CN105319313B - Liquid chromatogram-tandem mass spectrum detection method of toxoflavin - Google Patents
Liquid chromatogram-tandem mass spectrum detection method of toxoflavin Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 17
- 238000001819 mass spectrum Methods 0.000 title claims abstract description 16
- 239000007788 liquid Substances 0.000 title claims abstract description 10
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- 235000019253 formic acid Nutrition 0.000 claims description 15
- 238000005342 ion exchange Methods 0.000 claims description 15
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 15
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Abstract
The invention discloses a liquid chromatogram-tandem mass spectrum detection method of toxoflavin. The liquid chromatogram-tandem mass spectrum detection method comprises the following steps: (1) pretreating a sample; (2) enriching and purifying; and (3) carrying out LC-MS/MS detection. The method provided by the invention is simple and convenient to operate, has relatively strong qualitative and quantitative capabilities, and can be used as a reliable method for detecting the quality of ferment corn flour and evaluating the safety risk of foods.
Description
Technical field
The present invention relates to a kind of liquid chromatography-tandem mass of toxoflavin, belong to instrument detection technique field.
Background technology
Coconut palm poison Burkholderia (burkholderia cocovenans) the primary Salmonella of abbreviation coconut palm ferment, is that China finds
A kind of new food poisoning bacteria, it is present in frumentum fermented product and (includes fermented maize face, Zea mayssinesis Kulesh dumpling flour, corn starch
Deng), rotten Tremella, sorghum rice vermicelli product, potato class product (as Rhizoma Solani tuber osi bean noodles, sweet potato starch, sweet potato starch etc.) and surrounding
In environment, it is the pathogen of fermented flour and deteriorated tremella poisoning.The toxin that this bacterium produces, has toxicity to humans and animals cell
Effect.General cooking method can not destroy, and absorbed by digestive tract and spread to whole body, incubation period is short, is 20min～24h, suffers from
Person starts to feel stomach discomfort, general weakness, and diarrhoea in small number of patients, but gastrointestinal symptoms are slight；Vomitus mostly is coffee color,
Severe patient occur jaundice, stupor, delirium, tic of the limbs, hematuria, have blood in stool, the symptom such as hepatomegaly, liver function has obvious change.Acute
Poisoner's morbidity is anxious, assumes toxic shock, average case fatality rate is up to 41.80%.
The primary Salmonella of coconut palm ferment produces two kinds of toxin: 3-carboxymethyl-17-methoxy-6,18,21-trimethyldocosa-2,4,8,12,14,18,20-heptaenedioic acid and toxoflavin, are its pathogenesiss.Toxoflavin
(toxflavin, txf) is soluble small molecular fatty acid toxin, and toxicity is extremely strong, and its chemical toxicity reaches malicious chemicals mark by force
Accurate.The molecular formula of toxoflavin is c7h7n5o2, molecular mass is 193.17, and structure is 1,6 dimethyl 1,7- dioxy -1,5,
The asymmetric triazine of 6,7- tetrahydro-pyrimidine base (5,4e).Pure toxoflavin is glassy yellow flaky crystal, 172-173 DEG C of decomposition, ultraviolet
Absworption peak is in 256.7nm and 394nm (ε=16400,2500).Mice ld50L.7mg/kg intravenous injection is, is administered orally as 8.4mg/
kg.
Research currently, with respect to toxoflavin is concentrated mainly on physio-biochemical characteristics and poisoning detoxication mechanisms.Relevant poison is yellow
The research of plain extraction in food and detection method still belongs to blank, hence sets up toxoflavin in a kind of fermented flour of fast and reliable
Detection method, to the safety ensureing frumentum rice and flour food, promote the technological innovation of manufacturing enterprise, improve food quality and shelves
Secondary, ensure that the people's is healthy significant.
Content of the invention
The technical problem to be solved is: provides a kind of easy and simple to handle, toxoflavin that qualitative, quantitative ability is stronger
Liquid chromatography-tandem mass.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
The liquid chromatography-tandem mass of the toxoflavin that the present invention provides, comprises the following steps:
(1) sample pre-treatments
Accurately weigh the fermented flour 1.0g through pulverizing, homogenizing is crossed, extract toxin with 5ml ultra-pure water solution, 200rpm shakes
Bed concussion 30min, less than 20 DEG C 8000rpm are centrifuged 10min, take supernatant, with 2mol/l formic acid regulation supernatant ph value to 6～
7；Repeat to extract once with 5ml ultra-pure water, merge supernatant twice, be settled to 10ml with ultra-pure water.Now obtain more pure
Crude extract.
(2) it is enriched with and purify
Enrichment purification uses solid-phase extraction column, and solid-phase extraction column is weak cation exchange post, makees with weak cation exchange
With the polymer absorbant of mechanism, can be in high and low ph condition eluting.33 μm of particle diameter, 85 μm of aperture, surface area 800m2/ g,
Ion capacity 1.0meq/g.
First with 1ml methanol and 1ml ultra-pure water activating ion exchange column, activation process flow speed control, at 1-2 drop/sec, takes
2ml crude extract injection ion exchange column carries out purification enrichment, and flow speed control, at 1 drop/sec, then uses 1ml ultra-pure water eluent ion
Exchange column, to go the removal of impurity, finally with 1.0ml methanol solution eluting toxoflavin at twice, nitrogen is blown to closely do, with 50% acetonitrile
Aqueous solution is settled to 0.4ml, treats that liquid chromatography-tandem mass spectrometry (lc-ms/ms) detects；
(3) lc-ms/ms detection
Liquid-phase condition phenomenex gemini c18 chromatographic column (3.0mm × 150mm, 5 μm), 30 DEG C of column temperature；Sample size
20μl；Mobile phase a be water (containing 0.1% formic acid, 5mmol/l ammonium formate), b be 95% acetonitrile (containing 0.1% formic acid,
5mmol/l ammonium formate)；Condition of gradient elution is shown in Table 1:
Table 1 condition of gradient elution
| Time/min | Flow velocity/μ l min-1 | A/% | B/% |
| 0.0 | 250 | 90 | 10 |
| 3.0 | 250 | 40 | 60 |
| 3.01 | 500 | 40 | 60 |
| 5.0 | 500 | 40 | 60 |
| 5.01 | 250 | 90 | 10 |
| 10.0 | 250 | 90 | 10 |
Mass Spectrometry Conditions Ionization mode is electro-spray ionization negative mode (esi-)；Multiple-reaction monitoring pattern (mrm)；Collision
Atmospheric pressure (cad): 6；Gas curtain atmospheric pressure (cur): 10；Electron spray voltage (is): -4500v；Ion source temperature (tem): 600 DEG C.
The multiple-reaction monitoring ion pair of compound and mass spectrum relevant parameter are as shown in table 2.
The multiple-reaction monitoring ion pair of table 2 compound and mass spectrum relevant parameter table
* it is quota ion
During liquid chromatography-mass spectrography detection, due to the impact of matrix effect, target compound is led to occur ion to strengthen or press down
Make use, using without target determinand obtain matrix blank solution prepare gradient hybrid standard product solution (0.1,0.2,0.5,
1.0,2.0mg/kg).Condition measures according to the above analysis, with peak area to concentration linear regression.Sample beyond the range of linearity
Redeterminate after needing dilution.Standard curve see Fig. 5,6.
Table 3 standard curve and linear relationship
In sample, the retention time of toxoflavin chromatographic peak is compared with the retention time of respective standard chromatographic peak, excursion
Should be within ± 2.5%.The signal to noise ratio at the reconstructed ion chromatogram peak of the qualitative ion of toxoflavin should be greater than equal to 3 (s/n >=3),
The signal to noise ratio at the reconstructed ion chromatogram peak of quota ion should be greater than equal to 10 (s/n >=10).The detection lower bound of toxoflavin is
0.2mg/kg.
Beneficial effect:
The method of the present invention is easy and simple to handle, qualitative, quantitative ability is stronger, can make to detect fermented flour quality and assessment food peace
The reliable method of full blast danger.
Brief description
Below in conjunction with the accompanying drawings the specific embodiment of the present invention is described in further detail.
Fig. 1 is [m-h] of toxoflavin-Parent ion mass spectrum.
Fig. 2 is [m-h] of toxoflavin-The feature daughter fragment ion spectrogram producing for parent ion.
Fig. 3 is the total ion current figure of toxoflavin.
Fig. 4 is the selection daughter ion flow graph of toxoflavin.
The standard curve of Fig. 5 ion pair 192.1/107.8.
The standard curve of Fig. 6 ion pair 192.1/163.7.
Specific embodiment
The selection of embodiment 1 parent ion
Detection method about toxoflavin still belongs to blank, and the present invention compares [m-h] of toxoflavin-With [m-2h]2-Two grades
The response value of fragment ion, result shows [m-h] of toxoflavin-Ion as parent ion can meet with a response higher two grade from
Sub- fragment, final choice [m-h] of the present invention-, as toxoflavin parent ion, its Mass Spectrometer Method sensitivity is apparently higher than existing for ion
Detection method.The parent ion of toxoflavin selects [m-h] with a negative charge-Ion.
Fig. 1 is [m-h] of toxoflavin-Parent ion mass spectrum.Fig. 2 is [m-h] of toxoflavin-The feature producing for parent ion
Daughter fragment ion spectrogram.Carry out secondary fragment scanning after mass spectrometry parameters are optimized, find [m-h] of toxoflavin-Ion
The secondary ion fragment that can meet with a response higher as parent ion.Toxoflavin is with [m-h]-In the case of ion is as parent ion,
Determine two to ion pair, 192.1/163.7 and 192.1/107.8；Mrm optimization and ion are carried out respectively to above two class ion pairs
After source optimization, under respective optimal conditionss, liquid chromatography-tandem mass spectrometry detection is carried out to the mc-lr standard substance of same concentrations,
By the comparative analysiss to mass spectrum, find toxoflavin with [m-h]-Ion is as the two pairs of ion pairs determining in the case of parent ion
There is higher response value, sensitivity is higher.
Embodiment 2 sample pre-treatments condition
Sample pre-treatments
The present invention is based on fermented flour, and the substrate of fermented flour is more complicated, and it is higher that enrichment purifies pre-treatment requirement.
Accurately weigh the fermented flour 1.0g through pulverizing, homogenizing is crossed, extract toxin with 5ml ultra-pure water solution, 200rpm shakes
Bed concussion 30min, less than 20 DEG C 8000rpm are centrifuged 10min, take supernatant, with 2mol/l formic acid regulation supernatant ph value to 6～
7；Repeat to extract once with 5ml ultra-pure water, merge supernatant twice, be settled to 10ml with ultra-pure water.Now obtain more pure
Crude extract.
Enrichment and purification
First with 1ml methanol and 1ml ultra-pure water activating ion exchange column, activation process flow speed control, at 1-2 drop/sec, takes
2ml crude extract injection ion exchange column carries out purification enrichment, and flow speed control, at 1 drop/sec, then uses 1ml ultra-pure water eluent ion
Exchange column, to go the removal of impurity, finally with 1.0ml methanol solution eluting toxoflavin at twice, nitrogen is blown to closely do, with 50% acetonitrile
Aqueous solution is settled to 0.4ml, treats that liquid chromatography-tandem mass spectrometry (lc-ms/ms) detects.
The determination of embodiment 3 lc-ms/ms testing conditions
According to pertinent regulations in No. 657/2002/eec resolution of cac and eu, select two to carry out mrm to ion and monitor
Meet, the selection of daughter ion simultaneously is main to consider that wherein parent ion and daughter ion are special according to the mass spectrum of every kind of compound and structure
Property choose, and actual sample analysis mesostroma interference less.Determine the molecular ion of various materials, then respectively with various
The molecular ion of compound is parent ion, its daughter ion is carried out full scan choose that abundance is relatively strong, disturb less two antithetical phrases from
Son is qualitative ion, and toxoflavin second order mses figure is as shown in Fig. 2 selection ion flow graph such as Fig. 4 of the final two pairs of ions determining
Shown.Finally various mass spectrometry parameters are optimized with multiple-reaction monitoring (mrm) positive ion mode.
Lc-ms/ms testing conditions:
Liquid-phase condition phenomenex gemini c18 chromatographic column (3.0mm × 150mm, 5 μm), 30 DEG C of column temperature；Sample size
20μl；Mobile phase a be water (containing 0.1% formic acid, 5mmol/l ammonium formate), b be 95% acetonitrile (containing 0.1% formic acid,
5mmol/l ammonium formate)；Condition of gradient elution is shown in Table 1:
Table 1 condition of gradient elution
| Time/min | Flow velocity/μ l min-1 | A/% | B/% |
| 0.0 | 250 | 90 | 10 |
| 3.0 | 250 | 40 | 60 |
| 3.01 | 500 | 40 | 60 |
| 5.0 | 500 | 40 | 60 |
| 5.01 | 250 | 90 | 10 |
| 10.0 | 250 | 90 | 10 |
Mass Spectrometry Conditions Ionization mode is electro-spray ionization negative mode (esi-)；Multiple-reaction monitoring pattern (mrm)；Collision
Atmospheric pressure (cad): 6；Gas curtain atmospheric pressure (cur): 10；Electron spray voltage (is): -4500v；Ion source temperature (tem): 600 DEG C.
The multiple-reaction monitoring ion pair of compound and mass spectrum relevant parameter are as shown in table 2.
The multiple-reaction monitoring ion pair of table 2 compound and mass spectrum relevant parameter table
Embodiment 4 response rate is tested
(1) sample adds:
Blank fermented flour sample is taken to carry out the interpolation of three levels, the pitch-based sphere of toxoflavin is 0.2mg/kg, 0.5mg/
Kg, 1.0mg/kg, each level do 6 parallel.
(2) sample pre-treatments
Accurately weigh the fermented flour 1.0g having added toxin, extract toxin with 5ml ultra-pure water solution, 200rpm shaking table shakes
Swing 30min, less than 20 DEG C 8000rpm are centrifuged 10min, take supernatant, with 2mol/l formic acid regulation supernatant ph value to 6～7；With
5ml ultra-pure water repeats to extract once, merges supernatant twice, is settled to 10ml with ultra-pure water.Now obtain more pure thick
Extract.
(3) it is enriched with and purify
First with 1ml methanol and 1ml ultra-pure water activating ion exchange column, activation process flow speed control, at 1-2 drop/sec, takes
2ml crude extract injection ion exchange column carries out purification enrichment, and flow speed control, at 1 drop/sec, then uses 1ml ultra-pure water eluent ion
Exchange column, to go the removal of impurity, finally with 1.0ml methanol solution eluting toxoflavin at twice, nitrogen is blown to closely do, with 50% acetonitrile
Aqueous solution is settled to 0.4ml, treats that liquid chromatography-tandem mass spectrometry (lc-ms/ms) detects；
(4) lc-ms/ms detection
Liquid-phase condition phenomenex gemini c18 chromatographic column (3.0mm × 150mm, 5 μm), 30 DEG C of column temperature；Sample size
20μl；Mobile phase a be water (containing 0.1% formic acid, 5mmol/l ammonium formate), b be 95% acetonitrile (containing 0.1% formic acid,
5mmol/l ammonium formate)；Condition of gradient elution is shown in Table 1:
Table 1 condition of gradient elution
| Time/min | Flow velocity/μ l min-1 | A/% | B/% |
| 0.0 | 250 | 90 | 10 |
| 3.0 | 250 | 40 | 60 |
| 3.01 | 500 | 40 | 60 |
| 5.0 | 500 | 40 | 60 |
| 5.01 | 250 | 90 | 10 |
| 10.0 | 250 | 90 | 10 |
Mass Spectrometry Conditions Ionization mode is electro-spray ionization negative mode (esi-)；Multiple-reaction monitoring pattern (mrm)；Collision
Atmospheric pressure (cad): 6；Gas curtain atmospheric pressure (cur): 10；Electron spray voltage (is): -4500v；Ion source temperature (tem): 600 DEG C.
The multiple-reaction monitoring ion pair of compound and mass spectrum relevant parameter are as shown in table 2.
The multiple-reaction monitoring ion pair of table 2 compound and mass spectrum relevant parameter table
* it is quota ion
Experimental result shows, the response rate of this experimental technique is 92.0%～94.5%, the coefficient of variation is 1.2%～
6.3%.
Embodiment 5
In 2 parts of fermented flour samples of market stochastic buying.Using this method, 2 kinds of products are carried out with the detection of toxoflavin.
(1) sample pre-treatments
Accurately weigh fermented flour 1.0g, with 5ml ultra-pure water solution extract toxin, 200rpm shaking table shake 30min, 20 DEG C with
Lower 8000rpm is centrifuged 10min, takes supernatant, with 2mol/l formic acid regulation supernatant ph value to 6～7；Repeated with 5ml ultra-pure water
Extract once, merge supernatant twice, be settled to 10ml with ultra-pure water.
Blank fermented flour is taken to be added recovery experiment, toxoflavin pitch-based sphere is 0.5mg/kg, pre-treating method and sample
Product method is identical.
(2) it is enriched with and purify
First with 1ml methanol and 1ml ultra-pure water activating ion exchange column, activation process flow speed control, at 1-2 drop/sec, takes
2ml crude extract injection ion exchange column carries out purification enrichment, and flow speed control, at 1 drop/sec, then uses 1ml ultra-pure water eluent ion
Exchange column, to go the removal of impurity, finally with 1.0ml methanol solution eluting toxoflavin at twice, nitrogen is blown to closely do, with 50% acetonitrile
Aqueous solution is settled to 0.4ml, treats that liquid chromatography-tandem mass spectrometry (lc-ms/ms) detects；
(3) lc-ms/ms detection
Liquid-phase condition phenomenex gemini c18 chromatographic column (3.0mm × 150mm, 5 μm), 30 DEG C of column temperature；Sample size
20μl；Mobile phase a be water (containing 0.1% formic acid, 5mmol/l ammonium formate), b be 95% acetonitrile (containing 0.1% formic acid,
5mmol/l ammonium formate)；Condition of gradient elution is shown in Table 1:
Table 1 condition of gradient elution
| Time/min | Flow velocity/μ l min-1 | A/% | B/% |
| 0.0 | 250 | 90 | 10 |
| 3.0 | 250 | 40 | 60 |
| 3.01 | 500 | 40 | 60 |
| 5.0 | 500 | 40 | 60 |
| 5.01 | 250 | 90 | 10 |
| 10.0 | 250 | 90 | 10 |
Mass Spectrometry Conditions Ionization mode is electro-spray ionization negative mode (esi-)；Multiple-reaction monitoring pattern (mrm)；Collision
Atmospheric pressure (cad): 6；Gas curtain atmospheric pressure (cur): 10；Electron spray voltage (is): -4500v；Ion source temperature (tem): 600 DEG C.
The multiple-reaction monitoring ion pair of compound and mass spectrum relevant parameter are as shown in table 2.
The multiple-reaction monitoring ion pair of table 2 compound and mass spectrum relevant parameter table
* it is quota ion
(4) testing result
The response rate of method is 92.5%.
After the testing result of sample is corrected by the response rate, obtain final testing result: No. 1 sample poisoning of fermented flour is yellow
Cellulose content is 0.32mg/kg, and in No. 2 samples of fermented flour, toxoflavin content is not detect.
Claims (1)
1. a kind of liquid chromatography-tandem mass of toxoflavin is it is characterised in that comprise the following steps:
(1) sample pre-treatments
Accurately weigh through pulverizing, detected sample 1.0 g that crosses of homogenizing, extract toxoflavins with 5 ml ultra-pure water solutions, 200
Rpm shaking table shakes 30 min, and less than 20 DEG C 8000 rpm are centrifuged 10 min, take supernatant, adjust supernatant with 2 mol/l formic acid
Liquid ph value is to 6 ~ 7；Repeat to extract once with 5 ml ultra-pure waters, merge supernatant twice, be settled to 10ml with ultra-pure water；Obtain pure
Net crude extract；
(2) it is enriched with and purify
Enrichment purification uses solid-phase extraction column, and solid-phase extraction column is weak cation exchange post, 33 μm of particle diameter, aperture 85, surface
Long-pending 800 m2/ g, ion capacity 1.0 meq/g；Comprise the concrete steps that:
First with 1 ml methanol and 1 ml ultra-pure water activating ion exchange column, activation process flow speed control, at 1-2 drop/sec, takes 2
Ml crude extract injection ion exchange column carry out purification enrichment, flow speed control at 1 drop/sec, then with 1 ml ultra-pure water eluent ion
Exchange column, to go the removal of impurity, finally with 1.0 ml methanol solution eluting toxoflavins at twice, nitrogen is blown to closely do, with 50 % second
Nitrile aqueous solution (v/v) is settled to 0.4 ml, treats that liquid chromatography-tandem mass spectrometry (lc-ms/ms) detects；
(3) lc-ms/ms detection
Liquid-phase condition phenomenex gemini c18 reversed phase chromatographic column, 3.0 mm × 150 mm, 5 μm；30 DEG C of column temperature；Enter
Sample amount 20 μ l；Mobile phase a is water, containing 0.1% formic acid (v/v) and 5 mmol/l ammonium formates；B is 95 % acetonitriles, contains 0.1%
Formic acid (v/v) and 5 mmol/l ammonium formates；Gradient elution；Condition of gradient elution is as follows:
Mass Spectrometry Conditions Ionization mode is electro-spray ionization negative mode (esi-)；Multiple-reaction monitoring pattern (mrm)；Collision gas
Pressure (cad): 6psi；Gas curtain atmospheric pressure (cur): 10 psi；Electron spray voltage (is): -4500 v；Ion source temperature (tem):
600 ℃；The multiple-reaction monitoring ion pair of toxoflavin and mass spectrum relevant parameter are as follows:
* it is quota ion.
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