CN113466374A - Method for measuring chlorpromazine residual quantity in honey - Google Patents
Method for measuring chlorpromazine residual quantity in honey Download PDFInfo
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- CN113466374A CN113466374A CN202110753741.7A CN202110753741A CN113466374A CN 113466374 A CN113466374 A CN 113466374A CN 202110753741 A CN202110753741 A CN 202110753741A CN 113466374 A CN113466374 A CN 113466374A
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- ZPEIMTDSQAKGNT-UHFFFAOYSA-N chlorpromazine Chemical compound C1=C(Cl)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 ZPEIMTDSQAKGNT-UHFFFAOYSA-N 0.000 title claims abstract description 103
- 229960001076 chlorpromazine Drugs 0.000 title claims abstract description 100
- 235000012907 honey Nutrition 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000012224 working solution Substances 0.000 claims abstract description 36
- 239000000243 solution Substances 0.000 claims abstract description 35
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 238000004458 analytical method Methods 0.000 claims abstract description 6
- 238000000746 purification Methods 0.000 claims abstract description 6
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 5
- 238000000605 extraction Methods 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 101
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 61
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 25
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000012071 phase Substances 0.000 claims description 15
- 239000011550 stock solution Substances 0.000 claims description 15
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 150000002500 ions Chemical class 0.000 claims description 11
- 238000012417 linear regression Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- GHQPBDDZGPAVJP-UHFFFAOYSA-N azanium;methanol;hydroxide Chemical compound N.O.OC GHQPBDDZGPAVJP-UHFFFAOYSA-N 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- 238000004364 calculation method Methods 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 239000003643 water by type Substances 0.000 claims description 5
- 239000000908 ammonium hydroxide Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 238000007664 blowing Methods 0.000 claims description 3
- 239000003640 drug residue Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 14
- 239000003814 drug Substances 0.000 abstract description 6
- 238000005259 measurement Methods 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 5
- 235000013305 food Nutrition 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 description 9
- 241000257303 Hymenoptera Species 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- 201000010000 Agranulocytosis Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- GJSURZIOUXUGAL-UHFFFAOYSA-N Clonidine Chemical compound ClC1=CC=CC(Cl)=C1NC1=NCCN1 GJSURZIOUXUGAL-UHFFFAOYSA-N 0.000 description 1
- 102000015554 Dopamine receptor Human genes 0.000 description 1
- 108050004812 Dopamine receptor Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000003474 anti-emetic effect Effects 0.000 description 1
- 230000000561 anti-psychotic effect Effects 0.000 description 1
- 239000002111 antiemetic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229960002896 clonidine Drugs 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011491 glass wool Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000000147 hypnotic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000002990 phenothiazines Chemical class 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000932 sedative agent Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application relates to the field of detection methods of residual medicines in food, in particular to a method for determining the residual amount of chlorpromazine in honey. A method for measuring the chlorpromazine residual quantity in honey comprises the following steps: (1) the method comprises the steps of (1) sample preparation, (2) sample extraction, (3) sample purification, (4) preparation of a sample blank extracting solution of a chlorpromazine standard working solution, (6) liquid chromatography-tandem mass spectrometry, (7) standard working curve drawing, and (8) measurement of the residual quantity of a measured object in a pure sample. The method is high in accuracy and good in precision, can meet the requirement of chlorpromazine residue analysis, and provides a powerful tool for detecting chlorpromazine in animal-derived food.
Description
Technical Field
The application relates to the field of detection methods of residual medicines in food, in particular to a method for determining the residual amount of chlorpromazine in honey.
Background
In recent years, with the improvement of the living standard of people, the animal husbandry is gradually standardized and commercialized, and veterinary drugs and feed additives are widely applied to animal husbandry. For example, in order to obtain more economic benefits, bee farmers need to use the bee resources excessively, so that the body resistance of bees is reduced, and the bees are vulnerable to stress and diseases. Therefore, in order to quickly recover the bee productivity, bee farmers can treat bees by a large amount of medicines and even prevent bee diseases by using medicines in advance, so that the veterinary medicine residue in bee products exceeds the standard.
Chlorpromazine is one of veterinary drugs, is called chlorpromazine, clonidine, phenothiazines drugs and central dopamine receptor blockers, has pharmacological effects of antipsychotic, cooling, antiemetic, hypnosis, anesthesia and the like, is clinically used as a sedative drug, has a first-pass elimination effect, is combined with plasma protein after being absorbed by 95 percent, and is metabolized by the liver.
In the related literature at present, when the addition level of chlorpromazine in animal derived food is detected to be within the range of 5-50 mug/kg, the detection limit LOD is 2.5 mug/kg, the determination lower limit LOQ is 5.0 mug/kg, the recovery rate is 81.9-110.7%, and the RSD is 5.5-8.4%. However, chlorpromazine is slowly metabolized and easily accumulates and remains in the body, so that part of chlorpromazine also remains in honey produced by bees using chlorpromazine, and when people eat the honey with the part of chlorpromazine remaining, leucopenia and agranulocytosis of human bodies can be caused, thus bringing adverse effects to human health. Therefore, in order to improve the safety of people eating honey, the precision of detecting the chlorpromazine residual quantity in honey is urgently needed to be improved.
Disclosure of Invention
In order to detect the precision of the chlorpromazine residual quantity in honey and improve the safety of eating honey by people, the application provides a method for measuring the chlorpromazine residual quantity in honey.
The application provides a method for measuring the residual amount of chlorpromazine in honey, which adopts the following technical scheme:
a method for measuring the chlorpromazine residual quantity in honey comprises the following steps:
(1) pretreatment of the sample: weighing 0.5kg of honey, directly sampling when the honey is not crystallized, heating in water bath under sealed condition when the honey is crystallized, stirring after the honey is completely melted, cooling to 25 ℃, and sealing for later use;
(2) sample preparation: weighing 2.00g of sample, adding 20ml of 0.05mol/L phosphate buffer solution, and uniformly stirring to obtain a sample solution;
(3) sample purification: an OasisHLB solid phase extraction column from waters corporation was used;
before the Oasis HLB solid-phase extraction column is used, washing the column once with 6mL of methanol, 10mL of water and 5mL of phosphate buffer solution in sequence, and keeping the column wet;
adjusting the flow rate of an Oasis HLB solid phase extraction column to be less than or equal to 3mL/min, extracting a sample solution by using the solid phase extraction column, washing the column by using 5mL of water and 5mL of methanol aqueous solution in sequence after the sample solution completely flows out, and discarding all effluent liquid; under the pressure of-65 kPa, after the solid phase extraction column is decompressed and dried, 5mL of ammonium hydroxide methanol solution is used for elution, and eluent is collected; blowing the eluent by a nitrogen concentrator at 50 ℃ to obtain a crude sample; adding 1.0mL of constant volume solution to dissolve the crude sample, filtering with a 0.2 μm filter membrane, and collecting the pure sample for analysis and determination;
the methanol water solution is prepared by mixing methanol and water according to the volume ratio of 3: 7;
the ammonium hydroxide methanol solution is prepared by mixing ammonium hydroxide and methanol according to the volume ratio of 1: 19;
the constant volume solution is prepared by mixing acetonitrile and 0.01mol/L acetic acid solution according to the volume ratio of 1: 4;
(4) preparing a sample blank extracting solution of a chlorpromazine standard working solution: taking a blank honey sample without a target object, and obtaining a sample blank extracting solution of a chlorpromazine standard working solution after (2) sample extraction and (3) purification;
(5) preparing a chlorpromazine standard working solution: taking the sample blank extracting solution of the chlorpromazine standard working solution obtained in the step (4) as a solvent, and preparing chlorpromazine standard working solutions with different concentrations;
(6) liquid chromatography-tandem mass spectrometry: the chromatographic column adopts BEH C181.7 μm 100mm × 2.1mm i.d; column temperature: 30 ℃; the sample injection amount is 5 mu L; the mobile phase A is an aqueous solution containing 1% by volume of formic acid, and the mobile phase B is an acetonitrile solution containing 100% by volume; gradient elution was used: 0-0.5min, 95-85% mobile phase A; 0.5-1.5min, 85-75% of mobile phase A; 1.5-2.5min, 75-20% of mobile phase A; 2.5-4min, 20-95% mobile phase A; mass spectrum conditions: the instrument mode is MRM +; the capillary voltage is 4.0 kV; the voltage of the taper hole is 20V; the ion source temperature is 120 ℃; the temperature of the desolventizing gas is 350 ℃; the qualitative ion pair is 319.1/58.2, 319.1/86.2 m/z; the quantitative ion pair is 319.1/86.2 m/z; the residence time is 100 ms; the cone hole voltage of an ion pair 319.1/58.2m/z is 28V, and the collision energy is 25V; the cone hole voltage of an ion pair 319.1/86.2m/z is 28V, and the collision energy is 20V;
(7) drawing a chlorpromazine standard working curve: sequentially carrying out the operation (6) on the chlorpromazine standard working solutions with different concentrations prepared in the step (5), and drawing and fitting to obtain a linear regression equation and a correlation coefficient of a chlorpromazine standard working curve by taking the concentration mu g/mL of the chlorpromazine standard working solution as a horizontal coordinate and the area of a responding chromatographic peak as a vertical coordinate;
(8) and (3) measuring the residual quantity of the measured object in the pure sample: carrying out operation (6) on the pure sample collected in the step (3), reading out the chromatographic peak area of the detected object in the pure sample from the chromatogram, substituting the chromatographic peak area of the detected object in the pure sample into the linear regression equation of the chlorpromazine standard working curve in the step (7), calculating the concentration of the detected object in the pure sample, substituting the chromatographic peak area of the detected object in the pure sample and the concentration of the detected object in the pure sample into the determination formula of the chlorpromazine drug residue, and calculating the result;
the calculation formula of the residual quantity of the measured object in the pure sample is as follows:
X＝Cs×(A/As)×(V/m)×(1000/1000)
in the formula: x is the residual quantity of the tested object in the pure sample, and the unit is mu g/kg; the Cs is the concentration of the detected object in the sample pure product read from the standard working curve, and the unit is ng/mL; a is the chromatographic peak area of the measured object in the pure sample; as is the chromatographic peak area of the object to be measured in the chlorpromazine standard working solution; v is the final constant volume of the pure sample, and the unit is mL; m is the mass of the sample represented by the sample pure product, and the unit is g.
Preferably, in the preparation (5) of the chlorpromazine standard working solution, the preparation method is as follows:
s1: weighing chlorpromazine, dissolving the chlorpromazine in methanol, and preparing a standard stock solution with the mass concentration of 1 mg/mL;
s2: sucking 0.1mL of standard stock solution, putting the standard stock solution into a 100mL volumetric flask, and fixing the volume to a scale with methanol to obtain a chlorpromazine standard stock solution with the mass concentration of 1000 ng/mL;
s3: and (4) respectively sucking chlorpromazine standard stock solutions (S2) with different volumes, and preparing with the sample blank extracting solution prepared in the step (4) to obtain chlorpromazine standard working solutions with different concentrations.
Preferably, the concentrations of the prepared chlorpromazine standard working solution are 1ng/mL, 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL and 100ng/mL respectively.
Preferably, in the drawing of the chlorpromazine standard working curve (7), the obtained linear regression equation is y-22904 x-9854.88, and r-0.999992; wherein x is: the chlorpromazine in the pure sample matches the concentration of a chlorpromazine standard working solution; y is: chromatographic peak area; r is a correlation coefficient.
The application has the following beneficial effects: the method is established for determining the residual amount of chlorpromazine in honey by adopting a liquid chromatography-tandem mass spectrometry method, wherein the detection limit LOD of chlorpromazine is 0.5 mug/kg and the determination lower limit LOQ is 1.0 mug/kg within the addition level of 0.5-50 mug/kg, the recovery rate range of the method is 98-111%, and the RSD is 0.21-8.34%. Therefore, the method is high in accuracy and good in precision, can meet the requirement of chlorpromazine residue analysis, and provides a powerful tool for detecting chlorpromazine in animal-derived food.
Drawings
FIG. 1 is an MRM chromatogram of a chlorpromazine standard working solution in an example of the present application;
FIG. 2 is a standard working curve diagram of chlorpromazine in an example of the present application;
FIG. 3 is an MRM chromatogram of the detection limit in an embodiment of the present application;
FIG. 4 is an MRM chromatogram of the assay lower limit in the examples of the present application.
Detailed Description
The present application is described in further detail below with reference to figures 1-4 and examples.
The raw materials used in the examples of the present application are premium grade pure water, which is the first grade water specified in GB/T6682, and others are commercially available, except for the following special descriptions:
chlorpromazine CAS number 50-53-3;
the solid phase extraction column is an Oasis HLB solid phase extraction column, the specification of which is 500mg and 6mL, and the solid phase extraction column is collected from waters;
the methanol is chromatographically pure, and the CAS number is 67-56-1;
ammonium hydroxide CAS number 1336-21-6;
acetonitrile chromatography pure grade, CAS number 75-05-8;
acetic acid CAS number 64-19-7;
the chromatographic column is BEH C181.7 μm 100mm × 2.1mm i.d, collected from waters;
formic acid CAS number 64-18-6;
disodium phosphate CAS number 7558-79-4;
the CAS number of the monopotassium phosphate is 7778-77-0;
the blank honey sample without the target is rape honey, is collected from Sichuan Jianyang and provided by Shanghai Guanshengyuan bee products Limited, and has the product number of 1932;
the liquid mixer is a rotary mixer with the model of RT200 and is collected from Shanghai Daam industry Co., Ltd;
the vacuum pump is a DRP series Roots vacuum pump, the model is DRP540, and the vacuum pump is collected from Desaier;
the nitrogen concentrator is a full-automatic nitrogen concentrator JC-D12 with the model number of JC-D12, and is self-healing and environment-friendly.
Examples
Example 1: a method for measuring the chlorpromazine residual quantity in honey is prepared by the following steps:
(1) pretreatment of the sample: weighing 0.5kg of Mel, directly sampling when there is no crystal in Mel, heating in water bath of 40-60 deg.C under sealed condition when there is crystal in Mel, stirring after Mel is completely melted, cooling to 25 deg.C, and sealing for use.
(2) Sample preparation: weighing 2.00g of sample by using an analytical balance, adding 20mL of 0.05mol/L phosphate buffer solution, and uniformly mixing for 1min by using a liquid mixer at the rotating speed of 60-70r/min to obtain sample liquid;
wherein, the preparation process of the phosphate buffer solution comprises the following steps: 5.68g of disodium hydrogenphosphate and 1.36g of potassium dihydrogenphosphate were weighed on an analytical balance, placed in a 1000mL beaker, dissolved in 800mL of water, adjusted to pH 3.0 with phosphoric acid (4.3), and made up to 1000mL with water.
(3) Sample purification: adopting an Oasis HLB solid phase extraction column of waters company, the specification of which is 500mg and 6 mL;
before the Oasis HLB solid-phase extraction column is used, washing the column once with 6mL of methanol, 10mL of water and 5mL of phosphate buffer solution in sequence, and keeping the column wet;
connecting a 50mL glass liquid reservoir filled with glass wool to an Oasis HLB solid-phase extraction column, pouring all sample liquid into the glass liquid reservoir, adjusting the flow rate to be less than or equal to 3mL/min, allowing the sample liquid to pass through the Oasis HLB solid-phase extraction column, washing the column once by sequentially using 5mL water and 5mL methanol aqueous solution after the sample liquid completely flows out, and discarding all effluent liquid; under the pressure of-65 kPa, the solid phase extraction column is decompressed and dried, and then is eluted once by 5mL of ammonium hydroxide methanol solution, and the eluent is collected by a 10mL nitrogen blow-dry tube; blowing the eluent by a nitrogen concentrator at 50 ℃ to obtain a crude sample; adding 1.0mL of constant volume solution to dissolve the crude sample, filtering the crude sample through a 0.2-micron filter membrane once, and collecting a pure sample for analysis and determination;
wherein the methanol aqueous solution is prepared by mixing methanol and water according to the volume ratio of 3: 7;
the ammonium hydroxide methanol solution is prepared by mixing ammonium hydroxide and methanol according to the volume ratio of 1: 19;
the constant volume solution is prepared by mixing acetonitrile and 0.01mol/L acetic acid solution according to the volume ratio of 1: 4.
(4) Preparing a sample blank extracting solution of a chlorpromazine standard working solution: weighing 2.00g of blank honey sample without target substances, and obtaining a sample blank extracting solution of the chlorpromazine standard working solution after (2) extracting the sample and (3) purifying.
(5) Preparing a chlorpromazine standard working solution: taking the sample blank extracting solution prepared in the step (4) as a solvent, and preparing chlorpromazine standard working solutions with different concentrations;
the preparation method of the chlorpromazine standard working solution comprises the following steps:
s1: weighing 50.00mg of chlorpromazine standard substance in a 50mL volumetric flask by using an analytical balance, dissolving by using methanol, and fixing the volume to a scale to prepare a standard stock solution with the mass concentration of 1 mg/mL;
s2: sucking 0.1mL of standard stock solution by a pipette, putting the standard stock solution into a 100mL volumetric flask, and fixing the volume to a scale by using methanol to obtain a chlorpromazine standard stock solution with the mass concentration of 1000 ng/mL;
s3: and (4) respectively sucking the chlorpromazine standard stock solutions (S2) with the volumes of 1 mu L, 2 mu L, 4 mu L, 10 mu L, 20 mu L and 100 mu L by using a pipette, and fixing the volume to 1000 mu L by using the sample blank extracting solution prepared in the step (4) to obtain chlorpromazine standard working solutions with the concentrations of 1ng/mL, 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL and 100 ng/mL.
(6) Liquid chromatography-tandem mass spectrometry: wherein, the chromatographic column adopts BEH C181.7 μm 100mm × 2.1mm i.d; column temperature: 30 ℃; the sample injection amount is 5 mu L; the mobile phase A is a formic acid aqueous solution with the volume concentration of 1 percent, and the mobile phase B is an acetonitrile solution with the volume concentration of 100 percent; the mobile phase gradient elution conditions are shown in table 1. Mass spectrum conditions: the instrument mode is MRM +; the capillary voltage is 4.0 kV; the voltage of the taper hole is 20V; the ion source temperature is 120 ℃; the temperature of the desolventizing gas is 350 ℃; other parameters are shown in table 2 and fig. 1.
TABLE 1 gradient elution conditions
| Time (min) | Flow rate (mL/min) | A: 0.1% formic acid solution (%) | B: acetonitrile (%) |
| 0.00 | 0.3 | 95.0 | 5.0 |
| 0.50 | 0.3 | 85.0 | 15.0 |
| 1.50 | 0.3 | 75.0 | 25.0 |
| 2.50 | 0.3 | 20.0 | 80.0 |
| 4.00 | 0.3 | 95.0 | 5.0 |
TABLE 2 Mass Spectrometry Condition parameters
(7) Drawing a chlorpromazine standard working curve: sequentially carrying out the operation (6) on the chlorpromazine standard working solutions with different concentrations prepared in the step (5), drawing and fitting a chlorpromazine standard working curve (see figure 2) by taking the concentration (mu g/mL) of the chlorpromazine standard working solution as a horizontal coordinate and the responding chromatic peak area as a vertical coordinate, and obtaining a linear regression equation and a correlation coefficient; the linear regression equation is in the range of 0.5-50 mu g/kg, and has good linearity. The retention time, addition level concentration, linear regression equation, correlation coefficient, parameters are shown in table 3.
TABLE 3 Retention time, addition level concentration, Linear regression equation, correlation coefficient
Wherein x is: the chlorpromazine in the pure sample matches the concentration of a chlorpromazine standard working solution; y is: chromatographic peak area; r is a correlation coefficient.
(8) And (3) measuring the residual quantity of the measured object in the pure sample: carrying out operation (6) on the pure sample collected in the operation (3), reading out the chromatographic peak area of the detected object in the pure sample from the chromatogram, substituting the chromatographic peak area of the detected object in the pure sample into the linear regression equation of the chlorpromazine standard working curve in the operation (7), calculating the concentration of the detected object in the pure sample, substituting the chromatographic peak area of the detected object in the pure sample and the concentration of the detected object in the pure sample into the determination formula of the chlorpromazine drug residue, and calculating the result;
the calculation formula of the residual quantity of the measured object in the pure sample is as follows:
X＝Cs×(A/As)×(V/m)×(1000/1000)
in the formula: x is the residual quantity of the tested object in the pure sample, and the unit is mu g/kg; the Cs is the concentration of the detected object in the sample pure product read from the standard working curve, and the unit is ng/mL; a is the chromatographic peak area of the measured object in the pure sample; as is the chromatographic peak area of the object to be measured in the chlorpromazine standard working solution; v is 1.0 mL; m is 2.00 g.
Test examples
1. Limit of detection (LOD) and lower limit of measurement (LOQ)
The limit of detection is the minimum concentration at which the analyte can be detected, i.e., the minimum concentration of analyte in the sample that can be detected qualitatively in a certain way with a certain degree of confidence.
The lower limit of measurement is the lowest concentration of chemical contaminants in the sample that can be reliably quantified by the additive method through the entire course of the measurement method.
Detection method (calculation method): the lowest concentration (amount) of the component to be detected that can be detected in the sample yields a sample concentration at which the signal (peak height) is 5 times the baseline noise standard deviation, the method being such that the concentration at which the signal-to-noise ratio (S/N) is 5:1 is the detection limit and the signal-to-noise ratio (S/N) is 10:1 is the measurement limit. The signal-to-noise ratio can be obtained by automatically analyzing the map through a workstation. As is clear from the analysis in FIGS. 3 and 4, the limit of detection (LOD) of chlorpromazine was 0.5. mu.g/kg, and the lower Limit (LOQ) was 1.0. mu.g/kg.
2. Precision and accuracy experiment of pure sample
The recovery rate is used as an accuracy evaluation index, and the Relative Standard Deviation (RSD) of the detection result of a sample with a certain concentration is repeatedly measured is used as a precision evaluation index. The calculation formula is as follows:
the recovery rate is the residual amount of the detected object/standard chlorpromazine solution concentration multiplied by 100%
Average value X is the sum of measured value and/or number of measurements
Relative standard deviation RSD ═ SD/X × 100%
Wherein SD is a standard deviation and X is an average value
The detection method comprises the following steps: chlorpromazine was added to a blank sample at 0.5. mu.g/kg, 1. mu.g/kg, 5. mu.g/kg, 50. mu.g/kg, and the measurement was repeated 10 times for each addition level, the result was detected in step (8) in example 1 to obtain the measured value of the residual amount of the object in the sample, and the recovery rate, average value, and relative standard deviation of chlorpromazine were calculated and the results are shown in Table 4.
TABLE 4 precision and accuracy test of pure samples
The data in the table 4 are analyzed, and according to the detection method, chlorpromazine with four concentrations of 0.5 mug/kg, 1 mug/kg, 5 mug/kg and 50 mug/kg is added into a blank honey sample, wherein when the concentration of chlorpromazine in the blank honey sample is 0.5 mug/kg, the recovery rate is 90.0-111%, and the RSD is 8.34%; when the chlorpromazine concentration in the blank honey sample is 1 mug/kg and 5 mug/kg, the recovery rate is between 98% and 105.6%, and the RSD is between 1.51% and 3.94%; when the chlorpromazine concentration in the blank honey sample is 5 mug/kg and 50 mug/kg, the recovery rate is between 97.6% and 100.16%, and the RSD is 0.21-2.94%.
In the related technology, when the addition level is in the range of 5-50 mug/kg, the detection limit LOD is 2.5 mug/kg, the determination lower limit LOQ is 5.0 mug/kg, the recovery rate is 81.9-110.7%, and the RSD is 5.5-8.4%, thereby showing that the method has higher accuracy and better precision.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (4)
1. A method for measuring the chlorpromazine residual quantity in honey is characterized by comprising the following steps:
(1) pretreatment of the sample: weighing 0.5kg of honey, directly sampling when the honey is not crystallized, heating in water bath under sealed condition when the honey is crystallized, stirring after the honey is completely melted, cooling to 25 ℃, and sealing for later use;
(2) sample preparation: weighing 2.00g of sample, adding 20mL of 0.05mol/L phosphate buffer solution, and uniformly stirring to obtain a sample solution;
(3) sample purification: adopting an Oasis HLB solid phase extraction column of waters company;
before the Oasis HLB solid-phase extraction column is used, washing the column once with 6mL of methanol, 10mL of water and 5mL of phosphate buffer solution in sequence, and keeping the column wet;
adjusting the flow rate of an Oasis HLB solid phase extraction column to be less than or equal to 3mL/min, extracting a sample solution by using the solid phase extraction column, washing the column by using 5mL of water and 5mL of methanol aqueous solution in sequence after the sample solution completely flows out, and discarding all effluent liquid; under the pressure of-65 kPa, after the solid phase extraction column is decompressed and dried, 5mL of ammonium hydroxide methanol solution is used for elution, and eluent is collected; blowing the eluent by a nitrogen concentrator at 50 ℃ to obtain a crude sample; adding 1.0mL of constant volume solution to dissolve the crude sample, filtering with a 0.2 μm filter membrane, and collecting the pure sample for analysis and determination;
the methanol water solution is prepared by mixing methanol and water according to the volume ratio of 3: 7;
the ammonium hydroxide methanol solution is prepared by mixing ammonium hydroxide and methanol according to the volume ratio of 1: 19;
the constant volume solution is prepared by mixing acetonitrile and 0.01mol/L acetic acid solution according to the volume ratio of 1: 4;
(4) preparing a sample blank extracting solution of a chlorpromazine standard working solution: taking a blank honey sample without a target object, and obtaining a sample blank extracting solution of a chlorpromazine standard working solution after (2) sample extraction and (3) purification;
(5) preparing a chlorpromazine standard working solution: taking the sample blank extracting solution of the chlorpromazine standard working solution obtained in the step (4) as a solvent, and preparing chlorpromazine standard working solutions with different concentrations;
(6) liquid chromatography-tandem mass spectrometry: the chromatographic column adopts BEH C181.7 μm 100mm × 2.1mm i.d; column temperature: 30 ℃; the sample size is 5 muL; the mobile phase A is an aqueous solution containing 1% by volume of formic acid, and the mobile phase B is an acetonitrile solution containing 100% by volume; gradient elution was used: 0-0.5min, 95-85% mobile phase A; 0.5-1.5min, 85-75% of mobile phase A; 1.5-2.5min, 75-20% of mobile phase A; 2.5-4min, 20-95% mobile phase A; mass spectrum conditions: the instrument mode is MRM +; the capillary voltage is 4.0 kV; the voltage of the taper hole is 20V; the ion source temperature is 120 ℃; the temperature of the desolventizing gas is 350 ℃; the qualitative ion pair is 319.1/58.2, 319.1/86.2 m/z; the quantitative ion pair is 319.1/86.2 m/z; the residence time is 100 ms; the cone hole voltage of an ion pair 319.1/58.2m/z is 28V, and the collision energy is 25V; the cone hole voltage of an ion pair 319.1/86.2m/z is 28V, and the collision energy is 20V;
(7) drawing a chlorpromazine standard working curve: sequentially carrying out the operation (6) on the chlorpromazine standard working solutions with different concentrations prepared in the step (5), and drawing and fitting to obtain a linear regression equation and a correlation coefficient of a chlorpromazine standard working curve by taking the concentration ng/mL of the chlorpromazine standard working solution as a horizontal coordinate and the area of a corresponding chromatographic peak as a vertical coordinate;
(8) and (3) measuring the residual quantity of the measured object in the pure sample: carrying out operation (6) on the pure sample collected in the step (3), reading out the chromatographic peak area of the detected object in the pure sample from the chromatogram, substituting the chromatographic peak area of the detected object in the pure sample into the linear regression equation of the chlorpromazine standard working curve in the step (7), calculating the concentration of the detected object in the pure sample, substituting the chromatographic peak area of the detected object in the pure sample and the concentration of the detected object in the pure sample into the determination formula of the chlorpromazine drug residue, and calculating the result;
the calculation formula of the residual quantity of the measured object in the pure sample is as follows:
X＝Cs×(A/As)×(V/m)×(1000/1000)
in the formula: x is the residual amount of a detected object in a sample pure product, and the unit is microgram/kg; the Cs is the concentration of the detected object in the sample pure product read from the standard working curve, and the unit is ng/mL; a is the chromatographic peak area of the measured object in the pure sample; as is the chromatographic peak area of the object to be measured in the chlorpromazine standard working solution; v is the final constant volume of the pure sample, and the unit is mL; m is the mass of the sample represented by the sample pure product, and the unit is g.
2. The method for determining the chlorpromazine residual quantity in honey according to claim 1, wherein the preparation method of the chlorpromazine standard working solution (5) is as follows:
s1: weighing chlorpromazine, dissolving the chlorpromazine in methanol, and preparing a standard stock solution with the mass concentration of 1 mg/mL;
s2: sucking 0.1mL of standard stock solution, putting the standard stock solution into a 100mL volumetric flask, and fixing the volume to a scale with methanol to obtain a chlorpromazine standard stock solution with the mass concentration of 1000 ng/mL;
s3: and (4) respectively sucking chlorpromazine standard stock solutions (S2) with different volumes, and preparing with the sample blank extracting solution prepared in the step (4) to obtain chlorpromazine standard working solutions with different concentrations.
3. The method for determining the chlorpromazine residual quantity in honey according to claim 2, wherein the concentrations of the prepared chlorpromazine standard working solution are 1ng/mL, 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL and 100ng/mL respectively.
4. The method for determining the residual amount of chlorpromazine in honey as claimed in claim 1, wherein in the drawing of a chlorpromazine standard working curve (7), a linear regression with y =22904x-9854.88 and r =0.999992 is obtained; wherein x is: the chlorpromazine in the pure sample matches the concentration of a chlorpromazine standard working solution; y is: chromatographic peak area; r is a correlation coefficient.
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