Method for simultaneously detecting three steroid hormone substances in raw milk

Abstract

The invention relates to the technical field of dairy product detection, in particular to a method for simultaneously detecting three steroid hormone substances in raw milk. The three steroid hormone substances are estradiol, ethinyl estradiol and estrone respectively, and the detection method comprises the following steps: 1) removing protein in raw milk to obtain a sample solution to be detected; (2) carrying out qualitative and quantitative analysis on the three steroid hormone substances in the sample solution to be detected by adopting a high performance liquid chromatography-ultraviolet detection method; wherein the qualitative analysis is based on the retention time of each substance, and the quantitative analysis is based on a standard curvilinear equation to calculate the concentration. According to the detection method, the dependence on expensive instruments and professional operators is remarkably reduced while high sensitivity and accuracy are guaranteed, the cost is low, and the method is particularly suitable for rapid screening of grassroots units and large-batch samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of dairy product detection, in particular to a method for simultaneously detecting three kinds of steroid hormone substances in raw cow milk. BACKGROUND

[0002] Raw cow milk is one of the important sources of human nutrition, and its quality and safety is directly related to the health of consumers. In recent years, with the potential misuse of steroid hormone substances in animal husbandry, the problem of steroid hormone residues in raw cow milk has increasingly attracted attention. Steroid hormones are a class of compounds with endocrine disrupting effects, even trace residues can accumulate in the human body through the food chain, leading to a series of health risks. For example, long-term intake of dairy products containing steroid hormones may interfere with normal endocrine function in the human body, causing abnormal reproductive system, developmental disorders, and even increasing the incidence of cancer.

[0003] Among steroid hormones, estradiol, ethinyl estradiol and estrone are three common and significant compounds. Estradiol, as a natural estrogen, excessive intake may cause hormone imbalance in the human body, leading to menstrual disorders in women, breast hyperplasia, and increasing the risk of endometrial cancer and ovarian cancer; for men, it may inhibit testosterone secretion, affecting reproductive function. Ethinyl estradiol is a synthetic estrogen, its residues are highly persistent in the environment, can enter the human body through dairy products, causing endocrine system disorders, especially threatening the development of adolescents and the health of fetuses of pregnant women. Estrone also has estrogenic activity, its accumulation may exacerbate the development of hormone-dependent tumors, and is associated with cardiovascular disease and osteoporosis and other health problems. Given these hazards, regulatory agencies in various countries have set strict limit standards for steroid hormone residues in raw cow milk, highlighting the need for rapid and accurate detection methods.

[0004] Currently, the detection of steroid hormone compounds in raw cow milk mainly relies on chromatography tandem mass spectrometry technology, including gas chromatography tandem mass spectrometry (GC-MS / MS) and liquid chromatography tandem mass spectrometry (LC-MS / MS). These methods have high sensitivity and specificity, and can realize multi-residue analysis, but have significant defects. First, GC-MS / MS and LC-MS / MS have very high requirements for instruments and equipment, requiring expensive mass spectrometers and chromatography systems, and the maintenance cost is high; second, the sample pretreatment process is complex, and the technical level and experience of the operator are strongly dependent, which is easy to introduce human error. SUMMARY

[0005] In view of the above-mentioned defects of the prior art, in order to solve the problem of high cost and high requirement for operators of the existing detection method, the purpose of the present application is to provide a method for simultaneously detecting three kinds of steroid hormone substances in raw cow milk, to solve the problems in the prior art.

[0006] To achieve the above object and other related objects, the present application provides a method for simultaneously detecting three kinds of steroid hormone substances in raw cow milk, the three kinds of steroid hormone substances being estradiol, ethinyl estradiol and estrone, and the method has the following steps:

[0007] 1) removing the protein in the raw cow milk to obtain a sample solution to be detected;

[0008] 2) using high performance liquid chromatography-ultraviolet detection method to qualitatively and quantitatively analyze the three kinds of steroid hormone substances in the sample solution to be detected;

[0009] In the method, the qualitative analysis is based on the retention time of each substance, and the quantitative analysis is based on a standard curve equation to calculate the concentration.

[0010] Compared with the prior art, the present application has the following beneficial effects:

[0011] 1) The present application uses a reversed-phase C18 chromatographic column with a filler particle size of 3 μm to complete the detection of the three kinds of steroid compounds in raw cow milk, and the quantitative limit is 0.03 μg / mL. The method has the advantages of rapidity, high efficiency and solvent saving.

[0012] 2) The present application can be realized using a common high performance liquid chromatography instrument, without using an expensive ultra-high performance liquid chromatography instrument. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The present application detects the chromatograms of the three kinds of steroid hormone substances in the standard sample and the sample with added standard. DETAILED DESCRIPTION

[0014] In order to make the object, technical scheme and beneficial effects of the present application clearer, the present application will be further described below in combination with examples. It should be understood that the examples are only used to explain the present application, and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure.

[0015] The first aspect of the present application provides a method for simultaneously detecting three kinds of steroid hormone substances in raw cow milk, the three kinds of steroid hormone substances being estradiol, ethinyl estradiol and estrone, and the method has the following steps:

[0016] 1) removing the protein in the raw cow milk to obtain a sample solution to be detected;

[0017] 2) using high performance liquid chromatography-ultraviolet detection method to qualitatively and quantitatively analyze the three kinds of steroid hormone substances in the sample solution to be detected;

[0018] The qualitative analysis is based on the retention time of each substance, and the quantitative analysis is based on the concentration calculated by the standard curve equation.

[0019] In some specific embodiments, in step 1), the protein in raw cow milk is removed by using a protein precipitant.

[0020] In the present application, the "protein precipitant" refers to a chemical reagent capable of forming an insoluble precipitate of protein in a sample solution by destroying the hydration layer of the protein, neutralizing its surface charge or denaturing its spatial structure, and separating it from the liquid phase. Its role is to remove protein interference in the sample matrix, improve the extraction efficiency and detection accuracy of the target analyte, including but not limited to: trichloroacetic acid solution, perchloric acid solution, acetonitrile, methanol, acetone, ammonium sulfate, zinc sulfate solution and potassium ferrocyanide solution.

[0021] In some specific embodiments, the protein precipitant is a trichloroacetic acid solution.

[0022] Preferably, the concentration of the trichloroacetic acid solution is 30-50% (w / v). More preferably, the concentration of the trichloroacetic acid solution is 40% (w / v).

[0023] In some specific embodiments, in step 2), the determination conditions of the high performance liquid chromatography include that the chromatographic column is a reversed-phase C18 chromatographic column.

[0024] Preferably, the particle size of the reversed-phase C18 chromatographic column is 3 μm.

[0025] In some specific embodiments, in step 2), the determination conditions of the high performance liquid chromatography include that the mobile phase A is water and the mobile phase B is acetonitrile.

[0026] Preferably, the mobile phase is subjected to gradient elution.

[0027] More preferably, the gradient elution is performed according to the following gradient:

[0028] 0-18 min, the volume percentage of the mobile phase B is 35%-65%, and the rest is the mobile phase A;

[0029] 18-20 min, the volume percentage of the mobile phase B is 65%B-95%B, and the rest is the mobile phase A;

[0030] 20-22 min, the volume percentage of the mobile phase B is 95%B-95%B, and the rest is the mobile phase A.

[0031] In some specific embodiments, in step 2), the determination conditions of the high performance liquid chromatography include that the flow rate is 0.4-0.8 mL / min. Preferably, the flow rate is 0.6 mL / min.

[0032] In some specific embodiments, in step 2), the determination condition of the high performance liquid chromatography comprises: the column temperature is 25-35℃. Preferably, the column temperature is 30℃.

[0033] In some specific embodiments, in step 2), the determination condition of the high performance liquid chromatography comprises: the injection volume is 3-7μL. Preferably, the injection volume is 5μL.

[0034] In some specific embodiments, in step 2), the determination condition of the high performance liquid chromatography comprises: the injection tray temperature is 3-5℃. Preferably, the injection tray temperature is 4℃.

[0035] In some specific embodiments, in step 2), the UV detection wavelength of the UV detection method is 205 nm.

[0036] In some specific embodiments, the retention time corresponding to estradiol is 9.2-9.5min.

[0037] In some specific embodiments, the retention time corresponding to ethinyl estradiol is 11.1-11.4min.

[0038] In some specific embodiments, the retention time corresponding to estrone is 11.5-11.8min.

[0039] In some specific embodiments, the standard curve equation corresponding to estradiol is y = 531.03x -1.1265.

[0040] In some specific embodiments, the standard curve equation corresponding to ethinyl estradiol is y = 438.26x + 1.2157.

[0041] In some specific embodiments, the standard curve equation corresponding to estrone is y = 486.67x +0.2313.

[0042] In which, the ordinate y is the chromatographic peak area, and the abscissa x is the concentration (μg / mL).

[0043] The application is further illustrated by the following examples, but the scope of the application is not limited by the examples.

[0044] When the embodiments give numerical ranges, it is understood that unless the embodiments expressly state otherwise, the two endpoints and any number between the two endpoints of every numerical range is available. Unless otherwise defined, all technical and scientific terms used in the embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. Unless specific conditions are indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are used. Unless the manufacturer is indicated, all reagents or instruments are conventional products that can be purchased on the market. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to the methods, devices and materials described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.

[0045] Example 1: Detection of three kinds of steroid hormone compounds in raw milk

[0046] 8 g of sample was weighed; 2 mL of 40% (w / v) trichloroacetic acid solution was added, shaken for 10 minutes, and then centrifuged at 4.355G for 15 minutes; the supernatant was poured into a 10 mL volumetric flask, and pure water was added to the mark. An appropriate amount of liquid was passed through a 0.45 μm nylon filter membrane, and then analyzed by a chromatograph.

[0047] The determination conditions are as follows:

[0048] Chromatographic column: Waters Atlantis T3 (3.0 mm x 150 mm, 3 μm); mobile phase: mobile phase A is water, and mobile phase B is acetonitrile; gradient elution: 0-18 min, 35%B-65%B; 18-20 min, 65%B-95%B; 20-22 min, 95%B-95%B; flow rate: 0.6 mL / min; column temperature: 30°C; injection volume: 5 μL; injection tray temperature: 4°C; UV detection wavelength: 205 nm.

[0049] Example 2: System suitability test

[0050] In order to verify the effectiveness and reliability of the chromatographic system of the present application, standard solutions of three target analytes (estradiol, ethinyl estradiol and estrone) with a concentration of 40% were prepared, and analyzed according to the above chromatographic conditions. The results are shown in Table 1.

[0051] Table 1: System suitability test results

[0052]

[0053] As can be seen from Table 1, the three target substances can be effectively separated under the chromatographic conditions of the present application, the symmetry factor of each chromatographic peak is between 0.95 and 0.99, indicating that the peak shape is symmetrical, and there is no obvious tailing or fronting; the theoretical plate number is higher than 49000, indicating that the column efficiency is high and the separation efficiency is good. The results prove that the chromatographic system established in the present application meets the analysis requirements and can ensure the accuracy of the analysis.

[0054] Example 3: Methodology verification test

[0055] To comprehensively evaluate the analysis performance of the method of the present application, the specificity, linear range, limit of quantification, limit of detection, precision, stability, repeatability and the like of the method are further verified. The verification results are shown in Table 2.

[0056] Table 2 Methodology verification results

[0057]

[0058] In terms of sensitivity, the detection limit and the limit of quantification of the three hormones are all 0.05 μg / mL and 0.03 μg / mL, indicating that the method has high sensitivity. And in the concentration range of 0.05-10 μg / mL, the linear relationship is good, and all the correlation coefficients (R²) are greater than 0.999, proving that the method has excellent quantitative ability.

[0059] In terms of accuracy and precision, the spiked recovery rate of the method is between 97.10%-105.90%, which meets the requirements of trace analysis (usually 80%-120%). The precision (calculated by the relative standard deviation RSD% of peak area) is not higher than 1.00%, indicating that the method has good repeatability and the data is stable and reliable.

[0060] In terms of stability, the sample solution is stable within 24 hours (RSD% ≤ 1.14%), and the method repeatability also meets the detection requirements.

[0061] The above system suitability test and methodology verification results fully show that the high performance liquid chromatography-ultraviolet detection method established in the present application can quickly, accurately and stably detect the residues of the three kinds of steroid hormones, estradiol, ethinyl estradiol and estrone in raw cow milk at the same time. Compared with the chromatography tandem mass spectrometry technology mentioned in the background art, the detection method of the present application significantly reduces the dependence on expensive instruments and professional operators while ensuring high sensitivity and accuracy, is low in cost, and is especially suitable for rapid screening of primary units and large quantities of samples.

[0062] The above embodiments are only illustrative of the principles of the present application and its effects, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. A method for simultaneously detecting three kinds of steroid hormone substances in raw cow milk, the three kinds of steroid hormone substances being estradiol, ethinyl estradiol and estrone respectively, comprising the following steps: 1) removing proteins in raw cow milk to obtain a sample solution to be detected; 2) performing qualitative and quantitative analysis on the three kinds of steroid hormone substances in the sample solution to be detected by high performance liquid chromatography-ultraviolet detection method; wherein the qualitative analysis is based on the retention time of each substance, and the quantitative analysis is based on a standard curve equation to calculate the concentration thereof.

2. The method of claim 1, wherein, In step 1), the removal of proteins in raw cow milk is achieved by using a protein precipitant.

3. The method of claim 2, wherein, The protein precipitant is a trichloroacetic acid solution; preferably, the concentration of the trichloroacetic acid solution is 30-50% (w / v).

4. The method of claim 1, wherein, In step 2), the determination conditions of the high performance liquid chromatography include: the chromatographic column is a reversed-phase C18 chromatographic column; and / or, the mobile phase A is water, and the mobile phase B is acetonitrile; and / or, the flow rate is 0.4-0.8 mL / min; and / or, the chromatographic column temperature is 25-35℃; and / or, the injection volume is 3-7 μL; and / or, the injection tray temperature is 3-5℃.

5. The method of claim 4, wherein, The packing particle size of the reversed-phase C18 chromatographic column is 3 μm.

6. The method of claim 4, wherein, The mobile phase is subjected to gradient elution.

7. The method of claim 6, wherein, The gradient elution is performed according to the following gradient: 0-18 min, the volume percentage of the mobile phase B is 35%-65%, and the rest is the mobile phase A; 18-20 min, the volume percentage of the mobile phase B is 65%B-95%B, and the rest is the mobile phase A; 20-22 min, the volume percentage of the mobile phase B is 95%B-95%B, and the rest is the mobile phase A.

8. The method of claim 1, wherein, In step 2), the ultraviolet detection wavelength of the ultraviolet detection method is 205 nm.

9. The method of claim 1, wherein, The retention time corresponding to the estradiol is 9.2-9.5 min; and / or, the retention time corresponding to the ethinyl estradiol is 11.1-11.4 min; and / or, the retention time corresponding to the estrone is 11.5-11.8 min.

10. The method of claim 1, wherein, The standard curve equation corresponding to the estradiol is y = 531.03x - 1.1265; and / or, the standard curve equation corresponding to the ethinyl estradiol is y = 438.26x + 1.2157; and / or, the standard curve equation corresponding to the estrone is y = 486.67x + 0.2313; wherein the ordinate y is the chromatographic peak area, and the abscissa x is the concentration (μg / mL).

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