CN108519456A - It is a kind of to analyze a variety of remaining methods of aminoglycoside compound in agriculture beast product simultaneously - Google Patents
It is a kind of to analyze a variety of remaining methods of aminoglycoside compound in agriculture beast product simultaneously Download PDFInfo
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- CN108519456A CN108519456A CN201810249355.2A CN201810249355A CN108519456A CN 108519456 A CN108519456 A CN 108519456A CN 201810249355 A CN201810249355 A CN 201810249355A CN 108519456 A CN108519456 A CN 108519456A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention discloses a kind of while analyzing the assay methods of a variety of aminoglycoside compounds in agriculture beast product, it is to weigh sample, add ammonium acetate buffer solution, it after mechanical shaking extraction, is centrifuged, divides and the extracting solution obtained after centrifugation is taken to be adjusted to suitable range with pH meter, it is purified using dispersive solid-phase extraction and solid phase extraction column, ultra performance liquid chromatography tandem mass spectrum (UPLC MS/MS), electron spray ionisation (ESI), multiple-reaction monitoring (MRM) scan pattern.The present invention is easy to operate compared with other congenic methods, sample throughput is high, realizing streptomysin, dihydrostreptomycin, spectinomycin, gentamicin, apramycin, kanamycins, amikacin, neomycin, totally 8 kinds of glucoside-containing components detect together, greatly improve detection efficiency, it can be rapidly performed by qualitative, quantitative, detection limit is fully able to meet domestic and international regulation and limitation requirement, and strong support is provided for the product food security guarantee of China's agriculture beast and inlet and outlet.
Description
Technical field
The invention belongs to food detection method technical fields, more particularly to one kind analyzing a variety of amino in agriculture beast product simultaneously
The remaining method of glycoside compounds.
Background technology
Aminoglycoside antibiotics is a kind of antibiotic containing amino sugar glycosidic bond, has a broad antifungal spectrum, to aerobic gram-negative
Property bacillus there is powerful antibacterial activity, aminoglycoside antibiotics is and long by food due to toxic side effects such as ear kidneys
Human body will be made to generate drug resistance after the intake of phase is internal, therefore its residue detection becomes more and more important.
Aminoglycoside antibiotics have alkalinity, the big characteristic of polarity, retain on a cl 8 column it is very weak, along with lack hair
The characteristics such as color and fluorophor, are detected relatively difficult with conventional method.Our countries do not have standard detection agriculture still at present
Aminoglycoside compound in product, for the examination criteria of animal food, such as GB 21323-2007《In animal tissue
Measurement High Performance Liquid Chromatography/Mass Spectrometry/mass spectrography of aminoglycoside medicaments residual quantity》、GB/T 22969-2008《Milk powder and ox
The measurement Liquid Chromatography-Tandem Mass Spectrometry of milk streptomycin, dihydrostreptomycin and yapamicin relict amount》、GB/T 22945-
2008《The measurement Liquid Chromatography-Tandem Mass Spectrometry of royal jelly streptomycin dihydrostreptomycin and yapamicin relict amount》, according to
When aminoglycoside compound in above three standard detection food remains, ion-pairing agent is used, operation is relatively complicated, and
It is also easy to produce ion depression effect or incompatible with mass spectrum, maintenance cost increases.
The report that the country is detected simultaneously for aminoglycosides compounds such as streptomysins is only limitted in animal food, is lacked and is answered
For the research of the correlation technique of detection simultaneously in agriculture beast product.Therefore it establishes quick, effective, sensitive, reliable and practical residual
Analysis method is stayed to have important practical significance food security.
Invention content
The present invention in view of the above-mentioned deficiencies in the prior art, provides a kind of utilization dispersive solid-phase extraction and Solid Phase Extraction
Technology pre-processes sample, using the detection of hydrophilic Interaction Chromatography mass spectrometric hyphenated technique rapidly and efficiently and while result is accurate
Analyze a variety of remaining methods of aminoglycoside compound in agriculture beast product.
The technical solution that the present invention solves above-mentioned technical problem is as follows：It is a kind of to analyze a variety of amino sugars in agriculture beast product simultaneously
The remaining method of glycoside compound, includes the following steps：
(1) it extracts：It weighs in sample to plastic centrifuge tube, 15ml extraction ammonium acetate buffer solutions is added, are fully vortexed
Mixing is centrifuged, and in another 50mL plastic centrifuge tubes, residue repeats to extract once transfer supernatant, merges supernatant extremely
In centrifuge tube, the extracting solution divided after taking 10mL to merge adjusts pH value to 6.50 ± 0.05 with pH meter, obtains aqueous solution.
(2) it purifies：1g diatomite vortex mixings, centrifugation is added in the aqueous solution that step (1) is obtained；Supernatant is weak through mixed type
The Solid Phase Extraction column purification that cation exchanges；The solid-phase extraction column uses 3mL methanol, 3mL water to activate in advance, and loading discards outflow
Liquid, then with 3mL water, the elution of 3ml methanol discards leacheate, drains, first 2mL eluents 1 used to elute, and eluent 1 is 10% first
Acid, methanol+water=1+1 collect eluent in 10mL centrifuge tubes, add the elution of 2mL eluents 2, eluent 2 is 10% formic acid
Methanol is drained, and eluent, whirlpool mixing are merged.Eluent after collection crosses 0.22 μm of PTFE filter membrane, by following instruments
Condition, upper machine testing：
UPLC/MS/MS is detected：
Instrument condition：
Ultra high efficiency liquid phase mobile phase：A：250mM formic acid aqueous ammoniums, formic acid aqueous ammonium are in 200mL water plus 800uL first
Acid；B：200mM ammonium formate organic phase solutions, ammonium formate organic phase solution be 200mL in plus formic acid 400uL, organic phase be acetonitrile+
Methanol+water=6+1+3；
Ion source：ESI+
Chromatographic column：CORTECS HILIC 2.1cm×100mm×1.6μm Column
Chromatographic condition：
| Time-min | Flow-ml/min | %A | %B |
| Initial | 0.25 | 0 | 100 |
| 0.2 | 0.25 | 0 | 100 |
| 1.5 | 0.25 | 35 | 65 |
| 4.5 | 0.25 | 35 | 65 |
| 4.52 | 0.25 | 0 | 100 |
Mass Spectrometry Conditions：Multiple-reaction monitoring (MRM) scan pattern, band * are quota ion.
Further, the extraction described in step (1) is to weigh the sample 3.0g of uniform sample preparation in 50mL centrifuge tubes, is added
The 15mL extraction ammonium acetate buffer salting liquids of 0.01mol/L, be fully vortexed mixing, and 4000r/min centrifuges 5min；Shift supernatant
In another 50mL plastic centrifuge tubes, residue repeats to extract once liquid, merges in supernatant to centrifuge tube, divides after taking 10mL to merge
Extracting solution adjust pH value to 6.50 ± 0.05 with pH meter；1g diatomite vortex mixings are added in the aqueous solution that step (1) is obtained,
4000r/min centrifuges 5min；Solid Phase Extraction column purification of the supernatant through mixed type weak cation exchange：The solid-phase extraction column is used in advance
3mL methanol, the activation of 3mL water, loading discards efflux, then uses 3mL water, and 3ml methanol elutes, and discards leacheate, drains, first uses
2mL eluents 1 elute, and eluent 1 is 10% formic acid, and methanol+water=1+1 is collected eluent in 10mL centrifuge tubes, added
2mL eluents 2 elute, and eluent 2 is 10% formic acid methanol, is drained, and merge eluent, whirlpool mixing.Eluent after collection,
Cross 0.22 μm of PTFE filter membrane, upper machine testing.
The beneficial effects of the invention are as follows：The present invention locates sample using dispersive solid-phase extraction and solid phase extraction techniques in advance
Reason, it is rapidly and efficiently and a variety of in analysis agriculture beast product while result is accurate using the detection of hydrophilic Interaction Chromatography mass spectrometric hyphenated technique
The remaining method of aminoglycoside compound.Dispersive solid-phase extraction is by the way that adsorbent is added directly into sample solution, is utilized
Adsorbent adsorptive hindrance is removed by the method for centrifugation.Solid phase extraction techniques basic operation is exactly to utilize solid absorbent will
Target compound in fluid sample or impurity absorption make the matrix of sample and interfering compound be detached with target compound, reach
To the purpose for detaching and being enriched with target compound；Hydrophilic Interaction Chromatography technology be it is a kind of in reverse-phase chromatography retention property it is poor
The pattern that is effectively retained of highly polar analyte, the present invention uses 8 kinds of aminoglycoside antibiosis of CORTECS HILIC columns pair
Element, which is analyzed, can get good reservation, and sensitivity meets relevant laws and regulations requirement, to conventional amino glycoside antibiotic
It can carry out accurate qualitative and quantify, and improve detection and analysis flux.
The present invention is extracted using ammonium acetate buffer salting liquid, is accurately adjusted to using pH meter after suitable pH value and is first used silicon
Diatomaceous earth handles sample liquid, and diatomite is a kind of very general adsorbent and filter aid, can adsorb some polarity chaff interferents, keep filtrate clear
It is bright, be conducive to subsequent Solid phase extraction.Then the solid-phase extraction column for using mixed type weak cation exchange, can effectively go
Except the neutral and alkaline chaff interferent of some organic acids and part in sample solution, the UPLC-MS/MS inspections of scavenging solution high sensitivity
It surveys, external standard method qualitative and quantitative analysis.
Specific implementation mode
Principles and features of the present invention are described with reference to embodiments, the given examples are served only to explain the present invention,
It is not intended to limit the scope of the present invention.
Prepare spectinomycin, gentamicin, kanamycins, amikacin, apramycin, streptomysin, double hydrogen strepto-s
Element and 8 kinds of aminoglycoside hybrid standard product solution of neomycin (use apple, root-mustard, Chinese cabbage, spinach, green stalk to negative fruits and vegetables
Dish, potato, taro son, French bean, carrot, round onions, great Jiang, garlic stems, chive totally 13 kinds of fruits and vegetables uniformly made of mixed-matrix), it is cloudy
Property pig lean meat, negative chicken, negative pork liver and negative mackerel in add the standard items of detection limit level respectively, pitch-based sphere is equal
Equal to or less than the limit standard in China, each contents level is repeated 6 times.
Embodiment 1：
Above-mentioned negative fruits and vegetables sample is taken, that is, uses apple, root-mustard, Chinese cabbage, spinach, green stem vegetable, potato, taro son, green knife
Beans, carrot, round onions, great Jiang, garlic stems, chive totally 13 kinds of fruits and vegetables uniformly made of mixed-matrix, detection streptomysin and double hydrogen strepto-s
Plain two kinds of aminoglycoside compound residual contents, steps are as follows：
(1) it extracts：In the fruits and vegetables sample 3.0g to plastic centrifuge tube for weighing uniform sample preparation, 15mL extractions are added and use
The ammonium acetate buffer salting liquid of 0.01mol/L, be fully vortexed mixing, and 4000r/min centrifuges 5min；Supernatant is shifted in another
In 50mL plastic centrifuge tubes, residue repeats to extract once, merges in supernatant to centrifuge tube, divides the extracting solution after taking 10mL to merge
It is 6.50 ± 0.05 to adjust pH value using pH meter：First use 10mol/L sodium hydroxide solution coarse adjustment, then with 1mol/L sodium hydroxides with
0.1mol/L sodium hydroxides are finely tuned, and beyond being adjusted back using 6mol/L hydrochloric acid solutions or 0.1mol/L hydrochloric acid solutions, obtain aqueous solution；
(2) it purifies：1g diatomite vortex mixings, centrifugation is added in the aqueous solution that step (1) is obtained；Supernatant is weak through mixed type
The Solid Phase Extraction column purification that cation exchanges；The solid-phase extraction column uses 3mL methanol, 3mL water to activate in advance, and loading discards outflow
Liquid, then with 3mL water, the elution of 3ml methanol discards leacheate, drains, first 2mL eluents 1 used to elute, and eluent 1 is 10% first
Acid, methanol+water=1+1 collect eluent in 10mL centrifuge tubes, add the elution of 2mL eluents 2, eluent 2 is 10% formic acid
Methanol is drained, and eluent, whirlpool mixing are merged.Eluent after collection crosses 0.22 μm of PTFE filter membrane, by following instruments
Condition, upper machine testing.
UPLC/MS/MS is detected：
Instrument condition：
Ultra high efficiency liquid phase mobile phase：A：250mM formic acid aqueous ammoniums, formic acid aqueous ammonium are in 200mL water plus 800uL first
Acid；B：200mM ammonium formate organic phase solutions, ammonium formate organic phase solution be 200mL in plus formic acid 400uL, organic phase be acetonitrile+
Methanol+water=6+1+3；
Ion source：ESI+
Chromatographic column：CORTECS HILIC 2.1cm×100mm×1.6μm Column
Chromatographic condition：
| Time-min | Flow-ml/min | %A | %B |
| Initial | 0.25 | 0 | 100 |
| 0.2 | 0.25 | 0 | 100 |
| 1.5 | 0.25 | 35 | 65 |
| 4.5 | 0.25 | 35 | 65 |
| 4.52 | 0.25 | 0 | 100 |
Mass Spectrometry Conditions：Multiple-reaction monitoring (MRM) scan pattern, band * are quota ion.
The average recovery rate and relative standard deviation of streptomysin and dihydrostreptomycin residual content in obtained fruits and vegetables
RSD is shown in Table 1.
Embodiment 2：
Above-mentioned pig lean meat sample is taken, detects its 8 kinds of aminoglycoside compound residual contents, steps are as follows：
(1) it extracts：In the pig lean meat sample 3.0g to plastic centrifuge tube for weighing uniform sample preparation, 15mL extractions are added and use
The ammonium acetate buffer salting liquid of 0.01mol/L, be fully vortexed mixing, and 4000r/min centrifuges 5min；Supernatant is shifted in another
In 50mL plastic centrifuge tubes, residue repeats to extract once, merges in supernatant to centrifuge tube, divides the extracting solution after taking 10mL to merge
It is 6.50 ± 0.05 to adjust pH value using pH meter：First use 10mol/L sodium hydroxide solution coarse adjustment, then with 1mol/L sodium hydroxides with
0.1mol/L sodium hydroxides are finely tuned, and beyond being adjusted back using 6mol/L hydrochloric acid solutions or 0.1mol/L hydrochloric acid solutions, obtain aqueous solution；
(2) it purifies：1g diatomite vortex mixings, centrifugation is added in the aqueous solution that step (1) is obtained；Supernatant is weak through mixed type
The Solid Phase Extraction column purification that cation exchanges；The solid-phase extraction column uses 3mL methanol, 3mL water to activate in advance, and loading discards outflow
Liquid, then with 3mL water, the elution of 3ml methanol discards leacheate, drains,
First use 2mL eluents 1 elute, eluent 1 be 10% formic acid, methanol+water=1+1, collect eluent in 10mL from
Heart pipe adds the elution of 2mL eluents 2, and eluent 2 is 10% formic acid methanol, is drained, and eluent, whirlpool mixing are merged.It collects
Eluent afterwards crosses 0.22 μm of PTFE filter membrane, by following instrument conditions, upper machine testing.
UPLC/MS/MS is detected：
Instrument condition：
Ultra high efficiency liquid phase mobile phase：A：250mM formic acid aqueous ammoniums, formic acid aqueous ammonium are in 200mL water plus 800uL first
Acid；B：200mM ammonium formate organic phase solutions, ammonium formate organic phase solution be 200mL in plus formic acid 400uL, organic phase be acetonitrile+
Methanol+water=6+1+3；
Ion source：ESI+
Chromatographic column：CORTECS HILIC 2.1cm×100mm×1.6μm Column
Chromatographic condition：
Mass Spectrometry Conditions：Multiple-reaction monitoring (MRM) scan pattern, band * are quota ion.
The average recovery rate and relative standard deviation of 8 kinds of aminoglycoside compound residual contents of obtained pig lean meat
RSD is shown in Table 1.
Embodiment 3：
Above-mentioned chicken meat sample is taken, detects its 8 kinds of aminoglycoside compound residual contents, steps are as follows：
(1) it extracts：In the chicken meat sample 3.0g to plastic centrifuge tube for weighing uniform sample preparation, 15mL extractions are added and use
The ammonium acetate buffer salting liquid of 0.01mol/L, be fully vortexed mixing, and 4000r/min centrifuges 5min；Supernatant is shifted in another
In 50mL plastic centrifuge tubes, residue repeats to extract once, merges in supernatant to centrifuge tube, divides the extracting solution after taking 10mL to merge
It is 6.50 ± 0.05 to adjust pH value using pH meter：First use 10mol/L sodium hydroxide solution coarse adjustment, then with 1mol/L sodium hydroxides with
0.1mol/L sodium hydroxides are finely tuned, and beyond being adjusted back using 6mol/L hydrochloric acid solutions or 0.1mol/L hydrochloric acid solutions, obtain aqueous solution；
(2) it purifies：1g diatomite vortex mixings, centrifugation is added in the aqueous solution that step (1) is obtained；Supernatant is weak through mixed type
The Solid Phase Extraction column purification that cation exchanges；The solid-phase extraction column uses 3mL methanol, 3mL water to activate in advance, and loading discards outflow
Liquid, then with 3mL water, the elution of 3ml methanol discards leacheate, drains, first 2mL eluents 1 used to elute, and eluent 1 is 10% first
Acid, methanol+water=1+1 collect eluent in 10mL centrifuge tubes, add the elution of 2mL eluents 2, eluent 2 is 10% formic acid
Methanol is drained, and eluent, whirlpool mixing are merged.Eluent after collection crosses 0.22 μm of PTFE filter membrane, by following instruments
Condition, upper machine testing.
UPLC/MS/MS is detected：
Instrument condition：
Ultra high efficiency liquid phase mobile phase：A：250mM formic acid aqueous ammoniums, formic acid aqueous ammonium are in 200mL water plus 800uL first
Acid；B：200mM ammonium formate organic phase solutions, ammonium formate organic phase solution be 200mL in plus formic acid 400uL, organic phase be acetonitrile+
Methanol+water=6+1+3；
Ion source：ESI+
Chromatographic column：CORTECS HILIC 2.1cm×100mm×1.6μm Column
Chromatographic condition：
| Time-min | Flow-ml/min | %A | %B |
| Initial | 0.25 | 0 | 100 |
| 0.2 | 0.25 | 0 | 100 |
| 1.5 | 0.25 | 35 | 65 |
| 4.5 | 0.25 | 35 | 65 |
| 4.52 | 0.25 | 0 | 100 |
Mass Spectrometry Conditions：Multiple-reaction monitoring (MRM) scan pattern, band * are quota ion.
The average recovery rate and relative standard deviation RSD of 8 kinds of aminoglycoside compound residual contents of obtained chicken
It is shown in Table 1.
Embodiment 4：
Above-mentioned Pig Liver is taken, detects its 8 kinds of aminoglycoside compound residual contents, steps are as follows：
(1) it extracts：In the Pig Liver 3.0g to plastic centrifuge tube for weighing uniform sample preparation, 15mL extractions are added and use
The ammonium acetate buffer salting liquid of 0.01mol/L, be fully vortexed mixing, and 4000r/min centrifuges 5min；Supernatant is shifted in another
In 50mL plastic centrifuge tubes, residue repeats to extract once, merges in supernatant to centrifuge tube, divides the extracting solution after taking 10mL to merge
It is 6.50 ± 0.05 to adjust pH value using pH meter：First use 10mol/L sodium hydroxide solution coarse adjustment, then with 1mol/L sodium hydroxides with
0.1mol/L sodium hydroxides are finely tuned, and beyond being adjusted back using 6mol/L hydrochloric acid solutions or 0.1mol/L hydrochloric acid solutions, obtain aqueous solution；
(2) it purifies：1g diatomite vortex mixings, centrifugation is added in the aqueous solution that step (1) is obtained；Supernatant is weak through mixed type
The Solid Phase Extraction column purification that cation exchanges；The solid-phase extraction column uses 3mL methanol, 3mL water to activate in advance, and loading discards outflow
Liquid, then with 3mL water, the elution of 3ml methanol discards leacheate, drains, first 2mL eluents 1 used to elute, and eluent 1 is 10% first
Acid, methanol+water=1+1 collect eluent in 10mL centrifuge tubes, add the elution of 2mL eluents 2, eluent 2 is 10% formic acid
Methanol is drained, and eluent, whirlpool mixing are merged.Eluent after collection crosses 0.22 μm of PTFE filter membrane, by following instruments
Condition, upper machine testing.
UPLC/MS/MS is detected：
Instrument condition：
Ultra high efficiency liquid phase mobile phase：A：250mM formic acid aqueous ammoniums, formic acid aqueous ammonium are in 200mL water plus 800uL first
Acid；B：200mM ammonium formate organic phase solutions, ammonium formate organic phase solution be 200mL in plus formic acid 400uL, organic phase be acetonitrile+
Methanol+water=6+1+3；
Ion source：ESI+
Chromatographic column：CORTECS HILIC 2.1cm×100mm×1.6μm Column
Chromatographic condition：
| Time-min | Flow-ml/min | %A | %B |
| Initial | 0.25 | 0 | 100 |
| 0.2 | 0.25 | 0 | 100 |
| 1.5 | 0.25 | 35 | 65 |
| 4.5 | 0.25 | 35 | 65 |
| 4.52 | 0.25 | 0 | 100 |
Mass Spectrometry Conditions：Multiple-reaction monitoring (MRM) scan pattern, band * are quota ion.
The average recovery rate and relative standard deviation RSD of 8 kinds of aminoglycoside compound residual contents of obtained pork liver
It is shown in Table 1.
Embodiment 5：
Above-mentioned mackerel sample is taken, detects its 8 kinds of aminoglycoside compound residual contents, steps are as follows：
(1) it extracts：In the mackerel sample 3.0g to plastic centrifuge tube for weighing uniform sample preparation, 15mL extractions are added and use
The ammonium acetate buffer salting liquid of 0.01mol/L, be fully vortexed mixing, and 4000r/min centrifuges 5min；Supernatant is shifted in another
In 50mL plastic centrifuge tubes, residue repeats to extract once, merges in supernatant to centrifuge tube, divides the extracting solution after taking 10mL to merge
It is 6.50 ± 0.05 to adjust pH value using pH meter：First use 10mol/L sodium hydroxide solution coarse adjustment, then with 1mol/L sodium hydroxides with
0.1mol/L sodium hydroxides are finely tuned, and beyond being adjusted back using 6mol/L hydrochloric acid solutions or 0.1mol/L hydrochloric acid solutions, obtain aqueous solution；
(2) it purifies：1g diatomite vortex mixings, centrifugation is added in the aqueous solution that step (1) is obtained；Supernatant is weak through mixed type
The Solid Phase Extraction column purification that cation exchanges；The solid-phase extraction column uses 3mL methanol, 3mL water to activate in advance, and loading discards outflow
Liquid, then with 3mL water, the elution of 3ml methanol discards leacheate, drains, first 2mL eluents 1 used to elute, and eluent 1 is 10% first
Acid, methanol+water=1+1 collect eluent in 10mL centrifuge tubes, add the elution of 2mL eluents 2, eluent 2 is 10% formic acid
Methanol is drained, and eluent, whirlpool mixing are merged.Eluent after collection crosses 0.22 μm of PTFE filter membrane, by following instruments
Condition, upper machine testing.
UPLC/MS/MS is detected：Instrument condition：
Ultra high efficiency liquid phase mobile phase：A：250mM formic acid aqueous ammoniums, formic acid aqueous ammonium are in 200mL water plus 800uL first
Acid；B：200mM ammonium formate organic phase solutions, ammonium formate organic phase solution be 200mL in plus formic acid 400uL, organic phase be acetonitrile+
Methanol+water=6+1+3；
Ion source：ESI+
Chromatographic column：CORTECS HILIC 2.1cm×100mm×1.6μm Column
Chromatographic condition：
| Time-min | Flow-ml/min | %A | %B |
| Initial | 0.25 | 0 | 100 |
| 0.2 | 0.25 | 0 | 100 |
| 1.5 | 0.25 | 35 | 65 |
| 4.5 | 0.25 | 35 | 65 |
| 4.52 | 0.25 | 0 | 100 |
Mass Spectrometry Conditions：Multiple-reaction monitoring (MRM) scan pattern, band * are quota ion.
The average recovery rate and relative standard deviation RSD of 8 kinds of aminoglycoside compound residual contents of obtained mackerel
It is shown in Table 1.
The aminoglycoside compound rate of recovery and relative standard deviation (n=6) in 1 agriculture beast product of table
8 kinds of aminoglycosides in agricultural product streptomycin, dihydrostreptomycin and animal foodstuff it can be seen from data in table 1
39.34%~109.79%, relative standard deviation RSD is returned the remaining average recovery rate of compound 2.20%~16.82%
Yield and relative standard deviation meet agricultural and veterinary chemicals residue detection standard.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (2)
1. a kind of analyzing a variety of remaining assay methods of aminoglycoside compound in agriculture beast product simultaneously, it is characterised in that including with
Lower step：
(1) it extracts：It weighs in sample to plastic centrifuge tube, 15ml extraction ammonium acetate buffer solutions is added, be fully vortexed mixing,
It is centrifuged, for transfer supernatant in another 50mL plastic centrifuge tubes, residue repeats to extract primary, merging supernatant to centrifuge tube
In, the extracting solution divided after taking 10mL to merge adjusts pH value to 6.50 ± 0.05 with pH meter, obtains aqueous solution；
(2) it purifies：1g diatomite vortex mixings, centrifugation is added in the aqueous solution that step (1) is obtained；Supernatant through mixed type it is weak sun from
The Solid Phase Extraction column purification that son exchanges：The solid-phase extraction column uses 3mL methanol, 3mL water to activate in advance, and loading discards efflux, then
With 3mL water, the elution of 3ml methanol discards leacheate, drains, first 2mL eluents 1 used to elute, and eluent 1 is 10% formic acid, methanol
+ water=1+1 collects eluent in 10mL centrifuge tubes, adds the elution of 2mL eluents 2, eluent 2 is 10% formic acid methanol, is taken out
It is dry, merge eluent, whirlpool mixing；Eluent after collection crosses 0.22 μm of PTFE filter membrane, by following instrument conditions, on
Machine testing：
UPLC/MS/MS is detected：
Instrument condition：
Ultra high efficiency liquid phase mobile phase：A：250mM formic acid aqueous ammoniums, formic acid aqueous ammonium are in 200mL water plus 800uL formic acid；
B：200mM ammonium formate organic phase solutions, ammonium formate organic phase solution are in 200mL organic phases plus formic acid 400uL, and organic phase is second
Nitrile+methanol+water=6+1+3；
Ion source：ESI+
Chromatographic column：CORTECS HILIC 2.1cm×100mm×1.6μm Column
Chromatographic condition：
Mass Spectrometry Conditions：Multiple-reaction monitoring (MRM) scan pattern, wherein band * is quota ion
2. it is according to claim 1 while analyzing a variety of remaining assay methods of aminoglycoside compound in agriculture beast product,
It is characterized in that the extraction described in step (1) is to weigh the sample 3.0g of uniform sample preparation in 50mL centrifuge tubes, 15ml is added
The extraction ammonium acetate buffer salting liquid of 0.01mol/L, be fully vortexed mixing, is centrifuged, and transfer supernatant is in another 50mL
In plastic centrifuge tube, residue repeats to extract once, merges in supernatant to centrifuge tube, divides the extracting solution pH after taking 10mL to merge
Meter adjusts pH value to 6.50 ± 0.05, obtains aqueous solution.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN109932458A (en) * | 2019-04-22 | 2019-06-25 | 华中农业大学 | A kind of liquid chromatography-tandem mass spectrometry method detecting apramycin in pig ileal contents |
| CN109900841A (en) * | 2019-04-26 | 2019-06-18 | 上海市第一人民医院 | HPLC-MS/MS method that is a kind of while measuring aminoglycoside antibiotics concentration in blood plasma |
| CN112305091A (en) * | 2019-07-31 | 2021-02-02 | 天津市农业质量标准与检测技术研究所 | UPLC-MS/MS method for analyzing streptomycin and dihydrostreptomycin residues in Chinese cabbages and soil |
| CN110554110A (en) * | 2019-09-11 | 2019-12-10 | 中国检验检疫科学研究院 | method for measuring residual quantity of streptomycin and dihydrostreptomycin in plant food |
| CN113156025A (en) * | 2021-01-13 | 2021-07-23 | 上海凯宝药业股份有限公司 | Method for determining kanamycin in-vitro cultured bear gall powder |
| CN115201373A (en) * | 2022-07-13 | 2022-10-18 | 北京英太格瑞检测技术有限公司 | Method for detecting LC-MSMS (liquid chromatography-Mass Spectrometry) by hygromycin B in feed without using ion-pair reagent in mobile phase |
| CN115452974A (en) * | 2022-08-22 | 2022-12-09 | 浙江大学 | Method for determining spectinomycin in feed |
| CN115452974B (en) * | 2022-08-22 | 2024-03-29 | 浙江大学 | Determination method of spectinomycin in feed |
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