CN106526014A - Method for detecting cefquinome residual quantity in milk - Google Patents
Method for detecting cefquinome residual quantity in milk Download PDFInfo
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- CN106526014A CN106526014A CN201610962636.3A CN201610962636A CN106526014A CN 106526014 A CN106526014 A CN 106526014A CN 201610962636 A CN201610962636 A CN 201610962636A CN 106526014 A CN106526014 A CN 106526014A
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- cefquinome
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention relates to a method for detecting cefquinome residual quantity in milk through ultra-high performance liquid chromatography-electric spraying tandem mass spectrometry. A technological method is supplied for monitoring the cefquinome residual quantity in milk.
Description
Technical field
The invention belongs to cefquinome residual in drug residue detection field, particularly a kind of qualitative and detection by quantitative milk
The method of amount.
Background technology
Cephalosporinses are mainly used in treating the microbial various infection of gram, with has a broad antifungal spectrum, curative effect
The advantages of high, toxicity is low, anaphylaxiss are few, but the antibiotic of excess has toxic and side effects again, is mainly reflected in cranial nerve, listens
Feel and the infringement of kidney.Limitation of the China to Cephalosporins in the products such as beef, Carnis Sus domestica and milk is carried out
Regulation.
Cefquinome is the 4th generation cephalosporinses of first animal specific, by the synthesis for suppressing cell wall
Bactericidal effect is reached, and with broad spectrum antibacterial, antibacterial activity is strong, higher concentration can be reached in mammary gland tissue.Cefquinome
Permit for animals in China's registration at present, clinic is mainly used in treating the mammitis of cow that escherichia coli cause, killing property Pasteur more
The porcine respiratory disease that bacillus or Actinobacillus pleuropneumoniae cause.The examination criteria and method for cefquinome domestic at present
Mainly there are high performance liquid chromatography and Ultra Performance Liquid Chromatography-tandem mass spectrometry, focus mostly on animal products.With regard in milk
The most of documents of cefquinome adopt high performance liquid chromatography, and high performance liquid chromatography can only be qualitative by retention time, accurately
Property is poor and sensitivity is low.
The present invention establishes the Ultra Performance Liquid Chromatography-esi-msn instrument measure side of cefquinome residual in milk
Method, the method pre-treatment is simple and easy to operate, accuracy is good, sensitivity is high.
Ultra Performance Liquid Chromatography (Ultra Performance Liquid Chromatography UPLC) is separation section
A brand-new classification in, theoretical and principles of the UPLC by means of HPLC (efficient liquid phase color method), cover little particle filler,
The brand new technical such as very low system bulk and quick detection means, increased flux, sensitivity and the chromatograph peak capacity of analysis.Mesh
Before, Ultra Performance Liquid Chromatography instrument has begun to little by little put in liquid-phase assay.
Triple quadrupole bar tandem mass spectrum operation principle：
Compound molecule receives the effect of the additive methods such as " bombardment " or the highfield of electron stream under vacuum, is ionized into
Ion, while there is the regular fracture of some chemical bonds, generates the positively charged ion with different quality, these ions
By the collection of illustrative plates that the size of mass-to-charge ratio (m/z) is collected and records.
Electron spray ionisation (ESI)：
Sample solution forms the vaporific droplet with height electric charge in the presence of electric field, what is moved to mass analyzer
During, drop is reduced because solvent constantly volatilizees, and causes surface charge density constantly to increase, when the repulsive force foot between electric charge
To overcome during the surface tension of drop, drop splits point, is so repeated, finally obtain with single electric charge or multi-charge from
Son.
Triple quadrupole bar tandem mass spectrum advantage：
(1) it is not required to perform the derivatization.
(2) realize confirming quantitatively in single analysis.
(3) the low test limit in complicated very dirty matrix.
(4) it is more reliable and can trustworthy test result.
(5) mass range：5-1200amu
(6) sensitivity is high, quantitative favorable reproducibility
The content of the invention
Object of the present invention is to provide cephalo quinoline in a kind of easy, quick, sensitive, accurate and economic detection milk
The method of oxime residual quantity.
The present invention comprises the following steps：
Ultimate principle of the present invention using Ultra Performance Liquid Chromatography-esi-msn as detection, modern ultrahigh effect liquid
Phase chromatograph-esi-msn system configuration has chem workstation, computer and software system at interior data acquisition and place
Reason system, can accurately detect the residual quantity of cefquinome in milk.
Ultra Performance Liquid Chromatography-esi-msn is divided into two steps：Data acquisition and data processing.In reservoir
Mobile phase system is squeezed into by high-pressure pump, sample solution Jing injectors enter mobile phase, are loaded into chromatographic column by mobile phase (fixed
Phase) in, as each component in sample solution has different partition coefficients in biphase, when performing relative motion in biphase,
Through the assigning process of repeated multiple times absorption-desorption, each component produces larger difference in translational speed, is separated into list
Individual component is flowed out from post successively, and during by detector, sample concentration is converted into the signal of telecommunication and is sent to monitor, and data are scheming
Spectrum form is printed.
The configuration of standard solution：
Standard reserving solution：Appropriate cefquinome standard substance are weighed, and the standard inventory of 1.0mg/mL are configured to respectively with methanol
Liquid, the lucifuge storage at -18 DEG C.
Interstitial fluid in standard：The each 1mL of above-mentioned standard storing solution is taken, and it is 10.0mg/L's mass concentration to be diluted to respectively with methanol
Interstitial fluid in standard, the lucifuge storage at -18 DEG C.
Standard hybrid working liquid：The each 1mL of interstitial fluid in above-mentioned standard is taken, is 1.0mg/L into mass concentration with methanol dilution
Standard hybrid working liquid, the lucifuge storage at -4 DEG C.
This method is qualitative and quantitative according to selection ion.Blank milk sample is taken, adds the cefquinome of different quality concentration
Standard solution, is detected according to optimization experiment condition mass concentration from high to low, until signal to noise ratio be equal to 3 (S/N=3) and
When signal to noise ratio is equal to 10 (S/N=10), determine that the detection limit (LOD) of cefquinome in Pilus Sus domestica and chicken feather sample is 1.0 μ g/L, it is fixed
Amount limit (LOQ) is 3.0 μ g/L.
Standard working curve
Prepare the series standard that cefquinome standard solution mass concentration is 2.0,5.0,10.0,15.0,20.0 μ g/L molten
Liquid, does standard curve to concentration (x, μ g/L) with quota ion peak area (y).As a result show, cefquinome is in 2～20.0 μ g/L
In the range of the good (r of linear relationship2＞ is 0.999).In milk, cefquinome equation of linear regression and correlation coefficient are shown in Table 1.
Cefquinome equation of linear regression and correlation coefficient in 1 milk of table
Accuracy and precision：
Blank milk sample is taken, addition cefquinome standard solution is respectively 2.0,5.0,10.0 μ g/L, then according to this
Method carries out sample pre-treatments and measure, and each pitch-based sphere is determined 6 times, and withinday precision is by determining 3 mark-on water in 1d
6 parallel samples under flat are obtained, and day to day precision is that continuous 6d (determining 1 time daily) determines the sample under 3 mark-on levels
Obtaining, as a result show, when cefquinome addition concentration is 2.0,5.0,10.0 μ g/L, the average recovery rate of method is 89.2%～
102.5%, in a few days relative standard deviation is 1.88%～4.24%, and relative standard deviation is 3.69%～6.85% in the daytime.It is accurate
Exactness, precision and relative standard deviation are shown in Table 2.
Add in the blank milk of table 2 cefquinome in a few days with day to day precision and relative standard deviation (n=6)
In sample, in terms of mass fraction X, with micro- gram per liter (μ g/L), numerical value represents that single point correction is pressed to cefquinome residual quantity
Formula (1), (2) calculate：
In formula：
CiCefquinome concentration (μ g/L) in sample solution；
CsCefquinome concentration (μ g/L) in contrast solution；
AiCefquinome peak area in sample solution；
AsCefquinome peak area in contrast solution；
The residual quantity (μ g/kg) of cefquinome in X samples；
V1The final constant volume of sample liquid (mL)；
V sample sizes (g)；
Standard curve calibration is calculated by formula (3)：
CiCefquinome concentration (μ g/L) in the sample checked on standard curve；；
V1The final constant volume of sample liquid (mL)；
V sample sizes (g).
Represented with the arithmetic mean of instantaneous value of the measurement result independent twice obtained under the conditions of repeatability, as a result retain three effectively
Numeral.
Under the conditions of repeatability, the absolute difference of the result of independent test twice of acquisition is not more than the arithmetic of two measured values
The 20% of meansigma methodss.
The optimization of Mass Spectrometry Conditions：
The cefquinome standard solution of 1.0mg/L is prepared respectively, ion source is not directly entered by chromatographic column, in ESI+Mould
Carry out the optimization of Mass Spectrometry Conditions under formula respectively.First mass spectrometric figure full scan is first carried out, the parent ion of each target compound is determined, and
Optimization obtains fragmentation voltage in source, and then under fragmentation voltage conditions in the source selected, the parent ion to selecting carries out daughter ion
Scanning, chooses 1 pair of relatively most strong fragment ion of abundance as quota ion, and secondary 1 couple or 2 pairs of fragment ions by force is used as qualitative
Ion, and respectively the impact energy of daughter ion is optimized.It is last under MRM patterns respectively to atomization gas pressure, be dried temperature
The parameter such as degree and flow is optimized.Mass Spectrometry Conditions after optimization are shown in Table 3.
The mass spectrum optimal conditions of amantadine, rimantadine, Moroxydine and D15- amantadines under 3 MRM monitoring patterns of table
* quota ion
The characteristics of to further illustrate the present invention and effect, are further described to invention below in conjunction with accompanying drawing.
Description of the drawings
Fig. 1：Add 2.0 μ g/L cefquinomes TIC figures in freeze-dried milk sample
Embodiment
Below by embodiment, the invention will be further described.It should be understood that embodiment of the present invention methods described
It is only used for the explanation present invention, rather than limitation of the present invention, to preparation side of the invention under the concept thereof of the present invention
The simple modifications of method belong to the scope of protection of present invention.
Milk 2.00mL samples are accurately measured in 50mL polypropylene centrifuge tubes, 2mL water is added, is added 1% second of 10mL
Sour acetonitrile, supersound extraction 3min, under the conditions of 4 DEG C, 4500r/min centrifugations 10min, supernatant is transferred in 100mL Cor Gigeriae Galli bottles,
Residue repeats to extract once with 10mL acetonitrile solutions, merges extracting solution twice.Extracting solution is evaporated in 40 DEG C of water-baths
It is near dry, add upper machine after 5mL water dissolutioies.
Set liquid phase chromatogram condition as：
Mobile phase：A:0.1% formic acid water, B:Acetonitrile,
Gradient elution：0-2min keeps 8%B, 2-4min 8%B linear changes to 70%B, 4-8min to keep 70%B, 8-
8.5min 70%B linear changes keep 8%B to 8%B, 8.5-12min.
Flow velocity：0.3mL/min
Sample size：5.00μL
Mass Spectrometry Conditions：Ionization pattern：Electron spray ionisation positive ion mode (ESI+)；Detection mode：Multiple-reaction monitoring
(MRM)；Capillary voltage：4 000V；Ion source temperature (TEM)：325℃；It is dried temperature degree：300℃；Dry gas stream amount：
15L/min；Atomization gas pressure：50psi；Sheath temperature degree：400℃；Sheath throughput：12L/min.
Certainly, those of ordinary skill in the art is it should be appreciated that the embodiment of the above is intended merely to explanation originally
Illustrate, and be not intended as the restriction of the present invention, as long as in the essential scope of the present invention, change to embodiment described above,
Modification will all fall in the range of claims of the present invention.
Claims (1)
1. it is a kind of detection milk in cefquinome residual quantity method, it is characterised in that comprise the following steps：
1st, milk 2.00mL samples are accurately measured in 50mL polypropylene centrifuge tubes, 2mL water is added, is added 1% acetic acid of 10mL
Acetonitrile, supersound extraction 3min, under the conditions of 4 DEG C, 4500r/min centrifugations 10min, supernatant is transferred in 100mL Cor Gigeriae Galli bottles, residual
Slag repeats to extract once with 10mL acetonitrile solutions, merges extracting solution twice.Extracting solution is evaporated in 40 DEG C of water-baths closely
It is dry, add upper machine after 5mL water dissolutioies.
2nd, liquid-phase condition：
Mobile phase：A:0.1% formic acid water, B:Acetonitrile,
Gradient elution：O-2min keeps 8%B, 2-4min 8%B linear changes to 70%B, 4-8min to keep 70%B, 8-
8.5min 70%B linear changes keep 8%B to 8%B, 8.5-12min.Flow velocity：0.3mL/min
Sample size：5.00μL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610962636.3A CN106526014A (en) | 2016-11-04 | 2016-11-04 | Method for detecting cefquinome residual quantity in milk |
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| CN201610962636.3A CN106526014A (en) | 2016-11-04 | 2016-11-04 | Method for detecting cefquinome residual quantity in milk |
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| CN106526014A true CN106526014A (en) | 2017-03-22 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108387536A (en) * | 2018-01-31 | 2018-08-10 | 佛山市南海东方澳龙制药有限公司 | Cefquinome sulfate breast injection（Lactation period）The rapid assay methods of content |
| CN108490103A (en) * | 2018-03-30 | 2018-09-04 | 佛山市南海东方澳龙制药有限公司 | A kind of detection method of Cefquinome sulfate breast injection content |
| CN108802211A (en) * | 2018-04-19 | 2018-11-13 | 佛山市南海东方澳龙制药有限公司 | The liquid phase detection method of related substances in a kind of Cefquinome sulfate breast injection |
| CN111141837A (en) * | 2018-11-06 | 2020-05-12 | 江西省农业科学院农产品质量安全与标准研究所 | Method for detecting residual quantity of cefquinome in vegetables |
-
2016
- 2016-11-04 CN CN201610962636.3A patent/CN106526014A/en active Pending
Non-Patent Citations (6)
| Title |
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| GERTRAUD SUHREN ET AL.: "Detection of cefquinome in milk by liquid chromatography and screening methods", 《ANALYTICA CHIMICA ACTA》 * |
| MATTHIAS BECKER ET AL.: "Residue analysis of 15 penicillins and cephalosporins in bovine muscle,kidney and milk by liquid chromatography–tandem mass spectrometry", 《ANALYTICA CHIMICA ACTA》 * |
| 王晓燕: "HLB固相净化-液相色谱串联质谱法测定奶粉中7种头孢菌素残留量", 《食品安全导刊》 * |
| 王林 等: "牛奶和牛血浆中头孢喹肟HPLC-MS/MS检测方法的建立", 《畜牧与兽医》 * |
| 董晓云 等: "超高效液相色谱一串联质谱法测定牛奶中头孢喹肟残留量", 《分析试验室》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108387536A (en) * | 2018-01-31 | 2018-08-10 | 佛山市南海东方澳龙制药有限公司 | Cefquinome sulfate breast injection（Lactation period）The rapid assay methods of content |
| CN108490103A (en) * | 2018-03-30 | 2018-09-04 | 佛山市南海东方澳龙制药有限公司 | A kind of detection method of Cefquinome sulfate breast injection content |
| CN108802211A (en) * | 2018-04-19 | 2018-11-13 | 佛山市南海东方澳龙制药有限公司 | The liquid phase detection method of related substances in a kind of Cefquinome sulfate breast injection |
| CN111141837A (en) * | 2018-11-06 | 2020-05-12 | 江西省农业科学院农产品质量安全与标准研究所 | Method for detecting residual quantity of cefquinome in vegetables |
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