CN105842379A - Method for measuring phenolic estrogen by means of derivation - Google Patents
Method for measuring phenolic estrogen by means of derivation Download PDFInfo
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- CN105842379A CN105842379A CN201610387235.XA CN201610387235A CN105842379A CN 105842379 A CN105842379 A CN 105842379A CN 201610387235 A CN201610387235 A CN 201610387235A CN 105842379 A CN105842379 A CN 105842379A
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
Abstract
The invention discloses a method for measuring phenolic estrogen by means of derivation. The method includes simultaneously deriving six types of phenolic estrogen; quickly measuring the phenolic estrogen by the aid of ultrahigh-performance liquid chromatography-mass spectrometry detectors. Compared with the prior art, the method has the advantages that the sensitivity of the six types of phenolic estrogen can be improved, the method is good in repeatability and low in detection cost and only has small variable coefficients, and measurement requirements on recovery rates can be met.
Description
Technical field
The invention belongs to wild animal resources detection technique field, reacted by pre-column derivatization, ultra high efficiency liquid
Phase chromatography-mass spectroscopy detector measures female alcohol and female phenol simultaneously.The present invention can simultaneously to alcohol female in animal tissue and
The multi-residue analysis of female phenolic estrogen measures, it is possible to be analyzed other Polyphenols Organic substances measuring.
Background technology
Estrogen is that a class has extensive bioactive sterid, it is possible to promotes and maintains women phase
Close physiological action, also can Endocrine system, cardiovascular system, body metabolism, skeleton development and
The each side such as skin play special effect.Natural steroid hormones is mainly by the female or Male sexual device of animal
Official, the organ such as epinephrine produces, and is one of the important substance of animal growth, as estradiol, female three
Alcohol, estrone etc..The steroid hormone of a lot of similar structures of synthetic, as diethylstilbestrol, hexestrol and
Dienestrols etc., these hormones are difficult in animal body decompose, after entering human body by food chain, can upset
Human endocrine system, causes the decline of feminization, male fecundity, women with breast cancer and ovary
Cancer, female precocious puberty etc..European Union has forbidden its use in animal feeding, the Ministry of Agriculture of China 235
Also specifying in bulletin, the estrogen such as diethylstilbestrol must not detect in animal derived food.The most quick and precisely
In detection animal tissue, these estrogen residual is critically important for ensuring food safety.
Derivatization is directly to measure that the target compound existed is unstable, Ionization Efficiency is low etc. lack to make up
Point, methylates such as silane or acetylization reaction is converted into volatile ingredient estrogen and just can carry out GC-MS survey
Fixed.LC-MS/MS is one of common technology of estrogenic residue detection, and its testing cost is high, to part
Difficult ionising compounds such as estradiol, estriol, need derivative reaction to improve Ionization Efficiency, domestic foreign language
Offer and all have been reported that (1, M.Reza Anari, Ray Bakhtiar, Bing Zhu et al, Anal.
Chem.2002,74,4136-4144,2, Hiroyuki Yamada, Yasuhiro Kuwahara, 2
Yasuo Takamatsu et al, Biomed.Chromatogr.2000,14:333 337,3,
Robert E.Nelson,Stefan K.Grebe,Dennis J.O’Kane et al.,Clinical
Chemistry 2004,50 (2), 373 384,4, Zhao Huaixin, Sun Xuejun, Sun Zhiwei etc., analyze
Chemistry, 2009,37 (2), 187-193).These methods are all for estradiol, estriol target chemical combination
Thing, has no the report about female phenolic estrogen derivative reaction, and country measures female alcohol and female the most simultaneously
The standard of phenol.
Ultra Performance Liquid Chromatography (UPLC), as a kind of new chromatograph, covers little granular filler, low system
The system brand new technical such as volume and fast data collection, has that post effect is high, disengaging time is short, solvent consumption is few
Advantage.UPLC system is with a portable mass detector (QDa) at present, and sensitivity and accuracy are better than purple
External detector (PDA), has broken away from the condition optimizing of conventional mass spectrometer, easy and simple to handle, starts shooting the pumpdown time
Short (as long as 5min), plug and play, it is suitable for the detection of daily batch samples, but for female alcohol, female phenols
Compound there is also the shortcomings such as Ionization Efficiency is low, sensitivity is low, directly measures and can only achieve mg/kg residual water
Flat, it is impossible to meet trace detection analysis requirement.
Summary of the invention
It is an object of the invention to provide a kind of by using Ultra Performance Liquid Chromatography-Mass Spectrometer Method after pre-column derivatization
Device, can quickly, low cost, sensitivity highland measure female alcohol and the one of the many residuals of female phenolic estrogen simultaneously
The method of Derivatization Determination phenolic estrogen.
Technical scheme is as follows:
1. the method for a Derivatization Determination phenolic estrogen, it is characterised in that comprise the following steps:
(1) preparation of estrogen standard substance and mensuration
1. the preparation of standard reserving solution: by 17 beta estradiols, estriol, estrone, diethylstilbestrol, hexane is female
Phenol, after 6 kinds of estrogen solid etalon product of dienestrol dissolve with methanol respectively, being made into concentration with methanol respectively is
6 kinds of standard reserving solutions of 1mg/mL, are placed in-20 DEG C of refrigerator storage；
2. the preparation of five series concentration hybrid standard liquid: measuring the concentration that step (1) 1. prepared is 1mg/mL
17 beta estradiol standard reserving solutions, concentration be the estriol standard reserving solution of 1mg/mL, concentration be 1mg/mL
Estrone standard reserving solution, concentration be the diethylstilbestrol standard reserving solution of 1mg/mL, concentration be oneself of 1mg/mL
Alkane female phenol standard reserving solution, concentration are that the dienestrol standard reserving solution of 1mg/mL respectively takes 0.5mL and is mixed into
50mL volumetric flask, obtains hybrid working liquid by methanol constant volume to 50mL, then divides and takes hybrid working liquid methanol and divide
It is not diluted to hybrid standard liquid A, hybrid standard liquid B, hybrid standard liquid C, hybrid standard liquid D, hybrid standard
Five series concentration hybrid standard liquid of liquid E, wherein:
17 beta estradiols that contain in hybrid standard liquid A, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 5ng/mL；
17 beta estradiols that contain in hybrid standard liquid B, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 50ng/mL；
17 beta estradiols that contain in hybrid standard liquid C, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 100ng/mL；
17 beta estradiols that contain in hybrid standard liquid D, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 500ng/mL；
17 beta estradiols that contain in hybrid standard liquid E, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 1000ng/mL；
Prepared by interior target: after deuterated 17 beta estradiols (d3) being dissolved with methanol, be made into concentration with methanol
For the internal standard standard reserving solution of 1mg/mL, measuring internal standard standard reserving solution, add methanol being configured to concentration is 500
The internal standard of ng/mL；
4. the preparation of dansyl Cl derivative reagent: solid dansyl Cl is dissolved in organic solvent, organic with this
It is 1mg/mL dansyl Cl derivative reagent that solvent is configured to concentration, loads brown bottle, is placed in-20 DEG C of refrigerator storages
Deposit；
Take the hybrid standard liquid of five series concentration that 1mL step (1) is 2. prepared the most respectively, mix at each
Adding the internal standard that concentration the is 500ng/mL 100 μ L that step (1) is 3. prepared in standardization liquid, each mixes
Standardization liquid dries up with nitrogen after adding internal standard mix homogeneously, respectively obtains five mixed samples of correspondence；Mixed
Mixed sample corresponding for standardization liquid A is designated as mixed sample A ＇, and mixed sample corresponding for hybrid standard liquid B is designated as
It is corresponding that mixed sample B ＇, mixed sample corresponding for hybrid standard liquid C are designated as mixed sample C ＇, hybrid standard liquid D
Mixed sample be designated as mixed sample D ＇, mixed sample corresponding for hybrid standard liquid E is designated as mixed sample E'；
6. derivative reaction and Ultra Performance Liquid Chromatography-mass detector measure
5. described five mixed samples of step (1) perform the derivatization reaction the most as follows: each mixing
Standard specimen adds 1mg/mL dansyl Cl the derivative reagent 10～1000 μ L that step (1) is 4. prepared, uses bicarbonate
Sodium or sodium hydroxide adjust pH to be 8.0～11.0, vortex mixing 30s, and temperature 45 C～70 DEG C, derivatization is anti-
Answer 10～30min；
After solution after each derivative reaction is cooled to room temperature naturally, nitrogen dries up, and adds mixed liquor molten
Solving, described mixed liquor is mixed by 0.65mL methanol solution and 1mmol/L ammonium acetate solution 0.35mL；
In solution after dissolving, organic facies membrane filtration obtains filtrate, and gained filtrate enters Ultra Performance Liquid Chromatography-mass spectrum inspection
Survey device measures, and Ultra Performance Liquid Chromatography-mass detector condition determination is as follows:
Chromatographic column: waters BEH C18 2.1 × 50mm.Chromatogram column temperature: 35 DEG C.Sampling volume:
10μL；
Flowing is methanol mutually: 1mmol/L ammonium acetate, arranges gradient elution, and condition of gradient elution presses table 1:
Table 1 condition of gradient elution
Mass detector scan mode is set: choice ion pattern, as shown in table 2, mass detector measures
The condition of derivative products, negative ions scans simultaneously, and quality of scanning number scope is m/z=40～600,
Taper hole voltage 15V, sampling rate 8 points/second, capillary voltage 0.8kv；
Table 2 mass detector measures the condition of derivative products
7. measure through Ultra Performance Liquid Chromatography-mass detector and obtain 17 beta estradiols, estriol, estrone,
Diethylstilbestrol, hexestrol, the peak area of 6 kinds of estrogen of dienestrol and interior target peak area, respectively with 6
The peak area of kind of estrogen is vertical coordinate with the ratio of internal standard peak area, step (1) 2. in five mixing marks
The concentration value of quasi-liquid is abscissa, draws six corresponding standard working curves；
(2) measuring samples detection
The most accurately weighing 5g animal tissue measuring samples, adding concentration 3. prepared for 100 μ L step (1) is
The internal standard of 500ng/mL, uses 25mL acetonitrile extraction, 5000rpm, centrifugal 5min, collects supernatant, nitrogen
Dry up；
2., after adding the 1mg/mL dansyl Cl derivative reagent 10-1000 μ L that step (1) is 4. described, carbonic acid is used
Hydrogen sodium or sodium hydroxide adjust pH to be 8.0～11.0, vortex mixing 30s, at temperature 45 C～70 DEG C, derivatization
Reaction 10～30min；
3. after the solution after derivative reaction is cooled to room temperature naturally, nitrogen dries up, and adds mixed liquor and dissolves,
Described mixed liquor is mixed by 0.65mL methanol solution and 1mmol/L ammonium acetate solution 0.35mL；Dissolve
After solution in organic facies membrane filtration obtain filtrate, gained filtrate enters Ultra Performance Liquid Chromatography-mass detector
Measuring, the Ultra Performance Liquid Chromatography-mass detector condition determination 6. described by step (1) measures；
4. measure through Ultra Performance Liquid Chromatography-mass detector, obtain retention time and the face, peak at measuring samples peak
Long-pending, compare with the retention time of 6 kinds of estrogen standard specimens of step (1) 6. gained, determine measuring samples
In the kind of contained estrogen, with step (1) 7. in the standard working curve of corresponding estrogen compare,
Obtain the content of contained estrogen.
2. according to the method for the Derivatization Determination phenolic estrogen described in technical scheme 1, it is characterised in that: step
(1) 4. described in organic solvent be acetone or benzene.
Compared with prior art, the invention has the beneficial effects as follows:
1, by the pre-column derivatization reaction method of the present invention, improve 17 beta estradiols, estriol, female
The ionizing of ketone, diethylstilbestrol, hexestrol, dienestrol and internal standard deuterated 17 beta estradiols (d3)
Efficiency, sensitivity is improved.
2, there is presently no employing Ultra Performance Liquid Chromatography-mass detector simultaneously to 17 beta estradiols, female three
Alcohol, estrone, diethylstilbestrol, hexestrol, the detection of 6 kinds of many residuals of estrogen of dienestrol, the present invention adopts
Be set for qualitative and quantitative analysis with Ultra Performance Liquid Chromatography-mass detector and parameter thereof, quantitative limit up to
To 1～2 μ g/kg, result repeatability is good, and 3 are added the concentration response rate is 65%～121%, repeats for 6 times
The coefficient of variation of experiment is less than 13%.
3, the inventive method testing cost is low.Existing tandem mass spectrum instrument price is up to 2,500,000 yuan, and superelevation
The price of effect liquid phase chromatogram-mass detector 700,000 yuan, compares tandem mass spectrum instrument, and the inventive method detects
Low cost.
Accompanying drawing explanation
Fig. 1 is the chromatogram of 17 beta estradiol standard specimen derivants, and in figure, vertical coordinate is intensity, and abscissa is
Time.
Fig. 2 is the chromatogram of estriol standard specimen derivant, and in figure, vertical coordinate is intensity, and abscissa is the time.
Fig. 3 is estrone standard specimen derivant and the chromatogram of diethylstilbestrol standard specimen derivant, and in figure, vertical coordinate is strong
Degree, abscissa is the time.
Fig. 4 is the chromatogram of hexestrol standard specimen derivant, and in figure, vertical coordinate is intensity, when abscissa is
Between.
Fig. 5 is the chromatogram of dienestrol standard specimen derivant, and in figure, vertical coordinate is intensity, when abscissa is
Between.
Fig. 6 is the chromatogram of internal standard standard specimen derivant, and in figure, vertical coordinate is intensity, and abscissa is the time.
Fig. 7 is the chromatogram of 17 beta estradiol derivants of milk mark-on sample, and in figure, vertical coordinate is strong
Degree, abscissa is the time.
Fig. 8 is the chromatogram of the estriol derivant of milk mark-on sample, and in figure, vertical coordinate is intensity, horizontal seat
Mark is the time.
Fig. 9 is estrone derived steroids thing and the chromatogram of diethylstilbestrol derivant of milk mark-on sample, vertical seat in figure
Mark is intensity, and abscissa is the time.
Figure 10 is the chromatogram of the hexestrol derivant of milk mark-on sample, and in figure, vertical coordinate is intensity,
Abscissa is the time.
Figure 11 is the chromatogram of the dienestrol derivant of milk mark-on sample, and in figure, vertical coordinate is intensity,
Abscissa is the time.
Figure 12 is the chromatogram of the internal standard derivant of milk mark-on sample, and in figure, vertical coordinate is intensity, horizontal seat
Mark is the time.
Detailed description of the invention
All raw materials used by following example, reagent etc. are commercially available.Without specified otherwise in each embodiment
For conventional method.
Embodiment 1 derivative reaction condition
1. the preparation of dansyl Cl derivative reagent: solid dansyl Cl is dissolved in acetone solvent, compound concentration
1mg/mL dansyl Cl derivative reagent, loads brown bottle, is placed in-20 DEG C of refrigerator storage.
2. derivative reaction condition 1: add the 1mg/mL dansyl Cl derivative reagent that step (1) is 1. prepared
10 μ L, are 8.0 with the pH of sodium bicarbonate regulation mixing mixing, vortex mixing 30s, at temperature 45 C,
Derivative reaction 10min.
3. derivative reaction condition 2: add the 1mg/mL dansyl Cl derivative reagent that step (1) is 1. prepared
100 μ L, are 9.0 with the pH of sodium bicarbonate regulation mixing mixing, vortex mixing 30s, temperature 55 DEG C,
Derivative reaction 10min.
4. derivative reaction condition 3: add the 1mg/mL dansyl Cl derivative reagent that step (1) is 1. prepared
200 μ L, are 10.0 with the pH of sodium bicarbonate regulation mixed solution, and vortex mixing 30s, in temperature
55 DEG C, derivative reaction 30min.
5. derivative reaction condition 4: add the 1mg/mL dansyl Cl derivative reagent that step (1) is 1. prepared
500 μ L, are 11.0 with the pH of sodium bicarbonate regulation mixed solution, and vortex mixing 30s, in temperature
65 DEG C, derivative reaction 20min.
6. derivative reaction condition 5: add the 1mg/mL dansyl Cl derivative reagent that step (1) is 1. prepared
1000 μ L, are 10.0 with the pH of sodium bicarbonate regulation mixed solution, and vortex mixing 30s, in temperature
70 DEG C, derivative reaction 10min.
The preparation of embodiment 2 estrogen standard sample and estrogen Specification Curve of Increasing
(1) preparation of estrogen standard sample
1. the preparation of standard reserving solution: by following 6 kinds of estrogen solid etalon product: 17 beta estradiols, female three
Alcohol, estrone, diethylstilbestrol, hexestrol, dienestrol, dissolves with methanol respectively, is made into concentration respectively
Following 6 kinds of standard reserving solutions for 1mg/mL:
The 17 beta estradiol standard reserving solutions of 1mg/mL；
The estriol standard reserving solution of 1mg/mL；
The estrone standard reserving solution of 1mg/mL；
The diethylstilbestrol standard reserving solution of 1mg/mL；
The hexestrol standard reserving solution of 1mg/mL；
The dienestrol standard reserving solution of 1mg/mL；
Above 6 kinds of standard reserving solutions are placed in-20 DEG C of refrigerator storage.
2. the preparation of five series concentration hybrid standard liquid: measuring the concentration that step (1) 1. prepared is 1mg/mL
17 beta estradiol standard reserving solutions, concentration be the estriol standard reserving solution of 1mg/mL, concentration be 1mg/mL
Estrone standard reserving solution, concentration be the diethylstilbestrol standard reserving solution of 1mg/mL, concentration be oneself of 1mg/mL
Alkane female phenol standard reserving solution, concentration are that the dienestrol standard reserving solution of 1mg/mL respectively takes 0.5mL and is mixed into
50mL volumetric flask, obtains hybrid working liquid by methanol constant volume to 50mL, then divides and takes hybrid working liquid methanol and divide
It is not diluted to hybrid standard liquid A, hybrid standard liquid B, hybrid standard liquid C, hybrid standard liquid D, hybrid standard
The hybrid standard liquid of 5 variable concentrations of liquid E, wherein:
17 beta estradiols that contain in hybrid standard liquid A, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 5ng/mL；
17 beta estradiols that contain in hybrid standard liquid B, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 50ng/mL；
17 beta estradiols that contain in hybrid standard liquid C, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 100ng/mL；
17 beta estradiols that contain in hybrid standard liquid D, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 500ng/mL；
17 beta estradiols that contain in hybrid standard liquid E, estriol, estrone, diethylstilbestrol, hexane are female
Phenol, the concentration of 6 components of dienestrol are 1000ng/mL.
Prepared by interior target: after deuterated 17 beta estradiols (d3) being dissolved with methanol, be made into concentration with methanol
For the internal standard standard reserving solution of 1mg/mL, measuring internal standard standard reserving solution, add methanol being configured to concentration is 500
The internal standard of ng/mL.
4. the preparation of dansyl Cl derivative reagent: solid dansyl Cl is dissolved in acetone, prepares with acetone
Becoming concentration is 1mg/mL dansyl Cl derivative reagent, loads brown bottle, is placed in-20 DEG C of refrigerator storage.
Take the hybrid standard liquid of five series concentration that 1mL step (1) is 2. prepared the most respectively, mix at each
Adding the internal standard that concentration the is 500ng/mL 100 μ L that step (1) is 3. prepared in standardization liquid, each mixes
Standardization liquid dries up with nitrogen after adding internal standard mix homogeneously, respectively obtains five mixed samples of correspondence；Mixed
Mixed sample corresponding for standardization liquid A is designated as mixed sample A ＇, and mixed sample corresponding for hybrid standard liquid B is designated as
It is corresponding that mixed sample B ＇, mixed sample corresponding for hybrid standard liquid C are designated as mixed sample C ＇, hybrid standard liquid D
Mixed sample be designated as mixed sample D ＇, mixed sample corresponding for hybrid standard liquid E is designated as mixed sample E'.
(2) derivative reaction and Ultra Performance Liquid Chromatography-mass detector measure
1. five mixed samples that step (1) is 5. described perform the derivatization reaction the most as follows: each mixed
Close and standard specimen adds the 1mg/mL dansyl Cl derivative reagent 200 μ L that step (1) is 4. prepared, use sodium bicarbonate
Adjusting pH is 10.0, and vortex mixing 30s, at temperature 60 C, derivative reaction 10min；
After solution after each derivative reaction is cooled to room temperature naturally, nitrogen dries up, and adds mixed liquor molten
Solving, described mixed liquor is mixed by 0.65mL methanol solution and 1mmol/L ammonium acetate solution 0.35mL；
In solution after dissolving, organic facies membrane filtration obtains filtrate, and gained filtrate enters Ultra Performance Liquid Chromatography-mass spectrum inspection
Survey device measures, and Ultra Performance Liquid Chromatography-mass detector condition determination is as follows:
Chromatographic column: waters BEH C18 2.1 × 50mm.Chromatogram column temperature: 35 DEG C.Sampling volume:
10μL；
Flowing is methanol mutually: 1mmol/L ammonium acetate, arranges gradient elution, and condition of gradient elution presses table 1:
Table 1 condition of gradient elution
Mass detector scan mode is set: choice ion pattern, as shown in table 2, mass detector measures
The condition of derivative products, negative ions scans simultaneously, and quality of scanning number scope is m/z=40～600,
Taper hole voltage 15V, sampling rate 8 points/second；Capillary voltage 0.8kv；
Table 2 mass detector measures the condition of derivative products
2. measure through Ultra Performance Liquid Chromatography-mass detector and obtain 17 beta estradiols, estriol, estrone,
Diethylstilbestrol, hexestrol, the peak area of 6 kinds of estrogen of dienestrol and interior target peak area, each standard
Working curve is drawn as follows:
With the peak area difference that 5 concentration of every kind of estrogen in mixed sample A ＇ to mixed sample E ＇ are corresponding
Be vertical coordinate with 5 ratios obtained by the ratio of internal standard peak area, with step (1) 2. in five mixing mark
Concentration value 5ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL of quasi-liquid is horizontal seat
Mark, draws corresponding standard working curve；Refer to table 3.
The range of linearity of 6 standard curves is 5～1000ng/mL, coefficient R2> 0.995,6 kinds are female sharp
Element derivative products has good linear relationship, shows the inventive method and instrument condition reasonable.
17 beta estradiols, estriol, estrone, diethylstilbestrol, hexestrol, dienestrol derivant and interior
The chromatogram of mark derivant is shown in Fig. 1-Fig. 6.
36 kinds of estrogen regression equations of table, correlation coefficient and concentration range
Embodiment 3 milk pattern detection
(1) extraction of milk sample
It is experiment sample that market randomly chooses milk, and detects above-mentioned 6 kinds of estrogen residual simultaneously.Altogether
If 3 are added concentration, respectively 2,10,50 μ g/kg.Set a dummy, each concentration weight respectively
Multiple 6 times, carry out repeated experiment, calculate the response rate and the coefficient of variation respectively.
(2) derivative reaction
Accurately weigh 5g sample, add the internal standard that concentration is 500ng/mL that embodiment 2 step (1) is 3. prepared
100 μ L, collect eluent after extracted purification, nitrogen dries up, and adds volume 200 μ L embodiment 2 step
(1) the 1mg/mL dansyl Cl derivative reagent 4. prepared, is 10 with sodium bicarbonate regulation pH, and vortex mixes
30s, under temperature 60 C, the response time is 30min, obtains derivatization product.After taking-up nature is cooled to room temperature,
Nitrogen dries up, and adds mixed liquor and dissolves, and described mixed liquor is by 0.65mL methanol solution and 1mmol/L acetic acid
Ammonium salt solution 0.35mL mixes, and in the solution after dissolving, organic facies membrane filtration obtains filtrate, gained filtrate
Entering Ultra Performance Liquid Chromatography-mass detector to measure, condition determination is 1. described according to step (2) in embodiment 2
Ultra Performance Liquid Chromatography-mass detector condition determination.
(3) testing result
Testing result is shown in Table 4.Six kinds of estrogen is quantitatively limited to 1-in the interpolation concentration range of 2-50 μ g/kg
2 μ g/kg, response rate scope be 68%-121%, RSD scope be 4.3%-13.8%, show the inventive method pair
Low in the detection sensitivity of 6 kinds of estrogen, stability is preferable.
The response rate of 46 kinds of estrogen of table, RSD, quantitative limit n=6
Claims (2)
1. the method for a Derivatization Determination phenolic estrogen, it is characterised in that comprise the following steps:
(1) preparation of estrogen standard substance and mensuration
1. the preparation of standard reserving solution: by 17 beta estradiols, estriol, estrone, diethylstilbestrol, hexane is female
Phenol, after 6 kinds of estrogen solid etalon product of dienestrol dissolve with methanol respectively, being made into concentration with methanol respectively is
6 kinds of standard reserving solutions of 1mg/mL, are placed in-20 DEG C of refrigerator storage；
2. the preparation of five series concentration hybrid standard liquid: measuring the concentration that step (1) 1. prepared is 1mg/mL
17 beta estradiol standard reserving solutions, concentration be the estriol standard reserving solution of 1mg/mL, concentration be 1mg/mL
Estrone standard reserving solution, concentration be the diethylstilbestrol standard reserving solution of 1mg/mL, concentration be the hexane of 1mg/mL
Female phenol standard reserving solution, concentration are that the dienestrol standard reserving solution of 1mg/mL respectively takes 0.5mL and is mixed into 50mL
Volumetric flask, obtains hybrid working liquid by methanol constant volume to 50mL, then divides and takes hybrid working liquid methanol and dilute respectively
For hybrid standard liquid A, hybrid standard liquid B, hybrid standard liquid C, hybrid standard liquid D, hybrid standard liquid E five
Series concentration hybrid standard liquid, wherein:
17 beta estradiols that contain in hybrid standard liquid A, estriol, estrone, diethylstilbestrol, hexestrol,
The concentration of 6 components of dienestrol is 5ng/mL；
17 beta estradiols that contain in hybrid standard liquid B, estriol, estrone, diethylstilbestrol, hexestrol,
The concentration of 6 components of dienestrol is 50ng/mL；
17 beta estradiols that contain in hybrid standard liquid C, estriol, estrone, diethylstilbestrol, hexestrol,
The concentration of 6 components of dienestrol is 100ng/mL；
17 beta estradiols that contain in hybrid standard liquid D, estriol, estrone, diethylstilbestrol, hexestrol,
The concentration of 6 components of dienestrol is 500ng/mL；
17 beta estradiols that contain in hybrid standard liquid E, estriol, estrone, diethylstilbestrol, hexestrol,
The concentration of 6 components of dienestrol is 1000ng/mL；
Prepared by interior target: after deuterated 17 beta estradiols (d3) being dissolved with methanol, being made into concentration with methanol is
The internal standard standard reserving solution of 1mg/mL, measures internal standard standard reserving solution, and add methanol being configured to concentration is 500
The internal standard of ng/mL；
4. the preparation of dansyl Cl derivative reagent: solid dansyl Cl is dissolved in organic solvent, organic molten with this
It is 1mg/mL dansyl Cl derivative reagent that agent is configured to concentration, loads brown bottle, is placed in-20 DEG C of refrigerator storage；
Take the hybrid standard liquid of five series concentration that 1mL step (1) is 2. prepared the most respectively, mix at each
Titer adds the internal standard that concentration the is 500ng/mL 100 μ L that step (1) is 3. prepared, each mixing
Titer dries up with nitrogen after adding internal standard mix homogeneously, respectively obtains five mixed samples of correspondence；Mixing mark
Quasi-mixed sample corresponding for liquid A is designated as mixed sample A ＇, and mixed sample corresponding for hybrid standard liquid B is designated as mixing mark
Sample B ＇, mixed sample corresponding for hybrid standard liquid C are designated as mixed sample C ＇, mixing mark corresponding for hybrid standard liquid D
Sample is designated as mixed sample D ＇, mixed sample corresponding for hybrid standard liquid E is designated as mixed sample E'；
6. derivative reaction and Ultra Performance Liquid Chromatography-mass detector measure
5. described five mixed samples of step (1) perform the derivatization reaction the most as follows: each mixing
Standard specimen adds 1mg/mL dansyl Cl the derivative reagent 10～1000 μ L that step (1) is 4. prepared, uses bicarbonate
Sodium or sodium hydroxide adjust pH to be 8.0～11.0, vortex mixing 30s, at temperature 45 C～70 DEG C, derivative reaction
10～30min；
After solution after each derivative reaction is cooled to room temperature naturally, nitrogen dries up, and adds mixed liquor and dissolves,
Described mixed liquor is mixed by 0.65mL methanol solution and 1mmol/L ammonium acetate solution 0.35mL；Dissolve
After solution in organic facies membrane filtration obtain filtrate, gained filtrate enters Ultra Performance Liquid Chromatography-mass detector
Measuring, Ultra Performance Liquid Chromatography-mass detector condition determination is as follows:
Chromatographic column: waters BEH C18 2.1 × 50mm.Chromatogram column temperature: 35 DEG C.Sampling volume: 10
μL；
Flowing is methanol mutually: 1mmol/L ammonium acetate, arranges gradient elution, and condition of gradient elution presses table 1:
Table 1 condition of gradient elution
Mass detector scan mode is set: choice ion pattern, as shown in table 2, mass detector measures
The condition of derivative products, negative ions scans simultaneously, and quality of scanning number scope is m/z=40～600, cone
Hole voltage 15V, sampling rate 8 points/second, capillary voltage 0.8kv；
Table 2 mass detector measures the condition of derivative products
7. measure through Ultra Performance Liquid Chromatography-mass detector and obtain 17 beta estradiols, estriol, estrone, oneself
The female phenol of alkene, hexestrol, the peak area of 6 kinds of estrogen of dienestrol and interior target peak area, female with 6 kinds respectively
The ratio of the peak area of hormone and internal standard peak area is vertical coordinate, step (1) 2. in five hybrid standard liquid
Concentration value be abscissa, draw six corresponding standard working curves；
(2) measuring samples detection
The most accurately weighing 5g animal tissue measuring samples, adding concentration 3. prepared for 100 μ L step (1) is
The internal standard of 500ng/mL, uses 25mL acetonitrile extraction, 5000rpm, centrifugal 5min, collects supernatant, nitrogen
Dry up；
2., after adding the 1mg/mL dansyl Cl derivative reagent 10-1000 μ L that step (1) is 4. described, carbonic acid is used
Hydrogen sodium or sodium hydroxide adjust pH to be 8.0～11.0, vortex mixing 30s, and temperature 45 C～70 DEG C, derivatization is anti-
Answer 10～30min；
3. after the solution after derivative reaction is cooled to room temperature naturally, nitrogen dries up, and adds mixed liquor and dissolves, institute
State mixed liquor to be mixed by 0.65mL methanol solution and 1mmol/L ammonium acetate solution 0.35mL；After dissolving
Solution in organic facies membrane filtration obtain filtrate, gained filtrate is entered Ultra Performance Liquid Chromatography-mass detector and is surveyed
Fixed, the Ultra Performance Liquid Chromatography-mass detector condition determination 6. described by step (1) measures；
4. measure through Ultra Performance Liquid Chromatography-mass detector, obtain retention time and the face, peak at measuring samples peak
Long-pending, compare with the retention time of 6 kinds of estrogen standard specimens of step (1) 6. gained, determine in measuring samples
The kind of contained estrogen, with step (1) 7. in the standard working curve of corresponding estrogen compare,
Content to contained estrogen.
2. according to the method for the Derivatization Determination phenolic estrogen described in technical scheme 1, it is characterised in that: step (1)
4. the organic solvent described in is acetone or benzene.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404492A (en) * | 2016-11-04 | 2017-02-15 | 辽宁省检验检疫科学技术研究院 | Diethylstilbestrol, diethylstilbestrol and hexestrol standard sample in rabbit meat, preparation method and application |
| CN108709941A (en) * | 2018-07-24 | 2018-10-26 | 曲阜师范大学 | A kind of determination method of the neurosteroid of hydroxyl |
| WO2019037665A1 (en) * | 2017-08-22 | 2019-02-28 | 杭州谱景柏泰科技有限公司 | Method for detecting an aromatic compound, and kit |
| CN110824091A (en) * | 2019-12-17 | 2020-02-21 | 杭州度安医学检验实验室有限公司 | Steroid hormone detection method |
| CN111366671A (en) * | 2019-04-25 | 2020-07-03 | 中南民族大学 | Chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneously detecting 18 steroid hormones in serum |
| CN112611827A (en) * | 2020-12-23 | 2021-04-06 | 中南民族大学 | Chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneously detecting 3 estrogens in serum |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000088834A (en) * | 1998-09-16 | 2000-03-31 | Sumika Chemical Analysis Service Ltd | Analyzing method |
| JP2001116736A (en) * | 1999-10-21 | 2001-04-27 | Sumika Chemical Analysis Service Ltd | Method for analyzing estrogen |
| US6855703B1 (en) * | 2000-03-10 | 2005-02-15 | Endeavor Pharmaceuticals | Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds |
| US20100227412A1 (en) * | 2009-02-06 | 2010-09-09 | Perkinelmer Health Sciences, Inc. | Simultaneous Detection of Estrogen and Non Estrogen Steroids |
| JP2011163944A (en) * | 2010-02-10 | 2011-08-25 | Kao Corp | Method for quantifying estrogen |
| CN102636604A (en) * | 2012-02-09 | 2012-08-15 | 中国农业科学院农业质量标准与检测技术研究所 | Quantitative detecting method for estrogen in milk |
| CN103185762A (en) * | 2011-12-29 | 2013-07-03 | 复旦大学 | Method for analyzing and detecting a plurality of endocrine disruptors in food |
| CN103698414A (en) * | 2012-09-27 | 2014-04-02 | 中国科学院大连化学物理研究所 | Chemical derivatization based method for detection of steroid hormones in urine |
| CN104040332A (en) * | 2012-01-20 | 2014-09-10 | Dh科技发展私人贸易有限公司 | Analysis of estradiol and analytes with phenolic OH using labeling chemistry and LC-MSMS workflow |
-
2016
- 2016-06-02 CN CN201610387235.XA patent/CN105842379A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000088834A (en) * | 1998-09-16 | 2000-03-31 | Sumika Chemical Analysis Service Ltd | Analyzing method |
| JP2001116736A (en) * | 1999-10-21 | 2001-04-27 | Sumika Chemical Analysis Service Ltd | Method for analyzing estrogen |
| US6855703B1 (en) * | 2000-03-10 | 2005-02-15 | Endeavor Pharmaceuticals | Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds |
| US20100227412A1 (en) * | 2009-02-06 | 2010-09-09 | Perkinelmer Health Sciences, Inc. | Simultaneous Detection of Estrogen and Non Estrogen Steroids |
| JP2011163944A (en) * | 2010-02-10 | 2011-08-25 | Kao Corp | Method for quantifying estrogen |
| CN103185762A (en) * | 2011-12-29 | 2013-07-03 | 复旦大学 | Method for analyzing and detecting a plurality of endocrine disruptors in food |
| CN104040332A (en) * | 2012-01-20 | 2014-09-10 | Dh科技发展私人贸易有限公司 | Analysis of estradiol and analytes with phenolic OH using labeling chemistry and LC-MSMS workflow |
| CN102636604A (en) * | 2012-02-09 | 2012-08-15 | 中国农业科学院农业质量标准与检测技术研究所 | Quantitative detecting method for estrogen in milk |
| CN103698414A (en) * | 2012-09-27 | 2014-04-02 | 中国科学院大连化学物理研究所 | Chemical derivatization based method for detection of steroid hormones in urine |
Non-Patent Citations (4)
| Title |
|---|
| HE-XING WANG ET AL: "Enhanced screening efficiency for endocrine-disrupting chemicals in milk and powdered milk using UPLC/QTOF-MS by the introduction of dansyl chloride derivatisation", 《FOOD ADDITIVES & CONTAMINANTS: PART A》 * |
| XIAOJING DU ET AL: "Development of a HPLC-ECD method for the simultaneous determination of three synthetic estrogens in milk", 《ANALYTICAL METHODS》 * |
| 周建科 等: "豆奶粉中三种雌激素的高效液相色谱法测定", 《粮油食品科技》 * |
| 王小芳 等: "高效液相色谱荧光检测法测定鸡蛋中三种雌激素", 《中国卫生检验杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404492A (en) * | 2016-11-04 | 2017-02-15 | 辽宁省检验检疫科学技术研究院 | Diethylstilbestrol, diethylstilbestrol and hexestrol standard sample in rabbit meat, preparation method and application |
| WO2019037665A1 (en) * | 2017-08-22 | 2019-02-28 | 杭州谱景柏泰科技有限公司 | Method for detecting an aromatic compound, and kit |
| CN108709941A (en) * | 2018-07-24 | 2018-10-26 | 曲阜师范大学 | A kind of determination method of the neurosteroid of hydroxyl |
| CN108709941B (en) * | 2018-07-24 | 2020-11-17 | 曲阜师范大学 | Detection and analysis method of hydroxyl-containing neurosteroid |
| CN111366671A (en) * | 2019-04-25 | 2020-07-03 | 中南民族大学 | Chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneously detecting 18 steroid hormones in serum |
| CN111366671B (en) * | 2019-04-25 | 2020-10-30 | 中南民族大学 | Chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneously detecting 18 steroid hormones in serum |
| CN110824091A (en) * | 2019-12-17 | 2020-02-21 | 杭州度安医学检验实验室有限公司 | Steroid hormone detection method |
| CN112611827A (en) * | 2020-12-23 | 2021-04-06 | 中南民族大学 | Chemical derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry for simultaneously detecting 3 estrogens in serum |
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