CN111458442A - Method for measuring content of conjugated estrogen in pregnant mare urine - Google Patents
Method for measuring content of conjugated estrogen in pregnant mare urine Download PDFInfo
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Abstract
The invention relates to the technical field of chemical detection, and provides a method for detecting the content of conjugated estrogen in pregnant mare urine, wherein carbonate in the pregnant mare urine can form neutral salt, water and carbon dioxide gas when encountering inorganic acid, so that the column effect is improved, and the problems that the column pressure is increased, the column effect is reduced and the like when the content of the conjugated estrogen is detected by HP L C of the pregnant mare urine, 30 samples cannot be detected each time are solved, the accuracy of the content detection of the samples is ensured, and reliable data are provided for production.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to a method for determining the content of conjugated estrogens in pregnant mare urine.
Background
Conjugated estrogens are natural mixed estrogens extracted from pregnant mare's urine. The traditional Chinese medicine composition is mainly used for hormone replacement therapy in clinic and can relieve clinical symptoms caused by insufficient estrogen. The average content of the estrogen in the urine of each pregnant mare is 70-130 mg, and each pregnant mare can form 5-100 g of estrogen in the pregnancy period.
However, pregnant mare urine contains a large amount of soluble impurities, such as inorganic salts, urea, saccharides, flavone, isoflavone and the like, phenols and polyphenol components and the like, which are metabolites of pregnant mare, wherein phenols and polyphenols influence the detection separation degree of HP L C combined with estrogen components, and insoluble impurities, such as insoluble salts existing in urine, specifically precipitates generated in the existing process and deposits generated in the storage process, such as calcium carbonate, calcium bicarbonate, calcium phosphate, calcium hydrogen phosphate and the like, calcium carbonate, oxalate and the like, and the conventional treatment method comprises the following steps of 1 centrifugation method, dilution and sample injection after pregnant mare urine is centrifuged, wherein the method can only remove most of insoluble impurities aiming at mechanical impurities, 2 natural sedimentation method, filtering, diluting and sample injection of supernatant of settled clear pregnant mare urine, wherein only large mechanical impurities and part of other insoluble impurities can be removed, 3 organic dissolution sedimentation method, diluting and sample injection of pregnant mare urine with methanol, ethanol and acetone with a certain times of the insoluble impurities, filtering most of insoluble impurities and part of salts in urine, 4 filtration method, and the method can only remove most of insoluble impurities after the supernatant, and the pregnant mare urine with low HP separation rate, and can be directly detected.
Disclosure of Invention
The invention provides a method for measuring the content of conjugated estrogens in pregnant mare urine.
The invention provides a method for measuring the content of conjugated estrogens in pregnant mare urine, which comprises the following steps:
mixing pregnant mare urine and inorganic acid for pretreatment, and then adjusting the pH to 7.0-8.0 by using an alkaline substance to obtain a test solution;
performing high performance liquid chromatography detection on the test solution, determining the quality by retention time, and calculating by using a standard equation to obtain the content of the conjugated estrogens, wherein the standard equation is an equation with the concentration of the conjugated estrogens as an independent variable and a peak area as a dependent variable, and the conjugated estrogens comprise one or more of estrone sodium sulfate, equilenin sodium sulfate and 17 α -dihydroequilenin sodium sulfate;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: a C18 column;
the specification of the chromatographic column is 250mm × 4.6.6 mm, 5 μm;
the mobile phase comprises a phase A and a phase B, wherein the phase A is a mixed solution of sodium dihydrogen phosphate aqueous solution, acetonitrile and methanol, the concentration of the sodium dihydrogen phosphate aqueous solution in the phase A is 20 mmol/L, the pH value is 3.5, the volume ratio of the sodium dihydrogen phosphate aqueous solution in the phase A to the acetonitrile to the methanol is 17:2:1, the phase B is a mixed solution of disodium hydrogen phosphate aqueous solution and acetonitrile, the concentration of the disodium hydrogen phosphate aqueous solution in the phase B is 10 mmol/L, the pH value is 3.5, and the volume ratio of the disodium hydrogen phosphate aqueous solution to the acetonitrile in the phase B is 3: 7;
the mobile phase elution procedure was: the volume fraction of the phase A is gradually reduced from 70% to 67% in 0-18 min; for 18-23 min, gradually reducing the volume fraction of the phase A from 67% to 20%; the volume fraction of the phase A is gradually increased from 20% to 70% in 23-28 min; the volume fraction of the phase A is stabilized at 70 percent within 28-35 min;
the flow rate is 1.0m L/min;
column temperature: 40 ℃;
detection wavelength: 205 nm;
the sample injection amount is 20 mu L;
different conjugated estrogens peak at different retention times under the same chromatographic conditions.
Preferably, the inorganic acid is a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 0.1-3 mol/L.
Preferably, the volume ratio of the pregnant mare urine to the inorganic acid is 1: 1-20: 1.
Preferably, the alkaline substance is an NaOH aqueous solution, and the concentration of the NaOH aqueous solution is 0.1-3 mol/L.
Preferably, after the pH is adjusted to 7.0-8.0, filtering twice by using a 0.22 mu m filter membrane to obtain a subsequent filtrate as the sample solution.
Preferably, the pregnant mare urine is mixed with inorganic acid after being subjected to centrifugal treatment, the centrifugal treatment time is 10-30 min, and the rotating speed is 4000-6000 rpm.
Preferably, the determination of the standard equation comprises the steps of:
respectively preparing secondary standard substance solutions to be detected of 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate;
respectively carrying out high performance liquid chromatography detection on the solutions to be detected of the secondary standard substance to respectively obtain chromatograms of the secondary standard substance;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: a C18 column;
the specification of the chromatographic column is 250mm × 4.6.6 mm, 5 μm;
the mobile phase comprises a phase A and a phase B, wherein the phase A is a mixed solution of sodium dihydrogen phosphate aqueous solution, acetonitrile and methanol, the concentration of the sodium dihydrogen phosphate aqueous solution in the phase A is 20 mmol/L, the pH value is 3.5, the volume ratio of the sodium dihydrogen phosphate aqueous solution in the phase A to the acetonitrile to the methanol is 17:2:1, the phase B is a mixed solution of disodium hydrogen phosphate aqueous solution and acetonitrile, the concentration of the disodium hydrogen phosphate aqueous solution in the phase B is 10 mmol/L, the pH value is 3.5, and the volume ratio of the disodium hydrogen phosphate aqueous solution to the acetonitrile in the phase B is 3: 7;
the mobile phase elution procedure was: the volume fraction of the phase A is gradually reduced from 70% to 67% in 0-18 min; for 18-23 min, gradually reducing the volume fraction of the phase A from 67% to 20%; the volume fraction of the phase A is gradually increased from 20% to 70% in 23-28 min; the volume fraction of the phase A is stabilized at 70 percent within 28-35 min;
the flow rate is 1.0m L/min;
column temperature: 40 ℃;
detection wavelength: 205 nm;
the sample injection amount is 20 mu L;
according to the chromatogram of the secondary standard, respectively taking the concentrations of 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate as independent variables and peak areas as dependent variables to obtain a standard equation.
Preferably, the solvent of the solution to be detected of the secondary standard substance is a methanol-water mixed solution, the volume ratio of methanol to water in the mixed solution is 1:1, and the pH value of the mixed solution is 9.0.
Preferably, the pH of the mixture is adjusted to 9.0 with a Tris buffer.
Has the advantages that:
the invention provides a method for measuring the content of conjugated estrogen in pregnant mare urine, which comprises the following steps of mixing pregnant mare urine with inorganic acid for pretreatment, adjusting the pH to 7.0-8.0 by using an alkaline substance to obtain a test solution, carrying out high performance liquid chromatography detection on the test solution to obtain the content of the conjugated estrogen by using a standard equation, wherein the standard equation is an equation with the concentration of the conjugated estrogen as an independent variable and the peak area as a dependent variable, the conjugated estrogen comprises one or more of estrone sodium sulfate, equilenin sodium sulfate and 17 α -dihydroequilenin sodium sulfate, the combined estrogen in the pregnant mare urine is pretreated by using the inorganic acid, the pH value is adjusted by using an alkaline substance, so that a large amount of carbonate and oxalate contained in the pregnant mare urine can be removed, the detection efficiency is greatly improved, most of the conjugated estrogen in the pregnant mare urine is in a column state, the total amount of the conjugated estrogen in the pregnant mare urine can be measured by using an acid treatment method, the conjugated estrogen in the test result is released, the test result is stable, the combined estrogen in the test result, the result is stable, the result of the combined estrogen in the presence of a plurality of carbonate and the alkaline substance, the alkaline binding protein, the test result of the alkaline compound, the test protein is confirmed, the alkaline compound, the test protein is shown in the test protein, the test protein is shown in the test protein, the protein is shown in the test protein, the protein is shown in the protein, the test protein is shown in the protein, the protein is shown in the protein, the protein is shown in the test protein, the protein is shown in the protein, the protein is shown in the protein, the protein is shown in the protein.
Drawings
FIG. 1 is a standard curve of 17 a-dihydroequilenin sodium sulfate;
FIG. 2 is a standard curve of equilenin sodium sulfate;
FIG. 3 is a standard curve of sodium estrone sulfate;
FIG. 4 is a high performance liquid chromatogram of a test solution.
Detailed Description
The invention provides a method for measuring the content of conjugated estrogens in pregnant mare urine, which comprises the following steps:
mixing pregnant mare urine and inorganic acid for pretreatment, and then adjusting the pH to 7.0-8.0 by using an alkaline substance to obtain a test solution;
performing high performance liquid chromatography detection on the test solution, determining the quality by retention time, and calculating by using a standard equation to obtain the content of the conjugated estrogens, wherein the standard equation is an equation with the concentration of the conjugated estrogens as an independent variable and a peak area as a dependent variable, and the conjugated estrogens comprise one or more of estrone sodium sulfate, equilenin sodium sulfate and 17 α -dihydroequilenin sodium sulfate;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: a C18 column;
the specification of the chromatographic column is 250mm × 4.6.6 mm, 5 μm;
the mobile phase comprises a phase A and a phase B, wherein the phase A is a mixed solution of sodium dihydrogen phosphate aqueous solution, acetonitrile and methanol, the concentration of the sodium dihydrogen phosphate aqueous solution in the phase A is 20 mmol/L, the pH value is 3.5, the volume ratio of the sodium dihydrogen phosphate aqueous solution in the phase A to the acetonitrile to the methanol is 17:2:1, the phase B is a mixed solution of disodium hydrogen phosphate aqueous solution and acetonitrile, the concentration of the disodium hydrogen phosphate aqueous solution in the phase B is 10 mmol/L, the pH value is 3.5, and the volume ratio of the disodium hydrogen phosphate aqueous solution to the acetonitrile in the phase B is 3: 7;
the mobile phase elution procedure was: the volume fraction of the phase A is gradually reduced from 70% to 67% in 0-18 min; for 18-23 min, gradually reducing the volume fraction of the phase A from 67% to 20%; the volume fraction of the phase A is gradually increased from 20% to 70% in 23-28 min; the volume fraction of the phase A is stabilized at 70 percent within 28-35 min;
the flow rate is 1.0m L/min;
column temperature: 40 ℃;
detection wavelength: 205 nm;
the sample injection amount is 20 mu L;
different conjugated estrogens peak at different retention times under the same chromatographic conditions.
The method comprises the steps of mixing pregnant mare urine with inorganic acid for pretreatment, and then adjusting the pH to 7.0-8.0 by using an alkaline substance to obtain a test solution.
In the invention, the inorganic acid is preferably a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.1-3 mol/L, and more preferably 1 mol/L.
In the invention, the volume ratio of the pregnant mare urine to the inorganic acid is preferably 1: 1-20: 1, and more preferably 5: 1.
In the invention, the alkaline substance is preferably an NaOH aqueous solution, and the concentration of the NaOH aqueous solution is preferably 0.1-3 mol/L, and more preferably 1 mol/L.
In the invention, after the pH is adjusted to 7.0-8.0, the method preferably further comprises filtering twice by using a 0.22 μm filter membrane to obtain a subsequent filtrate as the sample solution.
In the invention, the pregnant mare urine is preferably subjected to centrifugal treatment and then mixed with inorganic acid, the centrifugal treatment time is preferably 10-30 min, and the rotation speed is preferably 4000-6000 rpm.
In the specific embodiment of the invention, preferably, 25m L pregnant mare urine is taken and placed in a 50m L centrifuge tube, after centrifugation is carried out for 10min (the rotation speed is 4000 rpm), 5.0m L pregnant mare urine is precisely absorbed and placed in a 100m L measuring flask, about 50m L of water is added, 1m L of 1 mol/L hydrochloric acid solution is added and then shaken rapidly until full reaction is achieved, the pH is adjusted to 7.0-8.0 by 1 mol/L NaOH solution, purified water is used for diluting to the scale, shaking is carried out uniformly, and after filtering twice by a 0.22 mu m filter membrane, filtrate is taken as sample solution.
After a test solution is obtained, performing high performance liquid chromatography detection on the test solution, determining the quality by retention time, and calculating by using a standard equation to obtain the content of conjugated estrogens, wherein the standard equation is an equation with the concentration of the conjugated estrogens as an independent variable and a peak area as a dependent variable, and the conjugated estrogens comprise one or more of estrone sodium sulfate, equilenin sodium sulfate and 17 α -dihydroequilenin sodium sulfate;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: a C18 column;
the specification of the chromatographic column is 250mm × 4.6.6 mm, 5 μm;
the mobile phase comprises a phase A and a phase B, wherein the phase A is a mixed solution of sodium dihydrogen phosphate aqueous solution, acetonitrile and methanol, the concentration of the sodium dihydrogen phosphate aqueous solution in the phase A is 20 mmol/L, the pH value is 3.5, the volume ratio of the sodium dihydrogen phosphate aqueous solution in the phase A to the acetonitrile to the methanol is 17:2:1, the phase B is a mixed solution of disodium hydrogen phosphate aqueous solution and acetonitrile, the concentration of the disodium hydrogen phosphate aqueous solution in the phase B is 10 mmol/L, the pH value is 3.5, and the volume ratio of the disodium hydrogen phosphate aqueous solution to the acetonitrile in the phase B is 3: 7;
the mobile phase elution procedure was: the volume fraction of the phase A is gradually reduced from 70% to 67% in 0-18 min; for 18-23 min, gradually reducing the volume fraction of the phase A from 67% to 20%; the volume fraction of the phase A is gradually increased from 20% to 70% in 23-28 min; the volume fraction of the phase A is stabilized at 70 percent within 28-35 min;
the flow rate is 1.0m L/min;
column temperature: 40 ℃;
detection wavelength: 205 nm;
the sample injection amount is 20 mu L;
different conjugated estrogens peak at different retention times under the same chromatographic conditions.
In the present invention, the determination of the standard equation preferably comprises the steps of:
respectively preparing secondary standard substance solutions to be detected of 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate;
respectively carrying out high performance liquid chromatography detection on the solutions to be detected of the secondary standard substance to respectively obtain chromatograms of the secondary standard substance;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: a C18 column;
the specification of the chromatographic column is 250mm × 4.6.6 mm, 5 μm;
the mobile phase comprises a phase A and a phase B, wherein the phase A is a mixed solution of sodium dihydrogen phosphate aqueous solution, acetonitrile and methanol, the concentration of the sodium dihydrogen phosphate aqueous solution in the phase A is 20 mmol/L, the pH value is 3.5, the volume ratio of the sodium dihydrogen phosphate aqueous solution in the phase A to the acetonitrile to the methanol is 17:2:1, the phase B is a mixed solution of disodium hydrogen phosphate aqueous solution and acetonitrile, the concentration of the disodium hydrogen phosphate aqueous solution in the phase B is 10 mmol/L, the pH value is 3.5, and the volume ratio of the disodium hydrogen phosphate aqueous solution to the acetonitrile in the phase B is 3: 7;
the mobile phase elution procedure was: the volume fraction of the phase A is gradually reduced from 70% to 67% in 0-18 min; for 18-23 min, gradually reducing the volume fraction of the phase A from 67% to 20%; the volume fraction of the phase A is gradually increased from 20% to 70% in 23-28 min; the volume fraction of the phase A is stabilized at 70 percent within 28-35 min;
the flow rate is 1.0m L/min;
column temperature: 40 ℃;
detection wavelength: 205 nm;
the sample injection amount is 20 mu L;
according to the chromatogram of the secondary standard, respectively taking the concentrations of 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate as independent variables and peak areas as dependent variables to obtain a standard equation.
In the invention, the solvents of the secondary standard substance solution to be tested are preferably methanol-water mixed solutions, the volume ratio of methanol to water in the mixed solutions is preferably 1:1, and the pH value of the mixed solutions is preferably 9.0.
In the present invention, the pH of the mixture is preferably adjusted to 9.0 with a Tris buffer.
In the specific embodiment of the invention, the secondary standard substances of 17 a-dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate are preferably taken respectively, precisely weighed and placed in a volumetric flask with the volume of 100m L respectively, diluted to the scale with methanol-water (1:1, V/V) (the pH is adjusted to 9.0 with Tris buffer solution), shaken to obtain control stock solutions with the concentration of about 17 a-dihydroequilenin sodium sulfate: 150 μ g/m L, 200 μ g/m L and 400 μ g/m L respectively, and the control stock solutions with the concentration of 2m L respectively are placed in a volumetric flask with the volume of 100m L respectively, diluted to the scale with methanol-water (1:1, V/V) (the pH is adjusted to 9.0 with Tris buffer solution), shaken to obtain control solutions with the concentration of 17 a-dihydroequilenin sodium sulfate: 3 μ g/m 63, equilenin sodium sulfate: 674 g/m, 868 μ g/m, and 868 μ g/m of estrone sodium sulfate.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
In the following examples, the test solutions were obtained from pregnant mare urine from different pastures and different pregnancy periods, and the contents of sodium 17 a-dihydroequilenin sulfate, sodium equilenin sulfate and sodium estrone sulfate in the pregnant mare urine from different pastures and different pregnancy periods were different.
Example 1
1 Experimental materials and instruments
(1) Reagent
Methanol (grade HP L C), acetonitrile (grade HP L C), purified water, sodium dihydrogen phosphate (analytically pure), disodium hydrogen phosphate (analytically pure), 1 mol/L hydrochloric acid solution, and 1 mol/L sodium hydroxide solution.
(2) Standard article
17 a-dihydroequilenin sodium sulfate, equilenin sodium sulfate standard and estrone sodium sulfate standard.
(3) Instrument for measuring the position of a moving object
High performance liquid chromatography (binary pump, autosampler, DAD detector), electronic balance (one hundred thousand), centrifuge (4000 rpm).
2 chromatographic conditions
Chromatographic column C18 column (250mm × 4.6.6 mm, 5 μm) mobile phase A of 20 mmol/L sodium dihydrogen phosphate, the pH value is adjusted to 3.5 by phosphoric acid, the sodium dihydrogen phosphate is acetonitrile, the methanol is 17:2:1, the mobile phase B of 10 mmol/L disodium hydrogen phosphate is adjusted to 6.5 by phosphoric acid, the disodium hydrogen phosphate is acetonitrile, the acetonitrile is 3:7, the elution procedure of the mobile phase is 0-18 min, 70% A-67% A, 18-23 min, 67% A-20% A, 23-28 min, 20% A-70% A, 28-35 min, 70% A-70% A, the flow rate is 1.0m L/min, the detection wavelength is 205nm, the sample injection amount is 20 μ L, the column temperature is 40 ℃, and the collection time is 35 min.
3 reference substance
Respectively taking secondary standard products of 17 a-dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate, precisely weighing, respectively placing in a volumetric flask with the volume of 100m L, diluting to a scale with methanol-water (1:1, V/V) (the pH is adjusted to be 9.0 by using Tris buffer solution), shaking up to obtain control stock solutions with the concentration of about 17 a-dihydroequilenin sodium sulfate: 150 mu g/m L, 200 mu g/m L for equilenin sodium sulfate, and 400 mu g/m L, respectively taking 2m L each control stock solution, placing in a volumetric flask with the volume of 100m L, diluting to a scale with methanol-water (1:1, V/V) (the pH is adjusted to be 9.0 by using the Tris buffer solution), shaking up to obtain control solutions with the concentration of 3 mu g/m sodium sulfate, 4 mu g/m sodium sulfate, 868 mu g/m for 17 a-dihydroequilenin estrone sodium sulfate, 6778 mu g/m.
4 preparation of test solutions
Placing 25m L pregnant mare urine (in a certain pastoral area) in a 50m L centrifuge tube, centrifuging for 10min (the rotation speed is 4000 rpm), precisely sucking 5.0m L pregnant mare urine, placing the pregnant mare urine in a 100m L measuring flask, adding water to about 50m L, adding 1m L of 1 mol/L hydrochloric acid solution, quickly shaking to fully react, adjusting the pH to 7.0-8.0 by using 1 mol/L NaOH solution, diluting with purified water to scale, shaking uniformly, filtering twice by using a 0.22 mu m filter membrane, and taking the subsequent filtrate as a test solution, wherein the test solution is two parallel samples.
5 measurement method
The separation degree of the estrone sodium sulfate, the equilin sodium sulfate and the 17 a-dihydroequilin sodium sulfate is not less than 2.0, the tailing factor calculated by the estrone sodium sulfate is not more than 1.3, and the theoretical plate number calculated by the estrone sodium sulfate is not less than 10000 by detecting through HP L C.
6 results of measurement
6.1 drawing of Standard Curve
Table 1 shows the preparation of control solutions of different concentrations.
TABLE 1 preparation of control solutions of different concentrations
Precisely sucking blank solution 1 needle and control solution at least 4 needles, and respectively performing 2 needles and 1 needle of control solution at each linear level for calibration. According to the above-mentioned chromatographic conditionsThe following measurements were carried out. In the results, the values of all the individual points should be reported; taking the peak area as a vertical coordinate and the concentration as a horizontal coordinate, and drawing a peak area-concentration curve of all the points; calculating a regression equation (x is concentration, and y is peak area); calculating a correlation coefficient R2Y-intercept, and regression curve slope. Coefficient of correlation R2Should not be less than 0.999; the y-axis intercept is within 2.0% of the 100% response value. (Y-intercept within 2.0% of the 100% response means that X is the concentration of the control solution specified by the standard, the resulting Y value, which should be less than 2.0%, is divided by the intercept divided by this Y value multiplied by 100%): the 3 components were calculated or plotted separately.
The standard curve of the obtained 17 a-dihydroequilenin sodium sulfate is shown in figure 1, and the standard curve equation is that y is 54858.7824x-138.9793, R21.0000; the standard curve of the equilenin sodium sulfate is shown in figure 2, and the standard curve equation is that y is 60422.1204x-30.7424, R21.0000; the standard curve of the estrone sodium sulfate is shown in figure 3, and the standard curve equation is that y is 66099.3479x +72.7473, R2The y-axis intercept at 100% response values were 0.1%, 0.02%, 0.01%, respectively, 1.0000.
6.2 results of detection
FIG. 4 is a high performance liquid chromatogram of a test solution.
The results of determining the amount of each conjugated estrogen component in the pregnant mare's urine according to the standard equation are shown in table 2.
TABLE 2 content of each conjugated Estrogen component in pregnant mare's urine
7 methodology examination
7.1 System applicability: the detection system can accurately detect the sample, and the data is accurate.
Acceptance criteria: the separation degree of the estrone sodium sulfate, the equilin sodium sulfate and the 17 a-dihydroequilin sodium sulfate is not less than 2.0; the tailing factor calculated by the sodium estrone sulfate is not more than 1.3; the theoretical plate number of the main peak is not less than 10000 calculated by sodium estrone sulfate. At least 4 of the control solutions should have a main peak RSD of not more than 3.0% by area of the sodium estrone sulfate.
The verification method comprises the following steps: (1) performing chromatographic sample injection detection according to a mobile phase blank (namely empty needle) 1 needle and at least 4 reference substance solutions respectively, and recording a chromatogram;
and (4) verification result:
TABLE 3 Agilent 1260(A) System applicability
7.2 accuracy
Acceptance criteria: the average recovery at each concentration should be between 90% and 110%; the RSD of the total recovery rate is not more than 15.0 percent, and the RSD of a sample with the same concentration is not more than 15.0 percent;
verification method
Each of the control solutions was prepared in 3 parts in parallel.
TABLE 4 preparation of solutions of different concentrations
Measurement of
(1) According to the mobile phase blank (namely empty needle) 1 needle, at least 4 needles of the reference solution, 50% concentration control, 100% concentration control and 200% concentration control, 3 needles of each reference solution with the same concentration are respectively arranged, the reference solution 1 is calibrated after every 10 needles of sample solution, and the solution is injected into a liquid chromatograph for measurement.
Measured value: c1/A1＝C2/A2
A1: the peak area of the reference sample is compared with the peak area,
C1: the actual concentration of the control substance,
A2: peak area of control at different concentrations
C2: actual concentration of different concentration reference substances
Recovery rate × 100% (theoretical value: stock solution concentration/dilution factor) measured value/theoretical value
As shown in tables 5 to 7, table 5 shows the results of verification of 17 α -dihydroequilenin sodium sulfate, table 6 shows the results of verification of equilenin sodium sulfate, and table 7 shows the results of verification of sodium estrone sulfate, and it is found that the recovery rates of 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate are all between 90.0% and 110.0%, and RSD values (n ═ 9) are 5.6%, 1.2% and 3.1%, respectively, and RSD values (n ═ 3) of 17 α -dihydroequilenin sodium sulfate under different concentration gradients (50%, 100% and 200%) are 0.3%, 0.9% and 1.0%, respectively, and RSD values (n ═ 3) of equilenin sodium sulfate are 0.3%, 1.6% and 0.5%, respectively, and RSD values (n ═ 3) of sodium estrone sulfate are 0.6%, 1.6% and 1.0% and less than 15.0%, respectively.
TABLE 5 accuracy verification results
TABLE 6 verification of equilenin sodium sulfate
TABLE 7 verification of estrone sodium sulfate
7.3 precision
Acceptance criteria: repeatability criteria: the RSD value of the content of 6 parts of parallel test sample solution (pregnant horse urine collected from a certain pasturing area) is not more than 15.0 percent,
intermediate precision standard: RSD should not be greater than 15.0%; RSD between different instruments and different testers at different time is not more than 15.0%.
Table 8 shows the results of precision.
TABLE 8 precision results
7.4 repeatability
A is a repetitive test solution.
B, respectively preparing 6 parts of test solution in parallel for the test
Respectively performing parallel sampling on 1 needle of the mobile phase blank (namely an empty needle), at least 4 needles of the reference substance solution, 6 parts of the test substance solution (pregnant mare urine collected from a certain pastoral area) and 1 needle of the reference substance solution, checking the 1 needle of the reference substance solution, injecting the check solution into a liquid chromatograph, and recording the chromatogram.
D, testing the result of the test,
table 9 shows the repeatability, and it can be seen from Table 9 that the RSD value between 6 parallel samples in the repeatability is 1.4% and less than 15.0%.
TABLE 9 repeatability results
7.4 detection and quantitation limits
Detection limit
a) And (3) taking a blank atlas for continuous sample injection for 3 times, recording the baseline noise level in the range of the main peak-off time of the working reference substance, and calculating the average noise.
b) Accurately preparing working standard stock solution, gradually diluting to a certain concentration and injecting. The ratio of the peak height to the average noise is calculated, and the signal-to-noise ratio is 3 +/-1: 1.
Quantitative limit (L OQ)
a) And (5) blank continuous sample introduction is carried out for 3 times, the baseline noise level in the peak emergence time range of the main peak of the reference substance is recorded, and the average noise is calculated.
b) And (3) accurately preparing a standard stock solution, gradually diluting to a certain concentration and injecting. The ratio of the peak height to the average noise is calculated, and the signal-to-noise ratio is 10 +/-2: 1.
And (4) conclusion:
l OD 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate of the Shimadzu 2030 are detected to have the concentrations of 0.0026 mu g/m L, 0.0035 mu g/m L and 0.0042 mu g/m L when the signal-to-noise ratios are 11, 11 and 12 respectively, and L OQ 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate of the Shimadzu 2030 are detected to have the concentrations of 0.0080 mu g/m L, 0.0086 mu g/m L and 0.0083 mu g/m L when the signal-to-noise ratios are 36, 36 and 33 respectively.
7.5 stability test
Although the pregnant mare urine acid-base treatment method effectively solves the problems of increased pressure of a chromatographic column, reduced column effect and the like, the stability investigation test of the determination result of the pregnant mare urine acid-base treatment method is carried out in consideration of the problem that the treatment result of the same method of the same sample is lack of repeatability reflected in the actual sample treatment process.
Test method
5 batches of pregnant mare urine samples are respectively taken 50m L, centrifuged for 10min at 4000r/min, supernatant liquid is taken and put in a clean centrifugal tube, and the samples are stored in a refrigerator to prevent the content from being reduced.
Sample treatment, each batch of pregnant mare urine is taken 2.5M L and placed in a 50M L volumetric flask, 0.5M L diluted hydrochloric acid is added to be shaken up (bubbles formed are eliminated), 0.5M L2M sodium hydroxide solution is rapidly added to be shaken up, water is added to be diluted to a scale mark, filtration and sample injection are carried out, and the sample number is X-1.
Pregnant horse urine dilution direct determination
Each batch of pregnant mare urine (collected from a certain pastoral area) is taken 2.5m L and placed in a 50m L volumetric flask, water is added to dilute to the scale, filtration and sample injection are carried out, and the sample is numbered X-2.
Two replicates of each batch were processed and the degree of deviation between the measurements of each replicate was calculated.
The results of the sample treatment on day 0 and the related statistics are shown in Table 10. from Table 10, it can be seen that the differences of the contents of the various replicates are small, the RSD value of 17 α -dihydroequilenin sodium sulfate is 2.64, the RSD value of equilenin sodium sulfate is 3.92, and the RSD value of the equilenin sodium sulfate is 2.36.
The method shows that the contents of 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate in the pregnant mare urine are measured by an acid-base treatment method, and the method has good repeatability.
TABLE 10 day 0 sample treatment assay results
Day 1 sample treatment
Sample treatment: two samples of each batch were processed by two different methods
Acid-base treatment
Each batch of pregnant mare urine is taken 2.5M L and placed in a 50M L volumetric flask, 0.5M L diluted hydrochloric acid is added to be shaken up (bubbles formed are eliminated), 0.5M L2M sodium hydroxide solution is rapidly added to be shaken up, water is added to be diluted to a scale mark, filtration and sample injection are carried out, and the sample number is X-1.
Pregnant horse urine dilution direct determination
Each batch of pregnant mare urine is taken 2.5m L and placed in a 50m L volumetric flask, water is added to dilute the pregnant mare urine to a scale, the pregnant mare urine is filtered and injected, and the sample is numbered X-2.
The results of the sample treatment and the related statistics on the day 1 are shown in the table 11. the conclusion is that the measurement result on the day 1 is higher than that on the day 0 due to the HP L C column, the samples treated by the two treatment methods are simultaneously measured after being stored in a refrigerator for 7 days, and the result shows that the difference of the measurement results of the two treatment methods is within an allowable range.
The content difference of samples treated by different methods is small, and the RSD value of 17 α -dihydroequilenin sodium sulfate is 2.60, the RSD value of equilenin sodium sulfate is 4.63, and the RSD value of the equilenin sodium sulfate is 5.79.
The result shows that the treatment method of the pregnant mare uric acid alkali is consistent with the result of the direct sample injection measurement of water dilution, the treatment result of the acid-alkali treatment sample is not parallel and the possibility of reducing the content of the conjugated estrogen is caused.
TABLE 11 day 1 sample treatment assay results
Day 11 sample treatment
Sample treatment: two samples of each batch were processed by two different methods
Acid-base treatment
Each batch of pregnant mare urine is taken 2.5M L and placed in a 50M L volumetric flask, 0.5M L diluted hydrochloric acid is added to be shaken up (bubbles formed are eliminated), 0.5M L2M sodium hydroxide solution is rapidly added to be shaken up, water is added to be diluted to a scale mark, filtration and sample injection are carried out, and the sample number is X-1.
Pregnant horse urine dilution direct determination
Each batch of pregnant mare urine is taken 2.5m L and placed in a 50m L volumetric flask, water is added to dilute the pregnant mare urine to a scale, the pregnant mare urine is filtered and injected, and the sample is numbered X-2.
The results of the sample treatment on the day 11 and the related statistical results are shown in Table 12. the conclusion is that the sample treated on the day two is not treated on time due to the HP L C column, the sample is uniformly stored in a refrigerator, and the sample is treated and calculated from the treatment of 0 day to the treatment of 11 days and is measured, so the content of the conjugated estrogen component in the original pregnant mare urine is reduced, so the measurement result is greatly different from the measurement result of 0 day, the content of the mare urine is reduced by about half of the original content, the content of the parallel measurement samples is greatly different due to the lower content of the sample, particularly, the RSD value of 17 α -dihydroequilenin content reaches 13.1, but the content of other two components is not greatly different between each parallel sample, and the RSD value is about 7.
TABLE 12 day 11 sample treatment assay results
From tables 10 to 12, it can be known that, before the determination of the pregnant mare urine HP L C, a large amount of carbonate contained in the pregnant mare urine can be removed after the pregnant mare urine sample is treated by acid and alkali, so that the detection efficiency is greatly improved, the problems that 30 samples are not determined each time, the column pressure is increased, the column efficiency is reduced and the like in the determination of the content of the conjugated estrogens in the pregnant mare urine HP L C are solved, the stability of the determination result of the method is preliminarily considered, the influence on the content of the conjugated estrogens in the pregnant mare urine is influenced, and the like are preliminarily considered, the result shows that the determination result after the treatment of the method is highly consistent with the determination result after the pregnant mare urine is diluted by water (the dilution times of the two methods are the same), the unstable determination result does not exist, and the peak shape of the sample HP L C treated by the acid and alkali treatment method is better than the peak shape of the water dilution, the interference condition of the impurity peak is reduced, so that the method can be used as a first-choice method for determining the components of the pregnant mare urine in HP L.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method for measuring the content of conjugated estrogens in pregnant mare urine is characterized by comprising the following steps:
mixing pregnant mare urine and inorganic acid for pretreatment, and then adjusting the pH to 7.0-8.0 by using an alkaline substance to obtain a test solution;
performing high performance liquid chromatography detection on the test solution, determining the quality by retention time, and calculating by using a standard equation to obtain the content of the conjugated estrogens, wherein the standard equation is an equation with the concentration of the conjugated estrogens as an independent variable and a peak area as a dependent variable, and the conjugated estrogens comprise one or more of estrone sodium sulfate, equilenin sodium sulfate and 17 α -dihydroequilenin sodium sulfate;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: a C18 column;
the specification of the chromatographic column is 250mm × 4.6.6 mm, 5 μm;
the mobile phase comprises a phase A and a phase B, wherein the phase A is a mixed solution of sodium dihydrogen phosphate aqueous solution, acetonitrile and methanol, the concentration of the sodium dihydrogen phosphate aqueous solution in the phase A is 20 mmol/L, the pH value is 3.5, the volume ratio of the sodium dihydrogen phosphate aqueous solution in the phase A to the acetonitrile to the methanol is 17:2:1, the phase B is a mixed solution of disodium hydrogen phosphate aqueous solution and acetonitrile, the concentration of the disodium hydrogen phosphate aqueous solution in the phase B is 10 mmol/L, the pH value is 3.5, and the volume ratio of the disodium hydrogen phosphate aqueous solution to the acetonitrile in the phase B is 3: 7;
the mobile phase elution procedure was: the volume fraction of the phase A is gradually reduced from 70% to 67% in 0-18 min; for 18-23 min, gradually reducing the volume fraction of the phase A from 67% to 20%; the volume fraction of the phase A is gradually increased from 20% to 70% in 23-28 min; the volume fraction of the phase A is stabilized at 70 percent within 28-35 min;
the flow rate is 1.0m L/min;
column temperature: 40 ℃;
detection wavelength: 205 nm;
the sample injection amount is 20 mu L;
different conjugated estrogens peak at different retention times under the same chromatographic conditions.
2. The method according to claim 1, wherein the inorganic acid is a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 0.1 to 3 mol/L.
3. The method according to claim 2, wherein the volume ratio of the pregnant mare urine to the inorganic acid is 1:1 to 20: 1.
4. The method according to claim 1, wherein the alkaline substance is an aqueous NaOH solution, and the concentration of the aqueous NaOH solution is 0.1 to 3 mol/L.
5. The method according to claim 1, wherein the adjusting the pH to 7.0 to 8.0 further comprises filtering twice with a 0.22 μm filter membrane, and collecting a subsequent filtrate as the sample solution.
6. The method according to claim 1, wherein the pregnant mare urine is centrifuged and then mixed with an inorganic acid, wherein the time for centrifugation is 10 to 30min, and the rotation speed is 4000 to 6000 rpm.
7. The assay of claim 1, wherein the assay of the standard equation comprises the steps of:
respectively preparing secondary standard substance solutions to be detected of 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate;
respectively carrying out high performance liquid chromatography detection on the solutions to be detected of the secondary standard substance to respectively obtain chromatograms of the secondary standard substance;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: a C18 column;
the specification of the chromatographic column is 250mm × 4.6.6 mm, 5 μm;
the mobile phase comprises a phase A and a phase B, wherein the phase A is a mixed solution of sodium dihydrogen phosphate aqueous solution, acetonitrile and methanol, the concentration of the sodium dihydrogen phosphate aqueous solution in the phase A is 20 mmol/L, the pH value is 3.5, the volume ratio of the sodium dihydrogen phosphate aqueous solution in the phase A to the acetonitrile to the methanol is 17:2:1, the phase B is a mixed solution of disodium hydrogen phosphate aqueous solution and acetonitrile, the concentration of the disodium hydrogen phosphate aqueous solution in the phase B is 10 mmol/L, the pH value is 3.5, and the volume ratio of the disodium hydrogen phosphate aqueous solution to the acetonitrile in the phase B is 3: 7;
the mobile phase elution procedure was: the volume fraction of the phase A is gradually reduced from 70% to 67% in 0-18 min; for 18-23 min, gradually reducing the volume fraction of the phase A from 67% to 20%; the volume fraction of the phase A is gradually increased from 20% to 70% in 23-28 min; the volume fraction of the phase A is stabilized at 70 percent within 28-35 min;
the flow rate is 1.0m L/min;
column temperature: 40 ℃;
detection wavelength: 205 nm;
the sample injection amount is 20 mu L;
according to the chromatogram of the secondary standard, respectively taking the concentrations of 17 α -dihydroequilenin sodium sulfate, equilenin sodium sulfate and estrone sodium sulfate as independent variables and peak areas as dependent variables to obtain a standard equation.
8. The method according to claim 7, wherein the solvent of the secondary standard solution to be tested is a methanol-water mixture, the volume ratio of methanol to water in the mixture is 1:1, and the pH value of the mixture is 9.0.
9. The method according to claim 8, wherein the pH of the mixture is adjusted to 9.0 with a Tris buffer.
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