liquid chromatography Chromatographic conditions: flow rate 1.0 mL / min, detection wavelength 210 nm, injection volume 20 μL~50 μL; Determination process: Hydroxymethionine standard solution is continuously injected into the high performance liquid chromatograph until a chromatographic peak with a stable baseline, symmetrical peak shape and reproducible peak area is obtained; the sample hydrolysate containing the standard and the first extract is injected sequentially, the peak area is obtained by integration, and single-point or multi-point calibration is performed using the standard series.

[0024] (4) Calculation of results The content of free hydroxymethionine (w1) in the first extract is expressed as a mass fraction (%). The mass content of free hydroxymethionine in the first extract is calculated using the formula w1 = (c1 × V1) / (m1 × 1000) × 100%, where c1 is the concentration of hydroxymethionine in the hydrolysate of the first extract (mg / mL) obtained from the standard curve, V1 is the volume (mL) of the 10wt% acetonitrile solution added to the sample, and m1 is the mass (g) of the sample. Parallel determination results are expressed as the arithmetic mean, rounded to two decimal places.

[0025] 2. The specific procedure for detecting the mass content w2 of metal ions in the second extract is as follows: (1) Preparation of standard solutions of copper, zinc, manganese and calcium Cu, Mn, and Zn standard solutions: Take 100 mL of water and 125 mL of analytical grade hydrochloric acid in a 1 L volumetric flask, mix well, weigh 392.9 mg of copper sulfate (CuSO4·5H2O), 439.8 mg of zinc sulfate (ZnSO4·7H2O), and 307.7 mg of manganese sulfate (MnSO4·H2O) and dissolve them in the volumetric flask. Dilute to volume with water to obtain a Cu, Mn, and Zn stock solution with a concentration of 100 μg / mL. Accurately transfer 20.0 mL of the Cu, Mn, and Zn stock solution to a 100 mL volumetric flask, dilute to volume with water, and obtain a Cu, Mn, and Zn standard solution with a concentration of 20 μg / mL. Prepare this standard solution on the day of use.

[0026] Ca standard solution: Place 2.497 g of calcium carbonate (CaCO3) in a beaker, add 50 mL of 6 mol / L hydrochloric acid solution, and after the reaction is complete, transfer the solution to a 1 L volumetric flask and dilute to volume with 0.6 mol / L hydrochloric acid solution to obtain a 1 mg / mL Ca stock solution. Accurately transfer 25.0 mL of the Ca stock solution to a 250 mL volumetric flask and dilute to volume with 0.6 mol / L hydrochloric acid solution to obtain a 100 μg / mL Ca standard solution. The prepared Ca standard solution can be stored in a polyethylene bottle and used within one week.

[0027] (2) Draw the standard curve The process of plotting the standard curves for Cu, Mn, and Zn is as follows: Dilute Cu, Mn, and Zn standard solutions with 0.6 mol / L hydrochloric acid solution to prepare a suitable set of Cu, Mn, and Zn standard solutions; measure the absorbance of the 0.6 mol / L hydrochloric acid solution and the Cu, Mn, and Zn standard solutions; subtract the absorbance of the 0.6 mol / L hydrochloric acid solution from the absorbance of the Cu, Mn, and Zn standard solutions, and plot standard curves for the content of Cu, Mn, and Zn metal ions using the absorbance correction values.

[0028] The process of plotting the standard curve for Ca is as follows: Dilute the Ca standard solution with water. Add 5 mL of lanthanum nitrate solution, 5 mL of cesium chloride solution, and 5 mL of 6 mol / L hydrochloric acid solution to every 100 mL of Ca standard solution to prepare a suitable set of Ca standard solutions. Measure the absorbance of the lanthanum / cesium blank solution. Measure the absorbance of the Ca standard solution and subtract the absorbance of the lanthanum / cesium blank solution. Plot a standard curve using the corrected absorbance against the Ca content.

[0029] The lanthanum nitrate solution was obtained by dissolving 133g of La(NO3)3·6H2O in 1L of water; the cesium chloride solution was obtained by dissolving 100g of cesium chloride CsCl in 1L of water; and the lanthanum / cesium blank solution was obtained by adding 5 mL of lanthanum nitrate solution, 5 mL of cesium chloride solution and 5 mL of 6mol / L hydrochloric acid solution to a 100 mL volumetric flask and then diluting with water.

[0030] (3) Preparation of solution The second extract solution was filtered through a 0.45 μm pore size filter membrane, and then heated in a constant temperature water bath at 80 °C to completely evaporate the ether in the filtrate. Next, 5 mL of 4 mol / L hydrochloric acid solution was added to the filtered and heated second extract solution, and the solution was heated to 90 °C and kept at a gentle boil to dissolve it. After cooling, the solution was transferred to a 100 mL volumetric flask and diluted to volume with water.

[0031] Blank solution: Take 5 mL of 4 mol / L hydrochloric acid solution into a 100 mL volumetric flask and dilute to volume with water.

[0032] (4) Atomic absorption spectrophotometry determination Adjust the instrument testing conditions of the atomic absorption spectrophotometer to ensure that the instrument is in optimal analytical condition in air-acetylene flame measurement mode; the measurement wavelengths of metal ions Cu, Mn, Zn, and Ca are as follows: Cu 324.8 nm, Mn 279.5 nm, Zn 213.8 nm, and Ca 422.6 nm.

[0033] The determination process of Cu, Mn, and Zn in the solution is as follows: Under the same measurement conditions used when plotting the Cu, Mn, and Zn standard curves, measure the absorbance of the solution of the second extract and the blank solution. Subtract the absorbance of the blank solution from the absorbance of the solution of the second extract, and obtain the concentration of the metal element in the solution of the second extract by referring to the Cu, Mn, and Zn standard curves. If necessary, dilute the solution of the second extract and the blank solution with 0.6 mol / L hydrochloric acid solution to ensure that their absorbance is within the linear range of the standard curve.

[0034] The procedure for determining Ca in the solution is as follows: Dilute the solution and blank solution of the second extract with water quantitatively. Add 5 mL of lanthanum nitrate solution, 5 mL of cesium chloride solution, and 5 mL of 6 mol / L hydrochloric acid solution to every 100 mL of solution. Under the same measurement conditions as when plotting the Ca standard curve, measure the absorbance of the solution and blank solution. Subtract the absorbance of the blank solution from the absorbance of the solution and obtain the concentration of the metal element in the solution of the second extract by referring to the Ca standard curve. If necessary, further dilute the solution and blank solution of the second extract with lanthanum / cesium blank solution to ensure that their absorbance is within the linear range of the standard curve. The lanthanum nitrate solution and cesium chloride solution used are the same as those used when plotting the Ca standard curve.

[0035] (5) Calculation of results The elements Cu, Zn, Mn, and Ca in the second extract, expressed as mass fraction w2 (%), are calculated using the formula w2 = [(c2 - c20) × 50 × N] / (m2 × 10⁻⁶). 6 Calculate the metal ion content w2 in the second extract, where c2 is the concentration of metal ions in the solution of the second extract (in μg / mL), c20 is the concentration of metal ions in the blank solution (in μg / mL), N is the dilution factor, and m2 is the mass of the sample to be tested (in g).

[0036] During atomic absorption measurements, if the absorbance of the second extract solution or blank solution exceeds the linear range of the standard curve, it needs to be diluted with an appropriate solution (such as 0.6 mol / L hydrochloric acid or lanthanum / cesium blank solution) to bring its absorbance within the linear range. N represents the dilution factor (N=1 if undiluted).

[0037] 3. The specific procedure for detecting the total hydroxymethionine content (w3) in the hydroxymethionine raw material sample is as follows: (1) Reagent preparation To prepare a 0.1 mol / L iodine standard solution: Weigh a certain amount of potassium iodide and iodine, dissolve them in an appropriate amount of water, and bring the volume up to the required level. Shake well.

[0038] To prepare a 0.1 mol / L sodium thiosulfate standard solution: Weigh sodium thiosulfate pentahydrate, dissolve it in freshly boiled and cooled distilled water, add a small amount of sodium hydroxide to adjust the pH, and store it in a dark glass bottle.

[0039] To prepare a 5g / L starch solution: Weigh 5g of soluble starch, mix it with a small amount of water to form a paste, then add 100mL of boiling water, stir well, and let it cool before use.

[0040] Prepare other auxiliary reagents, such as disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium iodide, etc., and accurately weigh and dissolve them according to the experimental requirements.

[0041] (2) Sample processing Accurately weigh 0.13g~0.15g of hydroxymethionine raw material sample, place it in an iodine flask, add 3mL of 3mol / L hydrochloric acid and 50mL of deionized water to dissolve the sample, then add 10mL of 500g / L dipotassium hydrogen phosphate and 10mL of 500g / L potassium dihydrogen phosphate to adjust the pH of the solution to 7, then add 2g of potassium iodide solid, shake to completely dissolve the sample, and obtain the sample solution.

[0042] (3) Titration operation Accurately measure 50 mL of 0.1 mol / L iodine standard solution and add it to the iodine flask containing the sample solution. Tighten the stopper, gently shake to mix, and let stand for 30 min. Titrate the excess iodine with 0.1 mol / L sodium thiosulfate standard solution until the blue color just disappears. Add 1 mL of starch indicator and continue titrating until colorless and hold for 30 s. Record the volume of sodium thiosulfate solution consumed.

[0043] (4) Blank test Following the steps above, without adding the sample, use only distilled water to perform the same titration operation and record the volume of sodium thiosulfate solution consumed in the blank test.

[0044] (5) Calculation of results The total hydroxymethionine content w3 is calculated using the formula w3=[(V30-V3)×c3×0.0751] / m3×100%, based on the volume of sodium thiosulfate solution consumed during the titration, the concentration of the sodium thiosulfate standard solution, and the sample mass. Here, V30 is the volume of sodium thiosulfate solution consumed in the blank test (mL), V3 is the volume of sodium thiosulfate solution consumed in the sample titration (mL), c3 is the concentration of the sodium thiosulfate standard solution (mol / L), M is the molar mass of hydroxymethionine (g / mol), m3 is the mass of the hydroxymethionine raw material sample (g), and 0.0751 is the mass of hydroxymethionine in grams equivalent to 1.00 mL of 1.000 mol / L sodium thiosulfate standard solution.

[0045] Example: Please see Figure 1 This invention provides a method for detecting the chelation rate of hydroxymethionine metal chelates, comprising the following steps: S1: Take two identical hydroxymethionine metal chelate samples and extract them with diethyl ether under the first and second preset conditions to obtain the first extract and the second extract. S2: After filtering and heating the first and second extracts, the mass content of free hydroxymethionine in the first extract was measured as w1, and the mass content of metal ions in the second extract was measured as w2. S3: Take the hydroxymethionine raw material sample for preparing the hydroxymethionine metal chelate test sample, measure the total hydroxymethionine mass content as w3, and satisfy: w2=0, calculate the chelation rate by formula = (w3- w1) / w3×100%.

[0046] In some embodiments, the two test samples in S1 have the same composition, and the test sample is any one of copper hydroxymethionine, zinc hydroxymethionine, manganese hydroxymethionine, and calcium hydroxymethionine.

[0047] In some embodiments, the quantity of each sample to be tested is 0.25g, and the first preset condition and the second preset condition are: take one sample to be tested and place it in a 25mL volumetric flask, dilute it with ether, and then extract it with 40KHz ultrasound for 20min.

[0048] It should be noted that the second preset condition is the same as the first preset condition, that is, the first extract and the second extract are the same extract.

[0049] In some embodiments, in S2, a filter membrane with a pore size of 0.45 μm is used for filtration, and the heating treatment is carried out under constant temperature water bath conditions of 80°C to remove the ether from the first extract and the second extract.

[0050] In some embodiments, the process of detecting the mass content w1 of free hydroxymethionine in the first extract in S2 includes: adding 25 mL of 10wt% acetonitrile solution to the first extract after filtration and heating and ultrasonically extracting it at 40 kHz for 20 min, adding 0.5 mL of 50wt% sodium hydroxide solution and shaking well, adding 1 mL of 50wt% phosphoric acid solution and shaking well, filtering it with a filter membrane with a pore size of 0.45 μm to obtain the hydrolysate of the first extract, and then determining the mass content of free hydroxymethionine by high performance liquid chromatography. The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are: flow rate 1.0 mL / min, detection wavelength 210 nm, and injection volume: 20 μL~50 μL. The mass content of free hydroxymethionine in the first extract, w1, is calculated using the formula w1 = (c1 × V1) / (m1 × 1000) × 100%, where c1 is the concentration of hydroxymethionine in the hydrolysate of the first extract (mg / mL) as determined by HPLC, V1 is the volume (mL) of the 10 wt% acetonitrile solution added to the sample, and m1 is the mass (g) of the sample. The specific detection process is as described in the detailed implementation method.

[0051] In this embodiment, the process of detecting the mass content w2 of metal ions in the second extract in S2 includes: adding 5 mL of 1~4 mol / L hydrochloric acid solution to the second extract after filtration and heating, then heating (heating to 90~100℃ and maintaining a gentle boil) to dissolve, cooling and transferring to a 100 mL volumetric flask, and making up to volume with water to obtain the solution of the second extract, and then determining the metal ion content in the second extract by atomic absorption spectrophotometry.

[0052] The measurement wavelengths for the atomic absorption spectrophotometry are: 324.8 nm for Cu, 279.5 nm for Mn, 213.8 nm for Zn, and 422.6 nm for Ca; measured using the formula w2 = [(c2 - c20) × 50 × N] / (m2 × 10⁻⁶). 6 The metal ion content w2 in the second extract is calculated by multiplying c2 by 100%, where c2 is the concentration of metal ions in the solution of the second extract (μg / mL), c20 is the concentration of metal ions in the blank solution (μg / mL), N is the dilution factor, and m2 is the mass of the sample to be tested (g). The specific detection process is as described in the specific implementation method.

[0053] In this embodiment, the third preset condition is as follows: 0.13g~0.15g of the hydroxymethionine raw material sample is placed in an iodine flask, 3mL of 3mol / L hydrochloric acid and 50mL of deionized water are added to dissolve the sample, then 10mL of 500g / L dipotassium hydrogen phosphate and 10mL of 500g / L potassium dihydrogen phosphate are added to adjust the pH of the solution to 7, and then 2g of potassium iodide solid is added. The mixture is shaken to completely dissolve the sample to obtain a sample solution. 50mL of 0.1mol / L iodine standard solution is added to the iodine flask containing the sample solution, the stopper is tightened, and the mixture is shaken well. After standing for 30min, excess iodine is titrated with 0.1mol / L sodium thiosulfate standard solution until the blue color just disappears. Then 1mL of starch indicator is added, and titration continues until colorless and maintained for 30s. The volume of sodium thiosulfate solution consumed is recorded. At the same time, distilled water is used instead of the hydroxymethionine raw material sample, and the same titration operation is performed with the sample solution. The volume of sodium thiosulfate solution consumed in the blank test is recorded. The total hydroxymethionine content w3 is calculated using the formula w3 = [(V30 - V3) × c3 × 0.0751] / m3 × 100%, where V30 is the volume of sodium thiosulfate solution consumed in the blank test (mL), V3 is the volume of sodium thiosulfate solution consumed in the sample titration (mL), c3 is the concentration of the sodium thiosulfate standard solution (mol / L), M is the molar mass of hydroxymethionine (g / mol), m3 is the mass of the hydroxymethionine raw material sample (g), and 0.0751 is the mass of hydroxymethionine (g) equivalent to 1.00 mL of 1.000 mol / L sodium thiosulfate standard solution. The specific detection process is as described in the detailed implementation method.

[0054] To better illustrate the accuracy of the chelation rate detection method for hydroxymethionine metal chelates involved in this embodiment, the following example verification experiments were conducted: Experimental Group 1: (1) Take two identical hydroxymethionine metal chelate test samples (hydroxymethionine copper, hydroxymethionine zinc, hydroxymethionine manganese, and hydroxymethionine calcium) 0.25 g each (accurate to 0.0001 g), place them in a 25 mL volumetric flask, dilute to volume with ether, and extract by ultrasonication at 40 kHz for 20 min to obtain the first extract and the second extract for later use. (2) The first extract was filtered through a 0.45 μm organic microporous membrane (nylon material). The ether in the filtrate was completely evaporated in an 80℃ constant temperature water bath. Then, 25 mL of 10 wt% acetonitrile solution was added and ultrasonically extracted at 40 kHz for 20 min. 0.5 mL of 50 wt% sodium hydroxide solution was added and shaken well. 1 mL of 50 wt% phosphoric acid solution was added and shaken well. After passing through a 0.45 μm filter membrane, the hydrolysate of the first extract was obtained. The mass content of free hydroxymethionine w1 was then determined by high performance liquid chromatography. The specific detection process is shown in the specific implementation method. (3) The second extract was filtered through a 0.45 μm organic microporous membrane (nylon material). The ether in the filtrate was completely evaporated in an 80℃ constant temperature water bath. Then, 5 mL of 4 mol / L dilute hydrochloric acid solution was added and heated (heated to 90℃ and kept at a gentle boil) to dissolve. After cooling, the solution was transferred to a 100 mL volumetric flask and diluted with water to obtain the solution of the second extract. The metal ion content w2 in the second extract was then determined by atomic absorption spectrophotometry. The specific detection process is shown in the specific implementation method. (4) Take 0.14g of hydroxymethionine raw material sample and determine the total hydroxymethionine content w3 according to the iodometric method; the specific detection process is shown in the specific implementation method. (5) Calculate the chelation rate using the formula chelation rate = (w3 - w1) / w3 × 100%.

[0055] Experimental Group 2: Under constant temperature of 25℃, 10g of 88wt% liquid hydroxymethionine sample (the remaining 12wt% being bound water) was mixed with 10mL of diethyl ether and shaken to reach dissolution equilibrium. After standing for 24h, the supernatant was collected as the extract, and the hydroxymethionine content in the supernatant was determined using the same method as for determining the free hydroxymethionine mass content w1 in the first extract. Then, the solubility of 88wt% liquid hydroxymethionine in diethyl ether was calculated. Solubility = mass of hydroxymethionine in saturated solution / volume of saturated solution (volume of the mixed solution of hydroxymethionine and diethyl ether).

[0056] Comparative Group 1: The reaction conditions in this experiment are the same as those in Experiment 1, except that the extraction solvent is acetonitrile.

[0057] Comparative Group 2: The reaction conditions in this experiment are the same as those in Experiment 1, except that the extraction solvent is dichloromethane.

[0058] Comparison Group 3: The reaction conditions in this experiment are the same as those in Experiment Group 1, except that the extraction solvent is n-hexane.

[0059] Comparative Group 4: The reaction conditions in this experiment were the same as those in Experiment 1. Samples of hydroxymethionine in metals such as copper, zinc, manganese, and calcium were used to determine the free hydroxymethionine six times in parallel.

[0060] Comparative Group 5: The reaction conditions in this experiment are the same as those in Experiment 2, except that the extraction solvent is acetonitrile.

[0061] Comparative Group 6: The reaction conditions in this experiment are the same as those in Experiment 2, except that the extraction solvent is dichloromethane.

[0062] Comparative Group 7: The reaction conditions in this experiment are the same as those in Experiment 2, except that the extraction solvent is n-hexane.

[0063] Comparative Group 8: The reaction conditions in this experiment are the same as those in Experiment 1. The difference lies in the source of the two samples of hydroxymethionine metal chelates. Specifically, the two samples of hydroxymethionine copper, zinc, manganese, and calcium are from different sources.

[0064] It should be noted that the experimental instruments required for the above experiments include: a P2300Ⅱ high performance liquid chromatograph (including an ultraviolet detection system, injection system, column oven, binary pump, etc.), an AA-6300C atomic absorption spectrophotometer, an AUW120D electronic balance (accuracy 0.00001g), a TG16-WS centrifuge (maximum speed 16000r / min), a KC-60A ultrasonic cleaner, a DZKW-D-2 electric thermostatic water bath, and an electric furnace.

[0065] The results of the above experiments are shown in Table 1, which shows the solubility of hydroxymethionine metal chelate in different extraction solvents; Table 2 shows the effect of different extraction solvents on the solubility of hydroxymethionine; Table 3 shows the content of free hydroxymethionine in diethyl ether determined by repeated verification experiments; and Table 4 shows the chelation rate of hydroxymethionine chelate products from different sources.

[0066] Table 1. Solubility of hydroxymethionine metal chelates in different extraction solvents

[0067] Table 2. Effect of different extraction solvents on the solubility of hydroxymethionine

[0068] Table 3. Determination of the mass content of free hydroxymethionine in diethyl ether by repeated validation experiments.

[0069] Table 4 Chelation rates of hydroxymethionine chelate products from different sources

[0070] Table 5. Solubility of hydroxymethionine in diethyl ether in Experiment 2

[0071] Table 1 shows that among the metal chelates of hydroxymethionine, such as copper, zinc, manganese, and calcium, the extracts in diethyl ether, n-hexane, and some dichloromethane do not contain metal ions. Among them, the diethyl ether extract has the highest average hydroxymethionine content, indicating that diethyl ether is more suitable for extracting free hydroxymethionine than n-hexane and dichloromethane. Although the n-hexane extract also does not contain metal ions, the free hydroxymethionine content is almost zero, which is related to the low polarity of n-hexane, which cannot dissolve free hydroxymethionine.

[0072] Furthermore, as shown in Table 2, hydroxymethionine is readily soluble in acetonitrile and dichloromethane, has good solubility in diethyl ether, and is almost insoluble in n-hexane.

[0073] Understandably, using hydroxymethionine metal chelates as raw materials, and following the basic principle of like compatibility, the selection principle of maximizing the solubility of hydroxymethionine and selecting extracts with zero metal atom content, diethyl ether is the best extraction solvent for free hydroxymethionine because it has the highest metal atom content and hydroxymethionine content in its extract.

[0074] As shown in Table 3, by using diethyl ether as the extraction solvent, the residual standard deviation (RSD) of six parallel determinations of free hydroxymethionine in samples of copper, zinc, manganese and calcium metals was less than 5%, which indicates that the parallel determinations have good repeatability. The method for detecting the chelation rate of hydroxymethionine metal chelates provided by this invention has high precision.

[0075] It should be noted that the method for determining the total hydroxymethionine content in Table 4 is as follows: Take 0.16 g of liquid hydroxymethionine with a content of 88 wt%, add 25 mL of diethyl ether, extract by ultrasonication for 10 min, filter, heat the filtrate in a constant temperature water bath at 80℃ to remove the diethyl ether, and then determine the total hydroxymethionine content according to GB / T 17810-2009.

[0076] Table 4 shows that free hydroxymethionine was extracted from the hydroxymethionine metal element chelate using diethyl ether, and the chelation rate of hydroxymethionine metal element was calculated based on the total hydroxymethionine content. Due to different sources of the same chelate, the measured chelation rates varied slightly. The error in the calcium chelation rate of hydroxymethionine was slightly higher, differing by 4.52%, while the errors among the other chelate products were small, all below 2%. This indicates that the present invention has a small error in detecting the chelation rate of hydroxymethionine chelate products from different sources.

[0077] The results in Table 5 show that the methionine content in the ether extract is basically consistent with the sample value (88 wt%), and the relative error between the six parallel determinations is very low.

[0078] The above experiments revealed that the chelation rate detection method for hydroxymethionine metal chelates provided in this embodiment, using diethyl ether as the extractant, demonstrates that free hydroxymethionine has high solubility in diethyl ether, while diethyl ether cannot dissolve hydroxymethionine metal chelates. This improves the detection accuracy and repeatability, mitigating the detection error caused by the difficulty in separating free and bound hydroxymethionine. Furthermore, the low boiling point and high volatility of diethyl ether avoid the influence of extractant residue on the methionine content determination results.

[0079] In the description of this specification, the reference to the term "some embodiments" means that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, those skilled in the art can combine and integrate the different embodiments and features described in this specification without contradiction.

[0080] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for detecting the chelation rate of hydroxymethionine metal chelates, characterized in that, Includes the following steps: Two identical samples of hydroxymethionine metal chelate were taken, and after being diluted with ether and extracted by ultrasound, respectively, a first extract and a second extract were obtained. The mass content of free hydroxymethionine in the first extract was measured as w1, and the mass content of metal ions in the second extract was measured as w2, and w2=0 was satisfied. The hydroxymethionine raw material sample used to prepare the hydroxymethionine metal chelate sample was taken, and the total hydroxymethionine mass content was measured as w3. The chelation rate was calculated using the formula chelation rate = (w3- w1) / w3×100%. The first extract and the second extract need to be filtered and heated before the content determination. The filtration is carried out using a filter membrane with a pore size of 0.45 μm, and the heating is carried out under a constant temperature water bath at 80°C to completely evaporate the ether in the filtrate. The method for detecting the mass content w2 of metal ions in the second extract is as follows: 5 mL of 1~4 mol / L hydrochloric acid solution is added to the second extract after filtration and heating treatment, and then the solution is heated and dissolved under constant temperature water bath at 80℃. After cooling, the solution is transferred to a 100 mL volumetric flask and diluted with water to obtain the solution of the second extract. Then, the metal ion content in the second extract is determined by atomic absorption spectrophotometry. The measurement wavelengths for the atomic absorption spectrophotometry are: 324.8 nm for Cu, 279.5 nm for Mn, 213.8 nm for Zn, and 422.6 nm for Ca.

2. The method for detecting the chelation rate of hydroxymethionine metal chelates according to claim 1, characterized in that, The extraction conditions for the first and second extracts are as follows: 0.25g of two identical hydroxymethionine metal chelate test samples are placed in 25mL volumetric flasks, diluted to volume with ether, and extracted by ultrasonication at 40KHz for 20min to obtain the first and second extracts. The extraction conditions for the first and second extracts are the same.

3. The method for detecting the chelation rate of hydroxymethionine metal chelates according to claim 1, characterized in that, The method for determining the mass content w1 of free hydroxymethionine in the first extract is as follows: 25 mL of 10wt% acetonitrile solution was added to the first extract after filtration and heating, and ultrasonic extraction was performed at 40 kHz for 20 min. 0.5 mL of 50wt% sodium hydroxide solution was added and shaken well. 1 mL of 50wt% phosphoric acid solution was added and shaken well. After filtration through a 0.45 μm filter membrane, the hydrolysate of the first extract was obtained. The mass content of free hydroxymethionine was then determined by high performance liquid chromatography.

4. The method for detecting the chelation rate of hydroxymethionine metal chelates according to claim 3, characterized in that, The chromatographic conditions for the high performance liquid chromatography method are: flow rate 1.0 mL / min, detection wavelength 210 nm, and injection volume 20 μL~50 μL. The mass content of free hydroxymethionine in the first extract was calculated using the formula w1=(c1×V1) / (m1×1000)×100%, where c1 is the concentration of hydroxymethionine in the hydrolysate of the first extract as determined by high performance liquid chromatography (mg / mL), V1 is the volume of 10wt% acetonitrile solution added to the sample (mL), and m1 is the mass of the sample (g).

5. The method for detecting the chelation rate of hydroxymethionine metal chelates according to claim 1, characterized in that, Using the formula w2=[(c2-c20)×50×N] / (m2×10 6 Calculate the metal ion content w2 in the second extract by multiplying c2 by 100%, where c2 is the concentration of metal ions in the solution of the second extract (μg / mL), c20 is the concentration of metal ions in the blank solution (μg / mL), N is the dilution factor, m2 is the mass of the sample to be tested (g), and 50 represents the final volume.

6. The method for detecting the chelation rate of hydroxymethionine metal chelates according to claim 1, characterized in that, The total hydroxymethionine content (w3) was determined by iodometric titration.

7. The method for detecting the chelation rate of hydroxymethionine metal chelates according to claim 6, characterized in that, The iodometric titration method is as follows: Take 0.13g~0.15g of the hydroxymethionine raw material sample and place it in an iodine flask. Add 3mL of 3mol / L hydrochloric acid and 50mL of deionized water to dissolve the sample. Then add 10mL of 500g / L dipotassium hydrogen phosphate and 10mL of 500g / L potassium dihydrogen phosphate to adjust the pH of the solution to 7. Then add 2g of potassium iodide solid and shake to completely dissolve the sample to obtain the sample solution. Add 50mL of 0.1mol / L iodine standard solution to the iodine flask containing the sample solution, tighten the stopper, shake well, and let stand for 30min. Titrate the excess iodine with 0.1mol / L sodium thiosulfate standard solution until the blue color just disappears as the endpoint. Then add 1mL of starch indicator and continue titrating until colorless and hold for 30s. Record the volume of sodium thiosulfate solution consumed. At the same time, replace the hydroxymethionine raw material sample with distilled water and perform the same titration operation as the sample solution. Record the volume of sodium thiosulfate solution consumed in the blank test. The total hydroxymethionine content w3 is calculated using the formula w3=[(V30-V3)×c3×0.0751] / m3×100%, where V30 is the volume of sodium thiosulfate solution consumed in the blank test (mL), V3 is the volume of sodium thiosulfate solution consumed in the sample titration (mL), c3 is the concentration of sodium thiosulfate standard solution (mol / L), M is the molar mass of hydroxymethionine (g / mol), and m3 is the mass of the hydroxymethionine raw material sample (g).

8. The method for detecting the chelation rate of hydroxymethionine metal chelates according to any one of claims 1-7, characterized in that, The hydroxymethionine metal chelate sample to be tested is any one of hydroxymethionine copper, hydroxymethionine zinc, hydroxymethionine manganese, and hydroxymethionine calcium.

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