CN102590364A - Method for simultaneously detecting fumonisins B1 and B2 in different matrix traditional Chinese medicines - Google Patents
Method for simultaneously detecting fumonisins B1 and B2 in different matrix traditional Chinese medicines Download PDFInfo
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Abstract
The invention relates to a method for simultaneously detecting fumonisins B1 and B2 in different matrix traditional Chinese medicines, namely an on-line derivation high-performance liquid phase fluorescent detection method. The method comprises the steps of extracting, purifying and detecting a sample and particularly comprises the following steps of: adding sodium chloride in the sample; performing oscillating extraction by using a horizontal table according to the volume ratio of methanol to water of 4:1; filtering; diluting a certain amount of filtrate by adding water; filtering by using a microfiber filtration membrane; purifying by using an immunoaffinity column; drying by using nitrogen; dissolving by using methanol; and sampling. A derivation agent is prepared by the following steps of: dissolving a proper amount of o-phthaldialdehyde by using methanol; adding into a proper amount of sodium tetraborate solution; adding 2-mercaptoethanol; mixing uniformly; and passing through a microfiltration membrane. The measurement method of the sample comprises the following steps of: separating by using C18 reversed phase chromatographic column; performing gradient elution by using methanol and sodium dihydrogen phosphate as mobile phase; reacting with the derivation agent; detecting by using a fluorescence detector; and confirming the positive sample by using high-performance liquid chromatography-mass spectrum.
Description
Technical field:
The present invention relates to a kind of fumonisin B in the different substrates Chinese medicine that detects simultaneously
1, B
2Method, particularly adopted the method for the online high performance liquid chromatogram fluoroscopic examination of deriving, detection fumonisin B that can be more easy
1, B
2
Background technology:
Fumonisin is one group of water-soluble metabolin that is mainly produced by fusarium moniliforme, is one type and has serious Cytotoxic mycotoxin, in numerous mycotoxins, is considered to the mankind and livestock are threatened one of comparatively serious toxin.China has abundant Chinese material medicine resource, Chinese crude drug grow, gather, process, PI fumonisin all in the process such as storage.Therefore, necessary Chinese medicine horse second of the three ten-day periods of the hot season toxin is studied, set up the detection method of toxin, work out its rational limit standard, to guarantee the safe and effective of Chinese medicine.
From mouldy corn, isolate fumonisin B to South Africa in 1988 and American Studies personnel
1, the fumonisin of up to the present finding has FA
1, FA
2, FB
1, FB
2, FB
3, FB
4, FC
1, FC
2, FC
3, FC
4, totally 11 kinds of FP etc.FB wherein
1Toxicity is the strongest, also is its key component, accounts for 70%～80% of total amount, secondly is FB
2Fumonisin mainly pollutes corn and goods thereof, also can pollute cereal crops and goods thereof such as wheat, rice.In addition, the report that detects fumonisin is also arranged at animal feed and some spice in like anistree, cassia bark and medicine food dual purpose plant such as asparagus.These spices and medicine food dual purpose plant are used in people's life widely.The fumonisin that therefore, possibly exist in these spices and the medicine food dual purpose plant brings harm will inevitably for people's health.Research confirms that fumonisin has neurotoxicity, lung toxicity, immune system toxicity, carcinogenicity etc.Fumonisin can cause the white encephalomalacia of horse (ELEM), pig pulmonary edema syndrome (PPE), and diseases such as cancer of the esophagus, liver cancer and fetal neural tube deformity of bringing out the mankind under a cloud.1993, in the Lyons, France, IARC (IARC) classified fumonisin as 2B class carcinogen (promptly human possibility carcinogenic substance).
Summary of the invention:
Up to the present, reported method is limited to fumonisin B in food, agricultural product and the feed mostly both at home and abroad
1, B
2Analysis, also accidental detection, but for fumonisin B to Chinese crude drug
1, B
2Detection, what adopt basically is that the mode of column front derivation is in the majority.Use online deriving method to Chinese medicine horse second of the three ten-day periods of the hot season toxin B
1, B
2Analysis, at present still blank.
Fumonisin B in existing mensuration food, agricultural product and the feed
1, B
2Method because sample substrate is simple relatively, disturbs less, generally all than being easier to mensuration.Fumonisin B behind the derivatization
1, B
2Stability is bad, and sample introduction just can obtain reappearance preferably in two minutes, and the toxicity of part derivatization reagent is bigger.The present invention overcomes the deficiency of prior art, has selected to be applicable to fumonisin B in the Chinese medicine
1, B
2Extraction, purification method, adopted the method for online derivatization, optimized liquid phase chromatogram condition, guarantee the sample preparation shortcut and simple, assay accuracy as a result is high, favorable reproducibility.So this method of employing can be more accurately to the fumonisin B in the different substrates Chinese medicine
1, B
2Measure.
Fumonisin B in the concrete different substrates Chinese medicine
1, B
2Method for measuring, can realize through following steps:
Instrument, medicine and material:
Instrument:
Tianjin, island LC-20AT high performance liquid chromatograph, day island proper Tianjin company;
Fluorescence detector RF-10A
XL, day island proper Tianjin company;
Post-column derivation reaction chamber CRB-6A, day island proper Tianjin company;
The triple level Four bar of API4000 mass spectrometer;
The Qilinbeier vortex mixer;
Heraeus Multifuge X3R low-temperature and high-speed hydro-extractor;
WH-861 type vortex mixer, blue instrument Manufacturing Co., Ltd is contained by Jintan City;
PGG-01D type dry type Nitrogen evaporator, Tianjin Ai Weiou development in science and technology company limited;
The horizontal shaking table of ZD-9556, Taicang science and education equipment factory;
Fumonisin Test immune affinity column, U.S. VICAM company;
Microfibril filter paper, U.S. VICAM company.
Reference substance:
Fumonisin B
1, B
2Standard items are purchased in sharp prosperous agricultural company, purity>=99%; OPA (OPA), U.S. AflaAesar company; Chromatographically pure methyl alcohol (B&J); Trifluoroacetic acid aqueous solution (B&J); Water (Wahaha); Sodium dihydrogen phosphate; Sodium hydrogen phosphate; Potassium dihydrogen phosphate; Sodium chloride; Potassium chloride; NaOH; Hydrochloric acid; Phosphoric acid; Sodium tetraborate; 2 mercapto ethanol.
Sample:
Institute's test sample article are respectively available from Jiangxi camphor tree, Hainan, Beijing and zunyi, guizhou and other places, and all samples is stochastic buying on the market, and is subsequent use after the low temperature drying.
1, measures fumonisin B in the Chinese medicine with the online high performance liquid chromatography of deriving-fluorescence detection
1, B
2
A. chromatographic condition:
Use Synergi C
18(column temperature is 40 ℃ to post for 4 μ m, 250mm * 4.6mm) separate, and the past column reaction case carries out derivatization (100 ℃ of temperature of reaction), and fluorescence detector detects.Moving phase is that pH is 3.15, and the sodium dihydrogen phosphate of 0.1mol/L and methyl alcohol, flow velocity are that 0.8ml/min carries out gradient elution.In 22 minutes, rise to 85% by 60% methyl alcohol at the beginning; Ran 5 minutes by 85% methyl alcohol again, in 2 minutes, reduce to 60% then, at last by 60% methyl alcohol balance 13 minutes by 85% methyl alcohol moment.The derivatization reagent flow velocity is 0.6ml/min, and reaction volume is 0.6ml.The excitation wavelength of fluorescence detector is 335nm, and emission wavelength is 440nm.
B. solution preparation:
Fumonisin B
1, B
2The preparation of standard items: respectively with 1mgFB
1, FB
2Pressed powder, with acetonitrile-water (1: 1, V/V) dissolving, constant volume is made into the storing solution of 100 μ g/mL in 10mL, 4 ℃ keep in Dark Place.
The preparation of derivatization reagent: take by weighing the OPA of 100mg, be dissolved in 20mL methyl alcohol, join in the sodium tetraborate solution of 0.05mol/L, be settled to 1000mL, add 2 mercapto ethanol 500 μ l again, mixing is crossed 0.45 μ m filter membrane, room temperature preservation.
The preparation of PBS damping fluid: take by weighing sodium hydrogen phosphate 1.2g, potassium dihydrogen phosphate 0.2g, sodium chloride 8g, potassium chloride 0.2g is dissolved in the 990mL water, and using hydrochloric acid to regulate pH is 7.0, and water dissolves to 1000mL surely.
The preparation of sodium dihydrogen phosphate: take by weighing the 15.6g sodium dihydrogen phosphate, the water dissolving is settled to 1000mL, is made into 0.1mol/L solution, and using phosphoric acid to regulate pH is 3.15.
C. the preparation of sample solution:
Sample extraction: precision takes by weighing sample 10g, and 2g sodium chloride adds in the 40ml methanol (4: 1), horizontal shaking table jolting 40 minutes, and groove line filter paper filtering is collected filtrating.Get above-mentioned filtrating 5ml in the 50ml conical flask, add the PBS of 20ml, the vibration mixing, the fento filter paper filtering is collected filtrate for later use.
Sample purification: the 10mL glass syringe barrels is linked to each other with affinity column, pipette step 5mL sample extracting solution in the glass syringe syringe, pull out cap at the bottom of the affinity column.Controlled pressure make kind liquid with 1 droplet/second flow velocity all through affinity column, get into until air.Take off affinity column, affinity column top is filled with, connect affinity column with glass syringe again,, get into, discard whole effluent until air with the flow velocity drip washing affinity column of 10mLPBS solution with 2 droplets/second with PBS.Repeat the drip washing pillar once with 10mLPBS again, discard whole effluent.Accurately add the flow velocity wash-out of 10mL methyl alcohol with 1mL/min, collect eluent, 60 ℃ of nitrogen dry up.Residue is used 1mL methyl alcohol: water (1: 1) dissolving, cross 0.45 μ m pin hole filter membrane, and wait for sample introduction.
D. determination method:
Accurate respectively reference substance and the sample solution 20 μ L of drawing inject high performance liquid chromatograph, measure fumonisin B
1, B
2Content.
2, with high performance liquid chromatography-mass spectrometry detection method positive findings is proved conclusively:
A. liquid-phase condition:
Chromatographic column is Phenomenex Gemini 3u C
18110A column (20mm*2.00mm, 3micron), mobile phase A is water (containing 0.1% formic acid), Mobile phase B is acetonitrile (containing 0.1% formic acid).Flow velocity is 0.4mL/min, and gradient is 0.01min 10%B, 0.6min 10%B, and 1.5min 95%B, 3.0min 95%B, 3.01min 10%B, 4.50min 10B% finishes.
B. mass spectrum condition:
Adopt the ESI ionization source, positive ion scanning, MRM detecting pattern, three ion pair monitorings.Fumonisin B
1: m/z722.4 → 352.5 collision voltages are 51eV; M/z 722.4 → 334.6 collision voltages are 55eV; M/z 722.4 → 704.5 collision voltages are 39eV.Fumonisin B
2: m/z 706.5 → 336.7 collision voltages are 53eV; M/z 706.5 → 354.5 collision voltages are 55eV; M/z 706.5 → 318.3 collision voltages are 55eV.
C. determination method:
Accurate respectively reference substance and the positive solution 10 μ L of drawing inject high performance liquid chromatography-mass spectrometry system, measure.
Through revision test repeatedly, confirmation method is easy, and the result is accurate, can be used as fumonisin B in the different substrates Chinese medicine
1, B
2Assay method.
The invention provides fumonisin B in the Chinese medicine
1, B
2The online high performance liquid chromatography-fluorescence detection of deriving; Fumonisin B in the Chinese medicine
1, B
2High performance liquid chromatography-mass spectrometry method positive findings is proved conclusively.The result is accurate, and favorable reproducibility can be used as fumonisin B in the different substrates Chinese medicine
1, B
2Assay method.
1, the online high performance liquid chromatography of deriving-fluorescence detection is measured the fumonisin B in the Chinese medicine
1, B
2
(1) selection of derivatization reagent:
For fumonisin B
1, B
2Owing to itself do not have fluorescent absorption, carry out to react after the derivative reaction.Fumonisin B
1, B
2Derivatization reagent fluorescamine, OPA (OPA), naphthalene-2 are arranged, 3-dicarboxylic acid (NDA), 9-fluorenyl methyl-chloro-carbonate (FMOC) or the like.The present invention is the most frequently used derivatization reagent OPA (OPA) of utilization.Reason is that this derivatization reagent sensitivity is high, and used reagent toxicity is less relatively, buys than being easier to.
(2) investigation of derivative reaction process:
Adopted orthogonal experiment to investigate the influence of three factors (derivative reagent flow velocity, reaction chamber temperature and reaction volume), three levels.With fumonisin B
1, B
2The peak area Comprehensive Assessment be that index is carried out orthogonal experiment, investigate the top condition of derivative reaction.Orthogonal experiment results sees Table 1-1, table 1-2.
Table 1-1 fumonisin B
1, B
2Orthogonal experiment results
1-1 carries out can finding out the intuitive analysis that the influence size of three kinds of factors is C＞A＞B, promptly for fumonisin B to experimental result from table
1, B
2The derivative reaction volume has the greatest impact to the derivatization result, and the optimal conditions that draws is A3B2C3, and promptly the derivative reaction temperature is that 100 ℃, derivatization reagent C flow rate pump are that 0.6mL/min, reactor coil volume are 0.6mL.
In order to ensure the reappearance under the optimal conditions, carried out parallel demonstration test three times, the result is following:
Table 1-2FB
1, FB
2Reappearance is investigated under the optimal conditions
(3) minimum detectability and quantitative limit
According to signal to noise ratio (S/N ratio) S/N>=3 is the minimum detectability of this material, FB
1Minimum detectability be 40 μ g/kg (5ng/mL), FB
2Minimum detectability be 40 μ g/kg (5ng/mL).
According to signal to noise ratio (S/N ratio) S/N>=10 is the minimum quantitative limit of this material, FB
1Quantitatively be limited to 100 μ g/kg (12.5ng/mL), FB
2Toxin quantitatively be limited to 100 μ g/kg (12.5ng/mL).
(4) typical curve
Get the fumonisin B of variable concentrations
1, B
2The difference sample introduction, the chromatographic peak peak area (A) that obtains the acquisition under the variable concentrations is to fumonisin B
1, B
2Mass concentration (C-μ g/mL) return.
Get FB
1Typical curve y=2566.0x-15244, r=0.9997, the range of linearity is 12.5～1000ng/ml, the result see the table 1-3
Get FB
2Typical curve y=2604.1x-21571, r=0.9996, the range of linearity is 12.5～1000ng/mL, the result see the table 1-4
Table 1-3FB
1Linear relationship is investigated the result
Reference substance concentration C (ng/mL) peak area A (average)
12.5 32954
25 66432
50 88653
200 465745
250 659226
500 1257262
800 2020021
1000 2568884
Table 1-4FB
2Linear relationship is investigated the result
Reference substance concentration C (ng/mL) peak area A (average)
12.5 25634
25 47236
50 136472
200 472095
250 608439
500 1250322
800 2101887
1000 2574485
(5) withinday precision experiment
Get same reference substance solution, press above-mentioned chromatographic condition continuous sample introduction 6 times, calculate fumonisin B
1, B
2Peak area value, the result see the table 1-5.
The experiment of table 1-5 withinday precision
(6) day to day precision experiment
Get same reference substance solution, press above-mentioned chromatographic condition sample introduction every day 1 time, continuous sample introduction 6 days calculates fumonisin B
1, B
2Peak area value, the result see the table 1-6
The experiment of table 1-6 day to day precision
(7) stability experiment
Get same reference substance solution, by above-mentioned chromatographic condition respectively 0,2,4,8,12h measures, the result sees table 1-7.
Table 1-7 stability experiment
(8) recovery experiment
The Chinese crude drug of Fructus Hordei Germinatus, Radix Codonopsis, semen armeniacae amarae, four kinds of different substrates of stir-baked MASSA FERMENTATA MEDICI-NALIS is adopted in this experiment; Four kinds of matrix of conventional Chinese medicine have been represented; And after these four kinds of medicinal materials are handled through immune affinity column; Advance high performance liquid chromatogram-fluoroscopic examination, noiseless to the standard items detection of adding, so four kinds of medicinal materials of adopter are done recovery experiment.Reference is in the world about fumonisin B
1, B
2Limit standard, this experimental design the application of sample recovery test of 5 levels.Level 1: fumonisin B
1, B
2Interpolation concentration be 3mg/kg; Level 2: fumonisin B
1, B
2Interpolation concentration be 2mg/kg; Level 3: fumonisin B
1, B
2Interpolation concentration be 1mg/kg; Level 4: fumonisin B
1, B
2Interpolation concentration be 0.5mg/kg; Level 5: fumonisin B
1, B
2Interpolation concentration be 0.2mg/kg, every group of experiment all repeats n=3 3 times.
Precision takes by weighing medicinal material 10.0g (being accurate to 0.1g), adds an amount of standard solution, is equipped with sample solution by text specimen preparation item below legal system, presses its content of external standard method, calculates the average recovery result and sees table 1-8.
Table 1-8 fumonisin B
1, B
2The mensuration of average recovery
(9) sample determination:
The utilization said method has been measured 93 Chinese crude drugs, measures the various Chinese medicine analyte sample fluids that prepare respectively with external standard method, each sample feeding 20 μ L.After the pre-treatment of all samples process immune affinity column, all there is chromatographic peak to occur, contains fumonisin B in institute's test sample article through the online high performance liquid chromatogram of deriving-fluoroscopic examination mensuration
1, B
2The positive of two kinds of toxin is 2, singly contains fumonisin B
1, positive be 3, singly contain fumonisin B
2Positive be 3.Remaining is negative sample.The fumonisin B that the chromatographic peak that the Chinese medicine that has occurs detects need
1, B
2Chromatographic peak is noiseless, with " * " expression; And the fumonisin B that the chromatographic peak that the Chinese medicine that has occurs detects need
1, B
2Chromatographic peak has interference, with "-" expression.The result sees table 1-9.
Fumonisin B in the table 1-9 sample
1, B
2Mensuration result
2, with high performance liquid chromatography-mass spectrometry detection method positive findings is proved conclusively;
Traditional Chinese medicine ingredients is complicated, and when adopting high performance liquid chromatogram to analyze, disturbing or false positive results appears in regular meeting.Therefore, only differentiate, might cause error to analysis result through the retention time of chromatographic peak.Disturb and eliminate error in order to get rid of, the present invention has adopted the HPLC-ESI-MS/MS technology through fumonisin B
1, B
2Retention time, and the fragment ion of analyte proves conclusively measuring the result, can see fumonisin B according to mass spectrogram
1, B
2Characteristic ion right: fumonisin B
1Parent ion be 722.4 ([M+H]
+), three daughter ions are m/z 704.5 ([M+H-H
2O]
+), m/z352.5 ([M+H-2TCA-H
2O]
+), m/z 334.6 ([M+H-2TCA-2H
2O]
+); Fumonisin B
2Parent ion be 706.5 ([M+H]
+), three daughter ions are m/z 354.5 ([M+H-2TCA]
+), m/z 336.7 ([M+H-2TCA-H
2O]
+), m/z 318.3 ([M+H-2TCA-2H
2O]
+).Can confirm not contain fumonisin B in the Radix Glycyrrhizae samples such as (Hainan) through chromatogram and corresponding mass spectrogram
1, B
2, confirm that the peak that liquid phase records is an Interference Peaks.
Description of drawings:
Fig. 1 is fumonisin B
1, B
2The high performance liquid chromatography of standard items-fluorescence detection chromatogram;
Fig. 2 is the high performance liquid chromatography-fluorescence detection chromatogram of the positive sterculia seed;
Fig. 3 is fumonisin B
1The HPLC-ESI-MS/MS second order ms figure of standard items;
Fig. 4 is fumonisin B
1Three pairs of ion pair chromatograms of the HPLC-ESI-MS/MS of standard items;
Fig. 5 is fumonisin B
2The HPLC-ESI-MS/MS second order ms figure of standard items;
Fig. 6 is fumonisin B
2Three couples of chromatography of ions figure of the HPLC-ESI-MS/MS of standard items
Fig. 7 is the HPLC-ESI-MS/MS mass spectrogram of the positive root of large-flowered skullcap
Embodiment:
1, the online high performance liquid chromatography of deriving-fluorescence detection is measured fumonisin B in the Chinese medicine
1, B
2
A. chromatographic condition: use Synergi C
18(column temperature is 40 ℃ to post for 4 μ m, 250mm * 4.6mm) separate, and the past column reaction case carries out derivatization (100 ℃ of temperature of reaction), and fluorescence detector detects.Moving phase is that pH is 3.15, and the sodium dihydrogen phosphate of 0.1mol/L and methyl alcohol, flow velocity are that 0.8ml/min carries out gradient elution.In 22 minutes, rise to 85% by 60% methyl alcohol at the beginning; Ran 5 minutes by 85% methyl alcohol again, in 0.01 second, reduce to 60% then, at last by 60% methyl alcohol balance 13 minutes by 85% methyl alcohol moment.The derivatization reagent flow velocity is 0.6ml/min, and reaction volume is 0.6ml.The excitation wavelength of fluorescence detector is 335nm, and emission wavelength is 440nm.
B. the preparation of solution:
Reference substance solution preparation: with 1mgFB
1, FB
2Pressed powder, with acetonitrile/water (1: 1, V/V) dissolving, constant volume is made into the storing solution of 100 μ g/mL in 10ml, 4 ℃ keep in Dark Place.
Derivatization reagent: take by weighing the OPA of 100mg, be dissolved in 20ml methyl alcohol, join in the sodium tetraborate solution of 0.05mol/L, be settled to 1000ml, add 2 mercapto ethanol 500 μ l again, mixing is crossed 0.45 μ m filter membrane, room temperature preservation.
The PBS damping fluid: take by weighing sodium hydrogen phosphate 1.2g, potassium dihydrogen phosphate 0.2g, sodium chloride 8g, potassium chloride 0.2g is dissolved in the 990ml water, and using hydrochloric acid to regulate pH is 7.0, and water dissolves to 1L surely.
Sodium dihydrogen phosphate: take by weighing the 15.6g sodium dihydrogen phosphate, the water dissolving is settled to 1L, is made into 0.1mol/L solution, and using phosphoric acid to regulate pH is 3.15.
C. the preparation of sample solution:
Sample extraction: precision takes by weighing sample 10g, and 2g sodium chloride adds in the 40ml methanol (4: 1), horizontal shaking table jolting 40 minutes, and groove line filter paper filtering is collected filtrating.Get above-mentioned filtrating 5ml in the 50ml conical flask, add the PBS of 20ml, the vibration mixing, the fento filter paper filtering is collected filtrate for later use.
Sample is evolved: the block of FumoniTest immune affinity column is taken off, cut the sealing end of block, cover block.The 10ml glass syringe barrels is linked to each other with affinity column, accurately pipette step 5ml sample extracting solution in the glass syringe syringe, pull out cap at the bottom of the affinity column.Controlled pressure make kind liquid with 1 droplet/second flow velocity all through affinity column, get into until air.Take off affinity column, affinity column top is filled with, connect affinity column with glass syringe again,, get into, discard whole effluent until air with the flow velocity drip washing affinity column of 10mlPBS solution with 2 droplets/second with PBS solution.Repeat the drip washing pillar once with 10mlPBS again, discard whole effluent.Accurately add 10ml methyl alcohol with 1 droplet/second flow velocity wash-out, collect eluent, 60 ℃ of nitrogen dry up.Residue is used 1ml methyl alcohol: (1: 1,0.45 μ m pin hole filter membrane was crossed in V/V) dissolving to water, waits for sample introduction.
D. accurate respectively reference substance and the sample solution 20 μ L of drawing inject high performance liquid chromatograph, detect fumonisin B
1, B
2
2, with high performance liquid chromatography-mass spectrometry detection method positive findings is proved conclusively:
A. liquid-phase condition:
Chromatographic column is Phenomenex Gemini 3u C
18110A column (20mm*2.00mm, 3micron), mobile phase A is water (containing 0.1% formic acid), Mobile phase B is acetonitrile (containing 0.1% formic acid).Flow velocity is 0.4mL/min, and gradient is 0.01min 10%B, 0.6min 10%B, and 1.5min 95%B, 3.0min 95%B, 3.01min 10%B, 4.50min 10B% finishes.
B. mass spectrum condition:
Adopt the ESI ionization source, positive ion scanning, MRM detecting pattern, three ion pair monitorings.Fumonisin B
1: m/z722.4 → 352.5 collision voltages are 51eV; M/z 722.4 → 334.6 collision voltages are 55eV; M/z 722.4 → 704.5 collision voltages are 39eV.Fumonisin B
2: m/z 706.5 → 336.7 collision voltages are 53eV; M/z 706.5 → 354.5 collision voltages are 55eV; M/z 706.5 → 318.3 collision voltages are 55eV.
C. determination method:
Accurate respectively reference substance and the positive solution 10 μ L of drawing inject high performance liquid chromatography-mass spectrometry system, measure.
Claims (3)
1. one kind is detected fumonisin B in the different substrates Chinese medicine simultaneously
1, B
2Method, it is characterized in that this method comprises the following steps:
(1) measures fumonisin B in the Chinese medicine with the online high performance liquid chromatography of deriving-fluorescence detection
1, B
2
(2) with high performance liquid chromatography-mass spectrometry detection method positive findings is proved conclusively;
2. detect fumonisin B in the different substrates Chinese medicine in the time of according to claim 1
1, B
2Method, it is characterized in that this method comprises the following steps:
(1) measures fumonisin B in the Chinese medicine with the online high performance liquid chromatography of deriving-fluorescence detection
1, B
2
A. chromatographic condition:
Use Synergi C
18(column temperature is 40 ℃ to post for 4 μ m, 250mm * 4.6mm) separate, and the past column reaction case carries out derivatization (100 ℃ of temperature of reaction), and fluorescence detector detects.Moving phase is that pH is 3.15, and the sodium dihydrogen phosphate of 0.1mol/L and methyl alcohol, flow velocity are that 0.8ml/min carries out gradient elution.In 22 minutes, rise to 85% by 60% methyl alcohol at the beginning; Ran 5 minutes by 85% methyl alcohol again, in 0.01 second, reduce to 60% then, at last by 60% methyl alcohol balance 13 minutes by 85% methyl alcohol moment.The derivatization reagent flow velocity is 0.6ml/min, and reaction volume is 0.6ml.The excitation wavelength of fluorescence detector is 335nm, and emission wavelength is 440nm.
B. solution preparation:
Fumonisin B
1, B
2The preparation of standard items: respectively with 1mgFB
1, FB
2Pressed powder, with acetonitrile-water (1: 1, V/V) dissolving, constant volume is made into the storing solution of 100 μ g/mL in 10mL, 4 ℃ keep in Dark Place.
The preparation of derivatization reagent: take by weighing the OPA of 100mg, be dissolved in 20mL methyl alcohol, join in the sodium tetraborate solution of 0.05mol/L, be settled to 1000mL, add 2 mercapto ethanol 500 μ l again, mixing is crossed 0.45 μ m filter membrane, room temperature preservation.
The preparation of PBS damping fluid: take by weighing sodium hydrogen phosphate 1.2g, potassium dihydrogen phosphate 0.2g, sodium chloride 8g, potassium chloride 0.2g is dissolved in the 990mL water, and using hydrochloric acid to regulate pH is 7.0, and water dissolves to 1000mL surely.
The preparation of sodium dihydrogen phosphate: take by weighing the 15.6g sodium dihydrogen phosphate, the water dissolving is settled to 1000mL, is made into 0.1mol/L solution, and using phosphoric acid to regulate pH is 3.15.
C. the preparation of sample solution:
Sample extraction: precision takes by weighing sample 10g, and 2g sodium chloride adds in the 40ml methanol (4: 1), horizontal shaking table jolting 40 minutes, and groove line filter paper filtering is collected filtrating.Get above-mentioned filtrating 5ml in the 50ml conical flask, add the PBS of 20ml, the vibration mixing, the fento filter paper filtering is collected filtrate for later use.
Sample purification: the 10mL glass syringe barrels is linked to each other with affinity column, pipette step 5mL sample extracting solution in the glass syringe syringe, pull out cap at the bottom of the affinity column.Controlled pressure make kind liquid with 1 droplet/second flow velocity all through affinity column, get into until air.Take off affinity column, affinity column top is filled with, connect affinity column with glass syringe again,, get into, discard whole effluent until air with the flow velocity drip washing affinity column of 10mLPBS solution with 2 droplets/second with PBS.Repeat the drip washing pillar once with 10mLPBS again, discard whole effluent.Accurately add 10mL methyl alcohol with 1 droplet/second flow velocity wash-out, collect eluent, 60 ℃ of nitrogen dry up.Residue is used 1mL methyl alcohol: water (1: 1) dissolving, cross 0.45 μ m pin hole filter membrane, and wait for sample introduction.
D. determination method:
Accurate respectively reference substance and the sample solution 20 μ L of drawing inject high performance liquid chromatograph, measure fumonisin B
1, B
2Content.
(2) with high performance liquid chromatography-mass spectrometry detection method positive findings is proved conclusively
A. liquid-phase condition:
Chromatographic column is Phenomenex Gemini 3u C
18110A column (20mm*2.00mm, 3micron), mobile phase A is water (containing 0.1% formic acid), Mobile phase B is acetonitrile (containing 0.1% formic acid).Flow velocity is 0.4mL/min, and gradient is 0.01min 10%B, 0.6min 10%B, and 1.5min 95%B, 3.0min 95%B, 3.01min 10%B, 4.50min 10B% finishes.
B. mass spectrum condition:
Adopt the ESI ionization source, positive ion scanning, MRM detecting pattern, three ion pair monitorings.Fumonisin B
1: m/z 722.4 → 352.5 collision voltages are 51eV; M/z 722.4 → 334.6 collision voltages are 55eV; M/z 722.4 → 704.5 collision voltages are 39eV.Fumonisin B
2: m/z 706.5 → 336.7 collision voltages are 53eV; M/z 706.5 → 354.5 collision voltages are 55eV; M/z 706.5 → 318.3 collision voltages are 55eV.
C. determination method:
Accurate respectively reference substance and the positive solution 10 μ L of drawing inject high performance liquid chromatography-mass spectrometry system, conclusive evidence fumonisin B
1, B
2
3. detect fumonisin B in the different substrates Chinese medicine when telling according to claim 2
1, B
2Method, it is characterized in that:
A. extract solvent adopt methanol (4: 1, V/V) horizontal shaking table jolting is extracted;
B. adopting moving phase during high performance liquid chromatogram-fluoroscopic examination is that pH is 3.15, the sodium dihydrogen phosphate of 0.1mol/L and methyl alcohol;
What c. high performance liquid chromatogram-fluoroscopic examination was adopted is online deriving mode of deriving;
D. the collocation method of the derivatization reagent of this method employing is: take by weighing the OPA of 100mg, be dissolved in 20mL methyl alcohol, join in the sodium tetraborate solution of 0.05mol/L; Be settled to 1000mL, add 2 mercapto ethanol 500 μ l again, mixing; Cross 0.45 μ m filter membrane, room temperature preservation.
E. adopt the ESI ionization source in high performance liquid chromatography-mass spectrometry method, positive ion scanning, MRM detecting pattern.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104931308A (en) * | 2015-05-18 | 2015-09-23 | 上海市农业科学院 | Method for simultaneously preparing standards of fumonisins B1, B2 and B3 |
| CN105543231A (en) * | 2016-01-11 | 2016-05-04 | 河南省农业科学院农业质量标准与检测技术研究所 | Screening and application of fumonisin B1 aptamer strand displacement probe |
| CN105865885A (en) * | 2016-04-06 | 2016-08-17 | 中国计量科学研究院 | Quick screening method for chloramphenicol in honey matrix |
| CN114235988A (en) * | 2021-11-27 | 2022-03-25 | 山东省烟台市农业科学研究院 | High performance liquid chromatography for determining content of maleic hydrazide |
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| WO2008141351A1 (en) * | 2007-05-21 | 2008-11-27 | Erber Aktiengesellschaft | Method for quantitatively determining analytes using a test element and test system and use thereof |
| CN101865924A (en) * | 2010-06-26 | 2010-10-20 | 上海交通大学 | Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008141351A1 (en) * | 2007-05-21 | 2008-11-27 | Erber Aktiengesellschaft | Method for quantitatively determining analytes using a test element and test system and use thereof |
| CN101865924A (en) * | 2010-06-26 | 2010-10-20 | 上海交通大学 | Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104931308A (en) * | 2015-05-18 | 2015-09-23 | 上海市农业科学院 | Method for simultaneously preparing standards of fumonisins B1, B2 and B3 |
| CN105543231A (en) * | 2016-01-11 | 2016-05-04 | 河南省农业科学院农业质量标准与检测技术研究所 | Screening and application of fumonisin B1 aptamer strand displacement probe |
| CN105543231B (en) * | 2016-01-11 | 2019-07-05 | 河南省农业科学院农业质量标准与检测技术研究所 | Fumonisin B1The screening and application of aptamer strand displacing probes |
| CN105865885A (en) * | 2016-04-06 | 2016-08-17 | 中国计量科学研究院 | Quick screening method for chloramphenicol in honey matrix |
| CN114235988A (en) * | 2021-11-27 | 2022-03-25 | 山东省烟台市农业科学研究院 | High performance liquid chromatography for determining content of maleic hydrazide |
| CN114235988B (en) * | 2021-11-27 | 2023-12-22 | 山东省烟台市农业科学研究院 | High performance liquid chromatography for determining content of anti-sprouting pellet |
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