CN101865886A - Method for measuring residual quantity of chloramphenicol in propolis by using high performance liquid chromatography tandem mass spectrum - Google Patents
Method for measuring residual quantity of chloramphenicol in propolis by using high performance liquid chromatography tandem mass spectrum Download PDFInfo
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Abstract
The invention relates to a method for measuring the residual quantity of chloramphenicol in propolis by using a high performance liquid chromatography tandem mass spectrum. At present, a measuring method with low detection cost and detection limit reaching 0.1mu g/kg is not found. The method comprises work procedures of standard solution preparation, standard curve preparation, propolis sample solution preparation and chloramphenicol residual quantity detection, wherein the work procedure of propolis sample solution preparation comprises the following steps of: adding 1mol/L NaOH solution to dissolve a propolis sample; diluting with water to dilute a target object in the propolis sample; acidizing with HCLO4 so as to precipitate propolis components in the propolis sample; adjusting the pH value of filtrate of the propolis sample solution to be 10.5 with the 1mol/L NaOH solution; and finally measuring the residual quantity of chloramphenicol in the propolis sample solution through a high performance liquid chromatography and a trebling quadrupole rod tandem mass spectrum. The invention has low detection cost and high detection efficiency, the detection limit energy reaches 0.1mu g/kg, the quantitation limit is 0.3mu g/kg and the linear correlation coefficient is 0.9999.
Description
Technical field
The present invention relates to a kind of method of measuring chloramphenicol residue in the propolis, especially relate to a kind of using high performance liquid chromatography tandem mass spectrum and measure the method for chloramphenicol residue in the propolis, be mainly used in chloromycetin drug residue in the propolis is checked, also be applicable to simultaneously chloromycetin drug residue in royal jelly, the honey is checked.
Background technology
Chloromycetin (Chloramphenicol) is the microbiotic that is produced by the Venezuela Streptothrix, is broad-spectrum antibacterial agent.Because its toxicity to hematological system is bigger, is forbidden being used for animal derived food by states such as European Union, Japan, the U.S..
The chloromycetin detection method mainly contains the LC/MS/MS method, the GC/MS method, the GC method, the ELISA method, HPLC method and CHARM II method etc., mensuration high performance liquid chromatography as chloramphenicol residue in the detection method of chloramphenicol residue in the import and export royal jelly among the SN/T 2063-2008 and the animal-derived food among No. 781 bulletin-2-2006 of the Ministry of Agriculture all belongs to the LC/MS/MS method, all belong to the GC/MS method as chloromycetin determination of drug residues in the mensuration vapor-phase chromatography of chloramphenicol residue in the honey among No. 781 bulletin-10-2006 of the Ministry of Agriculture and the animal derived food among the GB/T 22338-2008, detect vapor-phase chromatography as residual chloromycetin in the animal-derived food among No. 1025 bulletin-21-2008 of the Ministry of Agriculture and belong to the GC method, belong to the ELISA method as residual chloromycetin quantity measuring method in the import and export royal jelly among the SN/T 2058-2008, as by Wu Hua, Huang Hong and Ou Jianfeng etc. are published in the research of chloramphenicol residue in the high effective liquid chromatography for measuring honey on Sichuan chemical industry 2009 the 12nd volume the 3rd phase 40-42 page or leaf and by Pan Yingyu, Xu Qian and Kang Xuejun etc. are published in high performance liquid chromatography-fluorescence detection on chromatogram 2005 the 23rd volume the 6th phase 577-580 page or leaf and measure the residual quantity of chloromycetin in the milk and all belong to the HPLC method, belong to CHARM II method as residual chloromycetin detection method in the aquatic products on the SN/T1966-2007.
As from the foregoing, the liquid chromatography-tandem mass spectrometry method is extensively used by domestic and international government supervision inspection body as the conclusive evidence method, the unique advantage that it is had on sensitivity, accuracy, detection efficiency, become the residue detection instrument equipment of present superior property, when being used for detecting the animal derived food chloramphenicol residue, its detectability can reach 0.1 μ g/kg, and the pertinent literature report is a lot.But, because different types of sample composition is different, the sample purification degree is also different, when using the liquid chromatography-tandem mass spectrometry method, be not that all samples can both reach 0.1 this detection limit of μ g/kg, when for example using the existing liquid chromatography-tandem mass spectrometry method that can measure chloramphenicol residue to measure in the propolis chloramphenicol residue, its detectability does not far reach 0.1 μ g/kg, this is because the composition in the propolis has nearly thousand kinds, domestic also do not have desirable method as chloramphenicol residue examination criteria in the propolis, the domestic pertinent literature of not seeing that yet chloramphenicol residue detects in the propolis.
In sum, also do not have a kind of method simple, accurate at present, it is low to detect cost, the detection efficiency height, what detectability can reach 0.1 μ g/kg is used for measuring the method that the propolis residual chloromycetin is limited the quantity of, in propolis detects, be difficult to satisfy external to propolis in the residual chloromycetin requirement of limiting the quantity of.
Summary of the invention
The objective of the invention is to overcome above shortcomings in the prior art, and provide a kind of method simple, it is low to detect cost, and the using high performance liquid chromatography tandem mass spectrum that detection efficiency height, detectability can reach 0.1 μ g/kg is measured the method for chloramphenicol residue in the propolis.
The present invention addresses the above problem the technical scheme that is adopted: the characteristics that this using high performance liquid chromatography tandem mass spectrum is measured the method for chloramphenicol residue in propolis are: the used raw material of this method comprises that purity is greater than 99.0% chloromycetin standard items, concentration is the D5-chloromycetin standard items of 100mg/l, ethyl acetate for the residual level of farming, be analytically pure NaOH, be the pure normal hexane of top grade, be the pure acetonitrile of liquid chromatography, propolis sample;
The used equipment of this method comprises triple quadrupole bar tandem mass spectrometer, is furnished with the high performance liquid chromatograph of binary pump, online degasser, automatic sampler, data processing software; Ion source temperature in the described triple quadrupole bar tandem mass spectrometer is 600 ℃, and auxiliary heating gas is 55.00psi, and dry gas is 65.00psi, gas curtain gas is 25.00psi, collision gas is 5.00psi, and electron spray voltage is-4100.00V that the ionization method is a negative ion mode; Described high performance liquid chromatograph is Agilent 1200series, and chromatographic column is Hypersil ODS2, and the specification of chromatographic column is 4.6mm * 150mm, and 5 μ m, column temperature are 30 ℃, and sample size is 20 μ l;
This method comprises that standard solution preparation operation, standard curve making operation, propolis sample liquid preparation section and chloramphenicol residue detect operation; Described standard solution preparation operation is as follows: a, chloromycetin are stocked the standard solution preparation steps: take by weighing chloromycetin standard items 10mg and be settled to 10ml with acetonitrile to be made into concentration be that the chloromycetin of 1000mg/L is stocked standard solution, with this chloromycetin stock standard solution and place preserve below-18 ℃ stand-by; B, chloromycetin intermediate standard liquid preparation steps: get chloromycetin among a and stock standard solution 100 μ l and be settled to 10ml with acetonitrile and be made into the chloromycetin intermediate standard liquid that concentration is 10mg/L; C, chloromycetin deuterium interior mark intermediate liquid preparation steps of generation: adopt D5-chloromycetin standard items to replace the chloromycetin standard items, repeat above-mentioned a step and b step, making concentration is the chloromycetin deuterium interior mark intermediate liquid of generation of 10mg/L; D, interim standard are used the liquid preparation steps: extracting chloromycetin intermediate standard liquid volumetric concentration is that 30% acetonitrile solution is assigned to 5 μ g/kg and 50 μ g/kg, and the extracting chloromycetin deuterium is that 30% acetonitrile solution is assigned to 200 μ g/kg for interior mark intermediate liquid volumetric concentration;
In the described standard curve making operation; get the chloromycetin that makes in the standard solution preparation operation and stock standard solution, chloromycetin intermediate standard liquid, chloromycetin deuterium interior mark intermediate liquid of generation and interim standard use liquid, obtain typical curve by high performance liquid chromatograph and triple quadrupole bar tandem mass spectrometer;
In the described propolis sample liquid preparation section, getting propolis sample 2g and concentration and be in the D5-chloromycetin standard items of 200 μ g/kg mark 50ul places a centrifugal plastic bottle of 50ml and fully mixes, leave standstill more than the 5min then, in a centrifugal plastic bottle of 50ml, add the NaOH solution that 5ml concentration is 1mol/L again and constantly vibrate and extract 5min, the propolis sample is fully dissolved, in centrifugal plastic bottle of 50ml, add 5ml water then and shake up, in a centrifugal plastic bottle of 50ml, add 10ml concentration again and be 5% HCl0
4Carry out acidifying and fully shake up and make propolis liquid; This propolis liquid is ultrasonic Extraction 5min at normal temperatures earlier, is that centrifugal 5min obtains upper strata centrifugate under the 3000rpm at rotating speed again, then upper strata centrifugate is filtered and makes filtrate; Get filtrate 10ml and place the centrifugal plastic bottle of 50ml No. two, with concentration is that the NaOH solution of 1mol/L is adjusted to 10.5 with pH value of filtrate, in No. two centrifugal plastic bottles of 50ml, add 10ml ethyl acetate then and fully mix 3min, at rotating speed is that centrifugal 2min obtains ethyl acetate layer under the 3000rpm, ethyl acetate is placed in the 15ml plastic centrifuge tube then, this ethyl acetate layer carries out nitrogen and dries up under 35 ℃, adding volumetric concentration again in the 15ml plastic centrifuge tube is 30% acetonitrile solution 1ml and carries out ultrasonic dissolution, in the 15ml plastic centrifuge tube, add 2ml normal hexane and abundant the mixing then and make centrifugate, is that centrifugal 2min obtains lower floor's sample liquid under the 800rpm with this centrifugate at rotating speed, at last lower floor's sample liquid is crossed the filter membrane of 0.22 μ m and is made propolis sample liquid;
Described chloramphenicol residue detects in the operation, get the propolis sample liquid that makes in the propolis sample liquid preparation section, by high performance liquid chromatograph and triple quadrupole bar tandem mass spectrometer, and the typical curve that obtains in the combined standard curve plotting operation and record the residual quantity of chloromycetin in the propolis; The detection of this method is limited to 0.1 μ g/kg, quantitatively is limited to 0.3 μ g/kg.
The employed water of this method of the present invention is dual distilled water.
In the propolis sample liquid preparation section of the present invention, adding concentration in a centrifugal plastic bottle of 50ml is the NaOH solution dissolving propolis sample of 1mol/L, and dilute with water is used HClO again to disengage the residual chloromycetin thing in the propolis sample then
4Carry out acidifying with the propolis composition in the precipitation propolis sample.
In the propolis sample liquid preparation section of the present invention, propolis sample liquid concentration is that the NaOH solution of 1mol/L is adjusted to 10.5 with pH value of filtrate, ethyl acetate layer adopts Nitrogen evaporator to carry out nitrogen down at 35 ℃ and dries up, add volumetric concentration again and be 30% acetonitrile solution 1ml and adopt ultrasonoscope to carry out ultrasonic dissolution in the 15ml plastic centrifuge tube.
The present invention compared with prior art, have the following advantages and effect: the present invention is used for measuring the residual quantity of propolis chloromycetin medicine, comparing with the Solid-Phase Extraction method does not increase solvent load, do not adopt solid phase extraction column, sample water white transparency after the purification, noiseless on mass spectrogram, behind the continuous sample introduction 70 times, the ion gun gas curtain plate in the triple quadrupole bar tandem mass spectrometer still keeps clean.Detection of the present invention is limited to 0.1 μ g/kg, quantitatively is limited to 0.3 μ g/kg, and linearly dependent coefficient is 0.9999, the recovery is at 82.0-108.2%, relative standard deviation is at 5.35-14.2%, and precision is good, can satisfy the requirement that importer limits the quantity of to chloromycetin medicament residue in the propolis fully.
The present invention has given full play to mass spectral advantage, utilizes simply, sample purification method efficiently, by optimizing liquid phase, mass spectrum condition, reduces sample substrate and disturbs, and makes method reach enough sensitivity.This method is applicable to the detection of chloromycetin drug residue in royal jelly, the honey simultaneously, because sample pretreatment is with low cost, is particularly suitable for basic unit of enterprise purchase, production overall process are carried out quality control, helps improving the quality of royal jelly, honey.
Description of drawings
Fig. 1 is when the propolis negative sample adds 1.0 μ g/kg normal concentrations in the embodiment of the invention, measures the residual total ion current figure of nitroimidazoles medicine in the royal jelly with LC/MS/MS;
Fig. 2 is when the propolis negative sample adds 1.0 μ g/kg normal concentrations in the embodiment of the invention, measures the residual extraction ion flow graph of nitroimidazoles medicine in the royal jelly with LC/MS/MS.
Embodiment
The present invention is described in further detail below in conjunction with accompanying drawing and by embodiment, and following examples are explanation of the invention and the present invention is not limited to following examples.
Embodiment:
Referring to Fig. 1 and Fig. 2, the method that using high performance liquid chromatography tandem mass spectrum is measured chloramphenicol residue in the propolis in the present embodiment comprises that standard solution preparation operation, standard curve making operation, propolis sample liquid preparation section and chloramphenicol residue detect operation.
The used raw material of present embodiment comprises water, purity is greater than 99.0% chloromycetin standard items, concentration is that 100mg/l is all from the D5-chloromycetin standard items of German Dr.Ehrenstorfer company, ethyl acetate for the residual level of farming, be analytically pure NaOH, methyl alcohol, sodium chloride and anhydrous sodium sulfate, be top grade pure normal hexane and formic acid, be liquid chromatography pure acetonitrile and ammonium acetate, propolis sample to be detected.Wherein used water is dual distilled water.
The used equipment of present embodiment comprises API3200 triple quadrupole bar tandem mass spectrometer, the high performance liquid chromatograph of being furnished with binary pump, online degasser, automatic sampler, Analyst data processing software, Sartorius BS224S analytical balance, the accurate pH meter of PHS-3C thunder magnetic, the desk-top high capacity hydro-extractor of Ruijiang Analyzer Co. Ltd., Wuxi City's RJ-TDL-40B low speed, Organomation N-EVAP 111 Nitrogen evaporators, ultrasonoscope.Wherein the ion source temperature in the triple quadrupole bar tandem mass spectrometer is 600 ℃, and auxiliary heating gas is 55.00psi, and dry gas is 65.00psi, gas curtain gas is 25.00psi, collision gas is 5.00psi, and electron spray voltage is-4100.00V that the ionization method is a negative ion mode; Used high performance liquid chromatograph is Agilent 1200series, and chromatographic column is Hypersil ODS2, and the specification of chromatographic column is 4.6mm * 150mm, and 5 μ m, column temperature are 30 ℃, and sample size is 20 μ l.
Standard solution preparation operation in the present embodiment comprises the steps: that a, chloromycetin stocks the standard solution preparation steps: take by weighing chloromycetin standard items 10mg and place the 10ml volumetric flask, being settled to 10ml and being made into concentration with acetonitrile is that the chloromycetin of 1000mg/L is stocked standard solution, then this chloromycetin is stocked standard solution and places preserve below-18 ℃ stand-by; B, chloromycetin intermediate standard liquid preparation steps: get chloromycetin among a and stock standard solution 100 μ l and place the 10ml volumetric flask, be settled to 10ml and be made into the chloromycetin intermediate standard liquid that concentration is 10mg/L with acetonitrile; C, chloromycetin deuterium interior mark intermediate liquid preparation steps of generation: take by weighing D5-chloromycetin standard items 10mg and place the 10ml volumetric flask, being settled to 10ml and being made into concentration with acetonitrile is that the D5-chloromycetin of 1000mg/L is stocked standard solution, get D5-chloromycetin then and stock standard solution 100 μ l and place the 10ml volumetric flask, be settled to 10ml and be made into the chloromycetin deuterium interior mark intermediate liquid of generation that concentration is 10mg/L with acetonitrile; D, interim standard are used the liquid preparation steps: extracting chloromycetin intermediate standard liquid volumetric concentration be 30% acetonitrile solution be mixed with concentration be the interim standard of chloromycetin of 5 μ g/kg to use liquid and concentration be that the interim standard of chloromycetin of 50 μ g/kg is used liquid, extracting chloromycetin deuterium interior mark intermediate liquid volumetric concentration of generation is that to be mixed with concentration be that the chloromycetin deuterium of 200 μ g/kg is for the interim standard use of interior mark liquid for 30% acetonitrile solution.
In the standard curve making operation of present embodiment; get the chloromycetin that makes in the standard solution preparation operation and stock standard solution, chloromycetin intermediate standard liquid, chloromycetin deuterium interior mark intermediate liquid of generation and interim standard use liquid; measure correlation parameter by high performance liquid chromatograph and triple quadrupole bar tandem mass spectrometer, and then obtain typical curve.High performance liquid chromatograph adopts liquid phase gradient program, and table 1 be the liquid phase gradient elution program list of high performance liquid chromatograph, and the residual chloromycetin mensuration of triple quadrupole bar tandem mass spectrometer sees Table 2 with reference to the mass spectrum condition, the theing contents are as follows of table 1 and table 2:
The liquid phase gradient elution program list of table 1 high performance liquid chromatograph
The residual chloromycetin of table 2 triple quadrupole bar tandem mass spectrometer is measured with reference to mass spectrum condition table
Annotate: band underscore boldface type is a quota ion in the table.
In the propolis sample liquid preparation section of present embodiment, take by weighing propolis sample 2g earlier and place the centrifugal plastic bottle of 50ml No. one, getting concentration again and be the interim standard of chloromycetin deuterium interior mark of generation of 200 μ g/kg uses liquid 50ul to place the centrifugal plastic bottle of 50ml No. one, use liquid and propolis sample fully to mix the interim standard of chloromycetin deuterium interior mark of generation of 200 μ g/kg, leave standstill more than the 5min, in a centrifugal plastic bottle of 50ml, add the NaOH solution that 5ml concentration is 1mol/L then and constantly vibrate and extract 5min, the propolis sample is fully dissolved, in centrifugal plastic bottle of 50ml, add 5ml water again and shake up, in a centrifugal plastic bottle of 50ml, add 10ml concentration then and be 5% HClO
4Carry out acidifying and fully shake up and make propolis liquid.This propolis liquid is carried out ultrasonic Extraction 5min by ultrasonoscope earlier at normal temperatures, is centrifugal 5min under the condition of 3000rpm with the rotating speed by hydro-extractor again, and propolis liquid layering occurs and obtains upper strata centrifugate, then upper strata centrifugate is filtered and makes filtrate.Get filtrate 10ml and place the centrifugal plastic bottle of 50ml No. two, with concentration is that the NaOH solution of 1mol/L is adjusted to 10.5 with pH value of filtrate, reach the purpose of purifying filter liquor by adjusting to filtrate pH, and the whole process that filtrate purifies all need not to use solid phase extraction column, in No. two centrifugal plastic bottles of 50ml, add 10ml ethyl acetate then and fully mix 3min, be that centrifugal 2min obtains ethyl acetate layer under the condition of 3000rpm by hydro-extractor at rotating speed, ethyl acetate is placed in the 15ml plastic centrifuge tube then, and adopt down Nitrogen evaporators that ethyl acetate layer is carried out nitrogen at 35 ℃ to dry up, in the 15ml plastic centrifuge tube, add volumetric concentration again and be 30% acetonitrile solution 1ml and carry out ultrasonic dissolution by ultrasonoscope, in the 15ml plastic centrifuge tube, add 2ml normal hexane and abundant mixing degrease then and make centrifugate, be that 800rpm under centrifugal 2min obtain lower floor sample liquid with this centrifugate at rotating speed by hydro-extractor again, at last lower floor's sample liquid crossed the filter membrane of 0.22 μ m and made propolis sample liquid.
The present embodiment chloramphenicol residue detects in the operation, get the propolis sample liquid that makes in the propolis sample liquid preparation section, use high performance liquid chromatograph and triple quadrupole bar tandem mass spectrometer, and the typical curve that obtains in the combined standard curve plotting operation and record the residual quantity of chloromycetin in the propolis.
Present embodiment is optimized the purification method of propolis sample, because chloromycetin is insoluble in neutral aqueous solution, be dissolved in organic solvent, this provides theoretical support for liquid-liquid extraction purifies the propolis sample, make that the assay method of chloramphenicol residue can be without solid phase extraction techniques in the propolis, reduced the detection cost, and the consumption of solvent does not increase, the present invention is according to these characteristics, when medicine is electric neutrality, carry out liquid-liquid extraction, thereby reach the purpose that the propolis sample is purified with organic solvent.
The propolis sample is water insoluble, be dissolved in mass concentration and be 2% NaOH, methyl alcohol, ethanol etc., extract, the chloromycetin in purification and the enrichment propolis, must earlier the propolis sample be dissolved and can extract with solvent, because most documents and materials all adopt ethyl acetate to extract chloromycetin in the propolis sample, and ethyl acetate and methyl alcohol, ethanol dissolve each other, with water be immiscible, chloromycetin is more stable again under alkali condition, earlier the propolis sample is dissolved in the NaOH solution so the present invention preferentially selects for use, extracts with ethyl acetate again.
Through overtesting, when directly extracting chloromycetin in the propolis sample behind the NaOH dissolving propolis sample with ethyl acetate, impurity in the chloromycetin that extracts is many, this is because the molten part of the alkali in the propolis sample also is dissolved in ethyl acetate simultaneously, so consider the propolis sample of alkali dissolution is carried out acidifying, remove the molten component of most of alkali in the propolis sample, chloromycetin in the propolis sample still is dissolved in aqueous phase at this moment, the pH value of filtering back sample liquid is adjusted to 4 respectively, 6,8 and 10.5, re-using ethyl acetate extracts the chloromycetin in the propolis sample, the pH value of finding sample liquid is 10.5 o'clock, and the sample liquid of purification is colourless, shows that clean-up effect is good.
When carrying out quantitative test, if the propolis sample is carried out adopting water to come constant volume just to be easy to generate bubble after the acidifying,, then can cause impurity to increase if adopt methyl alcohol, ethanol to come froth breaking this moment, thus be difficult for adopting, but can consider to adopt other silicone defoamer.The mark method carried out quantitatively quantitatively adding reagent in the present invention adopted, because the propolis sample is insoluble to acid, so can ignore the volume influence that the propolis sample brings, the adding volume of reagent is sample liquid and amasss.In order further to purify the propolis sample, the sample liquid of the present invention before to sample introduction carries out ungrease treatment, has adopted the normal hexane in the hydro carbons as degreasing solvent.
Present embodiment is optimized the liquid phase chromatogram condition of high performance liquid chromatograph, in order to guarantee the separation efficiency that remains on the chromatographic column of different nature more, this method has been studied the chromatographic column of Hypersil, Akasil, Venusil MP, models such as Shim-pack VP, Intersil, finds that the degree of separation of Hypersil ODS2 chromatographic column is good, highly sensitive, peak shape is narrow and symmetrical.
Moving phase of the present invention has adopted acid water and organic phase commonly used and has made gradient elution, to improve degree of separation and sensitivity, common acid mainly contains formic acid, acetate, we are according to information and the actual conditions grasped, the formic acid of variable concentrations or acetate are to the influence of separating effect in the research moving phase, using volumetric concentration (V/V) respectively is 0.1%, 0.2%, 0.4%, 1.0% formic acid or acetate, and select suitable volatility salt, improve Ionization Efficiency, to obtain to separate well, the peak shape symmetry, signal intensity is big, highly sensitive chromatogram, the effective aspect volume concentrations is that 0.1% acetate is the strongest as the signal of moving phase, chromatographic signal is enhanced behind the ammonium acetate of adding 5mmol/L, has improved sensitivity.
The present invention adopts different gradient condition, analyzes chromatographic column to how residual chromatographic behavior, optimizes best gradient condition, optimizes flow velocity, the column temperature of moving phase simultaneously, to obtain optimum chromatogram, optimizes the result referring to table 1.
Present embodiment is optimized the mass spectrum condition of triple quadrupole bar tandem mass spectrometer, compound concentration is the various residue criterion solution of 1mg/L, enter mass spectrometer with the pin pump with the flow velocity of 5 μ L/mL, carry out Q1MS (Q1) full scan with positive ion mode, determine molecular ion peak, regulate ion gun voltage, DP, the FP parameter, again with molecular ion peak as parent ion, carry out daughter ion scanning, regulate the CE parameter, make the intensity of parent ion account for daughter ion intensity 1/3～1/4 for best, the daughter ion that 2-4 signal of selection is stronger from mass spectrogram is as qualitative ion, the daughter ion that abundance of ions is the strongest is a quota ion, adopts pin pump flow injection standard solution, manually moves RAMP and carries out parameter optimization.
After the mass spectrum parameter is determined, connect the liquid chromatography sample introduction, because parameters such as various residual ion source temperatures, auxiliary heating gas, dry gas, gas curtain gas, electron spray voltage are different under simple mass spectrum condition, therefore select one group of moderate parameter under liquid-phase condition, to be optimized again, the mixed mark of sample introduction 2 μ g/kg under the liquid-phase condition of present embodiment, be optimized one by one again and finely tune, make sensitivity, the peak shape of various materials reach best.
Come below the range of linearity of the present invention, detection limit, the recovery and precision are analyzed, with volumetric concentration is that 30% acetonitrile solution is mixed with the standard solution that chloramphenicol concentration is 0.3 μ g/kg, 0.5 μ g/kg, 1 μ g/kg, 5 μ g/kg, 10 μ g/kg series, mark 5 μ g/kg in containing measure according to the chromatographic condition sample introduction that the present invention sets.Represent peak area (A) with y, with x indicated concentration C (μ g/kg), try to achieve regression equation and linearly dependent coefficient with EXCEL software, obtain the range of linearity, the range of linearity of chloromycetin is 0.3 μ g/kg-10 μ g/kg, linearly dependent coefficient is 0.9999, determine that with signal to noise ratio (S/N ratio) S/N=3 corresponding concentration detecting of residual chloromycetin is limited to 0.1 μ g/kg, quantitatively be limited to 0.3 μ g/kg with what signal to noise ratio (S/N ratio) S/N=10 corresponding concentration was determined residual chloromycetin, said herein signal to noise ratio (S/N ratio) S/N is a prior art, adopt signal to noise ratio (S/N ratio) S/N to determine that in conjunction with corresponding concentration the detection limit of residual chloromycetin and quantitative limit are common practise for a person skilled in the art, no longer describe in detail herein.
In the royal jelly negative sample, add the residual chloromycetin standard items of 0.3 μ g/kg, 1 μ g/kg, 5 these 3 concentration of μ g/kg respectively, carry out the processing and the mensuration of sample by this method, determine the recovery and the relative standard deviation of this method, concrete outcome is referring to table 3.
Table 3 chloromycetin adds the recovery counting rate meter (n=3) of variable concentrations in the propolis sample
The present invention is with low cost to the propolis sample pretreatment, and is simple to operate, quick, and behind the continuous sample introduction 70 times, the ion gun gas curtain plate in the triple quadrupole bar tandem mass spectrometer still keeps totally, is particularly suitable for basic unit of enterprise purchase, production overall process are carried out quality control.Detection of the present invention is limited to 0.1 μ g/kg, quantitatively is limited to 0.3 μ g/kg, and linearly dependent coefficient is 0.9999, the recovery is at 82.0-108.2%, relative standard deviation is at 5.35-14.2%, and precision is good, can satisfy the requirement that importer limits the quantity of to chloromycetin medicament residue in the propolis fully.
Though the present invention with embodiment openly as above; but it is not in order to limit protection scope of the present invention; any technician who is familiar with this technology, change and the retouching done in not breaking away from design of the present invention and scope all should belong to protection scope of the present invention.
Claims (4)
1. a using high performance liquid chromatography tandem mass spectrum is measured the method for chloramphenicol residue in the propolis, it is characterized in that: the used raw material of this method comprises that purity is greater than 99.0% chloromycetin standard items, concentration is the D5-chloromycetin standard items of 100mg/l, ethyl acetate for the residual level of farming, be analytically pure NaOH, be the pure normal hexane of top grade, be the pure acetonitrile of liquid chromatography, propolis sample;
The used equipment of this method comprises triple quadrupole bar tandem mass spectrometer, is furnished with the high performance liquid chromatograph of binary pump, online degasser, automatic sampler, data processing software; Ion source temperature in the described triple quadrupole bar tandem mass spectrometer is 600 ℃, and auxiliary heating gas is 55.00psi, and dry gas is 65.00psi, gas curtain gas is 25.00psi, collision gas is 5.00psi, and electron spray voltage is-4100.00V that the ionization method is a negative ion mode; Described high performance liquid chromatograph is Agilent 1200series, and chromatographic column is Hypersil ODS2, and the specification of chromatographic column is 4.6mm * 150mm, and 5 μ m, column temperature are 30 ℃, and sample size is 20 μ l;
This method comprises that standard solution preparation operation, standard curve making operation, propolis sample liquid preparation section and chloramphenicol residue detect operation; Described standard solution preparation operation is as follows: a, chloromycetin are stocked the standard solution preparation steps: take by weighing chloromycetin standard items 10mg and be settled to 10ml with acetonitrile to be made into concentration be that the chloromycetin of 1000mg/L is stocked standard solution, with this chloromycetin stock standard solution and place preserve below-18 ℃ stand-by; B, chloromycetin intermediate standard liquid preparation steps: get chloromycetin among a and stock standard solution 100 μ l and be settled to 10ml with acetonitrile and be made into the chloromycetin intermediate standard liquid that concentration is 10mg/L; C, chloromycetin deuterium interior mark intermediate liquid preparation steps of generation: adopt D5-chloromycetin standard items to replace the chloromycetin standard items, repeat above-mentioned a step and b step, making concentration is the chloromycetin deuterium interior mark intermediate liquid of generation of 10mg/L; D, interim standard are used the liquid preparation steps: extracting chloromycetin intermediate standard liquid volumetric concentration is that 30% acetonitrile solution is assigned to 5 μ g/kg and 50 μ g/kg, and the extracting chloromycetin deuterium is that 30% acetonitrile solution is assigned to 200 μ g/kg for interior mark intermediate liquid volumetric concentration;
In the described standard curve making operation; get the chloromycetin that makes in the standard solution preparation operation and stock standard solution, chloromycetin intermediate standard liquid, chloromycetin deuterium interior mark intermediate liquid of generation and interim standard use liquid, obtain typical curve by high performance liquid chromatograph and triple quadrupole bar tandem mass spectrometer;
In the described propolis sample liquid preparation section, getting propolis sample 2g and concentration and be in the D5-chloromycetin standard items of 200 μ g/kg mark 50ul places a centrifugal plastic bottle of 50ml and fully mixes, leave standstill more than the 5min then, in a centrifugal plastic bottle of 50ml, add the NaOH solution that 5ml concentration is 1mol/L again and constantly vibrate and extract 5min, the propolis sample is fully dissolved, in centrifugal plastic bottle of 50ml, add 5ml water then and shake up, in a centrifugal plastic bottle of 50ml, add 10ml concentration again and be 5% HClO
4Carry out acidifying and fully shake up and make propolis liquid; This propolis liquid is ultrasonic Extraction 5min at normal temperatures earlier, is that centrifugal 5min obtains upper strata centrifugate under the 3000rpm at rotating speed again, then upper strata centrifugate is filtered and makes filtrate; Get filtrate 10ml and place the centrifugal plastic bottle of 50ml No. two, with concentration is that the NaOH solution of 1mol/L is adjusted to 10.5 with pH value of filtrate, in No. two centrifugal plastic bottles of 50ml, add 10ml ethyl acetate then and fully mix 3min, at rotating speed is that centrifugal 2min obtains ethyl acetate layer under the 3000rpm, ethyl acetate is placed in the 15ml plastic centrifuge tube then, this ethyl acetate layer carries out nitrogen and dries up under 35 ℃, adding volumetric concentration again in the 15ml plastic centrifuge tube is 30% acetonitrile solution 1ml and carries out ultrasonic dissolution, in the 15ml plastic centrifuge tube, add 2ml normal hexane and abundant the mixing then and make centrifugate, is that centrifugal 2min obtains lower floor's sample liquid under the 800rpm with this centrifugate at rotating speed, at last lower floor's sample liquid is crossed the filter membrane of 0.22 μ m and is made propolis sample liquid;
Described chloramphenicol residue detects in the operation, get the propolis sample liquid that makes in the propolis sample liquid preparation section, by high performance liquid chromatograph and triple quadrupole bar tandem mass spectrometer, and the typical curve that obtains in the combined standard curve plotting operation and record the residual quantity of chloromycetin in the propolis; The detection of this method is limited to 0.1 μ g/kg, quantitatively is limited to 0.3 μ g/kg.
2. using high performance liquid chromatography tandem mass spectrum according to claim 1 is measured the method for chloramphenicol residue in the propolis, and it is characterized in that: the employed water of this method is dual distilled water.
3. using high performance liquid chromatography tandem mass spectrum according to claim 1 is measured the method for chloramphenicol residue in the propolis, it is characterized in that: in the described propolis sample liquid preparation section, adding concentration in a centrifugal plastic bottle of 50ml is the NaOH solution dissolving propolis sample of 1mol/L, dilute with water is used HClO again to disengage the residual chloromycetin thing in the propolis sample then
4Carry out acidifying with the propolis composition in the precipitation propolis sample.
4. using high performance liquid chromatography tandem mass spectrum according to claim 1 and 2 is measured the method for chloramphenicol residue in the propolis, it is characterized in that: in the described propolis sample liquid preparation section, propolis sample liquid concentration is that the NaOH solution of 1mol/L is adjusted to 10.5 with pH value of filtrate, ethyl acetate layer adopts Nitrogen evaporator to carry out nitrogen down at 35 ℃ and dries up, add volumetric concentration again and be 30% acetonitrile solution 1ml and adopt ultrasonoscope to carry out ultrasonic dissolution in the 15ml plastic centrifuge tube.
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