CN104880522A - Method for determining residual quantity of chloramphenicol in bee wax by n-hexane pre-treatment-high performance liquid chromatography-tandem mass spectrometry - Google Patents

Abstract

The invention relates to a method for determining the residual quantity of chloramphenicol in bee wax by n-hexane pre-treatment-high performance liquid chromatography-tandem mass spectrometry. The method comprises the following steps: pre-dissolving a test sample by adopting n-hexane to form emulsion, and adding water and extracting; carrying out HLB (Hydrophile Lipophile Balance) solid-phase extraction column purification; separating by using an Accucore XL C18 column; and determining by using electric spraying ion source negative ion multi-reaction monitoring mode tandem mass spectrum. Extraction and purification methods, and instrument determination conditions and the like are optimized. A result shows that chloramphenicol has a relatively good linear relation in a range of 0.3ng/mL-10ng/mL, and the correlation coefficient is more than 0.998. The method qualitative limit (S/N>3) is 0.05 microgram/kilogram and the method quantitative limit (S/N>10) is 0.3 microgram/kilogram; the recycling rate of three adding levels of 0.3 microgram/kilogram, 1.0 microgram/kilogram and 2.0 microgram/kilogram is 80.2%-105.2%; and the relative standard deviation (RSD, n=6) is smaller than 8%. The method is rapid, sensitive and accurate.

Description

Normal hexane pre-service-using high performance liquid chromatography tandem mass spectrum method measures the method for chloramphenicol residue in beeswax

Technical field

The present invention relates to a kind of method measuring chloramphenicol residue in beeswax.

Background technology

Chloromycetin (chloramphenicol, CAP) is a kind of microbiotic of high-efficiency broad spectrum, has activity, be widely used in livestock breeding industry to various aerobic and anaerobe.But chloromycetin has certain toxic action to the mankind, as caused the agranulocytosis of people sick, the symptoms such as alpastic anemia and allergy.In FAO/WAO regulation food, must not remain of chloromycetin detects, in European Union (EEC) 96/23 instruction, CAP is listed in forbidding medicine, and specify that chloromycetin MRPL (minimum required performance limits) value is 0.3 μ g/ kg.

China is bee-keeping big country of the world, is also that bee product is produced and big export country, 2013 annual beeswax total productions about 8000 tons.Beeswax is a kind of material secreted by the four pairs of wax glands below worker bee belly, and its principal ingredient is the ester class etc. that alkanol and alkanoic acid are formed, and at cosmetics, food and medicine is industrial is widely used.But beekeeper sprays chloromycetin medicine for prevention and therapy honeybee disease in cultivation honeybee process, causes residual chloromycetin in beeswax.Because recycling of beeswax causes chloramphenicol residue in beeswax constantly to increase, meanwhile, by the contact of honeybee and honeycomb, chloromycetin medicament residue can be transferred in the bee products such as honey, royal jelly, propolis, and then causes other bee products to pollute.Chloromycetin remaining in beeswax is not only related to human body health, also produces huge risk by China's beeswax export trade.

In recent years, there is the report of the pesticide residue detection method such as germifuge in more beeswax both at home and abroad, but not yet have the report of residual chloromycetin quantity measuring method in beeswax.Because the matrix of beeswax is complicated, the method for chloromycetin in the bee product such as detection honey, royal jelly announced cannot be adopted both at home and abroad to detect.

Summary of the invention

In order to solve above-mentioned technical matters, the object of this invention is to provide the method that a kind of normal hexane pre-service-using high performance liquid chromatography tandem mass spectrum method measures chloramphenicol residue in beeswax, the advantages such as easy, the sensitive height of this method, matrix interference are little, and for qualitative and quantitative analysis that trace amount chloramphenicol in beeswax sample is residual.

In order to realize above-mentioned object, present invention employs following technical scheme:

Normal hexane pre-service-using high performance liquid chromatography tandem mass spectrum method measures the method for chloramphenicol residue in beeswax, and the method comprises the following steps:

1) preparation of standard solution

Take chloromycetin standard items 10 mg and be placed in the brown volumetric flask of 100 mL, be accurate to 0.1 mg, dissolve and be settled to scale with methyl alcohol, mixing is mixed with the standard reserving solution of 100 mg/L; Be mixed with the list mark storing solution of 1mg/L with methyl alcohol, be diluted to corresponding hybrid standard working solution by mobility according to actual needs; Chloromycetin-D5 is 10 ng/mL as the concentration of Isotopic Internal Standard; Various standard solution keeps in Dark Place in 4 DEG C of refrigerators;

2) beeswax pre-service

Sample can dissolve and mix under about 70 DEG C heating conditions;

3) extract

Take sample 2.0 g in 50 mL lid centrifuge tubes, be accurate to 0.01 g, add 10 mL normal hexanes, add the 10 mL eddies of waters and revolve mechanical shaking extraction after vortex ultrasonic dissolution, the centrifugal 5min of 8000 r/min, gets lower aqueous layer, to be clean;

4) purify

Pipette sample extract in OASIS HLB Solid phase cleaned-up pillar, OASIS HLB Solid phase cleaned-up pillar uses 3 mL methyl alcohol before using successively, 5 mL water activation, discard efflux, first use 5 mL ultrapure water drip washing, use 5 mL methanol/water again, the volume ratio of methanol/water solution is 2/8, solution drip washing pillar, discards efflux, finally uses 5 mL eluent ethyl acetates, collect eluent in centrifuge tube with a scale, nitrogen blows and is concentrated near doing, and is finally settled to 1 mL by methanol/water solution, analyzes after mixing after 0.22 μm of organic phase membrane filtration for upper machine;

5) chromatographic condition

Chromatographic column: Accucore XL C18 post, 150 mm × 4.6 mm, 4 μm; Column temperature: 35 DEG C; Sample size: 10 μ L; Flow velocity: 0.5 mL/min; Mobile phase: A is aqueous solution, and B is methanol solution; Gradient elution program: 0 ~ 0.5 min, 70%A; 0.5 ~ 7.0 min, 70%A ~ 10%A; 7.0 ~ 8.0 min, 10%A; 8.0 ~ 8.1 min, 10%A ~ 70%A; 8.1 ~ 12.0 min, 70%A;

6) Mass Spectrometry Conditions

Scan mode: negative mode scans; Detection mode: multiple-reaction monitoring (MRM); Electron spray voltage (Spray voltage) :-3000 V; Sheath gas (Sheath gas pressure): 207 kPa; Assisted gas (Auxiliary gas flow): 10 arbitrary units; Capillary temperature (Capillary Temperature): 350 DEG C; Evaporating temperature (Vaporizer Temperature): 300 DEG C; Collision gas: argon gas (1.5mtorr);

Chloromycetin and interior target Mass Spectrometry Conditions parameter:

* quota ion;

7) standard working solution of a series of variable concentrations is prepared, 0.3,0. 5,1.5,3.0,5.0,10.0 ng/mL, and sample introduction successively, respectively with the peak area Y of chloromycetin for ordinate, with corresponding concentration value for horizontal ordinate, make typical curve, result shows, object its concentration and peak area within the scope of 0.3 ~ 10 ng/mL are good linear relationship, and linear relationship equation is Y=0.286385*X-0.00152397, coefficient R ²be greater than 0.998.

The present invention is owing to have employed above-mentioned technical scheme, and chloromycetin has good linear relationship within the scope of 0. 3 ng/mL-10 ng/mL, and related coefficient is greater than 0.998.The qualitative limit of the method ( s/N>3) be 0.05 μ g/ kg, method quantitative limit ( s/N>10) being 0.3 μ g/kg, is 80.2%-105.2% in the recovery of 0.3 μ g/kg, 1.0 μ g/kg and 2.0 μ g/kg, tri-Pitch-based sphere, relative standard deviation ( rSD, n=6) all 8% is less than.This method is quick, sensitive, accurate, is applicable to the qualitative and quantitative analysis of residual chloromycetin in daily beeswax sample.

Accompanying drawing explanation

fig. 1 isthe SRM chromatogram of chloromycetin in actual sample.

Embodiment

1 experimental section

1.1 instruments and reagent

Ultimate 3000 Ultra Performance Liquid Chromatography-TSQ Vantage triple quadrupole bar tandem mass spectrum combined instrument (Thermo Scientific company of the U.S.); MS 3 digital eddy mixer (German IKA company); MD 200 Nitrogen evaporator (Hangzhou Ao Sheng instrument company)

Chloromycetin (CAS56-75-7, purity >=98%) and chloromycetin-D5(purity >=98%), Dr.Ehrenstorfer company.Methyl alcohol, normal hexane, ethyl acetate are chromatographically pure; OASIS HLB solid phase extraction column: 500 mg, 6 mL(Waters, US).

The preparation of 1.2 standard solution

Take chloromycetin standard items 10 mg(and be accurate to 0.1 mg) be placed in the brown volumetric flask of 100 mL, dissolve with methyl alcohol and be settled to scale, mixing is mixed with the standard reserving solution of 100 mg/L.Be mixed with the list mark storing solution of 1mg/L with methyl alcohol, be diluted to corresponding hybrid standard working solution by mobility according to actual needs.Chloromycetin-D5 is 10 ng/mL as the concentration of Isotopic Internal Standard.Various standard solution keeps in Dark Place in 4 DEG C of refrigerators.

1.3 beeswax pre-service

Sample can dissolve and mix under about 70 DEG C heating conditions.

1.4 extract

Take sample 1.0 g(and be accurate to 0.01 g) in 50 mL lid centrifuge tubes, add 10 mL normal hexanes, add the 10 mL eddies of waters and revolve mechanical shaking extraction after vortex ultrasonic dissolution, the centrifugal 5min of 8000 r/min, gets lower aqueous layer, to be clean.

1.5 purification

Pipette sample extract and (before use, use 3 mL methyl alcohol successively in OASIS HLB Solid phase cleaned-up pillar, 5 mL water activation) in, discard efflux, first use 5 mL ultrapure water drip washing, use 5 mL methanol/water (2/8:V/V) solution drip washing pillars again, discard efflux, finally use 5 mL eluent ethyl acetates, collect eluent in centrifuge tube with a scale, nitrogen blows and is concentrated near doing, finally use methanol/water (2/8, V/V) to be settled to 1 mL, analyze for upper machine after 0.22 μm of organic phase membrane filtration after mixing.

1.6 chromatographic condition

Chromatographic column: Accucore XL C18 post (150 mm × 4.6 mm, 4 μm); Column temperature: 35 DEG C; Sample size: 10 μ L; Flow velocity: 0.5 mL/min.Mobile phase: A is aqueous solution, and B is methanol solution.Gradient elution program: 0 ~ 0.5 min, 70%A; 0.5 ~ 7.0 min, 70%A ~ 10%A; 7.0 ~ 8.0 min, 10%A; 8.0 ~ 8.1 min, 10%A ~ 70%A; 8.1 ~ 11.0 min, 70%A.

1.7 Mass Spectrometry Conditions

Scan mode: negative mode scans; Detection mode: multiple-reaction monitoring (MRM); Electron spray voltage (Spray voltage) :-3000 V; Sheath gas (Sheath gas pressure): 207 kPa; Assisted gas (Auxiliary gas flow): 10 arbitrary units; Capillary temperature (Capillary Temperature): 350 DEG C; Evaporating temperature (Vaporizer Temperature): 300 DEG C; Collision gas: argon gas (1.5mtorr).

Table 1 chloromycetin and interior target Mass Spectrometry Conditions parameter

Note: * quota ion (Quantitative ion)

2 results and discussion

2.1 Extraction solvent are selected

The principal ingredient of beeswax has acid, free fatty acid, free alkyl alcohol, carbohydrates, carotenoid etc., belongs to liposoluble substance.Under normal temperature, be soluble in the weakly polar organic solvents such as normal hexane, chloroform and phenixin, do not allow the intensive polar solvents such as the Yishui River, methyl alcohol, acetonitrile.According to the chemical property of beeswax and chloromycetin, the present invention compares n-hexane dissolution and helps formulation and heating to help formulation.

Heating helps formulation: take sample 1.0 g in 50 mL tool lid centrifuge tubes, add 10 mL water or acetonitriles, heating water bath dissolves, vortex mixes, then continues heating for dissolving, repeatable operation 4 times, be cooled to room temperature, 30 more than min are placed, centrifugal 5 min of 8000 r/min, water intaking or acetonitrile layer in less than-10 DEG C refrigerators.

N-hexane dissolution helps formulation: take sample 1.0 g in 50 mL tool lid centrifuge tubes, add 10 mL normal hexanes, after vortex is mixed into emulsion, add 10 mL water or acetonitrile extraction of ocean eddies, with centrifugal 5 min of 8000 r/min, and water intaking or acetonitrile layer.

Test shows: because heating helps formulation needs to heat and the operation such as freezing, while freezing grease removal effect thorough not.And n-hexane dissolution helps formulation, there is the advantages such as easy and simple to handle, Liquid liquid Separation, grease removal be effective.

2.2 purification conditions are selected

According to sample with measure the character of object, HLB, the clean-up effect of silicagel column 2 kinds of Solid phase extraction pillars and organic efficiency are investigated.

Silicagel column method purifies: acetonitrile extract steams in bottle to revolving, and 45 DEG C are spin-dried for.With 2 mL ethyl acetate, 8 mL n-hexane dissolutions, cross silicagel column (5 mL n-hexane/ethyl acetate 8:2, V/V activates), 6 mL n-hexane/ethyl acetate (2:8, V/V) wash-outs, nitrogen blows, 1 mL mobile phase methanol/water (2:8, V/V) dissolve, add 2 mL normal hexanes, with centrifugal 5 min of 4000 r/min after vortex, take off layer and cross 0.45 μm of filter membrane, to be measured.The present invention 1.5 is shown in the purification of HLB post method.

Test shows: HLB and silica gel Solid phase extraction pillar all reach satisfactory result to the clean-up effect of object and the recovery.Because the purification of HLB method does not need to extract solution and upper purification pillar solvent switch, this method organic solvent use amount is also less simultaneously, so the present invention selects, operation is fast and convenient, solvent uses few HLB method purification.

2.3 matrix effect

Getting negative blank beeswax sample, to carry out by the present invention 1.4 and 1.5 joint the upper machine analytic liquid that pre-treatment obtains be the dilution of standard solution, with the peak area of chloromycetin, matrix mark-on typical curve is drawn to mass concentration, by its slope compared with the slope of the typical curve of standard items, slope ratio is 116.6%.Can find out, there is certain matrix impact to target compound in sample extracting solution, for ensureing quantitative test accuracy, reduce matrix effect, this method selects inner mark method ration.

The range of linearity of 2.4 methods and detection limit

Prepare the standard working solution (0.3,0. 5,1.5,3.0,5.0,10.0 ng/mL) of a series of variable concentrations, and sample introduction successively, respectively with the peak area of chloromycetin yfor ordinate, with corresponding concentration value for horizontal ordinate, make typical curve, result shows, object its concentration and peak area within the scope of 0.3 ~ 10 ng/mL are good linear relationship, and linear relationship equation is y=0.286385* x-0.00152397, related coefficient r ² be greater than 0.998.

In negative sample, add target compound, measure according to method of the present invention, with 3 times of signal to noise ratio (S/N ratio)s ( s/N) corresponding Pitch-based sphere as detection limit (LOD), 10 times of signal to noise ratio (S/N ratio)s ( s/N) corresponding Pitch-based sphere is as quantitative limit (LOQ).Result shows, this method LOD is 0.05 μ g/kg, LOQ is 0.3 μ g/kg.

2.5 the recovery of method and precision

Under 0.3 μ g/kg, 1.0 μ g/kg and 1.0 μ g/kg, tri-Pitch-based sphere, carry out blank recovery of standard addition test, each level repeats 6 times.During 0.3 μ g/kg Pitch-based sphere, the recovery of object is 87.9%-95.2%, rSDbe 5.2%; During 1.0 μ g/kg Pitch-based sphere, the recovery of object is 80.2%-98.1%, rSDbe 7.8%; During 1.0 μ g/kg Pitch-based sphere, the recovery of object is 101.6%, rSDbe 1.5 %, all meet the requirement that daily residual chloromycetin detects.

The table 2 method recovery and precision data ( n=6)

2.6 actual samples measure

In 25 parts of beeswax samples that application this method 15 parts of purchasing certain net purchase platform and certain bee product processing enterprise provide, chloramphenicol residue measures, result shows, in actual sample, the recall rate of chloromycetin is 41.5%, in all detection samples, chloromycetin total content has 10 between 0.3-1.0, accounts for 25.0%; Content has 7 being greater than 1.0, and wherein in 2 samples, chloromycetin content is respectively 40.3 ng/kg and 41.5 ng/kg.Fig. 1 is the SRM chromatogram of chloromycetin in actual sample.

3 conclusions

The present invention establishes the analytical approach that Solid phase extraction-Ultra Performance Liquid Chromatography-tandem mass spectrum detects trace amount chloramphenicol in beeswax.The method, as the confirmation method of residual chloromycetin in beeswax, has easy, sensitive, advantage accurately, may be used for the qualitative and quantitative analysis that in beeswax, trace amount chloramphenicol is residual.

Claims (1)

1. normal hexane pre-service-using high performance liquid chromatography tandem mass spectrum method measures the method for chloramphenicol residue in beeswax, it is characterized in that the method comprises the following steps:

1) preparation of standard solution

Take chloromycetin standard items 10 mg and be placed in the brown volumetric flask of 100 mL, be accurate to 0.1 mg, dissolve and be settled to scale with methyl alcohol, mixing is mixed with the standard reserving solution of 100 mg/L; Be mixed with the list mark storing solution of 1mg/L with methyl alcohol, be diluted to corresponding hybrid standard working solution by mobility according to actual needs; Chloromycetin-D5 is 10 ng/mL as the concentration of Isotopic Internal Standard; Various standard solution keeps in Dark Place in 4 DEG C of refrigerators;

2) beeswax pre-service

Sample can dissolve and mix under about 70 DEG C heating conditions;

3) extract

Take sample 2.0 g in 50 mL lid centrifuge tubes, be accurate to 0.01 g, add 10 mL normal hexanes, add the 10 mL eddies of waters and revolve mechanical shaking extraction after vortex ultrasonic dissolution, the centrifugal 5min of 8000 r/min, gets lower aqueous layer, to be clean;

4) purify

Pipette sample extract in OASIS HLB Solid phase cleaned-up pillar, OASIS HLB Solid phase cleaned-up pillar uses 3 mL methyl alcohol before using successively, 5 mL water activation, discard efflux, first use 5 mL ultrapure water drip washing, use 5 mL methanol/water again, the volume ratio of methanol/water solution is 2/8, solution drip washing pillar, discards efflux, finally uses 5 mL eluent ethyl acetates, collect eluent in centrifuge tube with a scale, nitrogen blows and is concentrated near doing, and is finally settled to 1 mL by methanol/water solution, analyzes after mixing after 0.22 μm of organic phase membrane filtration for upper machine;

5) chromatographic condition

Chromatographic column: Accucore XL C18 post, 150 mm × 4.6 mm, 4 μm; Column temperature: 35 DEG C; Sample size: 10 μ L; Flow velocity: 0.5 mL/min; Mobile phase: A is aqueous solution, and B is methanol solution; Gradient elution program: 0 ~ 0.5 min, 70%A; 0.5 ~ 7.0 min, 70%A ~ 10%A; 7.0 ~ 8.0 min, 10%A; 8.0 ~ 8.1 min, 10%A ~ 70%A; 8.1 ~ 12.0 min, 70%A;

6) Mass Spectrometry Conditions

Scan mode: negative mode scans; Detection mode: multiple-reaction monitoring (MRM); Electron spray voltage (Spray voltage) :-3000 V; Sheath gas (Sheath gas pressure): 207 kPa; Assisted gas (Auxiliary gas flow): 10 arbitrary units; Capillary temperature (Capillary Temperature): 350 DEG C; Evaporating temperature (Vaporizer Temperature): 300 DEG C; Collision gas: argon gas (1.5mtorr);

Chloromycetin and interior target Mass Spectrometry Conditions parameter:

* quota ion;

7) standard working solution of a series of variable concentrations is prepared, 0.3,0. 5,1.5,3.0,5.0,10.0 ng/mL, and sample introduction successively, respectively with the peak area Y of chloromycetin for ordinate, with corresponding concentration value for horizontal ordinate, make typical curve, result shows, object its concentration and peak area within the scope of 0.3 ~ 10 ng/mL are good linear relationship, and linear relationship equation is Y=0.286385*X-0.00152397, coefficient R ²be greater than 0.998.