CN114088858B - Detection method of lincosamide antibiotics - Google Patents
Detection method of lincosamide antibiotics Download PDFInfo
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- CN114088858B CN114088858B CN202111376545.9A CN202111376545A CN114088858B CN 114088858 B CN114088858 B CN 114088858B CN 202111376545 A CN202111376545 A CN 202111376545A CN 114088858 B CN114088858 B CN 114088858B
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- G—PHYSICS
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- 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
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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
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of analysis, and provides a detection method of lincosamide antibiotics. According to the detection method, an internal standard method is combined with a multi-reaction monitoring (MRM) -information association acquisition (IDA) -enhanced ion scanning (EPI) detection mode, the detected suspected positive sample is subjected to comparative analysis through chromatographic peaks, retention time, ion kurtosis ratio and an EPI screening database, then positive components are quantitatively analyzed by a standard addition method, and detection accuracy is improved. Compared with the time-of-flight mass spectrometry detection method, the detection method provided by the invention has low detection cost.
Description
Technical Field
The invention relates to the technical field of analysis, in particular to a detection method of lincosamide antibiotics.
Background
Along with the continuous improvement of the economic level of China, the aquaculture industry also develops towards diversification and intensification. The occurrence rate of various aquaculture diseases is also becoming serious while the economic benefit of aquaculture is improved. Therefore, the use of various fish inputs has become a conventional auxiliary means for aquaculture in order to prevent and control the occurrence of epidemic diseases in the aquaculture process. In order to pursue benefit and evasion supervision, illegal merchants occur when adding lincosamide antibiotics in fishery input products (non-medicines) illegally, and great potential safety hazards are brought to the aquaculture industry and the life health of people in China.
The lincomamide antibiotics mainly comprise clindamycin and lincomycin, have strong action on gram-positive bacteria and good anaerobic bacteria, and can be combined with 50S ribosomal subunits on ribosomes to block prokaryotic translation, so that bacteria die. At present, the detection method mainly comprises a triple quadrupole mass spectrometry, but the triple quadrupole mass spectrometry can not accurately distinguish substances with similar structures, and is easy to cause false positive of a detection result. In order to further improve the detection accuracy of lincoamide antibiotics, time-of-flight mass spectrometry is increasingly concerned, but time-of-flight mass spectrometers are expensive, resulting in high detection costs.
Disclosure of Invention
In view of the above, the invention aims to provide a detection method of lincosamide antibiotics, which has high accuracy and low detection cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a detection method of lincosamide antibiotics, which comprises the following steps:
carrying out ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection on a sample to be detected containing an internal standard substance, so as to realize detection of lincoamide antibiotics in the sample to be detected;
the ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection comprises ultra-high performance liquid chromatography detection and triple quadrupole composite linear ion trap mass spectrometry detection;
the parameters of the ultra performance liquid chromatography detection include:
the chromatographic column is Infnitylabporoshell SB-C 18 ；
The mobile phase system comprises a mobile phase A and a mobile phase B;
the mobile phase A is formic acid aqueous solution with volume concentration of 0.1%;
the mobile phase B is methanol;
the flow rate of the mobile phase system is 4.0mL/min:
mobile phase elution procedure:
0-2 min: the volume fraction of mobile phase a was 95%;
2-5 min: the volume fraction of the mobile phase A is changed from 95% to 75% at uniform speed;
5-6 min: the volume fraction of the mobile phase A is changed from 75% to 20% at uniform speed;
6-7 min: the volume fraction of the mobile phase A is changed from 20% to 95% at uniform speed;
7-10 min: the volume fraction of mobile phase a was 95%;
the sample injection amount is 10 mu L;
the column temperature is 25-40 ℃;
the triple quadrupole composite linear ion trap mass spectrum detection comprises the following steps: electrospray positive ion ionization mode, multi-reaction monitoring, information correlation acquisition and enhanced ion scanning;
parameters of the electrospray positive ion ionization mode include: the ionization voltage is 5000-5500V; the temperature of the ion source is 500-550 ℃; the air curtain gas is 30-35L/min; the atomization gas is 55-60L/min; the auxiliary heating gas is 55-60L/min;
the parameters for the multiple reaction monitoring are shown in table 1:
TABLE 1 parameters for multiple reaction monitoring
The parameters of the information association acquisition comprise: the scanning range is 100-500 Da, the ionic strength threshold for triggering the secondary scanning is 800-1200 cps, and the dynamic background subtraction is started;
the parameters of the enhanced ion scan include: the scanning type is Enhanced Product ion, the positive ion mode and the EPI collision energy is 40+/-20 eV;
the internal standard is caffeine 13 C or lincomycin 13 C-D 3 Hydrochloride salt.
Preferably, the concentration of the internal standard in the sample to be tested containing the internal standard is 100ng/mL.
Preferably, the sample to be measured comprises an aquatic product sample to be measured or a water sample to be measured.
Preferably, the method for obtaining the sample to be measured of the aquatic product comprises the following steps:
mixing an aquatic product to be detected, an internal standard substance and an extracting agent, and extracting to obtain an extracting solution;
evaporating the extracting solution to dryness and then re-dissolving to obtain the sample to be measured of the aquatic product.
Preferably, the extractant is acetonitrile, methanol or acetone; the dosage ratio of the aquatic product to be detected to the extractant is 2.0g: 15-25 mL.
Preferably, the method for obtaining the water sample to be measured comprises the following steps:
filtering the wastewater to be detected, adding an internal standard into the obtained filtrate, and then adjusting the pH value to be acidic to obtain acidic filtrate;
enriching the acidic filtrate by an HLB solid phase extraction column to obtain an enrichment solution;
evaporating the enrichment solution to dryness and then re-dissolving to obtain the water sample to be detected.
Preferably, the acidic pH is 3.0 to 6.0.
Preferably, the enriching process comprises: and (3) after passing the acidic filtrate through an HLB solid phase extraction column, sequentially eluting the HLB solid phase extraction column by using ultrapure water and an eluent, and collecting the eluent as the enrichment liquid.
Preferably, the eluent is methanol or ammonia water methanol solution; the volume ratio of the ammonia water to the methanol in the ammonia water methanol solution is 5:95 or 10:90; the mass concentration of the ammonia water is 25% -28%.
Preferably, the ultra performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection comprises quantitative detection and qualitative detection; the standard curve of quantitative detection is a concentration-peak area ratio standard curve; in the standard curve of quantitative detection, the peak area ratio is the ratio of the peak area of the parent ion of the compound to the peak area of the internal standard substance.
The invention provides a detection method of lincosamide antibiotics, which comprises the following steps: carrying out ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection on a sample to be detected containing an internal standard substance, so as to realize detection of lincoamide antibiotics in the sample to be detected; the ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection comprises ultra-high performance liquid chromatography detection and triple quadrupole composite linear ion trap mass spectrometry detection; the triple quadrupole composite linear ion trap mass spectrum detection comprises the following steps: electrospray positive ion ionization mode, multiple Reaction Monitoring (MRM), information-bearing acquisition (IDA), and enhanced ion scanning (EPI). In the invention, the MRM-IDA-EPI detection mode is adopted, the detected suspected positive sample is subjected to comparative analysis through chromatographic peaks, retention time, ion kurtosis ratio and screening EPI database, and then the positive component is quantitatively analyzed by adopting a standard addition method, so that the detection accuracy is improved. Compared with a time-of-flight mass spectrometry method, the detection method provided by the invention has low detection cost.
Drawings
FIG. 1 is a selective ion flow chromatogram of lincomycin, pirlimycin, and clindamycin in standard working fluid at a concentration of 20ng/mL (100 ng/mL of isotopic internal standard);
FIG. 2 is a mass spectrum of lincomycin EPI;
FIG. 3 is a mass spectrum of pirlimycin EPI;
FIG. 4 is a mass spectrum of clindamycin EPI;
FIG. 5 is an EPI diagram of a positive river crab sample;
FIG. 6 is an ion flow chromatogram of a positive river crab sample;
FIG. 7 is an ion flow chromatogram of a negative river crab sample.
Detailed Description
The invention provides a detection method of lincosamide antibiotics, which comprises the following steps:
and carrying out ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection on the sample to be detected containing the internal standard substance, thereby realizing detection of lincoamide antibiotics in the sample to be detected.
In the present invention, the internal standard is caffeine- 13 C or lincomycin 13 C-D 3 Hydrochloride, preferably lincomycin 13 C-D 3 Hydrochloride salt.
In the present invention, the concentration of the internal standard in the sample to be tested containing the internal standard is preferably 100ng/mL.
In the present invention, the sample to be measured preferably includes an aquatic product sample to be measured or a water sample to be measured.
In the invention, the method for acquiring the sample to be measured of the aquatic product preferably comprises the following steps:
mixing an aquatic product to be detected, an internal standard substance and an extracting agent, and extracting to obtain an extracting solution;
evaporating the extracting solution to dryness and then re-dissolving to obtain the sample to be measured of the aquatic product.
The invention mixes the aquatic product to be detected, the internal standard substance and the extracting agent, and extracts the mixture to obtain an extracting solution.
In the invention, the aquatic product to be detected is preferably a minced-shaped aquatic product to be detected; the preparation method of the minced-like aquatic product to be detected is not particularly limited, and can be well known to those skilled in the art.
In the present invention, the internal standard is preferably used in the form of an aqueous internal standard solution, and the concentration of the aqueous internal standard solution is preferably 1. Mu.g/mL.
In the present invention, the extractant preferably includes acetonitrile, methanol or acetone, and further preferably acetonitrile; the dosage ratio of the aquatic product to be tested to the extractant is preferably 2.0g:15 to 25mL, more preferably 2.0g:15mL.
In the present invention, the extraction is preferably performed under vortex oscillation conditions; the rotation speed of the vortex oscillation is preferably 2500-4000 rpm; the time of the swirling is preferably 1 to 10 minutes, more preferably 5 minutes.
After the extraction, the present invention preferably further comprises solid-liquid separation of the obtained extract system to obtain a filtrate as an extract. In the present invention, the solid-liquid separation is preferably performed by centrifugation, and the rotational speed of the centrifugation is preferably 8000rpm, and the time is preferably 10 minutes.
In the present invention, the number of times of the extraction is preferably 1 to 3 times, more preferably 2 times; when the number of times of the extraction is preferably 2, the extraction preferably includes the steps of: mixing an aquatic product to be detected, an internal standard substance and an extracting agent for first extraction, and carrying out solid-liquid separation on the obtained first extraction system to obtain first filtrate and first filter residues; mixing the first filter residue with an extractant, performing second extraction, and performing solid-liquid separation on the obtained second extraction system to obtain second filter residue and second filtrate; and combining the first filtrate and the second filtrate to obtain an extracting solution.
After the extracting solution is obtained, evaporating the extracting solution to dryness and then re-dissolving to obtain the sample to be measured of the aquatic product.
In the present invention, the evaporating mode is preferably spin evaporation, and the parameters of the spin evaporation are not particularly limited, so long as the solvent can be removed.
In the present invention, the redissolved solvent preferably includes a mixed solution of methanol and formic acid solution; the volume concentration of the formic acid solution is preferably 0.1%; the volume ratio of the methanol to the formic acid solution is preferably (5-9): (1 to 5), more preferably 9:1.
after the re-dissolution, the invention preferably further comprises filtering a re-dissolution system obtained after the re-dissolution; the filter membrane is preferably a 0.22 μm polytetrafluoroethylene filter membrane.
The sample pretreatment method for the aquatic product to be detected has simple steps, and the acetonitrile, the methanol or the acetone is used as the extraction solvent, so that the interference of grease, protein and amino acid is reduced, and the method has better solubility to the target object, thereby having higher extraction efficiency. The mixed solution of methanol-0.1% formic acid solution is used for re-dissolution and volume fixation, so that good chromatographic separation effect and mass spectrum response are obtained, and the detection accuracy is further improved.
In the present invention, the method for obtaining a water sample to be measured preferably comprises the steps of:
filtering the wastewater to be detected, adding an internal standard into the obtained filtrate, and then adjusting the pH value to be acidic to obtain acidic filtrate;
enriching the acidic filtrate by an HLB solid phase extraction column to obtain an enrichment solution;
evaporating the enrichment solution to dryness and then re-dissolving to obtain the water sample to be detected.
The invention filters the waste water to be tested, and adjusts the pH value to be acidic after adding an internal standard substance into the obtained filtrate to obtain acidic filtrate.
In the present invention, the pore size of the filtration membrane is preferably 0.22 μm, 0.45 μm or 0.65 μm, and more preferably 0.45 μm.
In the present invention, the internal standard is preferably used in the form of an aqueous internal standard solution, and the concentration of the aqueous internal standard solution is preferably 1. Mu.g/mL.
In the present invention, the acidic pH is preferably 3.0 to 6.0, more preferably 4.0.+ -. 0.05. In the present invention, the agent for adjusting pH to be acidic preferably includes a volatile acid; the volatile acid preferably comprises formic acid and/or hydrochloric acid, further preferably comprises formic acid; the concentration and the amount of the volatile acid are not particularly limited in the present invention, as long as the pH value can be adjusted to be acidic.
After the acid filtrate is obtained, the acid filtrate is subjected to enrichment through an HLB solid phase extraction column to obtain an enrichment solution.
In the present invention, the flow rate of the acidic filtrate through the HLB solid phase extraction column is preferably 3 to 5mL/min, and more preferably 4mL/min.
In the present invention, the HLB solid phase extraction column is preferably activated prior to use; the activating reagent preferably comprises methanol and water; the ratio of the activated reagent volume to the column volume of the HLB solid phase extraction column is preferably 1:1. in the present invention, the activation preferably includes the steps of: and sequentially adopting methanol and water to activate the HLB solid phase extraction column.
In the present invention, the enrichment process preferably includes: and (3) after passing the acidic filtrate through an HLB solid phase extraction column, sequentially eluting the HLB solid phase extraction column by using ultrapure water and an eluent, and collecting the eluent as the enrichment liquid.
In the present invention, the ratio of the volume of the ultrapure water to the column volume of the HLB solid phase extraction column is preferably 1:1. in the invention, the HLB solid phase extraction column is leached by ultrapure water, so that impurities with larger polarity can be removed.
In the present invention, the eluent is preferably methanol or aqueous ammonia methanol solution; the volume ratio of the ammonia water to the methanol in the ammonia water methanol solution is preferably 5:95 or 10:90, more preferably 5:95; the mass concentration of the ammonia water is preferably 25% -28%. In the present invention, the ratio of the volume of the eluent to the column volume of the HLB solid phase extraction column is preferably 1:1. in the present invention, when the eluent is rinsed, it is preferable to perform rinsing in two times; the volume ratio of the two leaches is preferably 1:1.
after obtaining the enrichment liquid, evaporating the enrichment liquid to dryness and then re-dissolving to obtain the water sample to be measured.
In the present invention, the evaporating method is preferably nitrogen blowing, and the parameters of the nitrogen blowing are not particularly limited as long as the solvent can be removed. In the present invention, the redissolved reagent is preferably consistent with the above technical solution, and will not be described herein.
After the re-dissolution, the invention preferably further comprises filtering a re-dissolution system obtained after the re-dissolution; the filter membrane is preferably a 0.22 μm polytetrafluoroethylene filter membrane.
In the invention, the ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection comprises ultra-high performance liquid chromatography detection and triple quadrupole composite linear ion trap mass spectrometry detection.
In the present invention, the parameters of the ultra performance liquid chromatography detection include:
the chromatographic column is Infnitylab poroshell SB-C 18 ；
The mobile phase system comprises a mobile phase A and a mobile phase B;
the mobile phase A is formic acid aqueous solution with volume concentration of 0.1%;
the mobile phase B is methanol;
the flow rate of the mobile phase system is 4.0mL/min:
mobile phase elution procedure:
0-2 min: the volume fraction of mobile phase a was 95%;
2-5 min: the volume fraction of the mobile phase A is changed from 95% to 75% at uniform speed;
5-6 min: the volume fraction of the mobile phase A is changed from 75% to 20% at uniform speed;
6-7 min: the volume fraction of the mobile phase A is changed from 20% to 95% at uniform speed;
7-10 min: the volume fraction of mobile phase a was 95%;
the sample injection amount is 10 mu L;
the column temperature is 25 to 40℃and preferably 35 ℃.
In the invention, the triple quadrupole composite linear ion trap mass spectrometry detection comprises: electrospray positive ion ionization mode, multi-reaction monitoring, information-dependent acquisition and enhanced ion scanning.
In the present invention, the parameters of the electrospray positive ion ionization mode include: the ionization voltage is 5000-5500V, preferably 5500V; the temperature of the ion source is 500-550 ℃, preferably 500 ℃; the air curtain gas is 30-35L/min, preferably 30L/min; the atomization gas is 55-60L/min, preferably 55L/min; the auxiliary heating gas is 55-60L/min, preferably 55L/min;
in the present invention, the parameters of the multiple reaction monitoring are shown in Table 1.
In the invention, the parameters of the information association acquisition comprise: the scanning range is 100-500 Da, the ionic strength threshold for triggering the secondary scanning is 800-1200 cps, preferably 1000cps, and the dynamic background subtraction is started.
In the present invention, the parameters of the enhanced ion scan include: the scan type was Enhanced Product ion, positive ion mode, EPI collision energy 40±20eV.
In the invention, the ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection comprises quantitative detection and qualitative detection. In the invention, the standard curve of quantitative detection is a concentration-peak area ratio standard curve; in the standard curve of quantitative detection, the peak area ratio is the ratio of the peak area of the parent ion of the compound to the peak area of the internal standard substance. In the present invention, the chromatographic peak retention time, parent ion and daughter ion of the reference compound are qualitatively detected.
In the invention, the ultra performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection is preferably performed on a 4500QTRAP ultra performance liquid chromatography composite linear ion trap mass spectrometer purchased from AB SCIEX company.
The following describes the method for detecting lincosamide antibiotics according to the present invention in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
1.1 the detection parameters of the following examples include;
the parameters of the ultra performance liquid chromatography detection include:
the chromatographic column is Infnitylab poroshell SB-C 18 ；
The mobile phase system comprises a mobile phase A and a mobile phase B;
mobile phase A is formic acid water solution with volume concentration of 0.1%;
mobile phase B is methanol;
the flow rate of the mobile phase system was 4.0mL/min:
mobile phase elution procedure:
0-2 min: the volume fraction of mobile phase a was 95%;
2-5 min: the volume fraction of the mobile phase A is changed from 95% to 75% at uniform speed;
5-6 min: the volume fraction of the mobile phase A is changed from 75% to 20% at uniform speed;
6-7 min: the volume fraction of the mobile phase A is changed from 20% to 95% at uniform speed;
7-10 min: the volume fraction of mobile phase a was 95%;
the sample injection amount is 10 mu L;
the column temperature is 35 ℃;
the triple quadrupole composite linear ion trap mass spectrum detection comprises the following steps: electrospray positive ion ionization mode, multi-reaction monitoring, information correlation acquisition and enhanced ion scanning;
parameters of the electrospray positive ion ionization mode include: the ionization voltage is 5500V; the temperature of the ion source is 500 ℃; the air curtain air is 30L/min; the atomization gas is 55L/min; the auxiliary heating gas is 55L/min;
the parameters of the multi-reaction monitoring are shown in table 1;
the parameters of the information association acquisition comprise: the scanning range is 100-500 Da, the ionic strength threshold for triggering the secondary scanning is 1000cps, and the dynamic background subtraction is started;
the parameters of the enhanced ion scan include: the scan type was Enhanced Product ion, positive ion mode, EPI collision energy 40±20eV.
Example 1
Preparation of standard solution: accurately preparing 100 mug/mL of lincomycin, clindamycin and pirlimycin mixed standard stock solution, accurately sucking 1mL of the mixed standard stock solution, transferring the mixed standard stock solution into a 100mL brown volumetric flask, accurately fixing the volume to a scale by using pure methanol, preparing 1 mug/mL of mixed standard intermediate solution, and storing the mixed standard intermediate solution at the temperature of minus 4 ℃ in a dark place.
Preparation of an isotope internal standard solution: accurate preparation of lincomycin 13 C-D3 hydrochloride solution 100 mug/mL, accurately sucking 1mL, transferring to a 100mL brown volumetric flask, accurately fixing the volume to the scale by using pure methanol to prepare 1 mug/mL isotope internal standard working solution, and preserving at-4 ℃ in a dark place.
Preparation of a standard working solution: and respectively sucking proper amounts of standard solution and isotope internal standard solution to prepare standard working solutions with the concentrations of 5, 10, 20, 50 and 100ng/mL (the concentration of the isotope internal standard substance is 100 ng/mL).
And (3) detecting each standard working solution by adopting a detection parameter of 1.1.
FIG. 1 is a selective ion flow chromatogram of lincomycin, pirlimycin, and clindamycin in standard working fluid at a concentration of 20ng/mL (100 ng/mL of isotopic internal standard); FIG. 2 is a mass spectrum of lincomycin EPI; FIG. 3 is a mass spectrum of pirlimycin EPI; FIG. 4 is a mass spectrum of clindamycin EPI. As can be seen from fig. 1 to 4: and an ion flow chromatogram and a secondary mass spectrum of each compound are obtained simultaneously through one sample injection, so that the judgment of the multi-reaction monitoring result is supplemented.
The concentration-peak area ratio standard curve is established by taking the concentrations of the compounds 1, 5, 10, 20, 50 and 100ng/mL as an abscissa X and the ratio of the parent ion peak area of the compound to the peak area of the internal standard substance as an ordinate Y, and the result is that:
the standard curve for lincomycin is: y=5.03×10 4 x+1.26×10 4 ；
The standard curve for clindamycin is: y=5.38×10 4 x+1.33×10 4 ；
The standard curve for pirlimycin is: y=5.25×10 4 x+1.29×10 4 。
The detection limit of lincomycin, clindamycin and pirlimycin is 0.3 mug/kg according to the signal to noise ratio (S/N) of 3 times, and the quantitative limit of lincomycin, clindamycin and pirlimycin is 1.0 mug/kg according to the signal to noise ratio (S/N) of 10 times.
Negative blank surimi was labeled to obtain 3 labeled levels of target concentrations of 1.0, 2.0 and 20.0 μg/kg, and the assay was repeated 6 times according to the optimized method. The standard recovery rate and the accuracy of 3 kinds of lincoamide antibiotics in minced fillet samples are shown in table 2.
TABLE 2 labeling recovery and precision results for 3 lincoamide antibiotics in blank surimi
| Target object | Addition level (μg/kg) | Recovery (%) | Relative standard deviation (%) |
| Lincomycin | 1；2；20 | 99.8；95.4；91.7 | 4.49；3.21；4.51 |
| Clindamycin | 1；2；20 | 84.2；83.9；87.5 | 7.10；4.24；2.48 |
| Pirlimycin | 1；2；20 | 81.5；89.7；78.5 | 10.3；3.71；3.92 |
As can be seen from table 2: the recovery rate of the three addition levels of the lincoamide antibiotics in the minced fillet sample is 78.5-99.8%, and the Relative Standard Deviation (RSD) is 2.48-10.3%.
Example 2
The differences from the labeling recovery and accuracy experiments in example 1 are: the external standard method is adopted in the embodiment, and the addition level is 10 mug/kg; the results of the labeling recovery rate and the precision of the obtained lincosamide antibiotics are shown in Table 3.
TABLE 3 labeling recovery and precision results for lincosamide antibiotics by external standard method
| Target object | Addition level (μg/kg) | Recovery rate of external standard method (%) | Relative standard deviation (%) |
| Lincomycin | 10 | 32.7 | 6.58 |
| Clindamycin | 10 | 45.6 | 10.3 |
| Pirlimycin | 10 | 54.7 | 11.6 |
As can be seen from table 3: the quantitative accuracy of lincomycin, clindamycin and pirlimycin is greatly reduced, and the recovery rate of each compound by an external standard method is 32.7-54.7%.
Example 3
The differences from the labeling recovery and accuracy experiments in example 1 are: in this example, lincomycin, an internal standard 13 Substitution of C-D3 hydrochloride with caffeine 13 C adopts an external standard method; the addition level is 10 mug/kg; the results of the labeling recovery rate and the precision of the obtained lincosamide antibiotics are shown in Table 4.
Table 4 internal standard caffeine- 13 C forestLabeling recovery rate and precision result of amide antibiotics
| Target object | Addition level (μg/kg) | Internal standard caffeine- 13 C recovery (%) | Relative standard deviation (%) |
| Lincomycin | 10 | 84.7 | 5.78 |
| Clindamycin | 10 | 62.7 | 9.55 |
| Pirlimycin | 10 | 63.6 | 10.5 |
As can be seen from table 4: with caffeine- 13 C is an internal standard, and the recovery rate of each compound is 62.7-84.7%. Thus, lincomycin is used 13 The C-D3 hydrochloride can be used as an internal standard substance to greatly improve the accuracy of detection and quantification of lincomycin, clindamycin and pirlimycin.
Example 4
(1) Sample holderTaking: 2.0g of meat emulsion is weighed into a 50mL centrifuge tube and 100 mu L of 1 mu g/mL lincomycin is added 13 C-D 3 The hydrochloride solution was added with 15mL of acetonitrile, vortexed at 2500rpm for 5min, centrifuged at 8000r/min at room temperature for 10min, and the supernatant was transferred. The residue was repeatedly extracted with 10mL of acetonitrile once more and the supernatants were combined.
(2) Sample purification: the supernatant is evaporated to dryness under reduced pressure of nitrogen at 45 ℃, the residual is fixed to 1.00mL by methanol-0.1% formic acid mixed solution (9:1), the mixture is mixed for 1min by vortex, and a sample to be detected of the aquatic product is obtained after passing through a polytetrafluoroethylene filter membrane with the thickness of 0.22 mu m, and the sample is taken as a sample liquid.
(3) And (3) detection: the obtained sample solution was subjected to detection under the detection conditions of 1.1 part.
(4) Data analysis:
4.1, detecting the obtained selective ion flow chromatogram, wherein the ratio of the retention time of the target compound in the sample solution to the retention time of the target compound in the standard solution is within +/-5%; and the detected relative kurtosis of the qualitative ions should be consistent with the relative abundance of the qualitative ions in the working solution with similar concentration, and the deviation should meet the requirements of Table 5. The mass numbers of the main fragment ions of the target compound in the sample liquid are consistent with those of the main fragment ions of the target compound in the standard solution, and the deviation of the mass numbers is not more than +/-0.1; the response value of each fragment ion is within + -20%.
TABLE 5 base peak to next highest fragment ion abundance ratio requirement
| Second strongest fragment ion relative abundance/(%) | Permissible deviation/(%) |
| ﹥50 | ±20 |
| 20-50 (without 20) | ±25 |
| 10-20 (without 10) | ±30 |
| ≤10 | ±50 |
4.2 quantitative determination
Extracting chromatographic information of the sample liquid, and quantifying target substances in the sample to be tested according to a formula 1 by referring to a standard curve obtained in the embodiment 1:
x=c×v/m formula:
x-the residual amount of the target compound in the aquatic product to be detected, mug/kg;
c-concentration of target compound in sample solution, ng/mL;
v-constant volume of final sample solution, mL;
m-the mass of the aquatic product to be measured, g.
75 batches of river crab samples extracted from each farmer market and the breeding base were examined by the method of example 4, and the results showed that lincomycin was detected in 3 batches of river crab samples, with the contents of 7.43. Mu.g/kg, 17.72. Mu.g/kg and 9.47. Mu.g/kg, respectively.
FIG. 5 is an EPI diagram of a positive river crab sample; FIG. 6 is an ion flow chromatogram of a positive river crab sample; FIG. 7 is an ion flow chromatogram of a negative river crab sample.
Example 5
(1) Preparing a water sample: measuring 600-800 mL of water sample, filtering the water sample by using a 1L-capacity suction filtration bottle device and a 0.45 mu m mixed cellulose filter membrane, and if the water sample is turbid, replacing the filter membrane or filtering for 2 times. And (4) hermetically preserving the filtered water sample at 4 ℃ and detecting as soon as possible.
(2) pH adjustment of a water sample: weighing 500mL of filtered water sample, and adding 100 mu L and 1 mu g/mL lincomycin 13 C-D 3 And adding a proper amount of 0.5mol/L formic acid into the hydrochloride to adjust the pH of the water sample to 4.0+/-0.05.
(3) Enriching and purifying a water sample: the HLB solid phase extraction column was activated with 6mL of methanol and 6mL of ultra pure water in sequence. And (3) passing 500mL of water sample with the pH value adjusted to 4.0 through a solid phase extraction column, wherein the flow rate is 4mL/min, after the water sample passes through the solid phase extraction column, rapidly flushing the solid phase extraction column by using 6mL of deionized water, drying the small column for 20min under the protection of nitrogen, eluting with 6mL of 5% ammonia water (the mass concentration is 26-28%) -methanol solution twice (the volume ratio is 1:1), and collecting eluent.
(4) Concentrating and fixing the volume of a water sample: and (3) drying the collected eluent in a water bath at 45 ℃ with mild nitrogen, and finally, fixing the volume to 1.00mL with a methanol-0.1% formic acid mixed solution (9:1), and then, passing through a polytetrafluoroethylene filter membrane with the thickness of 0.22 mu m to obtain a sample to be detected of the aquatic product, wherein the sample is taken as a sample solution of a sample solution on a machine.
(5) And (3) detection: the obtained sample solution was subjected to detection under the detection conditions of 1.1 part.
(6) Data analysis:
6.1, detecting the obtained selective ion flow chromatogram, wherein the ratio of the retention time of the target compound in the sample solution to the retention time of the target compound in the standard solution is within +/-5%; and the detected relative kurtosis of the qualitative ions should be consistent with the relative abundance of the qualitative ions in the working solution with similar concentration, and the deviation should meet the requirements of Table 5. The mass numbers of the main fragment ions of the target compound in the sample liquid are consistent with those of the main fragment ions of the target compound in the standard solution, and the deviation of the mass numbers is not more than +/-0.1; the response value of each fragment ion is within + -20%.
6.2 quantitative determination
25 water samples extracted from the culture base were tested by the method of example 5, and no linkman drug residues were detected.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. The detection method of the lincosamide antibiotics comprises the following steps:
carrying out ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection on a sample to be detected containing an internal standard substance, so as to realize detection of lincoamide antibiotics in the sample to be detected;
the ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection comprises ultra-high performance liquid chromatography detection and triple quadrupole composite linear ion trap mass spectrometry detection;
the parameters of the ultra performance liquid chromatography detection include:
the chromatographic column is Infnitylab poroshell SB-C18;
the mobile phase system comprises a mobile phase A and a mobile phase B;
the mobile phase A is formic acid aqueous solution with volume concentration of 0.1%;
the mobile phase B is methanol;
the flow rate of the mobile phase system is 4.0mL/min:
mobile phase elution procedure:
0-2 min: the volume fraction of mobile phase a was 95%;
2-5 min: the volume fraction of the mobile phase A is changed from 95% to 75% at uniform speed;
5-6 min: the volume fraction of the mobile phase A is changed from 75% to 20% at uniform speed;
6-7 min: the volume fraction of the mobile phase A is changed from 20% to 95% at uniform speed;
7-10 min: the volume fraction of mobile phase a was 95%;
the sample injection amount is 10 mu L;
the column temperature is 25-40 ℃;
the triple quadrupole composite linear ion trap mass spectrum detection comprises the following steps: electrospray positive ion ionization mode, multi-reaction monitoring, information correlation acquisition and enhanced ion scanning;
parameters of the electrospray positive ion ionization mode include: the ionization voltage is 5000-5500V; the temperature of the ion source is 500-550 ℃; the air curtain gas is 30-35L/min; the atomization gas is 55-60L/min; the auxiliary heating gas is 55-60L/min;
the parameters for the multiple reaction monitoring are shown in table 1:
TABLE 1 parameters for multiple reaction monitoring
The parameters of the information association acquisition comprise: the scanning range is 100-500 Da, the ionic strength threshold for triggering the secondary scanning is 800-1200 cps, and the dynamic background subtraction is started;
the parameters of the enhanced ion scan include: the scanning type is Enhanced Product ion, the positive ion mode and the EPI collision energy is 40+/-20 eV;
the internal standard is lincomycin- 13 C-D 3 A hydrochloride salt;
the sample to be measured comprises an aquatic product sample to be measured or a water sample to be measured;
the method for acquiring the sample to be detected of the aquatic product comprises the following steps:
mixing an aquatic product to be detected, an internal standard substance and an extracting agent, and extracting to obtain an extracting solution;
evaporating the extracting solution to dryness and then re-dissolving to obtain the sample to be measured of the aquatic product.
2. The method according to claim 1, wherein the concentration of the internal standard in the sample to be tested containing the internal standard is 100ng/mL.
3. The method according to claim 1, wherein the extractant is acetonitrile, methanol or acetone; the dosage ratio of the aquatic product to be detected to the extractant is 2.0 g:15-25 mL.
4. The method according to claim 1, wherein the method for obtaining the water sample to be measured comprises the steps of:
filtering the wastewater to be detected, adding an internal standard into the obtained filtrate, and then adjusting the pH value to be acidic to obtain acidic filtrate;
enriching the acidic filtrate by an HLB solid phase extraction column to obtain an enrichment solution;
evaporating the enrichment solution to dryness and then re-dissolving to obtain the water sample to be detected.
5. The method according to claim 4, wherein the acidic pH is 3.0 to 6.0.
6. The method of claim 4, wherein the enriching comprises: and (3) after passing the acidic filtrate through an HLB solid phase extraction column, sequentially eluting the HLB solid phase extraction column by using ultrapure water and an eluent, and collecting the eluent as the enrichment liquid.
7. The method according to claim 1, wherein the eluent is methanol or aqueous ammonia methanol solution;
the volume ratio of the ammonia water to the methanol in the ammonia water methanol solution is 5:95 or 10:90; the mass concentration of the ammonia water is 25% -28%.
8. The detection method according to claim 1, wherein the ultra-high performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry detection comprises quantitative detection and qualitative detection; the standard curve of quantitative detection is a concentration-peak area ratio standard curve; in the standard curve of quantitative detection, the peak area ratio is the ratio of the peak area of the parent ion of the compound to the peak area of the internal standard substance.
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