CN107748212A - A kind of method that medicine is detected in goat milk and its product - Google Patents

Abstract

The present invention provides 74 kinds of 8 major class in a kind of goat milk and its product and limits medication object detecting method, and methods described comprises the following steps：(1) sample pre-treatments：Testing sample is extracted using acidic organic solvent；And pass through SPE column purifications, scavenging solution is through initial flow phased soln, treat UPLC MS/MS detect (2) 74 kinds it is to be measured limit with drug monitoring, under instrument condition as defined in method, will be determined by pre-treatment sample using ultra performance liquid chromatography LC-MS.The present invention has detection medication amount more, species is enriched, detection time is short, the advantages of high sensitivity, result accurate stable, it also compensate in goat milk and dairy produce simultaneously without multiple types, multi-quantity medicine while detect blank, to ensureing consumer's health, improve goat milk and dairy produce export trade competitiveness is significant.

Description

A kind of method that medicine is detected in goat milk and its product

Technical field

The present invention relates to a kind of detection method limited with medicine, more particularly to 8 major classes 74 in goat milk and its product Kind limits medication object detecting method.

Background technology

Milk and dairy produce part indispensable in being formed as consumer's meals, its trophic component and quality safety by Increasing concern, wherein milk supply also progressively expands to goat milk and product from milk, and goat milk has fat granule compared to milk With protein particle is small, minerals and vitamins enrich, it is easier to the characteristics of being absorbed by the body；And goat milk is from foundation structure to nutrition Proportioning is especially suitable for infants, while the foreign protein content such as а S- caseins is less in goat milk, energy closer to people's milk Substantially reduce anaphylactoid generation.With the raising of levels of substance, the higher goat milk of nutritive value and dairy produce are by more next More pursues.

With cultivation industry large-scale development, it is Prevention Animal diseases, shortens the production cycle, promote growth of animal, The use of veterinary drug is extremely widespread, at present livestock and poultry cultivation industry often mainly have with veterinary drug sulfamido, quinolione class, macrolides, The medicines such as benzimidazole, sedative class, antiviral and pleuromulins, but due to the shortage and economy of scientific culture knowledge The trend of interests, overdose is especially prominent using phenomenon, and secondly to pursue curative effect, multiple types veterinary drug is answered for illegal veterinary drug manufacturing enterprise Side uses, and irrational off-drug period limit, causes in animal body while remain a variety of veterinary drugs in addition；Simultaneously for promote growth of animal and Weightening, addition forbidden drug of making a desperate move also are happened occasionally with the phenomenon played one's own game, ultimately result in and prohibit in animal derived product Medicament residue excessive problem is limited the use of frequently to occur, in recent years milk and dairy produce food safety affair also in rapid succession, to consumer Health causes potential threat, especially even more serious to infant and weak influence of group.

What forbidden drug remained in animal derived food mainly includes following aspect to human health damage：(1) acute and chronic Toxic reaction, will when a certain amount of because drug accumulation reaches when human body takes in the exceeded animal derived food of left drug for a long time Acute toxicity and chronic toxicity reaction is produced, as sulfa drugs can destroy human body hematopoietic function；Macrolides can cause acute liver toxicity； " clenbuterol hydrochloride " class medicine can cause blood vessel dilatation, drop in blood pressure, palpitating speed, breathing to aggravate so as to induce or aggravate heart patient The state of an illness.(2) teratogenesis, carcinogenic, mutagenesis, research show if residual " three cause " drugs with function food is eaten for a long time, most Human body may be caused to produce teratogenesis, carcinogenic and mutagenesis eventually, as benzimidazole class medicine has teratogenesis；Sulfanilamide (SN) diformazan The sulfa drugs such as pyrimidine can induce rodent thyroid hyperplasia, and with cause tumour tendency；Of QNS Other kind is found to have mutagenesis.(3) drug tolerant bacteria produces, long-term to limit with medicament residue exposure condition, people Internal sensitive strain is suppressed by selectivity, so that antibody-resistant bacterium amount reproduction, in addition, resistance to the action of a drug R plasmids are between bacterial strain Horizontal transfer makes many bacteriums develop into multidrug resistant by substance resistance, and the generation of drug tolerant bacteria causes some common drugs Curative effect, which declines, even loses curative effect, so that appearance effect is bad or without the available danger of medicine in human body diseases therapeutic process.This Limited in outer animal derived food and also result in allergic reaction with medicament residue, such as penicillin, Tetracyclines, sulfamido can make Part population occurs allergic reaction and even suffered a shock.

Therefore, many countries and tissue to limiting with medicament residue by being monitored to assess animal food safety Risk and formulate counter-measure, conventional monitoring method is mainly screening method and confirmatory analysis method, screening technique mainly with Based on ELISA quick detections, confirmation method mainly have liquid chromatogram, LC-MS, gas-chromatography, based on gas chromatography mass spectrometry, but with The development of Safety of Food Quality risk position, existing detection method is difficult to meet that same sample is a variety of to limit with medicine simultaneously Testing requirements, the problem of being primarily present have：(1) detection method and standard are to detect based on single kind medicine mostly, multiple types medicine During analyte detection, sample need to be repeatedly handled respectively, greatly waste human and material resources and financial resources (2) detection method and standard detection Matrix covering is not comprehensive, as animal product detection matrix is generally meat and liver specimens, rare kidney samples A；And milk and dairy produce Matrix mostly based on milk, have no goat milk and dairy produce sample (3) instrument and method sensitivity to be present low, it is poor reproducibility, preceding Handle the problems such as cumbersome, solvent consumption is big.Therefore to meet to sample milk and dairy produce supervision of quality safety, establish more kinds of The confirmation method that class, greater number veterinary drug detect simultaneously is particularly urgent.

It is presently used for limiting in animal derived product and mainly has ELISA method, liquid chromatogram with the method for medicine residue detection Method (HPLC), Liquid Chromatography/Mass Spectrometry (LC-MS/MS), gas chromatography (GC), gas chromatography mass spectrometry method (GC-MS/MS), biology sensor And chip technology.

The quick screening and analysis method that ELISA method is established based on reaction is specifically bound between Ag-Ab, have The advantages that high sensitivity, high specificity, analysis throughput are big, fast and safely reliable, its is simple to operate for this external cause, and analysis time is short to be It is applied to the Main Means of Site Detection at present.But ELISA method is a variety of because its high specificity determines that it can not be detected simultaneously simultaneously Class medicine；And reacted because its principle is antigen and antibody specific, cause its requirement of applicability to solvent just high, easily because of Matrix reason causes certain false positive and Problem of False Negative, in addition ELISA method not high, the stability difference etc. that sensitivity also be present Shortcoming, it is only capable of doing semi-quantitative analysis to analyte.

Bioactive substance is integrated in transducer face by biology sensor and chip technology, and by analyte and active matter Signal caused by certain specific reaction between matter changes into discernible signal, and then realizes to analyte content or concentration Measure.Such technology has simple to operate, high specificity, an advantage of high sensitivity, significant increase detection flux, this unofficial biography Sensor small volume, is easy to carry, the detection in real time of convenient scene.Therefore in recent years, sensor technology is residual with medicine applied to limiting Stay the important research field that analysis detection is food security；But biology sensor and chip technology be as emerging detection technique, Because its keep bioactive substance and it is integrated the characteristics of, to detect matrix processing requirement and existing integrated technology it is higher, because This easily causes the unstability of result, and the problems such as cost and service life be present.

Liquid chromatogram (HPLC), LC-MS (LC-MS/MS), gas-chromatography (GC) and gas chromatography mass spectrometry (GC-MS/MS) etc. Instrument analytical method, the higher sensitivity of the good stability of chromatographic technique and mass-spectrometric technique is combined, has given full play to instrument The characteristics of degree of accuracy of device analytic approach is high, and stability is good, high sensitivity, and selectivity is good, limits in animal derived product and uses medicine It is with the obvious advantage in terms of qualitative and quantitative analysis, accurately test knot also can be still obtained under matrix interference material complex situations Fruit, it is confirmation analysis means main at present；But current existing method is single in the presence of detection medicament categories, and medication amount waits lack less Point, easily cause the missing inspection of risk medicine；In addition it is not abundant enough to detect matrix, for example rare method and research on standard are increasingly by weight Depending on goat milk and dairy produce in limit with medicament residue situation, cause the blind area and dead angle of food safety Regulation link, be unfavorable for The lifting of China's food security integral level and the raising of export trade competitiveness.

Prior art CN103760269 discloses a kind of detection method of residue of veterinary drug, it is disclosed that 46 kinds of medicines Detection method, the species of detection medicine is extremely limited in the detection of this method, only including tetracycline medication, beta receptor excitement Agent class medicine, QNS and sulfa drugs, and for other class medicines without can detect.And actual production In, it is necessary to determine more kinds of, the left drug of particularly more classifications, therefore, exploitation is a kind of to detect larger class more simultaneously The method of a variety of medicines, turn into this area urgent problem to be solved.

The content of the invention

The present invention limits, species few the problem of few with medication amount for prior art detection, establishes and covers beta-receptor Activator (27 kinds), sulfanilamide (SN) (15 kinds), quinolione (20 kinds), macrolide (3 kinds), benzimidazole (3 kinds), sedative (2 kinds), The ultra performance liquid chromatography MS of 8 class, 74 kinds of veterinary drugs such as antiviral (2 kinds) and pleuromulins (2 kinds)；In addition institute The problem of method for building up compensate for existing detection method and not comprehensive enough standard for samples such as goat milk and its products.

Specifically, the present invention is achieved through the following technical solutions：

A kind of to detect the method for disabling or/and limiting the use of medicine in goat milk and its product, methods described comprises the following steps：

(1) processing of sample：

Testing sample is added after acidic organic solvent extraction and carries out the concussion extraction that is vortexed, centrifuging and taking supernatant, is centrifuged The precipitation arrived repeats to be vortexed concussion extraction once, merges supernatant, through SPE column purifications, the drying of scavenging solution nitrogen, uses initial flow It is used to UPLC-MS/MS after dynamic phased soln determine；

(2) disable or/and limit the use of the measure of medicine：

The sample that step (1) is obtained carries out UPLC-MS/MS measure, and condition determination is as follows：

Chromatographic condition：Chromatographic column is Waters Acquity UPLC BEH C18 posts；Mobile phase is that methanol and formic acid are water-soluble Liquid, type of elution are gradient elution；

Mass Spectrometry Conditions：Electric spray ion source (ESI+)；Capillary voltage is 3.0-3.5kV；Source temperature is 145 DEG C -155 DEG C； Desolventizing temperature is 500 DEG C -600 DEG C；Desolvention gas velocity degree is 850Lh^-1-950L·h^-1；Taper hole gas velocity degree is 45L·h^-1-55L·h^-1；Collision gas flow velocity：0.13mL·min^-1-0.15mL·min^-1。

Wherein, described limit includes beta-receptor activator, sulfanilamide (SN), quinolione, macrolide, benzimidazole, town with medicine Quiet dose, the antiviral and class medicine of pleuromutilin 8.

Wherein, described limit with medicine is orciprenaline, Cimaterol, Terbutaline, salbutamol, Zilpaterol, west Boot sieve, pirbuterol, Procaterol, Reproterol, fenoterol, ritodrine, methylol Clenbuterol, Ke Lunpu Sieve, Ke Lunsailuo, Clorprenaline, clenbuterol, Tulobuterol, Ractopamine, Mabuterol, metoprolol, horse spray special sieve, Bromine Boot sieve, Formoterol, bambuterol, Ao Dateluo, Bbu Tero, phenolethanolamine A, sulfacetamide, sulfamethazole, Sulphadiazine, sulfathiazole, sulfapryidine, sulfamerazine, bacteresulf, sulfadimidine, sulfamethoxypyridazine, sulphur How pungent amine is, sulfabenzamide, sulfamethoxazole, sulfadimethoxine, daimeton, sulfaquinoxaline, pipemidic acid, oxygen fluorine are husky Star, gatifloxacin, sarafloxacin, Orbifloxacin, trovafloxacin, T-3811, Norfloxacin, Ciprofloxacin, Pefloxacin, Lip river U.S. husky star, Danofloxacin, Enrofloxacin, gemifloxacin, MOXIFLOXACIN, Sparfloxacin, Difloxacin, Nadifloxacin, acidum nalidixicum, Flumequine, amantadine, Rimantadine, thiabendazole, Albendazole sulfone, oxfendazole, clindamycin, ROX, for rice Examine a kind of, several or whole in star, valnemulin, Tiamulin, chlorpromazine, stresnil.

Wherein, step (2) described gradient elution program is：

0-0.5min：The volume ratio of methanol is that 3.0% holding is constant；

0.5-2.0min：The volume ratio of methanol is 3.0%-25.0%；

2.0-3.5min：The volume ratio of methanol is that 25.0% holding is constant；

3.5-3.6min：The volume ratio of methanol is 25.0%-40.0%；

3.6-4.0min：The volume ratio of methanol is that 40.0% holding is constant；

4.0-5.5min：The volume ratio of methanol is 40.0%-90.0%；

5.5-8.0min：The volume ratio of methanol is that 90.0% holding is constant；

8.0-8.1min：The volume ratio of methanol is 90.0%-3.0%；

8.1-10.5min：The volume ratio of methanol is that 3.0% holding is constant.

Wherein, the temperature of step (1) the nitrogen drying supernatant is 35 DEG C -45 DEG C, preferably in 40 DEG C of progress.

Wherein, the rotating speed of step (1) described centrifugation is 9000rmin^-1-10000r·min^-1, the time of centrifugation is 3min-7min, preferably described centrifugal rotational speed are 9500rmin^-1, the time of centrifugation is 5min.

Wherein, step (1) the extractant volumetric concentration is 0.2%-1.0% formic acid acetonitrile solutions；It is preferred that formic acid volume Concentration is 0.5%.

Wherein, step (1) is described in SPE solid-phase extraction column purification process, flow rate of liquid 0.5ds-1-3ds- 1, preferably described flow rate of liquid is 1ds-1.

Wherein, the volumetric concentration of the aqueous formic acid of step (2) is 0.05%-0.2%, and preferred volume concentration is 0.1%.

Wherein, step (2) chromatographic condition：Chromatographic column is Waters Acquity UPLC BEH C18 posts；Mobile phase For methanol and aqueous formic acid, type of elution is gradient elution；Flow velocity is 0.3mLmin-1；Column temperature is 35 DEG C；

Mass Spectrometry Conditions：Electric spray ion source (ESI+)；Capillary voltage is 3.0KV；Extraction taper hole voltage is 3.0V；RF is saturating Mirror voltage is 0.5V；Source temperature is 150 DEG C；Desolventizing temperature is 600 DEG C；Desolvention gas velocity degree is 900Lh^-1；Taper hole air-flow Speed is 50Lh^-1；Impinging air flows speed is 0.14mLmin^-1；Sample collection mode：Mould is gathered using multichannel at times Formula.

More particularly, the present invention is achieved through the following technical solutions：

It is a kind of to detect the method for disabling or/and limiting the use of medicine in goat milk and its product, it is characterised in that methods described includes Following steps：

(1) sample pre-treatments：

Goat milk sample to be measured is carried out into pre-treatment, and (goat milk powder is dissolved into liquid milk in advance, at goat milk pre-treating method Reason), extraction of ocean eddies, centrifuging and taking supernatant are carried out after adding acidic organic solvent extraction, centrifugation repeats extraction once, merges Supernatant, through SPE column purifications, the drying of scavenging solution nitrogen, using after initial flow phased soln through measure；

(2) 74 kinds of to be measured limit use drug monitoring：

The sample that step (1) is obtained carries out UPLC-MS/MS measure, and condition determination is as follows：

Chromatographic condition：Chromatographic column is Waters Acquity UPLC BEH C₁₈(100mm × 2.1mm, 1.7 μm) post；Stream Dynamic is mutually methanol and aqueous formic acid, and type of elution is gradient elution；

Mass Spectrometry Conditions：Electric spray ion source (ESI+)；Capillary voltage is 3.0-3.5kV；Source temperature is 145 DEG C -155 DEG C； Desolventizing temperature is 500 DEG C -600 DEG C；Desolvention gas velocity degree is 850Lh^-1-950L·h^-1；Taper hole gas velocity degree is 45L·h^-1-55L·h^-1；Collision gas flow velocity：0.13mL·min^-1-0.15mL·min^-1。

Wherein, described limit includes beta-receptor activator, sulfanilamide (SN), quinolione, macrolide, benzimidazole, town with medicine Quiet dose, the 8 class medicines such as antiviral and pleuromutilin.

Wherein, described limit with medicine is orciprenaline, Cimaterol, Terbutaline, salbutamol, Zilpaterol, west Boot sieve, pirbuterol, Procaterol, Reproterol, fenoterol, ritodrine, methylol Clenbuterol, Ke Lunpu Sieve, Ke Lunsailuo, Clorprenaline, clenbuterol, Tulobuterol, Ractopamine, Mabuterol, metoprolol, horse spray special sieve, Bromine Boot sieve, Formoterol, bambuterol, Ao Dateluo, Bbu Tero, phenolethanolamine A, sulfacetamide, sulfamethazole, Sulphadiazine, sulfathiazole, sulfapryidine, sulfamerazine, bacteresulf, sulfadimidine, sulfamethoxypyridazine, sulphur How pungent amine is, sulfabenzamide, sulfamethoxazole, sulfadimethoxine, daimeton, sulfaquinoxaline, pipemidic acid, oxygen fluorine are husky Star, gatifloxacin, sarafloxacin, Orbifloxacin, trovafloxacin, T-3811, Norfloxacin, Ciprofloxacin, Pefloxacin, Lip river U.S. husky star, Danofloxacin, Enrofloxacin, gemifloxacin, MOXIFLOXACIN, Sparfloxacin, Difloxacin, Nadifloxacin, acidum nalidixicum, Flumequine, amantadine, Rimantadine, thiabendazole, Albendazole sulfone, oxfendazole, clindamycin, ROX, for rice Examine a kind of, several or whole in star, valnemulin, Tiamulin, chlorpromazine, stresnil.

Wherein, step (2) described gradient elution program is：

0-0.5min：The volume ratio of methanol is that 3.0% holding is constant；

0.5-2.0min：The volume ratio of methanol is 3.0%-25.0%；

2.0-3.5min：The volume ratio of methanol is that 25.0% holding is constant；

3.5-3.6min：The volume ratio of methanol is 25.0%-40.0%；

3.6-4.0min：The volume ratio of methanol is that 40.0% holding is constant；

4.0-5.5min：The volume ratio of methanol is 40.0%-90.0%；

5.5-8.0min：The volume ratio of methanol is that 90.0% holding is constant；

8.0-8.1min：The volume ratio of methanol is 90.0%-3.0%；

8.1-10.5min：The volume ratio of methanol is that 3.0% holding is constant.

Wherein, the temperature of step (1) the nitrogen drying supernatant is 35 DEG C -45 DEG C, preferably at 40 DEG C

Carry out.

Wherein, the rotating speed of step (1) described centrifugation is 9000rmin^-1-10000r·min^-1, the time of centrifugation is 3min-7min, preferably described centrifugal rotational speed are 9500rmin^-1, the time of centrifugation is 5min.

Wherein, step (1) the extractant volumetric concentration is 0.2%-1.0% formic acid acetonitrile solutions；It is preferred that

Formic acid volumetric concentration is 0.5%.

Wherein, step (1) is described in SPE solid-phase extraction column purification process, flow rate of liquid 0.5ds^-1-3d·s^-1, It is preferred that the flow rate of liquid is 1ds^-1

Wherein, the volumetric concentration of the aqueous formic acid of step (2) is 0.05%-0.2%, and preferred volume concentration is 0.1%.

Wherein, step (2) chromatographic condition：Chromatographic column is Waters Acquity UPLC BEH C₁₈(100mm× 2.1mm, 1.7 μm) post；Mobile phase is methanol and aqueous formic acid, and type of elution is gradient elution；Flow velocity is 0.3mLmin^-1； Column temperature is 35 DEG C.

Mass Spectrometry Conditions：Electric spray ion source (ESI+)；Capillary voltage is 3.0KV；Extraction taper hole voltage is 3.0V；RF is saturating Mirror voltage is 0.5V；Source temperature is 150 DEG C；Desolventizing temperature is 600 DEG C；Desolvention gas velocity degree is 900Lh^-1；Taper hole air-flow Speed is 50Lh^-1；Impinging air flows speed is 0.14mLmin^-1。

The advantageous effects of the present invention are as follows：

The present invention is high in UPLC stability, detection time is short and MS/MS high sensitivity on the basis of, the detection side of foundation Method, the two technical advantage is given full play to, wherein UPLC is the novel liquid-phase system developed on the basis of traditional HPLC system.

Compared with traditional high performance liquid chromatography (HPLC), UPLC has the advantage that：First, high separation, UPLC systems The chromatographic column particle diameter of carrying can reach 1.7 μm, and the system will be higher by 70% than traditional 5 μm of particle diameter chromatogram column system efficiency of HPLC, than It is 3.5 μm high by 40%；Second, at high speed, in the case where not influenceing separating degree, small particle can provide higher analyze speed, identical In time, UPLC can provide more peak capacities.Under the same terms, 1.7 μm of UPLC systems, relative to 5 μm of HPLC systems, divide 9 times are improved from speed；Third, high sensitivity, UPLC can improve post, reduce peak width, so as to improve sensitivity, improve post effect While, 70% and 40% has been respectively increased than the system sensitivity of 5 μm and 3.5 μm with 1.7 μm of UPLC systems.And UPLC And the combination of mass spectrometric hyphenated technique, the complementation of chromatogram and mass spectrum advantage is embodied, the high separation capacity by chromatogram to complex sample, There is high selectivity, high sensitivity with MS and relative molecular mass can be provided and combine with the advantages of structural information, be multiple Accurate qualitative and quantitative analysis provides possibility to multiple types medicine simultaneously in miscellaneous matrix.

The SPE solid phase extraction techniques that the present invention uses, using analysans, adsorption capacity difference is carried out in different medium Purification, is effectively analysed to thing and is separated with interfering component, greatly enhance the detection to analysans particularly trace analysis thing Ability, improves the rate of recovery of sample, while also reduces substrate background, improves detection sensitivity.

Experiment uses more Channels drainage pattern (multi-channel data acquisition analysis systems) at times, simultaneously as medicine Species is more, and appearance time more concentrates on the phenomenon in 2~6min, tests and carries out time slice to different channel, ensures Each medicine has sufficient scan frequency, and experiment is also carried out to every kind of medicine residence time between 0.005~0.05ms Optimization, ensures to be attained by 10-12 data collection point on each chromatographic peak, it is ensured that every kind of medicine can obtain higher sensitivity And accurate quantitative analysis.

The present invention is established suitable for goat milk and dairy produce for beta-receptor activator (27 kinds), sulfanilamide (SN) (15 kinds), quinoline Ketone (20 kinds), macrolide (3 kinds), benzimidazole (3 kinds), sedative (2 kinds), antiviral (2 kinds) and pleuromulins 8 class such as (2 kinds), 74 kinds of ultra performance liquid chromatography-mass spectrometry combination methods limited with medicine.Relative to current existing method, the party Method has the advantages of detection medication amount is more, and A wide selection of colours and designs, detection time is short, high sensitivity, result accurate stable, while also more Mend in goat milk and dairy produce without multiple types, multi-quantity medicine while detected blank, to ensureing consumer's health, improved sheep Milk and dairy produce export trade competitiveness are significant.

Detection method enables more medicines to extract and carry out by being improved pre-treating method Further detection, while a variety of methods for being advantageous to multi-medicament and detecting are employed in liquid phases separation, and adopted in result Multichannel drainage pattern at times is employed on collection, is advantageous to the accumulation of signal.

Brief description of the drawings

Fig. 1：The quota ion passage chromatogram of 27 kinds of beta-receptor stimulant medicine mixed standard solutions；

Fig. 2：The quota ion passage chromatogram of 15 kinds of sulfa drugs mixed standard solutions；

Fig. 3：The quota ion passage chromatogram of 20 kinds of QNS mixed standard solutions；

Fig. 4：Five classes are limited with medicine (3 kinds of macrolide, 3 kinds of benzimidazole, antiviral 2 kinds, 2 kinds of sedative, truncation side 2 kinds of ear element) the quota ion passage chromatogram of mixed standard solution；

Fig. 5:Different pharmaceutical substrate inhibition degree；

Above-mentioned each figure is the original figure spectrum of UPLC-MS/MS measurement results, and the translator of English in figure is as follows：

Channels：Determinand acquisition channel in mass spectrometry method

Orciprenaline：Orciprenaline, cimaterol：Cimaterol, terbutaline：Terbutaline, salbutamol：Salbutamol, zilpaterol：Zilpaterol, cimbuterol：Western Boot sieve, pyrbuterol：Pyrrole Boot Sieve, ritodrine：Ritodrine, procaterol：Procaterol, fenoterol：Fenoterol, reproterol： Reproterol, Hydroxymethylclenbuterol：Methylol Clenbuterol, clencyclohexerol：Ke Lunsailuo, clenproperol：Ke Lunpuluo, clorprenaline：Clorprenaline, clenbuterol：Clenbuterol, ractopamine：Ractopamine, tulobuterol：Tulobuterol, metoprolol：Metoprolol, mabuterol：Horse Boot sieve, mapenterol：Horse sprays special sieve, formoterol：Formoterol, brombuterol：Bromine Boot sieve, bambuterol：Bambuterol, olodaterol：Ao Dateluo, penbutolol：Bbu Tero, phenyl EA A：Benzene second Hydramine A.

Sulfadiazine：Sulphadiazine, sulfathiazole：Sulfathiazole, sulfacetamide：Sulfacetamide, sulfapyridine：Sulfapryidine, sulfamerazine：Sulfamerazine, sulfisoxazole：Bacteresulf, sulfamoxol：Sulfamethazole, sulfamethazine：Sulfadimidine, sulfamethoxypyridazine：Sulphur Amine methoxy pyridazine, sulfadoxine：Sulfadoxine, sulfabenzamide：Sulfabenzamide, sulfaquinoxaline：Sulfanilamide (SN) Quinoxaline, sulfamethoxazole：Sulfamethoxazole, sulfadimethoxine：Sulfadimethoxine, sulfamonomethoxine：Daimeton.

pipemidic acid：Pipemidic acid, ofloxacin：Ofloxacin, Gatifloxacin：Gatifloxacin, Sarafloxacin：Sarafloxacin, Orbifloxacin/；Orbifloxacin, Trovafloxacin：Trovafloxacin, Garenoxacin：T-3811, norfloxacin：Norfloxacin, Ciprofloxacin：Ciprofloxacin, pefloxacin： Pefloxacin, lomefloxacin：Lomefloxacin, Danofloxacin：Danofloxacin, Enrofloxacin：Enrofloxacin, Gemifloxacin：Gemifloxacin, Moxifloxacin：MOXIFLOXACIN, sparfloxacin spara：Sparfloxacin, difloxacin：Difloxacin, Nadifloxacin：Nadifloxacin, nalidixic acid：Acidum nalidixicum, Flumequine：Fluorine First quinoline.

Thiabendazole：Thiabendazole, Albendazole sulfone：Albendazole sulfone, Oxfendazole：It is difficult to understand Oxfendazole.

Clindamycin：Clindamycin, roxithromycin：ROX, Tilmicosin：Tilmicosin.

Taimulin：Tiamulin, valnemulin：Valnemulin.

Amantadine：Amantadine, rimantadine：Rimantadine.

Chlorpromazine：Chlorpromazine, azaperone：Stresnil.

Embodiment

The invention provides one kind to limit medication object detecting method, and methods described comprises the following steps：

1) sample pre-treatments：

Goat milk sample to be measured is carried out into pre-treatment, and (goat milk powder is dissolved into liquid milk in advance, at goat milk pre-treating method Reason), extraction of ocean eddies, centrifuging and taking supernatant are carried out after adding acidic organic solvent extraction, centrifugation repeats extraction once, merges Supernatant, through SPE column purifications, the drying of scavenging solution nitrogen, determined using after initial flow phased soln through UPLC-MS/MS；

The detection sample is goat milk, when sample is powdered milk sample, must quantitatively be dissolved into liquid milk in advance.

The Extraction solvent is that volumetric concentration is 0.2%-1.0% formic acid acetonitrile solutions；It is preferred that formic acid volumetric concentration is 0.5%.The selection of Extraction solvent is considered based on preferably removing removing protein and increase drug solubility.

It is described repeat extraction twice and centrifugation twice, be based on the abundant extraction to analysans, and precipitation is fully gone The consideration removed, so as to preferably reduce noise jamming and improve the sensitivity of analysans.

Preferably, the temperature of the nitrogen drying supernatant is 35 DEG C -45 DEG C, preferably in 40 DEG C of progress.

Preferably, the rotating speed of the centrifugation is 9000rmin^-1-10000r·min^-1, the time of centrifugation is 3min- 7min, preferably described centrifugal rotational speed are 9500rmin^-1, the time of centrifugation is 5min.

Preferably, it is described in SPE solid-phase extraction column purification process, flow rate of liquid 0.5ds^-1-3d·s^-1,

It is preferred that the flow rate of liquid is 1ds^-1。

During preceding processing, the concussion extraction that is vortexed is that matrix is scattered relative to ultrasonic wave extraction advantage and more filled Point, goat milk matrix can more fully contact with Extraction solvent during vortex, so that the extraction of drug target is more abundant.

The extractant wherein used is acidic organic solvent, and specially volumetric concentration is that 0.2%-1.0% formic acid acetonitriles are molten Liquid；It is preferred that formic acid volumetric concentration is 0.5%, the purposes of acidic organic solvent：Based on preferably going, removing protein and increase medicine are molten Solution property considers；Experimental selection formic acid Acidifying acetonitrile replaces trifluoroacetic acid acetonitrile solution, and advantage is that trifluoroacetic acid stimulates Property it is bigger, toxicity is stronger, has the mutagenic effect of teratogenesis, endangers larger, and extraction process to operator in operation Middle remaining trifluoroacetic acid also has a certain degree of infringement to mass spectrometer；And use formic acid that there is small toxicity as acidulant, It is comparatively safe to operator, and can improve improve determinand sensitivity to a certain extent, improve the effect of determinand peak type.

SPE column purifications are relative to only by filtering with microporous membrane, and SPE column purifications can more thoroughly place to go extraction be molten Impurity in liquid, it is especially obvious to the removal effect of generally existing phospholipid impurity in animal tissue, it is especially same in multiple types medicine When detection process in, can significantly reduce the ambient interferences of sample using the pre-treating method of SPE column purifications, improve medicine to be measured Sensitivity and the degree of accuracy of testing result.

Simultaneously using after initial flow phased soln through measure；

By the difference of the parameter of above-mentioned multiple pre-treatments, the medicine that can not most detect in the prior art at last and examine Measure, it is achieved thereby that the inventive method detects the purpose of a greater variety of medicines of more simultaneously, the present invention is relative to it Preceding prior art obtains obvious technological progress.

In summary, relative to prior art, this method is more with medicament categories are detected simultaneously, generic medicine covering Face is wider, and pretreatment process is safer to operator, as a result more accurately and reliably；Fill up most without multiple types in goat milk and dairy produce Measure the vacancy of left drug detection method.

Medicament categories in view of detection method detection and more and be improved to its pre-treating method, So that various different types of medicines can be extracted and then detect, this is also that the present invention does not solve relative to prior art The technical scheme that technical problem certainly provides.

Various kinds of drug is distinct in pretreatment process, compares the unsuitable multi-medicament of detection method of selectivity Detect simultaneously, therefore typically use universal Extraction solvent, on the premise of ensuring that result is accurate, ambient interferences are few while chase after The simple of pretreatment process is asked, this does not accomplish and it is expected what is solved in the prior art.

(2) 74 kinds of to be measured limit use drug monitoring：

The sample that step (1) is obtained carries out UPLC-MS/MS measure, and condition determination is as follows：

Chromatographic condition：Chromatographic column is Waters Acquity UPLC BEH C₁₈(100mm × 2.1mm, 1.7 μm) post；Stream Dynamic is mutually methanol and aqueous formic acid, and type of elution is gradient elution；

Preferably, the gradient elution program is：

0-0.5min：The volume ratio of methanol is that 3.0% holding is constant；

0.5-2.0min：The volume ratio of methanol is 3.0%-25.0%；

2.0-3.5min：The volume ratio of methanol is that 25.0% holding is constant；

3.5-3.6min：The volume ratio of methanol is 25.0%-40.0%；

3.6-4.0min：The volume ratio of methanol is that 40.0% holding is constant；

4.0-5.5min：The volume ratio of methanol is 40.0%-90.0%；

5.5-8.0min：The volume ratio of methanol is that 90.0% holding is constant；

8.0-8.1min：The volume ratio of methanol is 90.0%-3.0%；

8.1-10.5min：The volume ratio of methanol is that 3.0% holding is constant.

The proportion of mobile phase and elution program of the gradient elution can ensure that various limit is imitated with the good separation of medicine Fruit.

The gradient elution elution time that the present invention uses is shorter, and solvent conversion gradient is more, causes separating effect more preferable, institute Various limit with the good separating effect of medicine, gradient can be ensured by stating the proportion of mobile phase of gradient elution and elution program Relative to prior art graded faster, change trickleer, be mainly based upon in this method more, medicine of covering medication amount Polarity difference is larger, and gradient changes to high watr-proportion gamut again from high watr-proportion to high organic Phase Proportion, so as to ensure The different various kinds of drug of polarity can be eluted preferably in the chromatography column；And gradient is switched fast primarily to ensureing Medicine similar in polarity preferably can be separated and quantified, and finally ensure testing result accurately and reliably.

Mass Spectrometry Conditions：Electric spray ion source (ESI+)；Capillary voltage is 3.0kV；Source temperature is 150 DEG C；Desolventizing temperature For 600 DEG C；Desolvention gas velocity degree is 900Lh^-1；Taper hole gas velocity degree is 50Lh^-1；Collision gas flow velocity：0.14mL· min^-1。

The chromatographic condition with before " source temperature be 105 DEG C -115 DEG C；Desolventizing temperature is 340 DEG C -360 DEG C；Desolventizing gas Flow velocity degree is 600Lh^-1-700L·h^-1" change, be mainly based upon the consideration of which factor, carry out appropriate explanation.

The present invention uses Mass Spectrometry Conditions, and " source temperature is 105 DEG C -115 DEG C relative to of the prior art；Desolventizing temperature is 340℃-360℃；Desolvention gas velocity degree is 600Lh^-1-700L·h^-1" Mass Spectrometry Conditions, detection sensitivity is higher by (100 Times or so), reproduction type is more preferable.

Preferably, 74 kinds described of 8 major class limits medication object detecting method, wherein described limit includes beta-receptor with medicine The class medicine of activator, sulfanilamide (SN), quinolione, macrolide, benzimidazole, sedative, antiviral and pleuromutilin etc. 8.

The molecular weight of Macrocyclolactone lactone kind medicine is larger, and structure is extremely complex, and polarity is higher, is typically needed in liquid phase analysis Derivatization treatment is wanted, and the present invention uses LC-MS technologies, Macrocyclolactone lactone kind medicine does not have to derivatization.Therefore in detection process It is often difficult to other medicines while detects acquisition, and in actual production, macrolide antibiotics using and remaining It is more universal, therefore, it is detected simultaneously by with other drugs by same analysis system and led as Detecting Pesticide One problem in domain.

Preferably, it is described limit with medicine be orciprenaline, Cimaterol, Terbutaline, salbutamol, Zilpaterol, Western Boot sieve, pirbuterol, Procaterol, Reproterol, fenoterol, ritodrine, methylol Clenbuterol, Ke Lun General sieve, Ke Lunsailuo, Clorprenaline, clenbuterol, Tulobuterol, Ractopamine, Mabuterol, metoprolol, horse spray are special Sieve, bromine Boot sieve, Formoterol, bambuterol, Ao Dateluo, Bbu Tero, phenolethanolamine A, sulfacetamide, sulfanilamide (SN) diformazan are disliked Azoles, sulphadiazine, sulfathiazole, sulfapryidine, sulfamerazine, bacteresulf, sulfadimidine, kynix are rattled away Piperazine, sulfadoxine, sulfabenzamide, sulfamethoxazole, sulfadimethoxine, daimeton, sulfaquinoxaline, pipemidic acid, Ofloxacin, gatifloxacin, sarafloxacin, Orbifloxacin, trovafloxacin, T-3811, Norfloxacin, Ciprofloxacin, training fluorine Sha Xing, Lomefloxacin, Danofloxacin, Enrofloxacin, gemifloxacin, MOXIFLOXACIN, Sparfloxacin, Difloxacin, Nadifloxacin, Acidum nalidixicum, flumequine, amantadine, Rimantadine, thiabendazole, Albendazole sulfone, oxfendazole, clindamycin, Luo Hong are mould It is a kind of, several or whole in element, Tilmicosin, valnemulin, Tiamulin, chlorpromazine, stresnil.

It is wide that described 8 major class, 74 kinds of medicines cover medicament categories, basic common drug during covering livestock-raising, to taking precautions against Forbidden drug residual risk is significant in animal product, especially goat milk and dairy produce.

Wherein, multichannel collecting pattern, mainly in more, the same passage inspection that solves to detect medication amount in Same Way Medication amount is surveyed to be proposed by the problem that instrument condition itself is limited, and drainage pattern mainly solves full-time segment signal at times The problem of gatherer process medicine collection points to be measured not enough cause sensitivity and the inadequate degree of accuracy, drainage pattern ensures at times The period medicine can guarantee that more fully number of scan points, ensure to substantially increase the sensitivity of detection while the degree of accuracy. Therefore this method carries out drug data collection to be measured using multichannel timesharing stage mode.

In a preferred embodiment, the detection method of residue of veterinary drug of the present invention comprises the following steps：

(1) sample pre-treatments：Goat milk sample extraction：Accurately 2g (being accurate to 0.01g) goat milk is weighed in 50mL centrifuge tubes, Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-15min is centrifuged, takes supernatant to be centrifuged in a 50mL Guan Zhong, repeat extraction once, merge extract solution, treated decontaminating column.

Powdered milk sample extracts：It is accurate to weigh 5g (being accurate to 0.01g) powdered milk sample, suitable quantity of water dissolving is added, and it is finally fixed 20mL is dissolved in, after fully mixing, accurately weighs 2mL goat milk in 50mL centrifuge tubes, it is rear to add the formic acid acetonitriles of 8mL 0.5%, whirlpool Rotation mixing 5min, 9500rmin^-1Centrifugation 5min takes supernatant in a 50mL centrifuge tubes, repeats extraction once, merges extraction Liquid, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

(2) 74 kinds of measure limited with medicine

Liquid-phase condition：Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL· min^-1；Column temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~ 2.0min, 3%A linear change are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes To 40%；3.6~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A keeps constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Embodiment

Embodiment 1

1 instrument and material

1.1 instrument

Ultra Performance Liquid Chromatography instrument (H-Class UPLC, Waters companies)；Triple quadrupole mass spectrometer (xevo TQ- XS, Waters company)；The data processing softwares of MassLynx v 4.1 (Waters companies)；(Sigma is public for 3K15 types centrifuge Department)；Milli-Q pure water meters (millipore companies)；Nitrogen evaporator (Organomation companies).

1.2 material

The organic solvents such as methanol, acetonitrile, formic acid are chromatographically pure (Fisher companies)；SPE posts (60mg, 3cc, waters Company)；Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm, waters companies).

Beta-receptor activator, sulfanilamide (SN), quinolione, macrolide, benzimidazole, sedative, antiviral and pleuromutilin 8 class, 74 kinds of veterinary drugs such as class are purchased from German Dr.Ehrenstorfer companies and lark prestige company, and purity is more than 97%.

Goat milk and powdered milk sample are purchased from electric business supermarket, select to be analyzed to identify through LC-MS/MS without 74 kinds of medicines to appoint A kind of negative sample adds sample as matrix

Sample is as being goat milk：2g (being accurate to 0.01g) blank goat milk accurately is weighed in 50mL centrifuge tubes, is quantitatively adding institute 74 kinds are stated to limit with medicine, the addition formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-15min is centrifuged, is taken Clear liquid repeats extraction once in a 50mL centrifuge tubes, merges extract solution, treated decontaminating column.

Sample is as being goat milk powder：It is accurate to weigh 5g (being accurate to 0.01g) powdered milk sample, suitable quantity of water dissolving is added, and finally Constant volume accurately weighs 2mL goat milk in 50mL centrifuge tubes after 20mL, abundant mixing, rear to add the formic acid acetonitriles of 10mL 0.5%, Vortex mixed 5min, 9500rmin^-1Centrifugation 5min takes supernatant in a 50mL centrifuge tubes, repeats extraction once, merging carries Liquid is taken, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

2 methods and result

2.1 method

2.1.1 chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

2.1.2 Mass Spectrometry Conditions：

Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages：0.5V；Source Temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Impinging air flows Speed：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

2.2 Mass Spectrometry Conditions optimize

Beta-receptor activator, sulfanilamide (SN), quinolione, macrolide, benzimidazole, sedative, antiviral and pleuromutilin Contain amino structure in the class medicines structure of class etc. 8, therefore select positive ion mode gathered data, carried out under MS Scan patterns First mass spectrometric analysis, [M+H]+peak is obtained, and optimizing respective focus voltage makes its quasi-molecular ion peak response optimal, Ran Hou Second order mses scanning is carried out for respective [M+H]+peak under Daughter Scan patterns, obtains abundant fragment ion information.It is real Test and select respective quasi-molecular ion and the higher daughter ion of two pairs of intensity to meet in European Union 2002/957/EC resolutions on mass spectrum Analytic approach qualitative, quantitative is no less than 4 points of requirement, and optimizing collision voltage makes its response intensity optimal.

Limited by instrument condition, single channel can not disposably carry out scanning while all 74 kinds of medicines, therefore test Using more Channels patterns, simultaneously as medicament categories are more, appearance time more concentrates on the phenomenon in 2~6min, real Test and time slice is carried out to different channel, ensure that each medicine has sufficient scan frequency, test also to every kind of medicine Residence time optimizes between 0.005~0.05ms, ensures to be attained by 10-12 data collection point on each chromatographic peak, Ensure that every kind of medicine can obtain higher sensitivity and accurate quantitative analysis, when 8 74 kinds of major class medicines optimize mass spectrometry parameters and retained Between be shown in Table 1.

When 74 kinds of 1. 8 major class of table is limited with the qualitative/quantitative ion (*) pair of medicine and its corresponding mass spectrometry parameters and retained Between

2.3 matrix effects are investigated：

Animal derived matrix can have different degrees of and source matrix effect when carrying out quantitative analysis using mass spectrum, to comment Estimate the influence of matrix effect, can typically design matrix effect control experiment.Experimental result finds 8 major class medicines in goat milk matrix With the presence of different degrees of matrix effect, wherein beta-receptor activator ME is between 0.56~1.08, sulfanilamide (SN) ME 0.39~ Between 0.93, quinolione ME between 0.57~1.06, macrolide, benzimidazole, sedative, antiviral and pleuromutilin Class should use matrix mark-on solution between 0.32~1.06 in detection process, eliminate the influence of matrix effect.It is different Drug matrices inhibition level is shown in Fig. 5.

2.4 method validation

2.4.1 specificity is tested

Blank goat milk sample addition beta-receptor activator (27 kinds), sulfanilamide (SN) (15 kinds), quinolione (20 kinds), macrolide (3 Kind), benzimidazole (3 kinds), sedative (2 kinds), 74 kinds of 8 class such as antiviral (2 kinds) and pleuromulins (2 kinds) limit medication Thing, and compared with blank sample chromatogram, as a result find that 74 kinds of medicines be able to can separate well in 10.5 minutes, And interference is not present in blank sample chromatogram, 74 kinds limit medication and look for spectrogram to see Fig. 1-4.

2.4.2 linear relationship and test limit

Experiment uses blank goat milk sample through pre-treatment, in 1.0~80.0 μ gL^-174 kinds are added in concentration range respectively Mixed mark solution, prepares 1.0,5.0,10.0,20.0,80.0 μ gL^-1Matrix mark-on solution, sample introduction successively, using chromatographic peak area as Ordinate, compound concentration are that abscissa does standard curve；By the blank goat milk sample handled well working solution step by step, press 10 times of signal to noise ratio determine the quantitative limit (LOQ) of method.

8 major class, 74 kinds of medicine ranges of linearity, coefficient correlation and Quantitation Limit in the goat milk of table 6

Test result indicates that the class medicine of beta-receptor activator, sulfanilamide (SN), quinolione etc. 3 is in 1.0~40.0 μ gL^-1In the range of Linear good, coefficient correlation between 0.991-0.999, pick up the ears by macrolide, benzimidazole, sedative, antiviral and truncation The 5 class medicines such as element are in 1.0~80.0 μ gL^-1In the range of it is linear good, coefficient correlation is between 0.992-0.998,74 kinds of medicines Quantitative limit is respectively less than 1.0 μ gkg^-1, specifically it is shown in Table 2.

2.4.3 the rate of recovery and precision test

Blank goat milk sample is taken, addition concentration is 1.0,10.0,20.0 μ gkg^-1The mixed mark solution of three levels, according to 1.2 Sample-pretreating method item under handle sample, 6 parallel, 3 batches of different time replication in each concentration batch, carry out Addition recovery and Precision Experiment the results are shown in Table 3.

8 major class, 74 kinds of medicine TIANZHU XINGNAO Capsuls and the coefficient of variation in the goat milk of table 3

It can be seen from Table 3 that the inventive method average recovery rate, 70.1%~119.7%, variation within batch coefficient is 1.1%~17.6%, interassay coefficient of variation is 2.1%~18.5%, and the degree of accuracy of method and precision meet goat milk and dairy The requirement of kind detection technique.

Embodiment 2

(concentration is 27 kinds of beta-receptor stimulant medicine mixtures that 2g blank feminine gender goat milk sample adds described in 20.0 μ L 500ng/ml).Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-1Centrifuge 5min, take supernatant in In one 50mL centrifuge tubes, extraction is repeated once, merge extract solution, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram and Fig. 1 chromatogram homomorphosis are determined, illustrates to contain 27 kinds of beta-receptor activator classes in sample Medicine, while the content of each material is calculated between 3.67ng/g-5.23ng/g, the rate of recovery is between 73.4%-104.6, table The bright degree of accuracy is higher.

Embodiment 3

2g blank feminine gender goat milk sample adds 15 kinds of sulfa drugs mixture (concentration 500ng/ described in 20.0 μ L ml).Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-15min is centrifuged, takes supernatant in a 50mL In centrifuge tube, extraction is repeated once, merge extract solution, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram and Fig. 2 chromatogram homomorphosis are determined, illustrates to contain 15 kinds of sulfa drugs in sample, simultaneously The content of each material is calculated between 3.55ng/g-4.38ng/g, the rate of recovery shows the degree of accuracy between 71.0%-87.6% It is higher.

Embodiment 4

2g blank feminine gender goat milk sample adds 20 kinds of quinolone druge mixture (concentration 500ng/ described in 20.0 μ L ml).Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-15min is centrifuged, takes supernatant in a 50mL In centrifuge tube, extraction is repeated once, merge extract solution, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram and Fig. 3 chromatogram homomorphosis are determined, illustrates to contain 20 kinds of quinolone druges in sample, together When calculate the content 3.72ng/g-5.08ng/g of each material between, the rate of recovery shows the degree of accuracy between 74.4%-101.6% It is higher.

Embodiment 5

2g blank feminine gender goat milk sample adds 3 kinds of clindamycin, ROX and Tilmicosin big rings described in 20.0 μ L Lactone medicinal mixture (concentration 500ng/ml).Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500r min^-15min is centrifuged, takes supernatant in a 50mL centrifuge tubes, repeats extraction once, merges extract solution, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram chromatogram homomorphosis corresponding with Fig. 4 is determined, is illustrated in sample containing clindamycin, Luo Hong 3 kinds of Macrocyclolactone lactone kind medicines of mycin and Tilmicosin, at the same the content for calculating each material be respectively 3.86ng/g, 3.69ng/g and 5.34ng/g, the rate of recovery show that the degree of accuracy is higher between 73.8%-106.8%.

Embodiment 6

2g blank feminine gender goat milk sample adds 3 kinds of thiabendazole, Albendazole sulfone and oxfendazole benzene described in 20.0 μ L Imipramine class medicinal mixture (concentration 500ng/ml).The addition formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500r·min^-15min is centrifuged, takes supernatant in a 50mL centrifuge tubes, repeats extraction once, merges extract solution, treated purification Post.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram chromatogram homomorphosis corresponding with Fig. 4 is determined, is illustrated in sample containing thiabendazole, acetysalicylic acid phenobarbital Up to 3 kinds of benzimidazole class medicines of azoles sulfone and oxfendazole, while the content for calculating each material is respectively 3.87ng/g, 4.89ng/g And 4.05ng/g, the rate of recovery show that the degree of accuracy is higher between 77.4%-97.8%.

Embodiment 7

2g blank feminine gender goat milk sample adds the amantadine and the antiviral class mixture of 2 kinds of Rimantadine described in 20.0 μ L (concentration 500ng/ml).Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-15min is centrifuged, is taken Clear liquid repeats extraction once in a 50mL centrifuge tubes, merges extract solution, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram chromatogram homomorphosis corresponding with Fig. 4 is determined, illustrates to contain amantadine and gold in sample The antiviral class medicine of firm 2 kinds of ethamine, while the content for calculating each material is respectively 4.43ng/g and 4.12ng/g, the rate of recovery is distinguished For 88.6% and 82.4%, show that the degree of accuracy is higher.

Embodiment 8

Chlorpromazine and 2 kinds of sedative class mixtures of stresnil described in 20.0 μ L of 2g blank feminine gender goat milk sample addition is (dense Spend for 500ng/ml).Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-15min is centrifuged, takes supernatant Liquid repeats extraction once in a 50mL centrifuge tubes, merges extract solution, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram chromatogram homomorphosis corresponding with Fig. 4 is determined, illustrates to contain chlorpromazine and nitrogen piperazine in sample 2 kinds of sedative class medicines of ketone, while the content for calculating each material is respectively 3.72ng/g and 4.29ng/g, the rate of recovery is respectively 74.4% and 85.8%, show that the degree of accuracy is higher.

Embodiment 9

2 kinds of pleuromulins of Tiamulin and valnemulin that 2g blank feminine gender goat milk sample adds described in 20.0 μ L mix Compound (concentration 500ng/ml).Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-1Centrifuge 5min, Supernatant is taken in a 50mL centrifuge tubes, repeats extraction once, merges extract solution, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram chromatogram homomorphosis corresponding with Fig. 4 is determined, illustrates to contain Tiamulin in sample and irrigates Buddhist nun's 2 kinds of pleuromulins medicines of wonderful woods, while the content for calculating each material is respectively 3.67ng/g and 4.15ng/g, the rate of recovery Respectively 73.4% and 83.0%, show that the degree of accuracy is higher.

Embodiment 10

2g blank feminine gender goat milk sample adds mixture (the concentration 500ng/ of 8 74 kinds of medicines of major class described in 20.0 μ L ml).Add the formic acid acetonitriles of 10mL 0.5%, vortex mixed 5min, 9500rmin^-15min is centrifuged, takes supernatant in a 50mL In centrifuge tube, extraction is repeated once, merge extract solution, treated decontaminating column.

Purification：Whole extract solutions are transferred in the SPE posts activated in advance with 85% acetonitrile water, keep flow velocity 1ds^-1, Whole filtered solutions are collected, 40 DEG C of nitrogen, which are blown to, closely to be done, and 1mL, 9500rmin are settled to liquid phase^-15min is centrifuged, on Liquid layer uses 0.22 μm of membrane filtration, treats that machine determines.

Chromatographic condition

Chromatographic column：Acquity UPLC BEH C18 (100mm × 2.1mm, 1.7 μm)；Flow velocity：0.3mL·min^-1；Post Temperature：35℃；Mobile phase A:Methanol, B:0.1% aqueous formic acid, 0~0.5min, 3%A keep constant, 0.5~2.0min, 3% A linear changes are to 25%；2.0~3.5min, 25%A keep constant, 3.5~3.6min, 25%A linear changes to 40%；3.6 ~4.0min, 40%A keep constant；4.0~5.5min, 40%A linear change are to 90%；5.5~8.0min, 90%A are kept It is constant；For 8.0~8.1min, 90%A linear change to 3%, 8.1~10.5min, 3%A keep constant.

Mass Spectrometry Conditions：Ionization pattern：ESI+；Capillary voltage：3.0kV；Extract taper hole voltage：3.0V；RF lens voltages： 0.5V；Source temperature：150℃；Desolventizing temperature：600℃；Taper hole gas velocity：50L·h^-1；Desolvention gas velocity：900L·h^-1；Touch Hit gas velocity：0.14mL·min^-1；Drainage pattern：More multi-period MRM collections of Channels.

Obtained spectrogram chromatogram homomorphosis corresponding with Fig. 1-Fig. 4 is determined, illustrates to contain above-mentioned 74 kinds in sample Medicine, while calculate the content of each material respectively between 3.52ng/g-5.68ng/g, the rate of recovery 70.4%-113.6% it Between, show that the degree of accuracy is higher.

Claims (10)

1. a kind of detect the method for disabling or/and limiting the use of medicine in goat milk and its product, it is characterised in that methods described is included such as Lower step：

(1) processing of sample：

Testing sample is added after acidic organic solvent extraction and carries out the concussion extraction that is vortexed, centrifuging and taking supernatant, centrifuges what is obtained Precipitation repeats the concussion extraction that is vortexed, and merges supernatant, through SPE column purifications, the drying of scavenging solution nitrogen, uses initial flow phased soln It is used to UPLC-MS/MS afterwards determine；

(2) disable or/and limit the use of the measure of medicine：

The sample that step (1) is obtained carries out UPLC-MS/MS measure, and condition determination is as follows：

Chromatographic condition：Chromatographic column is Waters Acquity UPLC BEH C18 posts；Mobile phase is methanol and aqueous formic acid, Type of elution is gradient elution；

Mass Spectrometry Conditions：Electric spray ion source (ESI+)；

Sample collection mode：Using multichannel drainage pattern at times.

2. detection method as claimed in claim 1, wherein described limit includes beta-receptor activator, sulfanilamide (SN), quinoline with medicine Ketone, macrolide, benzimidazole, sedative, the antiviral and class medicine of pleuromutilin 8.

3. detection method as claimed in claim 1 or 2, wherein described limit with medicine is orciprenaline, Cimaterol, Te Bu His woods, salbutamol, Zilpaterol, western Boot sieve, pirbuterol, Procaterol, Reproterol, fenoterol, hydroxyl benzyl hydroxyl fiber crops Yellow alkali, methylol Clenbuterol, Ke Lunpuluo, Ke Lunsailuo, Clorprenaline, clenbuterol, Tulobuterol, Ractopamine, Mabuterol, metoprolol, horse spray special sieve, bromine Boot sieve, Formoterol, bambuterol, Ao Dateluo, Bbu Tero, benzene second Hydramine A, sulfacetamide, sulfamethazole, sulphadiazine, sulfathiazole, sulfapryidine, sulfamerazine, bacteresulf, Methoxy between sulfadimidine, sulfamethoxypyridazine, sulfadoxine, sulfabenzamide, sulfamethoxazole, sulfadimethoxine, sulfanilamide (SN) Pyrimidine, sulfaquinoxaline, pipemidic acid, Ofloxacin, gatifloxacin, sarafloxacin, Orbifloxacin, trovafloxacin, T-3811, Norfloxacin, Ciprofloxacin, Pefloxacin, Lomefloxacin, Danofloxacin, Enrofloxacin, gemifloxacin, MOXIFLOXACIN, department's pa Sha Xing, Difloxacin, Nadifloxacin, acidum nalidixicum, flumequine, amantadine, Rimantadine, thiabendazole, Albendazole sulfone, Austria It is a kind of, several in oxfendazole, clindamycin, ROX, Tilmicosin, valnemulin, Tiamulin, chlorpromazine, stresnil Kind is whole.

4. the detection method as described in claim any one of 1-3, step (2) described gradient elution program are：

0-0.5min：The volume ratio of methanol is that 3.0% holding is constant；

0.5-2.0min：The volume ratio of methanol is 3.0%-25.0%；

2.0-3.5min：The volume ratio of methanol is that 25.0% holding is constant；

3.5-3.6min：The volume ratio of methanol is 25.0%-40.0%；

3.6-4.0min：The volume ratio of methanol is that 40.0% holding is constant；

4.0-5.5min：The volume ratio of methanol is 40.0%-90.0%；

5.5-8.0min：The volume ratio of methanol is that 90.0% holding is constant；

8.0-8.1min：The volume ratio of methanol is 90.0%-3.0%；

8.1-10.5min：The volume ratio of methanol is that 3.0% holding is constant.

5. the temperature of the detection method as described in claim any one of 1-4, wherein step (1) the nitrogen drying supernatant is 35 DEG C -45 DEG C, preferably in 40 DEG C of progress.

6. the detection method as described in claim any one of 1-5, the rotating speed of wherein step (1) described centrifugation is 9000rmin^-1-10000r·min^-1, the time of centrifugation is 3min-7min, and preferably described centrifugal rotational speed is 9500rmin^-1, centrifugation when Between be 5min.

7. the detection method as described in claim any one of 1-6, wherein step (1) the extractant volumetric concentration are 0.2%- 1.0% formic acid acetonitrile solution；It is preferred that formic acid volumetric concentration is 0.5%.

It is 8. cleaned in SPE solid-phase extraction columns described in the detection method as described in claim any one of 1-7, wherein step (1) Cheng Zhong, flow rate of liquid 0.5ds^-1-3d·s^-1, preferably described flow rate of liquid is 1ds^-1。

9. the detection method as described in claim any one of 1-8, the volumetric concentration of the wherein aqueous formic acid of step (2) are 0.05%-0.2%, preferred volume concentration are 0.1%.

10. the detection method as described in claim any one of 1-9, wherein step (2) described chromatographic condition：Chromatographic column is Waters Acquity UPLC BEH C₁₈Post；Mobile phase is methanol and aqueous formic acid, and type of elution is gradient elution；Flow velocity For 0.3mLmin^-1；Column temperature is 35 DEG C；

Mass Spectrometry Conditions：Electric spray ion source (ESI+)；Sample collection mode；Capillary voltage is 3.0-3.5kV；Source temperature is 145 ℃-155℃；Desolventizing temperature is 500 DEG C -600 DEG C；Desolvention gas velocity degree is 850Lh^-1-950L·h^-1；Taper hole air-flow Speed is 45Lh^-1-55L·h^-1；Collision gas flow velocity：0.13mL·min^-1-0.15mL·min^-1；

It is preferred that Mass Spectrometry Conditions：Electric spray ion source (ESI+)；Capillary voltage is 3.0KV；Extraction taper hole voltage is 3.0V；RF is saturating Mirror voltage is 0.5V；Source temperature is 150 DEG C；Desolventizing temperature is 600 DEG C；Desolvention gas velocity degree is 900Lh^-1；Taper hole air-flow Speed is 50Lh^-1；Impinging air flows speed is 0.14mLmin^-1；

Sample collection mode：Using multichannel drainage pattern at times.