CN116338031A - Method for detecting pollen sex hormone - Google Patents
Method for detecting pollen sex hormone Download PDFInfo
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- CN116338031A CN116338031A CN202310095579.3A CN202310095579A CN116338031A CN 116338031 A CN116338031 A CN 116338031A CN 202310095579 A CN202310095579 A CN 202310095579A CN 116338031 A CN116338031 A CN 116338031A
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- acetonitrile
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- pollen
- solid phase
- sex hormone
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/06—Preparation
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Abstract
The invention discloses a method for detecting pollen sex hormone, which specifically comprises the following steps: (1) extraction of sample neutral hormone; (2) Purifying the extract (degreasing, washing, solid phase extraction); (3) constant volume; (4) detecting sex hormone by UPLC-MS/MS; (5) And (5) establishing a standard curve, and calculating the content of the neutral hormone in the sample. The invention establishes a method for simultaneously measuring 8 sex hormones (beta-estradiol, alpha-estradiol, estrone, testosterone, androstenedione, epitestosterone, androsterone and progesterone) in pollen, has high sensitivity and accuracy, can be applied to the actual inspection work of pollen or pollen foods, and provides a reference for the sex hormone detection of other plant materials.
Description
Technical Field
The invention relates to a detection method of pollen sex hormone, belonging to the fields of analytical chemistry and food safety.
Background
Sex hormones in living bodies exist in two forms, namely a free form and a bound form, and the bound sex hormone is mainly two, namely, glucuronic acid and sulfuric acid conjugate conjugates, and an enzymatic hydrolysis method and a chemical hydrolysis method are generally adopted for the release of the bound sex hormone. In view of the possible presence of sex hormones in free and/or bound form in pollen, extraction methods are critical in order to ensure the accuracy of their sex hormone detection.
The pollen neutral hormone content is extremely low, the matrix is complex, and the interference factors are large, so that the pollen sex hormone is difficult to measure, and therefore, the adoption of a proper impurity removal mode is very important for reducing the matrix effect of a sample and improving the sensitivity of a target object.
The sex hormone detection method comprises the following steps: immunoassay, high Performance Liquid Chromatography (HPLC), gas chromatography-tandem mass spectrometry (GC-MS/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). False positive results are easy to occur in an immunoassay method, and the detected hormone is limited; the ultraviolet detector of the HPLC is limited by sensitivity and accuracy, and is gradually replaced by a mass spectrum detector; GC-MS/MS requires that the analyte have a certain thermal stability and volatility, and that no derivatizing agent has been available to better derivatize all sex hormones; LC-MS/MS does not need to be derived, has high sensitivity, and can realize high-flux rapid detection.
In summary, it is necessary to optimize the method for detecting pollen sex hormone from aspects of sample extraction, purification, detection, etc., in order to improve accuracy and sensitivity of pollen sex hormone detection.
Disclosure of Invention
The invention aims to provide a method for detecting pollen sex hormone, which has the characteristics of good repeatability, high accuracy and high sensitivity.
The method for detecting pollen sex hormone provided by the invention comprises the following steps:
(1) Extracting: adding an internal standard into a sample to be detected, then adding acetonitrile for extraction, and collecting supernatant; purifying the supernatant, the purifying comprising the steps of: degreasing, washing with water and solid phase extraction, degreasing and washing with water, or degreasing and solid phase extraction:
(2a) Degreasing: adding normal hexane into the supernatant for extraction, and collecting acetonitrile phase;
(2b) Washing: removing the solvent of the acetonitrile phase, dissolving by adopting an organic reagent, washing by using brine, collecting and removing the organic phase reagent, adding a test agent to redissolve a sample, and purifying;
(2c) Solid phase extraction: activating and balancing the solid phase extraction column, adding a sample, eluting by using an eluent, and finally eluting and collecting a target object by using acetonitrile;
(3) Constant volume: adding a reagent to dissolve the target object and fixing the volume;
(4) And (3) detection: detecting sex hormone in the sample in the step (3) by using a UPLC-MS/MS method;
(5) Establishing a standard curve: preparing a sex hormone standard solution containing an internal standard, carrying out UPLC-MS/MS detection, taking the sex hormone concentration as an abscissa, taking the ratio of the detected sex hormone peak area to the internal standard peak area as an ordinate, drawing an internal standard curve, and calculating the content of neutral hormone in a sample to be detected according to the obtained internal standard curve.
Preferably, the ratio of the sample to acetonitrile in step (1) is 1: 5-20 g/mL.
Preferably, the volume ratio of acetonitrile to n-hexane in step (1) is 1-2: 1-2 mL/mL.
Preferably, the organic reagent in step (2 b) is ethyl acetate or and acetonitrile;
the brine is sodium carbonate aqueous solution or sodium chloride aqueous solution;
the volume ratio of the organic reagent to the saline is 1-5: 1-3 mL/mL;
the times of water washing are 2-4 times;
the reagent for dissolving the sample before purification is 5-40% methanol aqueous solution or 5-40% acetonitrile aqueous solution.
Preferably, the solid phase extraction column in step (2 c) is an HLB solid phase extraction column;
the eluent comprises water and organic eluent, wherein the organic eluent is 5% -40% of methanol aqueous solution or 5% -40% of acetonitrile aqueous solution.
Preferably, the constant volume reagent in the step (3) is a 50% -100% acetonitrile aqueous solution.
Preferably, in the steps (4) and (5), the UPLC-MS/MS analysis conditions are:
chromatographic conditions: acquisity (a Chinese character)
HSS T3 column (2.1 mm. Times.100 mm,1.8 μm); sample injection amount is 10uL; column temperature is 35 ℃; mobile phase A is 0.2% ammonia water, and B is acetonitrile; the flow rate was 0.38mL/min. Gradient elution: 0min,25% b; 0-6 min,25% -50% B; 6-10 min,50% -80% B; 10-11 min, 80-100% B; 11-13 min, 100-100% B; 13-14 min, 100-25% B; 14-16 min,25% -25% of B.
Mass spectrometry conditions: waters Xevo TQ mass spectrometry system, electrospray ion source (ESI), positive and negative ion scanning mode, multi-reaction monitoring (MRM) mode, ion source temperature of 150 ℃, capillary voltage of 2.7kV, desolventizing gas temperature of 500 ℃, desolventizing gas flow rate of 1000L/h, and cone hole gas flow rate of 50L/h.
Preferably, the sample is pollen or pollen food; the sex hormone comprises at least one of alpha-estradiol, beta-estradiol, estrone, testosterone, androstenedione, epididymis ketone, androsterone and progesterone.
The invention has the following beneficial technical effects:
the method for simultaneously measuring 8 sex hormones including estrogen (beta-estradiol, alpha-estradiol and estrone), androgen (testosterone, androstenedione, epitestosterone and androsterone) and progestin (progesterone) in pollen is established through optimizing sample extraction, purification and detection conditions, has high sensitivity and accuracy, can be applied to the actual detection work of pollen or pollen foods, and provides reference for the sex hormone detection of other plant materials.
Drawings
FIG. 1 is an MRM chromatogram of 8 sex hormones and 2 internal standards.
FIG. 2 is a flow chart of the method for detecting pollen sex hormone of the present invention.
FIG. 3 is an MRM chromatogram of 8 sex hormones and 2 internal standards in 'Paeonia ostii' pollen.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the attached drawings and the detailed description.
The reagents and instrumentation used in the following examples were as follows:
reagent: sodium hydroxide (the company of the chemical company of the ridge); acetonitrile, methanol, n-hexane (chromatographic grade, fisher); ethanol (Tianjin, metallocene chemical reagent plant); hydrochloric acid (national pharmaceutical group chemical reagent limited); ethyl acetate, magnesium sulfate (Shanghai Ala Biochemical technologies Co., ltd.); sodium chloride (schwann science); sodium acetate (Tianjin Fuchen chemical reagent factory); acetic acid and sodium carbonate (beijing chemical plant); beta-glucuronidase/arylsulfatase (Merck, germany); progesterone, beta-estradiol, alpha-estradiol (Shanghai derived leaf biotechnology limited); testosterone, androsterone (beijing wakame biotechnology limited); estrone, androstenedione (dr. Ehrentorfer, germany); epitestosterone, progesterone-D 9 (Tianjin Alta technologies Co., ltd.); estradiol-D 4 (MedChemExpress, USA); all standards had a purity of greater than 98%.
Instrument: ACQUITY TM Ultra-high performance liquid chromatography and Waters Xevo TM tQ MS triple quadrupole tandem mass spectrometer (UPLC-MS/MS, waters, USA); milli-Q ultra-pure water machine (Milibo Co., U.S.A.); AL104 analytical balance (mertrehler, germany); DK-8D electric heating constant temperature water tank (Shanghai-Hengsu science instruments Co., ltd.); vortex oscillator of Vortex-Genie2Us Scientific Industries); HN200 nitrogen blowing instrument (China sea energy); LD4-1.8 low speed centrifuge (Beijing genipin); THZ-C constant temperature oscillator (Taiku city experimental facility factory); type 3K30 high-speed cryocentrifuge (Sigma, germany); KQ-500DE type digital controlled ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); CNW Poly-Sery HLB Pro solid phase extraction column (6 mL/200mg, shanghai An Spectrometry laboratory technologies Co., ltd.).
Sample: the samples used in the following examples were 'Paeonia ostii' pollen: sampling anther before ten am, naturally ventilating and drying at room temperature (20-25 ℃), collecting pollen, and drying in a vacuum drying oven for 3 days. Before sample analysis, the sample is frozen in a refrigerator at the temperature of minus 80 ℃ for 24 hours, taken out and quickly put in a water bath kettle at the temperature of 95 ℃ for water bath for 10 minutes to carry out temperature difference wall breaking.
Example 1 detection of pollen neutral hormone
1. Sample pretreatment
1. The method comprises the following steps: degreasing, ethyl acetate washing, solid phase extraction and constant volume of 200 mu L acetonitrile
(1) Extracting: weighing 5g of broken pollen, placing into a centrifuge tube with a plug, accurately adding internal standard estradiol-D 4 100. Mu.L (33.33 ng/mL) and progesterone-D 9 100. Mu.L (2.22 ng/mL) and then 50mL acetonitrile were added, vortexed for 1min, and sonicated for 20min. Centrifuging at 8000r/min for 5min, and transferring the supernatant to a separating funnel. The residue was extracted with 50mL of acetonitrile and repeated 1 more times, and the two supernatants were combined into a separating funnel.
(2) Degreasing: 40mL of n-hexane was added to the separating funnel, and after separation by extraction, 40mL of n-hexane was added to the acetonitrile layer, and extraction was repeated 1 time. After standing and layering, transferring the acetonitrile at the lower layer into a rotary steaming bottle for multiple times, and rotary steaming at 35 ℃.
(3) Washing: the sample in the spin-flask was transferred to a 15mL centrifuge tube with 9mL ethyl acetate, 3mL5% aqueous sodium carbonate solution was added to vortex 20s,4000r/min was centrifuged for 5min, the aqueous phase was removed, and 3mL5% aqueous sodium carbonate solution was added to repeat the above operation 1 time. The upper organic phase was transferred to a new centrifuge tube and dried with nitrogen at 40 ℃. 10mL of a 10% aqueous acetonitrile solution was added to the mixture to be purified.
(4) Solid phase extraction: the HLB solid phase extraction column was previously activated and equilibrated with 5mL of methanol, 5mL of acetonitrile, 7mL of water at natural flow rate. Then, the sample solution was dropped at a natural flow rate, and the leacheate was eluted sequentially with 5mL of water and 10mL of 10% aqueous methanol, respectively. And pumping the liquid remained on the HLB extraction column by using a vacuum pump, eluting the target object by using 15mL of acetonitrile at a natural flow rate, collecting the eluent, and drying by using nitrogen at 40 ℃.
(5) Constant volume: accurately adding 2mL of acetonitrile to dissolve a sample, centrifuging for 10min at 8000r/min, accurately sucking 1.8mL of solution into a 2mL centrifuge tube, blowing the solution to dry by nitrogen, adding 200 mu L of acetonitrile, centrifuging for 10min at 12000r/min, and loading to be detected.
2. The second method is as follows: solid phase extraction +600mu.L acetonitrile constant volume
(1) Extracting: weighing 5g of broken pollen, placing into a centrifuge tube with a plug, accurately adding internal standard estradiol-D 4 300. Mu.L (33.33 ng/mL) and progesterone-D 9 300. Mu.L (2.22 ng/mL) and then 50mL acetonitrile was added, vortexed for 1min, and sonicated for 20min. Centrifuging at 8000r/min for 5min, transferring the supernatant to a rotary steaming bottle, extracting the residue with 50mL acetonitrile for 1 time, mixing the two supernatants, and drying at 35deg.C. 10mL of a 10% aqueous acetonitrile solution was added to the mixture to be purified.
(2) Solid phase extraction: and (3) solid phase extraction in the same method as in (4).
(3) Constant volume: accurately adding 2mL of acetonitrile to dissolve a sample, centrifuging for 10min at 8000r/min, accurately sucking 1.8mL of solution into a 2mL centrifuge tube, drying by nitrogen, adding 600 mu L of acetonitrile, centrifuging for 10min at 12000r/min, and loading to be detected.
3. And a third method: degreasing, solid phase extraction and 500 mu L acetonitrile constant volume
(1) Extracting: except for "Add internal standard estradiol-D 4 250. Mu.L (33.33 ng/mL) and progesterone-D 9 250. Mu.L (2.22 ng/mL) "is different from the extraction in method one (1), and the rest steps are the same as the extraction in method one (1).
(2) Degreasing: degreasing in the same method as in (2).
(3) Washing: the sample in the spin-flask was transferred to a 10mL centrifuge tube with 9mL ethyl acetate and dried with nitrogen at 40 ℃. 10mL of a 10% aqueous acetonitrile solution was added to the mixture to be purified.
(4) Solid phase extraction: and (3) solid phase extraction in the same method as in (4).
(5) Constant volume: accurately adding 2mL of acetonitrile to dissolve a sample, centrifuging at 8000r/min for 10min, accurately sucking 1.8mL of solution into a 2mL centrifuge tube, blowing the solution to dry by nitrogen, adding 500 mu L of acetonitrile, centrifuging at 12000r/min for 10min, and loading to be detected.
4. The method four: degreasing, ethyl acetate washing and 600 mu L acetonitrile constant volume
(1) Extracting: except for "Add internal standard estradiol-D 4 300. Mu.L (33.33 ng/mL) and progesterone-D 9 300. Mu.L (2.22 ng/mL) "is different from the extraction in method one (1), and the rest steps are the same as the extraction in method one (1).
(2) Degreasing: degreasing in the same method as in (2).
(3) Washing: the sample in the spin-flask was transferred to a 15mL centrifuge tube with 9mL ethyl acetate, 3mL5% aqueous sodium carbonate solution was added to vortex 20s,4000r/min was centrifuged for 5min, the aqueous phase was removed, and 3mL5% aqueous sodium carbonate solution was added to repeat the above operation 3 times. The upper organic phase was transferred to a new centrifuge tube and dried with nitrogen at 40 ℃.
(4) Constant volume: and (3) constant volume in the second method.
5. And a fifth method: degreasing, acetonitrile washing, solid phase extraction and constant volume of 200 mu L acetonitrile
(1) Extracting: extracting in the same way as in the first step (1).
(2) Degreasing: degreasing in the same method as in (2).
(3) Washing: the sample in the spin-flask was transferred to a 15mL centrifuge tube with 9mL acetonitrile, 3mL of saturated aqueous sodium chloride solution was added to the tube and vortexed for 20s, centrifuged at 4000r/min for 5min, the aqueous phase was removed, and 3mL of saturated aqueous sodium chloride solution was added to repeat the above operation 1 time. The upper organic phase was transferred to a new centrifuge tube and dried with nitrogen at 40 ℃. 10mL of a 10% aqueous acetonitrile solution was added to the mixture to be purified.
(4) Solid phase extraction: and (3) solid phase extraction in the same method as in (4).
(5) Constant volume: and (5) constant volume in the same method.
6. The method six: enzymolysis, degreasing, solid phase extraction and 600 mu L of 50% acetonitrile constant volume
(1) Extracting: weighing 5g of broken pollen, placing in triangular flask with stopper, adding sodium acetate buffer (9.5 g of sodium acetate with waterDiluting to 250mL, regulating pH to 5.2) with acetic acid, mixing with 40mL vortex, adding 200 μL of beta-glucuronidase/arylsulfatase, performing enzymolysis at 37deg.C with a constant temperature shaker at 110r/min for 12h, taking out, and cooling to room temperature. Accurate addition of internal standard estradiol-D 4 300. Mu.L (33.33 ng/mL) and progesterone-D 9 300. Mu.L (2.22 ng/mL) and then 50mL acetonitrile was added, vortexed for 1min, and sonicated for 20min. Centrifuging at 8000r/min for 5min, and transferring the supernatant to a separating funnel. The residue was taken up in 50mL of acetonitrile and extracted 1 more times and the two supernatants were combined in a separating funnel. To the separating funnel was added 20g total of sodium chloride and magnesium sulfate (1:1 g/g), and the separating funnel was shaken thoroughly to separate the organic phase and the aqueous phase thoroughly, leaving the organic phase.
(2) Degreasing: degreasing in the same method as in (2).
(3) Washing: and (3) washing with water in the same method.
(4) Solid phase extraction: and (3) solid phase extraction in the same method as in (4).
(5) Constant volume: accurately adding 2mL of acetonitrile to dissolve a sample, centrifuging at 8000r/min for 10min, accurately sucking 1.8mL of the solution into a 2mL centrifuge tube, blowing the solution to dry by nitrogen, adding 600 mu L of 50% acetonitrile aqueous solution, centrifuging at 12000r/min for 10min, and loading to be detected.
7. And a seventh method: enzymolysis, degreasing, ethyl acetate washing, solid phase extraction and 600 mu L of 50% acetonitrile constant volume
(1) Extracting: and (3) extracting in the same method as in the step (1).
(2) Degreasing: degreasing in the same method as in (2).
(3) Washing: ethyl acetate: 5% sodium carbonate aqueous solution (1:1, v:v) 18mL samples from the spin flask were transferred to a centrifuge tube, centrifuged at 4000r/min for 5min and the aqueous phase removed. 3mL of 5% sodium carbonate aqueous solution was added and vortexed for 20s, centrifuged at 4000r/min for 5min, the aqueous phase was removed, and 3mL of 5% sodium carbonate aqueous solution was added and the above procedure was repeated 2 times. The upper organic phase was transferred to a new centrifuge tube and dried with nitrogen at 40 ℃. 10mL of a 10% aqueous acetonitrile solution was added to the mixture to be purified.
(4) Solid phase extraction: and (3) solid phase extraction in the same method as in (4).
(5) Constant volume: and (5) constant volume in the same method.
8. Method eight: alkali hydrolysis, degreasing, solid phase extraction and 600 mu L acetonitrile constant volume
(1) Extracting: weighing 5g of broken pollen, placing into a centrifuge tube with a plug, accurately adding internal standard estradiol-D 4 300. Mu.L (33.33 ng/mL) and progesterone-D 9 300. Mu.L (2.22 ng/mL) was then added with 80mL of a 2moL/L sodium hydroxide-ethanol solution, saponified in a water bath at 80℃for 60min, and cooled to room temperature. Centrifuging at 8000r/min for 5min, sucking supernatant, regulating pH value to 6.5-7.5 with hydrochloric acid, centrifuging at 8000r/min for 10min, transferring supernatant to rotary steaming bottle, and rotary steaming at 35deg.C. The sample in the spin-flask was sonicated with 100mL acetonitrile and the solution was transferred to a separatory funnel.
(2) Degreasing: degreasing in the same method as in (2).
(3) Washing: and (3) washing with water in the same method.
(4) Solid phase extraction: and (3) solid phase extraction in the same method as in (4).
(5) Constant volume: and (3) constant volume in the second method.
2. UPLC-MS/MS analysis of sex hormone
1. Chromatographic conditions: acquisity (a Chinese character)
HSS T3 column (2.1 mm. Times.100 mm,1.8 μm); sample injection amount is 10uL; column temperature is 35 ℃; mobile phase A is 0.2% ammonia water, and B is acetonitrile; the flow rate was 0.38mL/min. Gradient elution: 0min,25% b; 0-6 min,25% -50% B; 6-10 min,50% -80% B; 10-11 min, 80-100% B; 11-13 min, 100-100% B; 13-14 min, 100-25% B; 14-16 min,25% -25% of B.
2. Mass spectrometry conditions: waters Xevo TQ mass spectrometry system, electrospray ion source (ESI), positive and negative ion scanning mode, multiple Reaction Monitoring (MRM) mode, ion source temperature 150 ℃; capillary voltage 2.7kV, desolventizing gas temperature 500 ℃, desolventizing gas flow rate 1000L/h and taper hole gas flow rate 50L/h.
3. Establishment of a Standard Curve
Respectively weighing 8 sex hormones in a volumetric flask, dissolving the sex hormones in methanol to fix the volume to scale marks, and gradually diluting the sex hormones into mixed standard solution with 6 concentrations. The alpha-estradiol concentration is 0.2,1,4, 20, 80, 200ng/mL; beta-estradiolAnd estrone concentrations of 0.8,4, 16, 80, 320, 800ng/mL; testosterone, androstenedione, epididymis, and progesterone concentrations 0.1,0.5,2, 10, 40, 100ng/mL; androsterone concentration is 0.5,2.5, 10, 50, 200, 500 μg/mL, and internal standard estradiol-D is added 4 And progesterone-D 9 The concentration was set to 15ng/mL (estradiol-D 4 ) And 1ng/mL (progesterone-D) 9 ) And (3) passing through a microporous filter membrane with the thickness of 0.22 mu m for testing.
Example 2 analysis of the results of the detection of pollen neutral hormone
1. Optimization results of analytical methods
1. Optimization of extraction method
In view of the possible presence of free and/or bound sex hormones in plant foods such as pollen, extraction methods are critical in order to ensure the accuracy of the detection. The invention adopts acetonitrile extraction, enzyme hydrolysis-acetonitrile extraction and alkali hydrolysis-acetonitrile extraction to examine the influence of the acetonitrile extraction on the pollen sex hormone content (method one to method eight), the result is shown in the table 1, and 'Paeonia ostii' pollen in 8 sex hormones only detects progesterone, and the alkaline hydrolysis method does not detect progesterone aiming at progesterone, thus being not applicable to pollen sex hormone extraction. Although the enzymatic hydrolysis method detects a progesterone content 26 times that of acetonitrile extraction, it is not possible to exist in the bound form due to the lack of hydroxyl groups in the progesterone structure, and there is no literature report on the bound form of progesterone, so that it is not possible to convert the bound progesterone into free progesterone by enzymatic hydrolysis. For this document (Li Xin-liquid chromatography-tandem mass spectrometry) there is a relevant explanation in the detection of 26 steroid hormone content in poultry products [ J ]. Chinese feed, 2015 (13): 29-37), which enzyme also has steroid hormone reductase activity, possibly reducing pregnenolone to progesterone, resulting in a rapid increase in progesterone content after enzymatic hydrolysis of pollen samples. Therefore, for the detection of pollen sex hormone, an enzyme hydrolysis method is not suitable, and the direct acetonitrile extraction is more reasonable.
TABLE 1 sex hormone content of Paeonia ostii pollen under different methods
2. Optimization of the purification method
Because the content of pollen neutral hormone is extremely low, the matrix is complex, and the interference factors are large, the impurity removal is very important, and the method comprises the steps of removing fat-soluble impurities, preventing a solid phase extraction column from being blocked in the subsequent column passing process, removing water-soluble impurities, reducing the matrix effect of a sample and improving the sensitivity of a target object. Therefore, the invention respectively examines the influence of methods such as normal hexane degreasing, ethyl acetate water washing, solid phase extraction and the like on sample purification. As shown in Table 1, the comparison of method two and method three shows that when no n-hexane is added for extraction, a large amount of insoluble precipitate is generated through the solid phase extraction column, so that the extraction column is blocked and the components are lost, and the step of degreasing the n-hexane is necessary for pollen samples. The comparison of the method III and the method IV shows that the solid phase extraction (the method III) has a slightly better impurity removing effect than the ethyl acetate water washing (the method IV) after degreasing, but the impurities cannot be removed completely by adopting one mode, and the method can be seen through the fixed-volume reagent amount, so that the two methods are needed to be adopted for removing the water-soluble impurities simultaneously. The influence of two different water washing methods on the impurity removal effect is examined, and the result shows that the organic reagent loss rate of the ethyl acetate water washing (method one) is lower than that of the acetonitrile water washing (method five), and the measured progesterone content is higher, so that the ethyl acetate water washing is selected. Although the enzymolysis method (method six and method seven) is not suitable for detecting pollen sex hormone, the step of washing the pollen enzymolysis sample with organic reagent is also necessary, otherwise, insoluble impurities are more when the pollen enzymolysis sample passes through the extraction column, and the extraction column is blocked. The method one is the most suitable method for pollen sex hormone detection in terms of comprehensive sample purification efficiency and progesterone content.
3. Selection of internal standards
Because the matrix of pollen is complex, the matrix effect is a problem frequently encountered in detecting trace components by a liquid chromatography-mass spectrometry method, and the result of the test is not accurate enough. In order to avoid matrix interference, sample purification is an important means, and isotope internal standards are also effective measures to reduce matrix effects. In consideration of the similarity of the structures of the internal standard and the target, the invention uses the estradiol-D 4 progesterone-D as an internal estrogen standard 9 As an internal standard of androgens and progestogens, the accuracy and precision of the method are improved.
4. Optimization of constant volume reagent and constant volume
Because the content of hormone components in the sample is very low, in order to improve the detection concentration of target components, the sample solubility of the acetonitrile-extracted sample (method one to five) is higher when the constant volume reagent is acetonitrile, the sample solubility of the enzymolysis sample (method six and seven) is higher when the constant volume reagent is 50% acetonitrile aqueous solution, and finally the constant volume reagent amount is adjusted to enable the sample to be completely dissolved, so that the target components are easier to detect. The fixed capacity of the 8 methods is shown in Table 1.
5. Optimization of chromatographic conditions
The composition of the mobile phase influences the peak shape and ionization efficiency of the compound, and experiments show that the signal response of each hormone by taking acetonitrile as the mobile phase is superior to that of methanol, so that a water-acetonitrile system is selected as the mobile phase. The effect of 4 sets of mobile phase systems (0.1% formic acid water-acetonitrile, 0.1% formic acid water 20mmol/L ammonium acetate water-acetonitrile, 0.2% ammonia water 20mmol/L ammonium acetate water-acetonitrile and 0.2% ammonia water-acetonitrile) on target response and separation effect was compared. The results show that the acidic mobile phase can enhance the ionization efficiency of the positive ion mode test object (androgen and progestogen) compared with the alkaline mobile phase, so that the response value is improved by 2-3 times, but the acidic mobile phase can inhibit the generation of negative ions, so that the response value of estrogen is reduced, and the estrogen does not show peaks. The alkaline mobile phase can enhance the ionization efficiency of the negative ion mode object to be detected (estrogen), even if the response values of the androgen and the progestogen are reduced, the detection requirement can be met, and the object to be detected shows peaks, so the alkaline mobile phase is selected. Compared with 20mmol/L ammonium acetate water-acetonitrile of 0.2% ammonia water, the response value of 0.2% ammonia water-acetonitrile to estrogen is higher, so that the mobile phase system of 0.2% ammonia water-acetonitrile is selected comprehensively. Optimizing gradient elution program to obtain optimal separation effect for each target. The MRM chromatograms of 8 sex hormones and 2 internal standards are shown in figure 1.
6. Optimization of mass spectrometry conditions
And under the condition of mobile connection, the experiment respectively adopts a positive ion mode and a negative ion mode to comprehensively scan the target object, and finally determines that the estrogen adopts a negative ion mode and the androgen and the progestogen adopt a positive ion mode. And (3) determining characteristic parent ions and child ions according to the characteristics of the parent ions and the two groups of fragment ions with highest response, and optimizing mass spectrum parameters such as taper hole voltage, collision energy, ion source temperature, desolvation gas temperature, flow, collision gas flow and the like, so that the ionization efficiency of each hormone in the sample liquid is optimal. The mass spectral parameters are shown in table 2.
Table 28 MRM mass spectrometry parameters for sex hormones
Note that: "" is a quantitative ion.
2. Linear range, detection limit and quantification limit
In consideration of different ionization modes, the response intensity of sex hormone is different, so that the estrogen (alpha-estradiol, beta-estradiol, estrone) in negative ion mode is treated by estradiol-D 4 To correct internal standard, androgens (testosterone, androstenedione, epididylesterol, androsterone) and progestins (progesterone) in positive ion mode are used as progesterone-D 9 To correct the internal standard. And drawing an internal standard curve by taking the concentration of the standard substance as an abscissa and the ratio of the peak area to the internal standard peak area as an ordinate. The results show that the 8 sex hormones all show good linear relationship in the concentration range, and the correlation coefficient (r 2 ) 0.9934 to 0.9998. The concentration of sex hormones corresponding to a signal to noise ratio (S/N) of 3 times is a detection Limit (LOD), the concentration of sex hormones corresponding to a signal to noise ratio (S/N) of 10 times is a quantitative Limit (LOQ), the detection limit of 8 sex hormones is 0.005-86.541 ng/mL, the quantitative limit is 0.017-288.461 ng/mL, and the results are shown in Table 3.
Table 38 Linear equation, linear range, correlation coefficient, detection limit and quantitative limit of sex hormone
3. Actual sample detection
The 8 methods are respectively adopted to detect 8 sex hormones in the 'Paeonia ostii' pollen, as shown in table 1, the first method is the optimal method for detecting the sex hormones in the 'Paeonia ostii' pollen, the detection flow chart is shown in figure 2, the progesterone content is 748.01ng/kg, and other sex hormones are not detected. The chromatogram of the actual sample of the Paeonia ostii pollen is shown in figure 3.
4. Conclusion(s)
Through the optimization of pollen sample extraction, purification, constant volume, liquid chromatography and mass spectrum, the invention successfully establishes a UPLC-MS/MS method (method I) for detecting 8 sex hormones in pollen, and the method has high accuracy and sensitivity and good reproducibility, and is suitable for detecting the sex hormones of pollen or pollen foods.
The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives can occur depending upon design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.
Claims (8)
1. A method for detecting pollen sex hormone comprises the following steps:
(1) Extracting: adding an internal standard into a sample to be detected, then adding acetonitrile for extraction, and collecting supernatant; purifying the supernatant, the purifying comprising the steps of: degreasing, washing with water and solid phase extraction, degreasing and washing with water, or degreasing and solid phase extraction:
(2a) Degreasing: adding normal hexane into the supernatant for extraction, and collecting acetonitrile phase;
(2b) Washing: removing the solvent of the acetonitrile phase, dissolving by adopting an organic reagent, washing by using brine, collecting and removing the organic phase reagent, adding a test agent to redissolve a sample, and purifying;
(2c) Solid phase extraction: activating and balancing the solid phase extraction column, adding a sample, eluting by using an eluent, and finally eluting and collecting a target object by using acetonitrile;
(3) Constant volume: adding a reagent to dissolve the target object and fixing the volume;
(4) And (3) detection: detecting sex hormone in the sample in the step (3) by using a UPLC-MS/MS method;
(5) Establishing a standard curve: preparing a sex hormone standard solution containing an internal standard, carrying out UPLC-MS/MS detection, taking the sex hormone concentration as an abscissa, taking the ratio of the detected sex hormone peak area to the internal standard peak area as an ordinate, drawing an internal standard curve, and calculating the content of neutral hormone in a sample to be detected according to the obtained internal standard curve.
2. The method of claim 1, wherein: in the step (1), the feed liquid ratio of the sample to acetonitrile is 1: 5-20 g/mL;
the volume ratio of the acetonitrile in the step (1) to the n-hexane in the step (2 a) is 1-2: 1-2 mL/mL.
3. The detection method according to claim 1 or 2, characterized in that: in the step (2 b), the organic reagent is ethyl acetate or acetonitrile;
the brine is sodium carbonate aqueous solution or sodium chloride aqueous solution;
the volume ratio of the organic reagent to the brine is 1-5: 1-3 mL/mL;
the times of water washing are 2-4 times;
the reagent for dissolving the sample before purification is 5-40% of methanol aqueous solution or 5-40% of acetonitrile aqueous solution.
4. A detection method according to any one of claims 1-3, characterized in that: in the step (2 c), the solid phase extraction column is an HLB solid phase extraction column;
the eluent comprises water and organic eluent, wherein the organic eluent is 5% -40% of methanol aqueous solution or 5% -40% of acetonitrile aqueous solution.
5. The detection method according to any one of claims 1 to 4, wherein: in the step (3), the solvent used for constant volume is 50% -100% acetonitrile aqueous solution.
6. The detection method according to any one of claims 1 to 5, wherein: in the steps (4) and (5), the chromatographic conditions of UPLC-MS/MS detection are as follows:
Acquity
HSS T3 chromatographic column;
the sample injection amount is 10uL, the column temperature is 35 ℃, and the flow rate is 0.38mL/min;
mobile phase A is 0.2% ammonia water, mobile phase B is acetonitrile;
gradient elution: 0min,25% b; 0-6 min,25% -50% B; 6-10 min,50% -80% B; 10-11 min, 80-100% B; 11-13 min, 100-100% B; 13-14 min, 100-25% B; 14-16 min,25% -25% of B.
7. The detection method according to any one of claims 1 to 5, wherein: in the steps (4) and (5), the mass spectrum conditions detected by the UPLC-MS/MS are as follows:
the ion source temperature of the Waters Xevo TQ mass spectrometry system is 150 ℃ in an electrospray ion source, a positive and negative ion scanning mode and a multi-reaction monitoring mode;
the capillary voltage is 2.7kV, the desolvation gas temperature is 500 ℃, the desolvation gas flow rate is 1000L/h, and the taper hole gas flow rate is 50L/h.
8. The detection method according to any one of claims 1 to 7, wherein the sample is pollen or pollen food; the sex hormone comprises at least one of alpha-estradiol, beta-estradiol, estrone, testosterone, androstenedione, epididymis ketone, androsterone and progesterone.
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