CN101696962A - Derivatization method of environmental estrogens - Google Patents
Derivatization method of environmental estrogens Download PDFInfo
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- CN101696962A CN101696962A CN200910095092A CN200910095092A CN101696962A CN 101696962 A CN101696962 A CN 101696962A CN 200910095092 A CN200910095092 A CN 200910095092A CN 200910095092 A CN200910095092 A CN 200910095092A CN 101696962 A CN101696962 A CN 101696962A
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- BFPYWIDHMRZLRN-UHFFFAOYSA-N 17alpha-ethynyl estradiol Natural products OC1=CC=C2C3CCC(C)(C(CC4)(O)C#C)C4C3CCC2=C1 BFPYWIDHMRZLRN-UHFFFAOYSA-N 0.000 abstract 1
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Abstract
The invention discloses a derivatization method of environmental estrogens (estrone, 17beta-estradiol, estriol, 17alpha-ethynylestradiol), which is a treatment method and a core technology before a gas chromatography-mass spectrum (GC-MS) is used for analyzing the samples of the substances. The method comprises the following steps: firstly, leading high-purity nitrogen in at a room temperature to dry a standard mixed solution containing environmental estrogens; adding derivative reagents N, O-BSTFA and pyridine to react at room temperature (20 DEG C)-50 DEG C to obtain derivative products; drying; adding a sampling solvent and an internal standard; sampling and analyzing. The method has the advantages of simple and convenient experimental operation, time saving and low energy consumption and cost, simultaneously derivates the four environmental estrogens without generating side products, has favorable linear correlation among various derivative products and achieves an instrument detection limit up to 0.5pg/ muL, thereby being applied to the GC-MS detection of environmental estrogens in water bodies, sediment, biological samples, and the like.
Description
Technical field
The invention belongs to the Environmental Analytical Chemistry field, the derivatization method that relates to a kind of environmental estrogens (oestrone, 17 beta estradiols, estriol, 17 α-ethinyl estradiol) is sample-pretreating method and the core technology that gas chromatograph-mass spectrometer (GCMS) is used to analyze this type of material.
Background technology
Environmental hormone (Environmental Hormones) is meant the xenobiotics that can disturb the mankind or all links of wild animal internal system and functions such as reproduction, nerve and immune system are exerted an influence that exists in the environment.Environmental hormone has very strong toxicity and biological accumulation, can disturb the internal system of animal and human's class by potable water and food chain enrichment, (ng/L level) promptly produces serious harm to biosome under extremely low concentration, not only influence human health and continue procreation, also threaten biological race's living or death.A series of environmental problems that environmental hormone caused have caused global great attention.Environmental hormone mainly comprises the hormonal compounds of natural hormone (estrogen and androgen), synthetic and has endocrine activity or the compound of anti-endocrine activity, with environmental estrogens harm maximum, also is focus and the forward position of studying both at home and abroad at present wherein.The present invention is a research object with ubiquity in the environment and four kinds of bigger typical environment estrogen of harm, comprise naturally occurring oestrone (Estrone in animal and human's body, E1), 17 beta estradiols (estradiol) (17 β-Estradiol, E2), estriol (Estriol, E3), 17 α that the are used for oral contraceptive-ethinyl estradiol of synthetic (17 α-ethynylestradoil, EE2).The chemical constitution of environmental estrogens E1, E2, EE2, E3 and derivatization product thereof is as follows:
This class material has steroid ring, chemical property is stable, in case enter environment and biological body accumulation, just be difficult to decompose, relative other environmental hormones, stronger endocrine interference effect is arranged, especially remarkable to the influence of ecologic environment and human health, even ambient concentration also can work the mischief to biosome below 0.1ng/L.
Because the concentration of above-mentioned this type of environmental estrogens in environment is extremely low, be generally ng/L even pg/L level, and the interference of complex matrices in the sample, make that its monitoring difficulty is bigger, also be in the starting stage with regard to world wide at present, do not have ready-made standard method of analysis.Therefore, R and D have high sensitivity and optionally the analysis and detection technology of low concentration environmental estrogens is significant.In the environmental sample detection of environmental estrogens generally comprise extraction, purification, concentrate, pre-treatment step and instrumental analysis such as derivatization, by Solid-Phase Extraction or solid-phase microextraction, the extraction of sample, purification, concentrate and can finish in a step.The most frequently used instrument analytical method is GC-MS(gas chromatography-mass spectrography) (GC-MS) and liquid chromatograph mass spectrography method (LC-MS) at present, yet LC-MS instrument price is comparatively expensive, limited it and be extensive use of, GC-MS is subjected to the favor that organic micro-pollutant is analyzed in the environment with its good detection sensitivity and relatively low operating cost always.But environmental estrogens all contains hydroxy functional group basically, polarity is bigger, directly analyze with GC-MS, in gas chromatographic column, be easy to generate absorption, thereby make the value of detecting more on the low side than actual value, sensitivity descends, and is not suitable for trace analysis, need to change into the derivant of lower, the more volatile and Heat stability is good of polarity, thereby improve detection sensitivity and improve chromatographic peak profile by derivatization.Therefore the development of environmental estrogens derivatization technology becomes GC-MS and is applied to the core technology that this type of species analysis detects.
At present, the derivatization about environmental estrogens does not still have patent at home.Abroad in the various derivatization reagents of paper report, with N, the two trimethyl silicon based trifluoroacetamides of O-(N, O-bis (trimethylsilyl) trifluoroacetamide is BSTFA) commonly used.(A.Shareef such as A.Shareef, M.J.Angove, J.D.Wells.Journal of Chromatography A, 2006,1108:121-128) with BSTFA be derivatization reagent, (Tert-butyldimethylsilyl-chlorosilane TMCS) is catalyzer with tert-butyl chloro-silicane, in 45-105 ℃ of scope, studied of the influence of derivatization temperature, pointed out that best derivatization temperature is 75-80 ℃ for E1 and EE2 derivatization.(A.Hibberd such as A.Hibberd, K.Maskaoui, Z.Zhang, et al.Talanta, 2009,77:1315-1321) in testing environment water sample and sediment sample during environmental estrogens, by adding 50 μ LBSTFA (containing catalyzer 1%TMCS) and 50 μ L pyridines, under the condition of 60-70 ℃ of heating 30min, E1, E2, EE2 have been carried out derivatization, and detected its content with GC-MS.(M.Esperanza such as M.Esperanza, M.T.Suidan, R.M.Vega, et al.Chemosphere, 2007,66:1535-1544) in detecting municipal sewage plant's water sample and solid sample during environmental estrogens, by adding BSTFA (containing catalyzer 10%TMCS) and pyridine, under the condition of 70 ℃ of heating 15h, E1, E2, EE2, E3 are carried out derivatization, detected its content with GC-MS then.More than studies show that, is E1, the E2 of derivatization reagent, the derivatization of EE2, E3 with BSTFA at present, all is to carry out under the condition of heating basically, and needs to add catalyzer, and experimental implementation is comparatively loaded down with trivial details and time-consuming.(Y.G.Zuo such as Y.G.Zuo, K.Zhang, Y.J.Lin.Journal of Chromatography A, 2007,1148:211-218) inquired into the derivatization of environmental estrogens E1, E2, EE2, E3 under the microwave-assisted heating condition, though can shorten to the derivative reaction time within the 1min, owing to introduced the microwave-assisted heating technique, further increased experimental procedure, made the derivatization process become complicated more.
In a word, the deficiency that the estrogenic derivatization method of current environment exists is: derivative reaction need carry out under the condition of heating, heating-up temperature is 60-80 ℃, and be 30min-15h heat time heating time, and needs to add catalyzer, this makes derivatization experiment energy consumption and cost rise, complex operation step, experimental period is longer, in the actual environment sample detection, strengthen the sample loss, reduced accuracy in detection.
Summary of the invention
The objective of the invention is to the deficiency that exists in the present environmental estrogens derivatization method, propose a kind of derivatization new method.This method have experimental implementation easy, save time, advantage that derivatization is effective.
For achieving the above object, the present invention under lower temperature (20-50 ℃, under the normal temperature condition also can), with N, the two trimethyl silicon based trifluoroacetamides (BSTFA) of O-are derivatization reagent, four kinds of environmental estrogens oestrone (E1), 17 beta estradiols (E2), estriol (E3), 17 α-ethinyl estradiol (EE2) have been carried out derivatization, and need not to add catalyzer, the derivatization time shortens to 3min-8min, simplified the derivatization experimental procedure greatly, shorten the reaction time, reduced energy consumption and cost, reduced the sample loss.Adopt method of the present invention to carry out environmental estrogens sample behind the derivatization, detect through gas chromatograph-mass spectrometer (GCMS) (GC-MS), can be with four kinds of environmental estrogens E1, E2, EE2, E3 while derivatization, no coupling product generates, the linear dependence of each derivatization product is good, and the instrument detection limit can reach 0.5pg/ μ L.
Be dissolved in the methyl alcohol after environmental estrogens standard items E1, E2, EE2, the accurate weighing of E3, be made into certain density standard mixed solution.Because the amount of analytic sample is generally trace (pg level), the precision of analytical balance is difficult to reach requirement, so the dilution certain multiple is beneficial to accurately quantitatively after must being dissolved in methyl alcohol.During actual the use,, make derivative reaction more fast, fully for fear of the interference of methanol solvate; the standard mixed solution that the present invention will contain finite concentration E1, E2, EE2, E3 uses high pure nitrogen slowly to dry up; in addition, because the protection of high pure nitrogen can reduce the loss of material.Then under the certain reaction temperature and time, add certain BSTFA and pyridine in the standard items that dry up and carry out derivatization, the addition of BSTFA is suitable, use very little, can make derivative reaction incomplete, can not get good derivatization effect, and can the chromatogram cornice be injured too much, shorten its serviceable life.With the derivatization process of BSTFA in add a certain amount of pyridine, not only can stop the generation of EE2 derivatization accessory substance TMS-E1 and mono-TMS-EE2, can also play the effect of solvent, help the abundant reaction of determinand and BSTFA, eliminate the little assorted peak that occurs in the chromatogram.For accurately quantitative, and can reduce the injury of derivatization reagent to chromatographic column, must dry up a certain amount of high-purity organic solvent of adding after derivative reaction is finished, sampling injection GC-MS detects then.
Above-mentioned environmental estrogens standard items E1, E2, EE2, E3 are solid, and purity is more than 97%, are dissolved in being made into the standard mixed solution in the methyl alcohol, and sealing is placed on below-20 ℃ and preserves, and methyl alcohol is chromatographically pure.
Above-mentioned high pure nitrogen purity is more than 99.99%.
Above-mentioned BSTFA rank is the derivatization level, and purity is greater than 98%, and pyridine is a chromatographically pure.
Above-mentioned sample introduction solvent is a chromatographically pure level high-purity organic solvent, can be methyl alcohol, methylene chloride, acetone, ethyl acetate or normal hexane.
The used chromatographic column of above-mentioned GC-MS is Supelco SPB
TM-5 or J﹠amp; W Scientific DB-5MS capillary column.
The present invention specifically comprises the steps:
1, the first step is at first got standard mixed solution that 100 μ L contain finite concentration (1pg/ μ L-100ng/ μ L) E1, E2, EE2, E3 in the 1.5mL chromatogram bottle, and chromatogram bottle is put into Nitrogen evaporator, feeds high pure nitrogen and slowly dries up.
Need not heating when using high pure nitrogen that the standard mixed solution is dried up in Nitrogen evaporator, normal temperature condition gets final product down.
2, in second step, add a certain amount of BSTFA and pyridine, vibration places normal temperature environment or puts into the heated at constant temperature case after mixing, and reacts 3min-8min down for-50 ℃ in room temperature (20 ℃), obtains the derivatization product.
Temperature of reaction is lower and comparatively wide in range, is-50 ℃ of room temperatures (20 ℃); Reaction time is shorter, is 3min-8min; Type of heating is the freeze-day with constant temperature heating.The vibration incorporation time is 20s-60s.
The BSTFA consumption is 15 μ L-50 μ L; The pyridine consumption is 30 μ L-60 μ L; The usage ratio of BSTFA and pyridine does not have strict regulation.
3, in the 3rd step, taking-up is cooled to room temperature, puts into Nitrogen evaporator, feeds high pure nitrogen and slowly dries up, and adding 90 μ L sample introduction solvents and 10 μ L concentration is the interior mark (androstane) of 1ng/ μ L, after vibration mixes, gets 1 μ L and injects the GC-MS analysis.
Need not heating when drying up in Nitrogen evaporator, normal temperature condition gets final product down.The vibration incorporation time is 20s-60s.
The derivatization product of E1, E2, EE2, E3 is respectively TMS-E1 (oestrone monohydroxy derivatization product), di-TMS-E2 (the two hydroxyl derivatization products of 17 beta estradiols), di-TMS-EE2 (the two hydroxyl derivatization products of 17 α-ethinyl estradiol), tri-TMS-E3 (estriol trihydroxy derivatization product).
GC is carrier gas with the helium, and flow velocity is 1mL/min; Shunting mode sample introduction not, injector temperature is 280 ℃, sampling volume 1 μ L; Heating schedule is as follows:
The MS interface temperature is 280 ℃, and the transmission line temperature is 300 ℃, and ion source temperature is 250 ℃, and the ionization mode is an electron impact ionization, i.e. EI, and the electron bombard energy is 70eV, the solvent delay time is 12min.GC-MS is quantitative with full scan pattern qualitative (sweep limit m/z 50～600), selection ion scan pattern (being the SIM pattern); Under the SIM pattern, TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3 choose m/z342,327,285 respectively, m/z416,401,285, m/z440,425,285, m/z504,489,285 is as the feature fragmention, and be 0.5s-0.7s sweep time.
The present invention has the following advantages:
(1) the derivative reaction temperature is lower and comparatively wide in range, under the normal temperature condition (20 ℃) also can owing to can save heating steps, make the derivatization experiment become easy and save trouble, reduced energy consumption and cost;
(2) the derivative reaction time shorter, be 3min-8min, whole derivatization experimental implementation can be finished in 10min, has reduced the sample loss, has improved detection efficiency and accuracy;
(3) need not to add catalyzer, further simplified the derivative reaction system, reduced experimental cost;
(4) under these conditions, the present invention can be with four kinds of environmental estrogens E1, E2, EE2, E3 while derivatization, and the derivatization product is respectively TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3, and no coupling product generates.The linear dependence of each derivatization product is good, is 0.99462-0.99942, and the range of linearity is 1pg/ μ L-100ng/ μ L, and the instrument detection limit can reach 0.5pg/ μ L.
The GC-MS that the present invention can be applied to environmental estrogens E1, E2, EE2, E3 in the samples such as water body, bed mud, biology detects.
Description of drawings
The full scan chromatogram (concentration is 10ng/ μ L) that GC-MS detected when Fig. 1 was environmental estrogens E1, E2, EE2, E3 underivatized.
Fig. 2 is the full scan chromatogram that environmental estrogens E1, E2, EE2, E3 (concentration is 10ng/ μ L) derivatization product TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3 detect through GC-MS.
Embodiment
Further specify flesh and blood of the present invention below in conjunction with drawings and Examples, but content of the present invention is not limited to this.
Embodiment 1:
1, at first gets standard mixed solution that 100 μ L contain finite concentration (10pg/ μ L) E1, E2, EE2, E3 in the 1.5mL chromatogram bottle, chromatogram bottle is put into Nitrogen evaporator, feed high pure nitrogen and slowly dry up.
2, add 15 μ LBSTFA and 30 μ L pyridines, after vibration mixes, place normal temperature environment (20 ℃) reaction 3min.
3, taking-up is cooled to room temperature, puts into Nitrogen evaporator, feeds high pure nitrogen and slowly dries up, and adding 90 μ L sample introduction solvents and 10 μ L concentration is the interior mark (androstane) of 1ng/ μ L, after vibration mixes, gets 1 μ L and injects GC-MS analysis (SIM pattern).
After testing, E1, E2, EE2, E3 are by whole derivatizations, the derivatization product is respectively TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3, no coupling product generates, RRF (relative response factor RelativeResponse Factor, the ratio of determinand peak area and interior mark peak area) is respectively 1.22,1.09,0.09,0.12.
Embodiment 2:
1, at first gets standard mixed solution that 100 μ L contain finite concentration (10ng/ μ L) E1, E2, EE2, E3 in the 1.5mL chromatogram bottle, chromatogram bottle is put into Nitrogen evaporator, feed high pure nitrogen and slowly dry up.
2, add 30 μ LBSTFA and 50 μ L pyridines, after vibration mixes, place normal temperature environment (20 ℃) reaction 5min.
3, taking-up is cooled to room temperature, puts into Nitrogen evaporator, feeds high pure nitrogen and slowly dries up, and adding 90 μ L sample introduction solvents and 10 μ L concentration is the interior mark (androstane) of 1ng/ μ L, after vibration mixes, gets 1 μ L and injects GC-MS analysis (SIM pattern).
After testing, E1, E2, EE2, E3 are by whole derivatizations, and the derivatization product is respectively TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3, and no coupling product generates, and RRF is respectively 1157.76,1074.82,146.65,187.13.
Embodiment 3:
1, at first gets standard mixed solution that 100 μ L contain finite concentration (8pg/ μ L) E1, E2, EE2, E3 in the 1.5mL chromatogram bottle, chromatogram bottle is put into Nitrogen evaporator, feed high pure nitrogen and slowly dry up.
2, add 15 μ LBSTFA and 20 μ L pyridines, vibration is put into the heated at constant temperature case after mixing, at 30 ℃ of reaction 3min.
3, taking-up is cooled to room temperature, puts into Nitrogen evaporator, feeds high pure nitrogen and slowly dries up, and adding 90 μ L sample introduction solvents and 10 μ L concentration is the interior mark (androstane) of 1ng/ μ L, after vibration mixes, gets 1 μ L and injects GC-MS analysis (SIM pattern).
After testing, E1, E2, EE2, E3 are by whole derivatizations, and the derivatization product is respectively TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3, and no coupling product generates, and RRF is respectively 0.99,0.88,0.06,0.08.
Embodiment 4:
1, at first gets standard mixed solution that 100 μ L contain finite concentration (83pg/ μ L) E1, E2, EE2, E3 in the 1.5mL chromatogram bottle, chromatogram bottle is put into Nitrogen evaporator, feed high pure nitrogen and slowly dry up.
2, add 30 μ LBSTFA and 50 μ L pyridines, vibration is put into the heated at constant temperature case after mixing, at 30 ℃ of reaction 5min.
3, taking-up is cooled to room temperature, puts into Nitrogen evaporator, feeds high pure nitrogen and slowly dries up, and adding 90 μ L sample introduction solvents and 10 μ L concentration is the interior mark (androstane) of 1ng/ μ L, after vibration mixes, gets 1 μ L and injects GC-MS analysis (SIM pattern).
After testing, E1, E2, EE2, E3 are by whole derivatizations, and the derivatization product is respectively TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3, and no coupling product generates, and RRF is respectively 9.67,8.94,1.16,1.49.
Embodiment 5:
1, at first gets standard mixed solution that 100 μ L contain finite concentration (365pg/ μ L) E1, E2, EE2, E3 in the 1.5mL chromatogram bottle, chromatogram bottle is put into Nitrogen evaporator, feed high pure nitrogen and slowly dry up.
2, add 50 μ LBSTFA and 60 μ L pyridines, vibration is put into the heated at constant temperature case after mixing, at 50 ℃ of reaction 8min.
3, taking-up is cooled to room temperature, puts into Nitrogen evaporator, feeds high pure nitrogen and slowly dries up, and adding 90 μ L sample introduction solvents and 10 μ L concentration is the interior mark (androstane) of 1ng/ μ L, after vibration mixes, gets 1 μ L and injects GC-MS analysis (SIM pattern).
After testing, E1, E2, EE2, E3 are by whole derivatizations, and the derivatization product is respectively TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3, and no coupling product generates, and RRF is respectively 42.32,39.25,5.30,6.77.
Claims (10)
1. the derivatization method of environmental estrogens is characterized in that comprising the steps:
(1) at first gets standard mixed solution that 100 μ L contain oestrone, 17 beta estradiols, estriol, 17 α-ethinyl estradiol in the 1.5mL chromatogram bottle, chromatogram bottle is put into Nitrogen evaporator, feed high pure nitrogen and slowly dry up;
(2) add N, two trimethyl silicon based trifluoroacetamides of O-and pyridine, vibration places normal temperature environment or puts into the heated at constant temperature case after mixing, and reaction 3min～8min obtains the derivatization product under 20 ℃～50 ℃;
(3) take out and to be cooled to room temperature, put into Nitrogen evaporator, feed high pure nitrogen and slowly dry up, the interior mark that adds 90 μ L sample introduction solvents and 10 μ L concentration and be 1ng/ μ L is an androstane, and vibration is got 1 μ L injection gas chromatography-GC-MS analysis after mixing.
2. the derivatization method of environmental estrogens as claimed in claim 1, it is characterized in that: in the described step (1), environmental estrogens oestrone, 17 beta estradiols, estriol, 17 α-ethinyl estradiol are represented with E1, E2, E3, EE2 respectively, E1, E2, E3, EE2 are solid, purity is more than 97%, be dissolved in and be made into the standard mixed solution in the methyl alcohol, sealing is placed on below-20 ℃ and preserves, methyl alcohol is chromatographically pure, and containing oestrone, 17 beta estradiols, estriol, 17 α-ethinyl estradiol is 1pg/ μ L-100ng/ μ L.
3. the derivatization method of environmental estrogens as claimed in claim 1, it is characterized in that: in described step (1) and (3), high pure nitrogen purity is more than 99.99%; Need not heating when drying up in Nitrogen evaporator, normal temperature condition gets final product down.
4. the derivatization method of environmental estrogens as claimed in claim 1 is characterized in that: in the described step (2), and N, the two trimethyl silicon based trifluoroacetamide ranks of O-are the derivatization level, and purity is greater than 98%, and consumption is 15 μ L-50 μ L; Pyridine is a chromatographically pure, and consumption is 30 μ L-60 μ L.
5. the derivatization method of environmental estrogens as claimed in claim 1, it is characterized in that: in the described step (2), temperature of reaction is 20 ℃～50 ℃; Reaction time is shorter, is 3min～8min; Type of heating is the freeze-day with constant temperature heating, and the vibration incorporation time is 20s～60s.
6. the derivatization method of environmental estrogens as claimed in claim 1, it is characterized in that: in the described step (3), the sample introduction solvent is a chromatographically pure level high-purity organic solvent, can be methyl alcohol, methylene chloride, acetone, ethyl acetate or normal hexane; The vibration incorporation time is 20s～60s.
7. the derivatization method of environmental estrogens as claimed in claim 2, it is characterized in that: in the described step (3), the derivatization product of E1, E2, EE2, E3 is respectively TMS-E1 (oestrone monohydroxy derivatization product), di-TMS-E2 (the two hydroxyl derivatization products of 17 beta estradiols), di-TMS-EE2 (the two hydroxyl derivatization products of 17 α-ethinyl estradiol), tri-TMS-E3 (estriol trihydroxy derivatization product).
8. the derivatization method of environmental estrogens as claimed in claim 1, it is characterized in that: in the described step (3), the used chromatographic column of gas chromatograph-mass spectrometer (GCMS) is Supelco SPB
TM-5 or J﹠amp; WScientific DB-5MS capillary column; Gas chromatograph is carrier gas with the helium, and flow velocity is 1mL/min; Shunting mode sample introduction not, injector temperature is 280 ℃, sampling volume 1 μ L; Heating schedule is as follows:
9. the derivatization method of environmental estrogens as claimed in claim 1, it is characterized in that: in the described step (3), the spectrometer interface temperature is 280 ℃, the transmission line temperature is 300 ℃, ion source temperature is 250 ℃, and the ionization mode is electron impact ionization, i.e. EI, the electron bombard energy is 70eV, and the solvent delay time is 12min.
10. the derivatization method of environmental estrogens as claimed in claim 1, it is characterized in that: in the described step (3), gas chromatograph-mass spectrometer (GCMS) is qualitative with the full scan pattern, and sweep limit m/z 50～600, quantitatively adopt ion scan pattern, i.e. the SIM pattern selected; Under the SIM pattern, TMS-E1, di-TMS-E2, di-TMS-EE2, tri-TMS-E3 choose m/z342,327,285 respectively, m/z416,401,285, m/z440,425,285, m/z504,489,285 is as the feature fragmention, and be 0.5s-0.7s sweep time.
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