CN107525855B - The screening method of antifungal drug chemical risk substance in a kind of washing product - Google Patents
The screening method of antifungal drug chemical risk substance in a kind of washing product Download PDFInfo
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Abstract
The invention discloses antifungal drug chemical risk substance screening method in a kind of washing product, include the following steps: that (1) establishes accurate mass database and the mass spectrum spectrum library of untested compound；(2) pre-treatment and detection of actual sample: pre-treatment steps, the obtained sample solutions such as washing product sample is extracted, is centrifuged, purifies, filters carry out ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection；(3) test result for obtaining step (2) and the accurate mass database established in step (1) and mass spectrum spectrum library compare and analyze, and carry out the screening of antifungal drug chemical risk substance.Antifungal drug chemical risk substance screening method accuracy height, high specificity, high sensitivity in the washing product that the present invention establishes are applicable to routine testing and the quality control of washing product.
Description
Technical field
The present invention relates to a kind of screening methods of chemical substance, are based on ultrahigh pressure liquid phase chromatography-four more particularly to one kind
The screening side of antifungal drug chemical risk substance in pole bar-electrostatic field orbit trap high resolution mass spectrum joint technology washing product
Method.
Background technique
1. there are potential hazard effects to consumer's health of human body for the chemical risk substance in washing product.
Washing product is exactly to pursue clean, health and fashion life for people to provide safeguard at the beginning of the birth, with society
Meeting makes constant progress, and people also constantly upgrade the pursuit of the life style of clean, health and fashion, washes shield to drive
Articles industry has stepped into the fast traffic lane of development and upgrading.Currently, washing product market product is dazzling, functionalization, sectionalization,
Specialized product continues to bring out, and meets the diversified demand of the majority of consumers.
Today of people's daily necessities is had become in washing product, safety is also more and more in widespread attention.
2014, Han Yi shampoo was detected by third party authority testing agency containing possible carcinogenic substance acrylamide, two of them
Preservative-methylisothiazolinone and methylchloroisothiazandnone are also detected exceeded nearly twice, easily cause human skin mistake
It is quick；As the baby shampoo of one of the significant product of Johnson & Johnson, maximum attraction is exactly not stimulate eyes, but the product is 2009
Year finds dioxanes containing noxious material by the U.S. and can release the quaternary ammonium salt component of formaldehyde；2010, Beijing's disease prevention
During spot-check domestic washing product, significant proportion product is detected phthalic acid ester, wherein the inspection of perfume for control centre
Extracting rate is up to 92.3% (phthalic acid ester is the environmental hormone for endangering human reproduction's ability)；In addition, three in bath accessory
Chlorine is raw and antibiotic in other washing products and hormone etc. are also potential chemical risk substance.
The shield product washed containing above-mentioned poisonous and harmful substance, which is used for a long time, can lead to that skin of face blackspot, atrophy be thinning, bone
The problems such as matter is loose, muscular atrophy, metabolic disorder, can seriously cause the generation of cancer.Businessman for its short-term bring effect,
To attract customer to buy, still use combined set system class, in certain or certain several prods added with the similar activity such as hormone at
Point, it is also convenient for escaping inspection.Therefore, harmfulness should not be underestimated.
2. the supervision of chemical risk substance relatively lags behind in washing product, prevent scarce capacity.
It is counted according to American National electronics nociceptive neurons system (NEISS), all kinds of washing products cause within 2001~2009 years
Security incident it is accumulative up to 7405.European Union's quick early warning system of the non-food stuff class consumer goods (RAPEX), U.S.'s Consumer Product Safety committee
Member's meeting (CPSC), U.S. Food and Drug Administration (FDA) have also issued to be directed in washing product adds chemicals in violation of rules and regulations
The Risk-warning of matter.
In recent years, China is put into terms of washing product security control financial resources, man power and material are more and more, but more
It drills in face of stronger product security incident, still seems unable to do what one wishes.Wherein main problem is the regulatory format in China mostly with subsequent
It based on remedying, works hard in terms of the prevention of product early period still inadequate, is mainly shown as that developed country puts into effect newly repeatedly
Rules and regulations, cause the product in China to be called back again and again, such as 2013, baby's washing product of Johnson & Johnson is called back 48 times；It is precious
The events such as problem mouthwash were recalled in 2011 by clean (China) Co., Ltd.
3. high resolution mass spectrum has advantage in terms of the screening of chemical risk substance.
Currently, domestic and international correlative study is mainly using triple quadrupole series connection matter for the quantitative detection of target compound
Spectrum.And the washing product detection method of high resolution mass spectrum technology is used to have not been reported.
Summary of the invention
It is increasingly strict with regulation, it is desirable that the chemical risk amount of material of detection is more and more, passes through multiple-reaction monitoring
The conventional method that mode is detected can no longer meet the requirement of this high flux examination, and the measurement of chemical risk substance should be to
Quickly develop with high throughput.Its resolution ratio of high resolution mass spectrum and Mass accuracy are substantially better than triple quadrupole mass spectrum, are that a kind of energy is same
When qualitative and quantitative mass spectrum；High-resolution can be realized in wide mass range, obtain substance accurate molecular weight；It obtains true same
Position element distribution；Have the function of highly sensitive tandem mass spectrum, realizes the accurate mass measurement of parent ion and daughter ion.Therefore, this hair
Bright technical problems to be solved, which are to provide one kind, has high-resolution and Mass accuracy, and substance essence can be obtained in wide mass range
True molecular weight information obtains true isotope distribution, has the function of highly sensitive tandem mass spectrum, realizes parent ion and daughter ion
Accurate mass measurement washing product in chemical risk substance screening method.
The detection method of antifungal drug chemical risk substance, includes the following steps: in a kind of washing product
The pre-treatment and detection of sample: the pre-treatment steps such as washing product sample is extracted, is centrifuged, purifies, filters obtain
Sample solution carry out ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection；The super-pressure
In liquid chromatogram-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection, chromatographic separation condition is as follows:
Chromatographic column: Waters ACQUITY UPLC BEH C8, length 50mm, internal diameter 2.1mm, 1.7 μm of partial size；Column temperature:
35℃；Flow velocity: 0.35mL min-1；Sample volume: 10 μ L；
The antifungal drug includes ketoconazole, clotrimazole, voriconazole and bifonazole.
The detection method of antifungal drug chemical risk substance in washing product of the present invention, wherein the superelevation
In pressure liquid chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection, mobile phase and gradient elution program are shown in Table 1:
1 chromatogram flow phase of table and gradient elution program
In the ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection, quadrupole rod-is quiet
Electric field orbit trap high resolution mass spectrum testing conditions are as follows:
Electron spray voltage: positive ion mode 3.5kV；Sheath atmospheric pressure: 35, arbitrary unit；Assist gas pressure power: 5, it is any single
Position；Ion source temperature: 350 DEG C；Transmission metal capillary temperature: 320 DEG C；Lens radio-frequency voltage: 50V；Scanning range: mass-to-charge ratio
100-800；First mass spectrometric full scan resolution ratio: 70000, full width at half maximum (FWHM)；Orbit trap maximum capacity: 1 × 106；Orbit trap is maximum
Injection length: 100ms；
The second level daughter ion full scan resolution ratio of data dependence: 17500, full width at half maximum (FWHM)；Window: mass-to-charge ratio ± 2 is isolated；Return
One changes collision energy: 20,40,60eV；Orbit trap maximum capacity: 1 × 105；Orbit trap maximum injection length: 100ms；Dynamic is arranged
Except the time: 6s.
The detection method of antifungal drug chemical risk substance in washing product of the present invention, wherein place before described
Reason includes the following steps:
The washing product sample of 0.2g is weighed into 10mL plastic centrifuge tube, 2mL saturated sodium chloride solution is added, is vortexed
30s carries out mixing demulsification, and 8mL methanol, ultrasonic extraction 30min is then added；Extracting solution is centrifuged under 10000rpm revolving speed
Supernatant after centrifugation is transferred in another 10mL plastic centrifuge tube by 10min, and 50mg carbon 18 and 50mg N- propyl is added
Ethylenediamine Solid Phase Extraction powder, vortex 30s draw supernatant extracting solution, and super-pressure liquid is carried out after 0.22 μm of filtering with microporous membrane
Phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection.
The screening method of antifungal drug chemical risk substance, includes the following steps: in a kind of washing product
(1) the accurate mass database and mass spectrum spectrum library, the accurate mass database for establishing untested compound include changing
Close name claim, the accurate mass number information of molecular formula, chromatographic retention and a precursor ion and two fragments characteristic ions,
The mass spectrum spectrum includes applying the second order ms figure generated after different collision energies respectively to untested compound in library；
(2) sample pre-treatments and detection method of the present invention；
(3) the accurate mass number of the untested compound of the test result and foundation that obtain sample pre-treatments and detection method
It is compared according to library and mass spectrum spectrum library, only when the accurate mass number of precursor ion and two fragments characteristic ions, chromatography
Retention time, the isotopic peak distribution of precursor ion and second order ms figure and accurate mass database and mass spectrum spectrum library information are whole
When matching, it may be determined that screening detects untested compound in actual sample.
The screening method of antifungal drug chemical risk substance in washing product of the present invention, wherein described to be measured
Accurate mass database and mass spectrum the spectrum library method for building up of compound specifically comprise the following steps:
Accurate mass database: compound concentration is the untested compound standard solution of 100 μ g/L respectively, using superelevation hydraulic fluid
The peristaltic pump direct injected that phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectroscope is equipped with, respectively in positive and negative ion
Analysis detection is carried out under mode, determines the precursor ion accurate mass number of corresponding untested compound；
Collision energy is applied to every kind of untested compound, obtains the fragment ion of every kind of compound, selects two responses strong
Higher fragment ion is spent as fragments characteristic ion；
In above process, the mass spectrums keys such as electron spray voltage, ion source temperature, sheath atmospheric pressure, resolution ratio are joined respectively
Number optimizes；
Compound concentration is the mixed standard solution of the untested compound of 100 μ g/L, optimizes ultrahigh pressure liquid phase chromatographic isolation item
Part obtains the chromatographic retention of every kind of compound；
It establishes accurate mass database: inputting title, molecular formula, the chemical abstracts number, precursor of every kind of compound respectively
Accurate mass number, chromatographic retention and the retention time window of exact mass of ion number, two fragments characteristic ions, in addition also
The response lag for inputting untested compound, when the response of the signal of untested compound is more than the threshold value, corresponding precursor ion is into one
Step carries out second mass analysis；Untested compound and its precursor ion and the accurate mass number information of fragment ion are shown in Table 2；
The accurate mass number information of 2 untested compound of table and its precursor ion and fragment ion
Establish mass spectrum spectrum library: compound concentration is the untested compound standard solution of 100 μ g/L respectively, using ultrahigh pressure liquid phase
The peristaltic pump direct injection analysis that chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectroscope is equipped with, is arranged a series of differences
Target compound is smashed the second order ms figure for obtaining every kind of compound by collision energy；It inputs, store all second order ms figures,
Obtain the mass spectrum spectrum library of whole untested compounds.
The mass spectrogram of antifungal drug chemical risk substance is as shown in Figure 1 in the present invention, wherein each code name refers to relationship such as
Under: 1. ketoconazoles；2. clotrimazole；3. voriconazole；4. bifonazole.
The screening method of chemical risk substance in washing product of the present invention, wherein untested compound using just from
Subpattern is analyzed by mass spectrometry；
The preparation of standard solution includes the following steps: to prepare 500-1000 μ g mL respectively-1Standard Stock solutions, in 4 DEG C
Under the conditions of be kept in dark place；Prepare 10 μ g mL-1Mixed standard solution be configured to a series of various concentrations then through methanol dilution
Matrix matching standard solution；The standard substance of antifungal drug is dissolved using acetonitrile solvent.Resist in washing product of the present invention true
The screening method difference from prior art of bacterium pharmaceutical chemistry specified risk material is:
The present invention is established based on ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum joint technology
Washing product in antifungal drug chemical risk substance screening method, will effectively solve antifungal drug in washing product
Technical problem is confirmed in screening, can be used to carry out the formulation work of the risk assessment of poisonous and harmful substance and limit standard in washing product
Make, to reduce the occurrence probability for damaging event to consumer by chemical risk substance, builds consumption ring in good health
Border；Research achievement can also guide enterprise to be avoided in process of producing product using the chemical risk substance for leading to security risk,
Consumer is set more clearly to recognize the potential hazard of chemical risk substance in washing product, to improve the general safety of society
Consumption view generates good social benefit.
The relevant laws and regulations and standard of washing product are to measure and control the main means and technology of product quality characteristics
Foundation, enterprise etc. have only known about these laws and regulations and standard, can just effectively facilitate the health and normal development for washing shield product,
To make product obtain good economic benefits.The screening method that the present invention establishes can provide effective technology branch for coherent detection mechanism
Every laws and regulations on the management standard with raising washing product, the market economy rule of specification washing product industry are further improved in support
With management order, the capacity for technological innovation of washing product industry is improved, shortens the technological gap with international washing product industry, it will
There is important economic significance to the sustainable and healthy development for accelerating Chinese relevant enterprise.
The method of the invention has high-resolution and Mass accuracy, and substance accurate molecular can be obtained in wide mass range
Amount, obtains true isotope distribution, has the function of highly sensitive tandem mass spectrum, realizes the accurate mass of parent ion and daughter ion
The screening method of chemical risk substance in the washing product of measurement.
The present invention establishes a kind of based on the combination of ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum
The rapid screening and quantitative analysis strategy of antifungal drug in accurate mass database and the washing product in spectrum library.By to this hair
The detection limit of bright method, quantitative limit, linear relationship, the investigation of stability and matrix effect and the detection of actual sample, this
Invent rapid analysis method accuracy height established, high specificity, high sensitivity, be applicable to the routine testing of washing product with
Quality of production control.
With reference to the accompanying drawing to the screening method of antifungal drug chemical risk substance in washing product of the invention make into
One step explanation.
Detailed description of the invention
Fig. 1 is the mass spectrogram of antifungal drug chemical risk substance in the present invention, wherein it is as follows that each code name refers to relationship:
1. ketoconazole；2. clotrimazole；3. voriconazole；4. bifonazole.
Specific embodiment
Embodiment 1
The detection method of antifungal drug chemical risk substance, includes the following steps: in a kind of washing product
Pre-treatment steps, the obtained sample solutions such as washing product sample is extracted, is centrifuged, purifies, filters carry out superelevation
Pressure liquid chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection；Ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field
In orbit trap high resolution mass spectrum analysis detection, chromatographic separation condition is as follows:
Chromatographic column: Waters ACQUITY UPLC BEH C8, length 50mm, internal diameter 2.1mm, 1.7 μm of partial size；Column temperature:
35℃；Flow velocity: 0.35mL min-1；Sample volume: 10 μ L, mobile phase and gradient elution program are shown in Table 1:
1 chromatogram flow phase of table and gradient elution program
In the ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection, quadrupole rod-is quiet
Electric field orbit trap high resolution mass spectrum testing conditions are as follows:
Electron spray voltage: positive ion mode 3.5kV；Sheath atmospheric pressure: 35, arbitrary unit；Assist gas pressure power: 5, it is any single
Position；Ion source temperature: 350 DEG C；Transmission metal capillary temperature: 320 DEG C；Lens radio-frequency voltage: 50V；Scanning range: mass-to-charge ratio
100-800；First mass spectrometric full scan resolution ratio: 70000, full width at half maximum (FWHM)；Orbit trap maximum capacity: 1 × 106；Orbit trap is maximum
Injection length: 100ms；
The second level daughter ion full scan resolution ratio of data dependence: 17500, full width at half maximum (FWHM)；Window: mass-to-charge ratio ± 2 is isolated；Return
One changes collision energy: 20,40,60eV；Orbit trap maximum capacity: 1 × 105；Orbit trap maximum injection length: 100ms；Dynamic is arranged
Except the time: 6s.
Pre-treatment includes the following steps:
The washing product sample of 0.2g is weighed into 10mL plastic centrifuge tube, 2mL saturated sodium chloride solution is added, is vortexed
30s carries out mixing demulsification, and 8mL methanol, ultrasonic extraction 30min is then added；Extracting solution is centrifuged under 10000rpm revolving speed
Supernatant after centrifugation is transferred in another 10mL plastic centrifuge tube by 10min, and 50mg carbon 18 and 50mg N- propyl is added
Ethylenediamine Solid Phase Extraction powder, vortex 30s draw supernatant extracting solution, and super-pressure liquid is carried out after 0.22 μm of filtering with microporous membrane
Phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection.
The antifungal drug includes ketoconazole, clotrimazole, voriconazole and bifonazole.
Using the method for the present embodiment 1 20 kinds of washing product samples have been carried out with the inspection of antifungal drug chemical risk substance
It surveys.Contain antifungal drug ingredient the result shows that being unscreened and having found sample.
Embodiment 2
One, instrument and reagent
Rapidly and efficiently liquid chromatographic system (the Thermo Fisher company, the U.S.) of Dionex UltiMate 3000；Q
Exactive Focus quadrupole rod-electrostatic field orbit trap high-resolution mass spectrometer (Thermo Fisher company, the U.S.)；Milli-Q
Ultrapure water instrument (Millipore company, the U.S.)；2 turbula shaker of Vortex-Genie (U.S. Scientific
Industries company).
Methanol, acetonitrile (chromatographically pure, Thermo Fisher company, the U.S.)；Ammonium hydroxide, formic acid (chromatographically pure, U.S. Dima
Technology company)；Use for laboratory water is deionized water.Carbon 18, N- propyl ethylenediamine and ketjenblack EC (Germany
Macherey-Nagel company)；Multi-walled carbon nanotube (German Miltenyi Biotec GmbH company)；Anhydrous magnesium sulfate, chlorine
Change sodium (Guangzhou West Gansu Province chemical industry Co., Ltd)；0.22 μm of miillpore filter (Pall company, the U.S.).
500-1000 μ g mL is prepared respectively-1Standard Stock solutions, be kept in dark place under the conditions of 4 DEG C.Prepare 10 μ g mL-1Mixed standard solution be configured to a series of matrix matching standard solution of various concentrations then through methanol dilution.
The information such as molecular formula, molecular weight, chemical abstracts number, Determination of oil-water partition coefficient and the preparation solvent of target compound are shown in
Table 3.
Title, molecular formula, molecular weight, chemical abstracts number, Determination of oil-water partition coefficient and the preparation solvent of 3 target compound of table
Etc. information
Two, the foundation of accurate mass database and mass spectrum spectrum library
Accurate mass database include compound name, molecular formula (for calculate isotopic peak distribution), retention time and
The accurate mass number (table 4) of precursor ion and two fragments characteristic ions.Compose in library includes for every kind of compound using different
The second order ms figure generated after collision energy through analysis.Spectrum library can be regarded as to sampled data through accurate mass database screening
Auxiliary confirmation means afterwards.
Accurate mass database and mass spectrum the spectrum library method for building up of untested compound specifically comprise the following steps:
Accurate mass database: compound concentration is the untested compound standard solution of 100 μ g/L respectively, using superelevation hydraulic fluid
The peristaltic pump direct injected that phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectroscope is equipped with, respectively in positive and negative ion
Analysis detection is carried out under mode, determines the precursor ion accurate mass number of corresponding untested compound；
Collision energy is applied to every kind of untested compound, obtains the fragment ion of every kind of compound, selects two responses strong
Higher fragment ion is spent as fragments characteristic ion；
In above process, the mass spectrums keys such as electron spray voltage, ion source temperature, sheath atmospheric pressure, resolution ratio are joined respectively
Number optimizes；
Compound concentration is the mixed standard solution of the untested compound of 100 μ g/L, optimizes ultrahigh pressure liquid phase chromatographic isolation item
Part obtains the chromatographic retention of every kind of compound；
It establishes accurate mass database: inputting title, molecular formula, the chemical abstracts number, precursor of every kind of compound respectively
Accurate mass number, chromatographic retention and the retention time window of exact mass of ion number, two fragments characteristic ions, in addition also
The response lag for inputting untested compound, when the response of the signal of untested compound is more than the threshold value, corresponding precursor ion is into one
Step carries out second mass analysis；
Establish mass spectrum spectrum library: compound concentration is the untested compound standard solution of 100 μ g/L respectively, using ultrahigh pressure liquid phase
The peristaltic pump direct injection analysis that chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectroscope is equipped with, is arranged a series of differences
Target compound is smashed the second order ms figure for obtaining every kind of compound by collision energy；It inputs, store all second order ms figures,
Obtain the mass spectrum spectrum library of whole untested compounds.
The mass spectrogram of antifungal drug chemical risk substance is as shown in Figure 1 in the present invention, wherein each code name refers to relationship such as
Under: 1. ketoconazoles；2. clotrimazole；3. voriconazole；4. bifonazole.
Three, sample pre-treatments
The washing product sample of 0.2g is weighed into 10mL plastic centrifuge tube, 2mL saturated sodium chloride solution is added, is vortexed
30s carries out mixing demulsification, and 8mL methanol, ultrasonic extraction 30min is then added；Extracting solution is centrifuged under 10000rpm revolving speed
Supernatant after centrifugation is transferred in another 10mL plastic centrifuge tube by 10min, and 50mg carbon 18 and 50mg N- propyl is added
Ethylenediamine Solid Phase Extraction powder, vortex 30s draw supernatant extracting solution, and super-pressure liquid is carried out after 0.22 μm of filtering with microporous membrane
Phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection.
Four, chromatographic separation condition
Chromatographic column: Waters ACQUITY UPLC BEH C8, length 50mm, internal diameter 2.1mm, 1.7 μm of partial size；Column temperature:
35℃；Flow velocity: 0.35mL min-1；Sample volume: 10 μ L, mobile phase and gradient elution program are shown in Table 1:
1 chromatogram flow phase of table and gradient elution program
Five, Mass Spectrometer Method condition
Electron spray voltage: positive ion mode 3.5kV；Sheath atmospheric pressure: 35, arbitrary unit；Assist gas pressure power: 5, it is any single
Position；Ion source temperature: 350 DEG C；Transmission metal capillary temperature: 320 DEG C；Lens radio-frequency voltage: 50V；Scanning range: mass-to-charge ratio
100-800；First mass spectrometric full scan resolution ratio: 70000, full width at half maximum (FWHM)；Orbit trap maximum capacity: 1 × 106；Orbit trap is maximum
Injection length: 100ms；
The second level daughter ion full scan resolution ratio of data dependence: 17500, full width at half maximum (FWHM)；Window: mass-to-charge ratio ± 2 is isolated；Return
One changes collision energy: 20,40,60eV；Orbit trap maximum capacity: 1 × 105；Orbit trap maximum injection length: 100ms；Dynamic is arranged
Except the time: 6s.
Six, experimental data compares analysis
By the accurate mass database and mass spectrum of sample detection experimental data and the untested compound of foundation compose library into
Row compares analysis, only when the accurate mass number of precursor ion and two fragments characteristic ions, chromatographic retention, precursor ion
Isotopic peak distribution and second order ms figure and accurate mass database and mass spectrum spectrum library in when all matching, be as a result just shown as
Positive sample.
Specifically comprise the following steps:
Mass number is set and extracts window, retention time window and isotope distribution threshold parameter, according to set parameter,
Sample data collected is compared:
First, according to the parameter of setting, as mass number extracts window ± 5ppm, chromatographic peak area is not less than 1 × 106, into
The extraction of precursor ion accurate mass number in row database；If there is targeted in database in sample full scan acquisition data
The precursor ion mass number of object is closed, and chromatographic peak area is not less than 1 × 106, then be considered as precursor ion accurate mass number compare at
Function；
Second, chromatographic retention, the retention time window standard deviation of setting is ± 3, if the chromatography of precursor ion
Retention time is fallen in corresponding ± 3 range of retention time standard deviation in the database, then is considered as retention time successful match；
Third carries out isotope distribution calculating according to the molecular formula of untested compound, and the threshold value of setting is 90%, works as sample
The isotope distribution of precursor ion and database compare matching degree 90% in data, then are considered as successful match；
4th, according to two fragment ions of two fragments characteristic ions and experimental result acquisition acquisition in database
Compare situation, if in experiment value and database fragment ion accurate mass number deviation be no more than ± 5ppm, be considered as match at
Function；
5th, the second order ms figure that experiment acquisition obtains is compared with mass spectrum spectrum library；At this stage, precursor ion,
Whole fragment ions and relative ion abundance are compared than, if all coincide, are considered as successful match.
Seven, result
Using the method for the present embodiment 2 20 kinds of washing product samples have been carried out with the inspection of antifungal drug chemical risk substance
It surveys.Contain antifungal drug ingredient the result shows that being unscreened and having found sample.
Eight, it discusses
1, the foundation of screening method
To give full play to advantage of the electrostatic field orbit trap high resolution mass spectrum in Screening analysis, this research uses full scan-
The second level daughter ion scanning mode of data dependence.In this acquisition mode, mass spectrum carries out total quality number (m/z 100- first
800) it scans, when the ion of target compounds certain in database is detected and its intensity is not less than a certain given threshold,
Then the ion, which will be admitted in energetic encounter pond, carries out fragmentation, and generates daughter fragment ion.
In the compound screening stage, the detection of target substance is mainly according to three big key parameters: precursor ion accurate mass
Number, retention time and isotopic peak distribution.Accurate mass number extracts detection sensitivity and selection of the setting for method of window
Property has vital influence.In the present invention, it is 100 μ g L that concentration, which has been respectively adopted,-1Mixed standard solution and 500 μ g
kg-1Standardized sample carry out the optimization of this parameter.The experimental results showed that mass number deviation is better than respectively in two kinds of solution
2ppm and 3.5ppm.In view of subsequent fragments characteristic ion and second level spectrogram are confirmed, while false negative result is also avoided as far as possible
Appearance, originally grind and set 5ppm for the extraction window of mass number, not only can guarantee the appearance of no false negative result, but also other side at this time
The selectivity of method detection and sensitivity do not make significant difference.For retention time parameter, retention time window it is reasonable setting for
The accuracy of screening results plays more important role.Due to the behaviour of different high resolution mass spectrum systematic differences and different personnel
Make, retention time may have gentle drift, and therefore, this research equipment retention time window is Average residence time ± 3
× retention time standard deviation.In the mass spectrum full scan stage, since the setting of different resolution is to the selectivity of method and sensitive
For degree there is also certain influence, this research has also carried out detailed optimization to mass resolution parameter.Three differences of experimental setup
Resolution ratio (30000,50000,70000 full width at half maximum (FWHM)) carry out data acquisition respectively, the results showed that, in 70000 fulls width at half maximum (FWHM)
Resolution ratio under, the Mass accuracy highest of all target compounds, sensitivity, without significant difference, and is stilld provide compared with the above two
10~15 scanning element is to obtain excellent chromatographic peak profile.
In the confirmation stage, this research acquires the second order ms figure of each target compound, and has recorded every kind of compound
Two fragments characteristic exact mass of ion numbers.In first mass spectrometric scan phase, the resolution ratio of 70000 fulls width at half maximum (FWHM) is had been provided enough
High selectivity, at this point, sensitivity should be the main aspect considered, therefore, in the confirmation stage, we set mass resolution
For lower and common 17500 full width at half maximum (FWHM).To obtain more complete second order ms figure, this research uses normalization impact energy
20,40 and 60eV is measured, and records the accurate mass number of the higher fragment ion of two of them response intensity into database.In high score
It distinguishes in mass spectral analysis, for some key parameters, such as spray voltage, capillary temperature, gas curtain gas, purge gass and lens radio frequency
Voltage has also carried out detailed optimization.
2, the optimization of sample extraction and purification method
The present invention uses the sample extraction mode of ultrasound assisted extraction.Firstly, to there are commonly solvent methanol, acetonitrile with
And the isometric ratio mixed solvent methanol+acetonitrile (1:1, v/v) of the two has carried out the investigation of rate of recovery effect.In view of solvent
To the efficient and harmonious of all target compounds extraction, this research chooses methanol as Extraction solvent first.
When only using solvent extraction, when extracting solution crosses upper machine analysis after miillpore filter, the matrix in washing product still can be to mesh
There are larger matrix interferences for the analysis of mark compound, and therefore, for further purification sample extracting solution, the present invention takes relatively simple
And efficient sample purification mode-dispersive solid-phase extraction.Experiment is respectively provided with 6 groups of dispersing agents, respectively 50mg carbon 18,
50mg N- propyl ethylenediamine, 25mg ketjenblack EC, 10mg multi-walled carbon nanotube, 18+50mg N- propyl second two of 50mg carbon
18+25mg ketjenblack EC of amine and 50mg carbon carries out rate of recovery experiment.The result shows that 18+50mgN- propyl second of 50mg carbon
Diamines is preferable to the clean-up effect of sample.Therefore, the present invention selects 18+50mg N- propyl ethylenediamine of 50mg carbon as sample
Cleanser.
3, linear relationship, detection limit and quantitative limit
Hybrid standard stock solution is successively diluted to the hybrid working of series of concentrations gradient with processed sample solution
Solution is measured, with the molecular ion peak area (y) of each target substance to concentration (x) under the conditions of the chromatographic mass spectrometry of optimization
It is linearly investigated, the range of linearity, detection limit and the quantitative limit result table 5 of target compound.The linear regression of target compound
Coefficient is all larger than 0.99, shows that the linear relationship of method is good, can carry out accurate quantification.Respectively with 3 times and 10 times of signal-to-noise ratio
The detection limit and quantitative limit of target compound are calculated, as a result such as table 5.
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.
Claims (5)
1. the detection method of antifungal drug chemical risk substance in a kind of washing product, characterized by the following steps:
Sample pre-treatments and detection method: weighing the washing product sample of 0.2g into 10mL plastic centrifuge tube, and 2mL saturation is added
Sodium chloride solution, vortex 30s carry out mixing demulsification, and 8mL methanol, ultrasonic extraction 30min is then added；Extracting solution is in 10000rpm
It is centrifuged 10min under revolving speed, the supernatant after centrifugation is transferred in another 10mL plastic centrifuge tube, 18 He of 50mg carbon is added
50mg N- propyl ethylenediamine Solid Phase Extraction powder, vortex 30s draw supernatant extracting solution, laggard through 0.22 μm of filtering with microporous membrane
Row ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection；
In the ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection, ultrahigh pressure liquid phase chromatography
Separation condition is as follows:
Chromatographic column: Waters ACQUITY UPLC BEH C8, length 50mm, internal diameter 2.1mm, 1.7 μm of partial size；Column temperature: 35 DEG C；
Flow velocity: 0.35mL min-1；Sample volume: 10 μ L；
The antifungal drug includes ketoconazole, clotrimazole, voriconazole and bifonazole；
Mobile phase and gradient elution program are shown in Table 1:
1 chromatogram flow phase of table and gradient elution program
In the ultrahigh pressure liquid phase chromatography-quadrupole rod-electrostatic field orbit trap high resolution mass spectrum analysis detection, quadrupole rod-electrostatic field
Orbit trap high resolution mass spectrum testing conditions are as follows:
Electron spray voltage: positive ion mode 3.5kV；Sheath atmospheric pressure: 35, arbitrary unit；Assist gas pressure power: 5, arbitrary unit；From
Source temperature: 350 DEG C；Transmission metal capillary temperature: 320 DEG C；Lens radio-frequency voltage: 50V；Scanning range: mass-to-charge ratio 100-
800；First mass spectrometric full scan resolution ratio: 70000, full width at half maximum (FWHM)；Orbit trap maximum capacity: 1 × 106；The injection of orbit trap maximum
Time: 100ms；
The second level daughter ion full scan resolution ratio of data dependence: 17500, full width at half maximum (FWHM)；Window: mass-to-charge ratio ± 2 is isolated；Normalization
Collision energy: 20,40,60eV；Orbit trap maximum capacity: 1 × 105；Orbit trap maximum injection length: 100ms；When dynamic excludes
Between: 6s.
2. the screening method of antifungal drug chemical risk substance in a kind of washing product, characterized by the following steps:
(1) accurate mass database and the mass spectrum spectrum library of untested compound are established, the accurate mass database includes compound
The accurate mass number information of title, molecular formula, chromatographic retention and a precursor ion and two fragments characteristic ions, it is described
It includes applying the second order ms figure generated after different collision energies respectively to untested compound that mass spectrum, which is composed in library,；
(2) sample pre-treatments and detection method described in claim 1；
(3) the accurate mass database of the untested compound of the test result and foundation that obtain sample pre-treatments and detection method
It is compared with mass spectrum spectrum library, only when the accurate mass number of precursor ion and two fragments characteristic ions, chromatography retain
Time, the isotopic peak distribution of precursor ion and second order ms figure are all matched with accurate mass database and mass spectrum spectrum library information
When, determine that screening detects untested compound in actual sample.
3. the screening method of antifungal drug chemical risk substance, feature exist in washing product according to claim 2
In: the accurate mass database and mass spectrum spectrum library method for building up specifically comprise the following steps:
Accurate mass database: compound concentration is the untested compound standard solution of 100 μ g/L respectively, using ultrahigh pressure liquid phase color
The peristaltic pump direct injected that spectrum-quadrupole rod-electrostatic field orbit trap high resolution mass spectroscope is equipped with, respectively in positive and negative ion mode
Lower carry out analysis detection determines the precursor ion accurate mass number of corresponding untested compound；
Collision energy is applied to every kind of untested compound, obtains the fragment ion of every kind of compound, select two response intensities compared with
High fragment ion is as fragments characteristic ion；
In above process, respectively to the mass spectrums key parameters such as electron spray voltage, ion source temperature, sheath atmospheric pressure, resolution ratio into
Row optimization；
Compound concentration is the mixed standard solution of the untested compound of 100 μ g/L, optimizes ultrahigh pressure liquid phase chromatographic separation condition, obtains
To the chromatographic retention of every kind of compound；
It establishes accurate mass database: inputting title, molecular formula, the chemical abstracts number, precursor ion of every kind of compound respectively
Accurate mass number, chromatographic retention and the retention time window of accurate mass number, two fragments characteristic ions, in addition also input
The response lag of untested compound, when untested compound signal response be more than the threshold value when, corresponding precursor ion further into
Row second mass analysis；Untested compound and its precursor ion and the accurate mass number information of fragment ion are shown in Table 2；
The accurate mass number information of 2 untested compound of table and its precursor ion and fragment ion
Establish mass spectrum spectrum library: compound concentration is the untested compound standard solution of 100 μ g/L respectively, using ultrahigh pressure liquid phase color
The peristaltic pump direct injection analysis that spectrum-quadrupole rod-electrostatic field orbit trap high resolution mass spectroscope is equipped with, is arranged a series of differences and touches
Energy is hit, target compound is smashed to the second order ms figure for obtaining every kind of compound；It inputs, store all second order ms figures, i.e.,
Obtain the mass spectrum spectrum library of whole untested compounds.
4. the screening method of antifungal drug chemical risk substance, feature exist in washing product according to claim 3
In: it is compared that specific step is as follows:
Mass number is set and extracts window, retention time window and isotope distribution threshold parameter, according to set parameter, to institute
The sample data of acquisition is compared:
First, according to the parameter of setting, as mass number extracts window ± 5ppm, chromatographic peak area is not less than 1 × 106, carry out data
The extraction of precursor ion accurate mass number in library；If there is target compound in database in sample full scan acquisition data
Precursor ion mass number, and chromatographic peak area is not less than 1 × 106, then it is considered as precursor ion accurate mass number and compares successfully；
Second, chromatographic retention, the retention time window standard deviation of setting is ± 3, if the chromatography of precursor ion retains
Time falls in corresponding ± 3 range of retention time standard deviation in the database, then is considered as retention time successful match；
Third carries out isotope distribution calculating according to the molecular formula of untested compound, and the threshold value of setting is 90%, works as sample data
The isotope distribution and database of middle precursor ion compare matching degree 90%, then are considered as successful match；
4th, according to the comparison of two fragment ions of two fragments characteristic ions and experimental result acquisition acquisition in database
Situation is considered as successful match if fragment ion accurate mass number deviation is no more than ± 5ppm in experiment value and database；
5th, the second order ms figure that experiment acquisition obtains is compared with mass spectrum spectrum library；At this stage, precursor ion, whole
Fragment ion and relative ion abundance are compared than, if all coincide, are considered as successful match.
5. the screening method of antifungal drug chemical risk substance, feature exist in washing product according to claim 4
In:
Untested compound is analyzed by mass spectrometry using positive ion mode；
The preparation of standard solution includes the following steps: to prepare 500-1000 μ g mL respectively-1Standard Stock solutions, in 4 DEG C of conditions
Under be kept in dark place；Prepare 10 μ g mL-1Mixed standard solution be configured to a series of base of various concentrations then through methanol dilution
Matter matching criteria solution；The standard substance of antifungal drug is dissolved using acetonitrile solvent.
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