WO2021093870A1 - 一种同时检测苯并[a]芘及其代谢产物的方法及其应用 - Google Patents

一种同时检测苯并[a]芘及其代谢产物的方法及其应用 Download PDF

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WO2021093870A1
WO2021093870A1 PCT/CN2020/128800 CN2020128800W WO2021093870A1 WO 2021093870 A1 WO2021093870 A1 WO 2021093870A1 CN 2020128800 W CN2020128800 W CN 2020128800W WO 2021093870 A1 WO2021093870 A1 WO 2021093870A1
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pyrene
benzo
sample
tested
solid phase
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French (fr)
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罗茜
李芳�
向彬彬
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中国科学院深圳先进技术研究院
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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
    • 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
    • G01N30/08—Preparation using an enricher
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

Definitions

  • the invention relates to the technical field of analysis and detection, in particular to a method for simultaneously detecting benzo[a]pyrene and its metabolites and its application.
  • Benzo[a]pyrene (Benzo[a]pyrene, B[a]P), molecular formula C 20 H 12 , is a strong carcinogen that exists widely in food and the environment, derived from cooking, smoking, coal and petroleum Processes such as incomplete combustion of natural gas and natural gas can be entered through diet (such as fried, smoked, grilled, grains, vegetables, fats and oils, etc.), breathing (such as air, tobacco smoke and automobile exhaust, etc.) and skin contact. in vivo.
  • diet such as fried, smoked, grilled, grains, vegetables, fats and oils, etc.
  • breathing such as air, tobacco smoke and automobile exhaust, etc.
  • B[a]P that enters the body is excreted in the feces in its original form, most of it is oxidized by the cytochrome P450 in the body into a series of epoxy compounds including phenols (such as 3-hydroxybenzo[a]pyrene (3-OH) -B[a]P)), ketones (such as benzo[a]pyrene-1,6-dione (1,6-B[a]P-dione), benzo[a]pyrene-3,6 -Dione (3,6-B[a]P-dione)) and dihydrodiols (such as benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8 -dihydrodiol)), and the benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]P-7,8-diol-9,10- e
  • the current B[a]P human exposure level assessment method mainly uses 3-hydroxybenzo[a]pyrene in urine as a marker. Since the metabolites in urine can only reflect the short-term exposure, and B[a]P exposure is a long-term process, the existing detection methods are still unable to fully and effectively monitor the human exposure level of B[a]P , And the metabolism of B[a]P in the body.
  • the present invention provides a method and application for the simultaneous detection of benzo[a]pyrene and its metabolites.
  • the method can simultaneously detect benzo[a]pyrene in a sample to be tested with high sensitivity and selectivity.
  • ]Pyrene and its metabolites can accurately and comprehensively characterize the exposure level of benzo[a]pyrene.
  • the present invention provides a method for simultaneously detecting benzo[a]pyrene and its metabolites, which includes the following steps:
  • the sample to be tested contains benzo[a]pyrene and the metabolites of the benzo[a]pyrene, and the metabolites include 3- Hydroxybenzo[a]pyrene, benzo[a]pyrene-1,6-dione, benzo[a]pyrene-3,6-dione and benzo[a]pyrene-7,8-dihydrodione One or more of alcohol-9,10-epoxide;
  • the step (3) using retention time locking and characteristic ion pair information locking means: the retention time and characteristic ion pair information of the processed sample to be side measured in the multi-reaction monitoring mode, respectively Compare with the standard retention time and characteristic ion pair information of benzo[a]pyrene and its metabolites, and then according to the comparison results, the actual benzo[a]pyrene and its metabolites contained in the sample to be side can be determined kind of.
  • the standard retention time and characteristic ion pair information of the benzo[a]pyrene and its metabolites are based on the determination of a standard solution containing benzo[a]pyrene and its metabolites under the same detection conditions. Data composition.
  • the processed sample to be tested can be detected by a university liquid chromatography-tandem mass spectrometer (UPLC-MS/MS).
  • UPLC-MS/MS university liquid chromatography-tandem mass spectrometer
  • the sample to be tested includes a mammalian body fluid sample
  • the body fluid sample includes at least one of a blood sample, a urine sample, a cerebrospinal fluid, and a tissue extract.
  • the mammals include humans.
  • the sample to be tested when the sample to be tested is a blood sample, the sample to be tested can be centrifuged at a high speed, the supernatant is taken, and stored at low temperature for later use.
  • the pretreatment process of the blood sample includes: centrifuging the blood sample, collecting the supernatant, and then extracting it with an organic solvent for multiple times to obtain the extract, concentrating the extract to near dryness, and then regenerating Dissolve in ultrapure water with a volume of less than or equal to 1mL
  • the organic solvent includes one or more of methyl tert-butyl ether, ethyl acetate, acetonitrile HE methanol.
  • the organic solvent may be methyl tert-butyl ether.
  • the ultra-high performance liquid chromatography conditions are: BEH C18 chromatographic column with a specification of 2.1mm ⁇ 100mm ⁇ 1.7 ⁇ m, the mobile phase is a mixed solution of water and methanol, and the gradient wash is De-program elution; injection volume is 5.0 ⁇ 20.0 ⁇ L; needle washing liquid is a mixed solution of acetonitrile and water with a volume ratio of 1:1.
  • the tandem mass spectrometry conditions are: ionization mode is electrospray ionization, positive ion and negative ion modes are mixed; detection mode is selective reaction monitoring; ion source temperature is 120-150°C, capillary The voltage is 3.0 ⁇ 4.0kV, the cone voltage is 20 ⁇ 40V, the desolventizing temperature is 300 ⁇ 500°C, the desolventizing gas flow rate is 500 ⁇ 700L/h, and the cone gas flow velocity is 30 ⁇ 80L/h.
  • the activation process of the C18 solid phase extraction column includes: sequentially activating with 5.0-10.0 mL of dichloromethane, methanol and ultrapure water. During the activation process, The C18 solid phase extraction column is kept dry.
  • the specific process of eluting the C18 solid phase extraction column includes: washing the sample to be tested with 5.0-20.0 mL ultrapure water The container and the C18 solid phase extraction column after the enrichment treatment, and then the residual water in the C18 solid phase extraction column after the enrichment treatment is drained; and 5.0-10.0 mL of methanol and dichloromethane are used for elution in sequence.
  • the eluent can be concentrated to near dryness with high-purity nitrogen, and directly subjected to instrumental analysis after reconstitution.
  • the step (2) before the step (3), it further includes preparing a plurality of standard solutions with a concentration gradient, and then quantitatively determining the standard solution and preparing a standard curve;
  • the standard solution includes benzene Bi[a]pyrene, 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-1,6-dione, benzo[a]pyrene-3,6-dione and benzo[a]pyrene -7,8-Dihydrodiol-9,10-epoxide.
  • the data obtained by measuring the processed sample to be tested in step (3) is substituted into the standard curve to quantitatively analyze the benzo[a]pyrene and the benzo[a]pyrene in the sample to be tested A metabolite of benzo[a]pyrene.
  • the method for simultaneous detection of benzo[a]pyrene and its metabolites adopts an ultra-high performance liquid chromatography-tandem mass spectrometry method, which can detect in a sample to be tested with high sensitivity, selectivity and rapidity.
  • the benzo[a]pyrene and its metabolites Compared with the traditional detection method of benzo[a]pyrene alone or its metabolites alone, the method of the present invention can more comprehensively detect the benzo[a]pyrene and its metabolism in the sample to be tested. The product more accurately reflects the exposure level of benzo[a]pyrene in the sample to be tested.
  • the method of the present invention is simple to operate and can be easily developed into a standardized detection method for benzo[a]pyrene and its metabolites.
  • the present invention also provides an application of the method for simultaneous detection of benzo[a]pyrene and its metabolites as described in the first aspect of the present invention in the field of biochemical analysis and detection or life sciences.
  • the method for simultaneously detecting benzo[a]pyrene and its metabolites according to the present invention can be used to detect human urine samples or blood samples to evaluate the exposure level of benzo[a]pyrene in the above samples,
  • the obtained detection data can be used in the fields of biochemical analysis and detection or life sciences, or other fields.
  • benzo[a]pyrene is a strong carcinogen widely present in food and the environment, it seriously endangers human health. Therefore, a comprehensive and accurate reflection of the exposure level of benzo[a]pyrene in the sample to be tested is of great significance for studying the exposure level of benzo[a]pyrene in the human body and assessing human health. It can also be toxicologically related. Research provides important technical support.
  • Fig. 1 is a process flow diagram of a method for simultaneously detecting benzo[a]pyrene and its metabolites according to an embodiment of the present invention
  • Figure 2 is an ion current chromatogram of benzo[a]pyrene and its metabolites provided by an embodiment of the present invention.
  • the raw materials and other chemical reagents used in the embodiments of the present invention are all commercially available products.
  • an embodiment of the present invention also provides a method for simultaneously detecting benzo[a]pyrene and its metabolites, including the following steps:
  • the sample to be tested contains benzo[a]pyrene and a metabolite of the benzo[a]pyrene, and the metabolite includes 3-hydroxyl Benzo[a]pyrene, benzo[a]pyrene-1,6-dione, benzo[a]pyrene-3,6-dione and benzo[a]pyrene-7,8-dihydrodiol One or more of -9,10-epoxides;
  • the detection method of ultra-high performance liquid chromatography-tandem mass spectrometry is adopted. After the processed sample is separated by liquid chromatography and detected in the multi-reaction monitoring mode, the retention time lock and the characteristic ion pair information lock are used to detect The benzo[a]pyrene and the metabolites of the benzo[a]pyrene contained in the processed sample to be tested, and the characteristic ion pair information includes parent ions and product ions.
  • the sample to be tested includes a mammalian body fluid sample, wherein the mammal includes a human; the body fluid sample includes at least one of a blood sample, a urine sample, a cerebrospinal fluid sample, and a tissue extract sample One kind.
  • the pretreatment process includes: centrifuging the blood sample, collecting the supernatant, and then extracting it with an organic solvent for multiple times to obtain the extract, and
  • the extract is concentrated to nearly dryness, and then reconstituted to a volume of less than or equal to 1 mL of ultrapure water.
  • the near dry means that the volume of the extract after the concentration process is negligible.
  • the volume of the extract after being concentrated is less than 0.1 mL.
  • the supernatant of the blood sample is extracted using methyl tert-butyl ether.
  • methyl tert-butyl ether can be used to more fully extract the benzo[a]pyrene and its metabolites contained in the sample to be tested.
  • the extract is concentrated to near dryness by means of nitrogen blowing and concentration.
  • a nitrogen blower is used to concentrate the extract.
  • the activation process of the C18 solid phase extraction column includes: sequentially activating with 5.0-10.0 mL of methylene chloride, methanol and ultrapure water. During the activation process, the The C18 solid phase extraction column is kept dry.
  • the specific process of eluting the C18 solid phase extraction column includes: washing the container containing the pretreated sample to be tested with 5.0-20.0 mL of ultrapure water and The C18 solid phase extraction column after the enrichment treatment is drained, and then the residual water in the C18 solid phase extraction column after the enrichment treatment is drained; 5.0-10.0 mL of methanol and dichloromethane are used for elution.
  • the residual moisture in the C18 solid phase extraction column after the enrichment treatment is drained by a vacuum draining method, and the draining time is 10-20 min.
  • the eluate is concentrated to near dryness by means of nitrogen blowing and concentration.
  • nitrogen blowing and concentration For example, a 40°C water bath nitrogen blowing instrument is used for nitrogen blowing, and the flow rate of the nitrogen gas is always maintained to make the liquid surface of the eluent concave.
  • the near dry means that the volume of the eluent after the concentration process is negligible.
  • the volume of the eluate after being concentrated is less than 0.1 mL.
  • methanol is used for constant volume.
  • membrane treatment is performed through a filter membrane with a pore size of 0.2 ⁇ m.
  • the constant volume can be but not limited to 500-1000 ⁇ L.
  • the ultra-high performance liquid chromatography conditions are: BEH C18 chromatographic column, specification 2.1mm ⁇ 100mm ⁇ 1.7 ⁇ m, mobile phase is a mixed solution of water and methanol, washing according to gradient elution procedure
  • the injection volume is 5.0 ⁇ 20.0 ⁇ L;
  • the needle washing solution is a mixed solution of acetonitrile and water with a volume ratio of 1:1.
  • the tandem mass spectrometry conditions are: ionization mode is electrospray ionization, positive ion and negative ion modes are mixed; detection mode is selective reaction monitoring; ion source temperature is 80-150°C, capillary voltage is 3.0 ⁇ 4.0kV, cone voltage is 20 ⁇ 40V, desolventizing temperature is 300 ⁇ 500°C, desolventizing gas flow rate is 500 ⁇ 700L/h, cone gas flow velocity is 30 ⁇ 80L/h.
  • the S20 before the S30, it further includes preparing a plurality of standard solutions with a concentration gradient, and then quantitatively determining the standard solution and preparing a standard curve;
  • the standard solution includes benzo[a]pyrene, 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-1,6-dione, benzo[a]pyrene-3,6-dione and benzo[a]pyrene-7,8-di Hydrogen diol-9,10-epoxide.
  • the data obtained by the measurement of the processed sample to be tested in the S30 is substituted into the standard curve, and the benzo[a]pyrene and the benzene in the sample to be tested are quantitatively analyzed And [a] metabolites of pyrene.
  • a method for simultaneous detection of benzo[a]pyrene and its metabolites including the following steps:
  • the C18 solid phase extraction cartridge was activated sequentially with 5.0mL dichloromethane, 5.0mL methanol, and 5.0mL ultrapure water.
  • the solid phase extraction cartridge was activated during the activation process.
  • the column is kept moist and not dry.
  • the liquid level of ultrapure water in the solid phase extraction cartridge is about 1mm away from the upper sieve plate, turn off the solid phase extraction device and fill the solid phase extraction cartridge with ultrapure water for use. Add the pretreated sample to the activated solid phase extraction cartridge to enrich the spiked blood sample.
  • the sample bottle and solid phase extraction cartridge After the enrichment is complete, rinse the sample bottle and solid phase extraction cartridge with 10.0 mL ultrapure water, and drain the cartridge by a vacuum pump 10min. Then the enriched solid-phase extraction cartridge was eluted.
  • the elution conditions were as follows: firstly add 5.0 mL methanol and 5.0 mL dichloromethane, collect the eluates separately, blow with nitrogen in a water bath at 40°C, and set the flow rate of nitrogen to the eluent liquid.
  • the processed sample to be tested is analyzed by UPLC-MS/MS (Waters Xevo TQD) to obtain multiple reaction monitoring (MRM) data and use retention time
  • MRM reaction monitoring
  • the method of locking and characteristic ion pair locking qualitatively screens the possible benzo[a]pyrene and its metabolites in the sample; adopts an ultra-high performance liquid chromatography column (ACQUITY UPLC) BEH C18 (specification 2.1 ⁇ 100mm, 1.7 ⁇ m), flowing Phase: A is water, B is methanol.
  • Gradient elution program initial ratio A is 50%, B is 50%; gradient elution program: initial ratio A is 50%, B is 50%, hold for 0.5min; 0.5 ⁇ 1.0min, A drops to 20% , B rose to 80%, maintained to 7.0min; 7.0 ⁇ 7.5min, A decreased to 0%, B rose to 100%, maintained to 12.0min; At 12.2min, it returned to the initial gradient equilibrium system to 18.0min.
  • the flow rate is 200 ⁇ L/min; the column temperature is room temperature; the injection volume is 10.0 ⁇ L; the needle wash is a mixed solution of acetonitrile and water (1:1, v/v).
  • Tandem mass spectrometry conditions ionization mode is electrospray ionization (ESI), positive ion and negative ion mode are mixed; detection method is selective reaction monitoring (MRM); ion source temperature is 120°C, capillary voltage is 3.5kV, cone voltage is 30V, The desolventizing temperature is 380°C, the desolventizing gas flow rate is 600L/h, and the cone gas flow rate is 50L/h. Then, record the monitoring ion pair, collision energy and other parameter data of benzo[a]pyrene and its metabolites.
  • ESI electrospray ionization
  • MRM selective reaction monitoring
  • Example 1 the monitored ion pair and collision energy parameter data of benzo[a]pyrene and its metabolites are shown in Table 1 below.
  • Table 1 The corresponding extracted ion current chromatograms of benzo[a]pyrene and its metabolites are shown in Figure 2 for further details.
  • the method described in the embodiments of the present invention can effectively solid-phase extract benzo[a]pyrene and its metabolites, obtain a clean eluate and remove the interference of impurities; then simultaneously extract and detect the content of the serum sample A variety of benzo[a]pyrene bodies and their metabolites.
  • the method of using retention time locking and characteristic ion pair locking described in the embodiment of the present invention can efficiently and quickly perform qualitative screening and quantitative analysis of the benzo[a]pyrene itself and its metabolites that may be present in the sample.
  • the methods described in the embodiments of the present invention have potential application value in the preliminary screening and quantitative analysis of benzo[a]pyrene and its metabolites in the sample to be tested; especially for mammals (Including human) body fluid samples, because benzo[a]pyrene will be partially metabolized when entering the body, the detection of benzo[a]pyrene alone or the detection of its metabolites alone cannot reflect comprehensively and accurately.
  • the exposure level of benzo[a] pyrene in the body fluid sample can be well improved by the method described in the embodiment of the present invention, and the method is more efficient and more sensitive. Further, the methods described in the embodiments of the present invention can provide important reference information for the effects of benzo[a]pyrene and its metabolites on human health and related toxicological studies.
  • the liquid-mass spectrometry method described in the embodiments of the present invention does not require derivatization to determine the metabolites of benzo[a]pyrene.
  • the method of the present invention can even directly inject samples without any pre-treatment, while the GC-MS method requires corresponding solvent conversion before injecting. .

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Abstract

一种同时检测苯并[a]芘及其代谢产物的方法，包括以下步骤：取待测样品，对待测样品进行预处理，待测样品含有苯并[a]芘和苯并[a]芘的代谢产物；用活化后的C18固相萃取柱对预处理后的待测样品进行富集处理，然后对C18固相萃取柱进行洗脱，收集洗脱液；并经浓缩至近干、定容和经过膜处理后，得到处理后的待测样品（S20）；采用超高效液相色谱-串联质谱联用检测方式，处理后的待测样品经液相色谱分离和多反应监测模式检测后，利用保留时间锁定和特征离子对信息锁定，检测出待测样品中含有的苯并[a]芘及其代谢产物。该方法能高选择性的检测苯并[a]芘及其代谢产物。

Description

一种同时检测苯并[a]芘及其代谢产物的方法及其应用 技术领域
本发明涉及分析检测技术领域，具体涉及一种同时检测苯并[a]芘及其代谢产物的方法及其应用。
背景技术
苯并[a]芘(Benzo[a]pyrene,B[a]P)，分子式C 20H 12，是一种食品和环境中广泛存在的强致癌物质，来源于烹饪加工、吸烟及煤、石油和天然气的不完全燃烧等过程，可通过饮食(如油炸烟熏烧烤类、谷类、蔬菜、脂肪和油类等)、呼吸(如空气、烟草烟雾和汽车尾气等)和皮肤接触等途径进入体内。进入体内的B[a]P除少部分以原形随粪便排出外，大部分被体内的细胞色素P450氧化成一系列环氧化合物包括酚类(例如3-羟基苯并[a]芘(3-OH-B[a]P))，酮类(例如苯并[a]芘-1,6-二酮(1,6-B[a]P-dione)、苯并[a]芘-3,6-二酮(3,6-B[a]P-dione))和二氢化二醇类(例如苯并[a]芘-7,8-二氢二醇(B[a]P-7,8-dihydrodiol))，以及进一步氧化生成的苯并[a]芘-7,8-二氢二醇-9,10-环氧化物(B[a]P-7,8-diol-9,10-epoxide，BPDE)，上述代谢物能与DNA、蛋白以及脂质等生物分子相互作用而表现出毒性。因此，对B[a]P进行定性及定量检测具有重要意义。
目前B[a]P的人体暴露水平评估方法主要是以尿液的中的3-羟基苯并[a]芘作为标志物。由于尿液中的代谢产物只能反应短期内暴露的情况，而B[a]P暴露是一个长期的过程，因此，现有检测手段仍无法全面有效地监测B[a]P在人体暴露水平，以及B[a]P在体内的代谢情况。
发明内容
有鉴于此，本发明提供了一种同时检测苯并[a]芘及其代谢产物的方法及其应 用，该方法能高灵敏的、高选择性的同时检测待测样品中的苯并[a]芘及其代谢产物，能准确、全面地表征苯并[a]芘的暴露水平。
第一方面，本发明提供了一种同时检测苯并[a]芘及其代谢产物的方法，包括以下步骤：
(1)取待测样品，对所述待测样品进行预处理，所述待测样品含有苯并[a]芘和所述苯并[a]芘的代谢产物，所述代谢产物包括3-羟基苯并[a]芘，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮和苯并[a]芘-7,8-二氢二醇-9,10-环氧化物中的一种或多种；
(2)用活化后的C18固相萃取柱对所述预处理后的待测样品进行富集处理，然后对所述C18固相萃取柱进行洗脱，收集洗脱液；然后对所述洗脱液浓缩至近干后，定容和经过膜处理后，得到处理后的待测样品；
(3)采用超高效液相色谱-串联质谱联用检测方式，所述处理后的待测样品经液相色谱分离和多反应监测模式检测后，利用保留时间锁定和特征离子对信息锁定，检测出所述处理后的待测样品中含有的所述苯并[a]芘和所述苯并[a]芘的代谢产物，所述特征离子对信息包括母离子和子离子。
本发明实施方式中，所述步骤(3)，利用保留时间锁定和特征离子对信息锁定是指：处理后的待侧样品在多反应监测模式下测得的保留时间和特征离子对信息，分别与苯并[a]芘及其代谢产物的标准保留时间和特征离子对信息比对，然后根据比对结果，可以确定所述待侧样品中实际含有的苯并[a]芘及其代谢产物的种类。
可选地，所述苯并[a]芘及其代谢产物的标准保留时间和特征离子对信息是基于在相同检测条件下，由含有苯并[a]芘及其代谢产物的标准溶液的测定数据组成。
本发明实施方式中，所述处理后的待测样品可以通过高校液相色谱-串联质谱联用仪(UPLC-MS/MS)进行检测。
可选地，所述待测样品包括哺乳动物体液样品，所述体液样品包括血液样品、尿液样品、脑脊液、组织提取液中的至少一种。其中，所述哺乳动物包括人类。
可选地，当所述待测样品为血液样品时，可以将所述待测样品经高速离心后，取上清，低温储存备用。其中，所述血液样品时的预处理过程包括：将所述血液样品进行离心处理，收集上清液，然后用有机溶剂多次萃取后得到萃取液，将所述萃取液浓缩至近干，然后复溶至体积小于等于1mL超纯水中。
进一步地，可选地，所述有机溶剂包括甲基叔丁基醚、乙酸乙酯、乙腈HE甲醇中的一种或多种。一实施方式中，所述有机溶剂可以为甲基叔丁基醚。
可选地，所述步骤(3)中，所述超高效液相色谱条件为：BEH C18色谱柱，规格为2.1mm×100mm×1.7μm，流动相为水和甲醇的混合溶液，按梯度洗脱程序洗脱；进样体积为5.0～20.0μL；洗针液为体积比1:1的乙腈和水的混合溶液。
可选地，所述梯度洗脱程序为：起始梯度按水：甲醇＝50：50比例洗脱，维持0.5min，然后在0.5～1.0min内，变化为水：甲醇＝20：80比例洗脱，维持至7.0min；然后在7.0～7.5min内，变化为水：甲醇＝0：100比例洗脱，维持至12.0min；然后在12.2min时恢复至所述起始梯度平衡系统至18.0min；流速为200μL/min。
可选地，所述步骤(3)中，所述串联质谱条件为：电离模式为电喷雾电离，正离子和负离子模式混合；检测方式为选择反应监测；离子源温度为120～150℃，毛细管电压为3.0～4.0kV，锥孔电压为20～40V，脱溶剂温度为300～500℃，脱溶剂气流速为500～700L/h，锥孔气流速为30～80L/h。
可选地，所述步骤(2)中，所述C18固相萃取柱的活化过程包括为：依次用5.0～10.0mL的二氯甲烷、甲醇和超纯水进行活化，所述活化过程中，所述C18固相萃取柱保持不干状态。
可选地，所述步骤(2)中，然后对所述C18固相萃取柱进行洗脱的具体过程包括：用5.0～20.0mL超纯水冲洗盛放所述预处理后的待测样品的容器及所述富集处理后的C18固相萃取柱，然后抽干所述富集处理后的C18固相萃取柱中残留水分；依次用5.0～10.0mL甲醇和二氯甲烷进行洗脱。其中，所述洗脱液可以用高纯氮气浓缩至近干，复溶后直接进行仪器分析。
可选地，所述步骤(2)之后，所述步骤(3)之前还包括配制多个浓度梯度的标准溶液，然后对所述标准溶液进行定量测定并制作标准曲线；所述标准溶液 包括苯并[a]芘、3-羟基苯并[a]芘，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮和苯并[a]芘-7,8-二氢二醇-9,10-环氧化物。
可选地，将步骤(3)中对所述处理后的待测样本测定获得的数据，代入所述标准曲线，定量分析所述待测样本中的所述苯并[a]芘和所述苯并[a]芘的代谢产物。
本发明第一方面提供的同时检测苯并[a]芘及其代谢产物的方法，采用超高效液相色谱-串联质谱联用方法，能高灵敏、高选择性、快速的检测待测样品中的苯并[a]芘及其代谢产物。相比于传统仅单独对苯并[a]芘，或单独针对其代谢产物的检测方法，本发明所述方法能更为全面地检测出待测样品内的苯并[a]芘及其代谢产物，更为准确地反映出待测样品的苯并[a]芘的暴露水平。同时，本发明所述方法操作简单，易发展成为一种苯并[a]芘及其代谢产物的标准化检测手段。
第二方面，本发明还提供了一种包含本发明第一方面所述同时检测苯并[a]芘及其代谢产物的方法在生化分析检测或生命科学领域的应用。
例如，本发明所述同时检测苯并[a]芘及其代谢产物的方法可以用于检测人体的尿液样品或血液样品，用于评价上述样品中的苯并[a]芘的暴露水平，获得的检测数据可以服务于生化分析检测或生命科学领域，或者其他领域。
由于苯并[a]芘是一种食品和环境中广泛存在的强致癌物质，严重危害人类的健康。因此，全面、准确地反映出待测样品内的苯并[a]芘的暴露水平对于研究人体内苯并[a]芘的暴露水平，评估人体健康具有重要意义，同时也可为毒理学相关研究提供重要的技术支撑。
本发明的优点将会在下面的说明书中部分阐明，一部分根据说明书是显而易见的，或者可以通过本发明实施例的实施而获知。
附图说明
为更清楚地阐述本发明的内容，下面结合附图与具体实施例来对其进行详细说明。
图1为本发明一实施例提供的同时检测苯并[a]芘及其代谢产物的方法的工艺 流程图；
图2为本发明一实施例提供的苯并[a]芘及其代谢产物的离子流色谱图。
具体实施方式
以下所述是本发明实施例的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明实施例原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明实施例的保护范围。
下面分多个实施例对本发明实施例进行进一步的说明。其中，本发明实施例不限定于以下的具体实施例。在不变主权利的范围内，可以适当的进行变更实施。
若无特别说明，本发明实施例所采用的原料及其它化学试剂皆为市售商品。
参见图1，本发明一实施例还提供了一种同时检测苯并[a]芘及其代谢产物的方法，包括以下步骤：
S10、取待测样品，对所述待测样品进行预处理，所述待测样品含有苯并[a]芘和所述苯并[a]芘的代谢产物，所述代谢产物包括3-羟基苯并[a]芘，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮和苯并[a]芘-7,8-二氢二醇-9,10-环氧化物中的一种或多种；
S20、用活化后的C18固相萃取柱对所述预处理后的待测样品进行富集处理，然后对所述C18固相萃取柱进行洗脱，收集洗脱液；然后对所述洗脱液浓缩至近干后，定容和经过膜处理后，得到处理后的待测样品；
S30、采用超高效液相色谱-串联质谱联用检测方式，所述处理后的待测样品经液相色谱分离和多反应监测模式检测后，利用保留时间锁定和特征离子对信息锁定，检测出所述处理后的待测样品中含有的所述苯并[a]芘和所述苯并[a]芘的代谢产物，所述特征离子对信息包括母离子和子离子。
其中，所述步骤S10中，所述待测样品包括哺乳动物体液样品，其中，所述哺乳动物包括人类；所述体液样品包括血液样品、尿液样品、脑脊液样品和组织提取液样品中的至少一种。
可选地，所述待测样品为血液样品时，所述预处理过程包括：将所述血液样 品进行离心处理，收集上清液，然后用有机溶剂多次萃取后得到萃取液，将所述萃取液浓缩近干，然后复溶至体积小于等于1mL超纯水中。其中，所述近干是指所述萃取液经所述浓缩过程后的体积忽略不计。例如，所述萃取液经浓缩处理后的体积小于0.1mL。
一实施方式中，使用甲基叔丁基醚对所述血液样品的上清液进行萃取。本发明所述实施方式中，采用甲基叔丁基醚可以更加充分地将待测样品中的含有的苯并[a]芘及其代谢产物萃取出来。
可选地，采用氮吹浓缩的方式将所述萃取液浓缩至近干。例如，使用氮吹仪对所述萃取液进行浓缩。
可选地，所述步骤S20中，所述C18固相萃取柱的活化过程包括为：依次用5.0～10.0mL的二氯甲烷、甲醇和超纯水进行活化，所述活化过程中，所述C18固相萃取柱保持不干状态。
可选地，所述步骤S20中，然后对所述C18固相萃取柱进行洗脱的具体过程包括：用5.0～20.0mL超纯水冲洗盛放所述预处理后的待测样品的容器及所述富集处理后的C18固相萃取柱，然后抽干所述富集处理后的C18固相萃取柱中残留水分；用5.0～10.0mL甲醇和二氯甲烷进行洗脱。
可选地，采用真空抽干方式抽干所述富集处理后的C18固相萃取柱中残留水分，抽干时间为10-20min。
可选地，所述S20中，采用氮吹浓缩的方式将所述洗脱液浓缩至近干。例如，使用40℃水浴氮吹仪进行氮吹，氮气的气流流速始终保持在使所述洗脱液液面呈下凹状态。其中，所述近干是指所述洗脱液经所述浓缩过程后的体积忽略不计。例如，所述洗脱液经浓缩处理后的体积小于0.1mL。
可选地，所述S20中，使用甲醇进行定容。所述定容过程后，通过孔径为0.2μm的滤膜进行过膜处理。可选地，所述定容体积可以但不限于至500～1000μL。
可选地，所述S30中，所述超高效液相色谱条件为：BEH C18色谱柱，规格为2.1mm×100mm×1.7μm，流动相为水和甲醇的混合溶液，按梯度洗脱程序洗脱；进样体积为5.0～20.0μL；洗针液为体积比1:1的乙腈和水的混合溶液。
可选地，所述梯度洗脱程序为：起始梯度按水：甲醇＝50：50比例洗脱，维持0.5min，然后在0.5～1.0min内，变化为水：甲醇＝20：80比例洗脱，维持至7.0min；然后在7.0～7.5min内，变化为水：甲醇＝0：100比例洗脱，维持至12.0min；然后在12.2min时恢复至所述起始梯度平衡系统至18.0min；流速为200μL/min。
可选地，所述S20中，所述串联质谱条件为：电离模式为电喷雾电离，正离子和负离子模式混合；检测方式为选择反应监测；离子源温度为80～150℃，毛细管电压为3.0～4.0kV，锥孔电压为20～40V，脱溶剂温度为300～500℃，脱溶剂气流速为500～700L/h，锥孔气流速为30～80L/h。
可选地，所述S20之后，所述S30之前还包括配制多个浓度梯度的标准溶液，然后对所述标准溶液进行定量测定并制作标准曲线；所述标准溶液包括苯并[a]芘、3-羟基苯并[a]芘，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮和苯并[a]芘-7,8-二氢二醇-9,10-环氧化物。
可选地，将所述S30中对所述处理后的待测样本测定获得的数据，代入所述标准曲线，定量分析所述待测样本中的所述苯并[a]芘和所述苯并[a]芘的代谢产物。
下面分多个实施例对本发明实施例进行进一步的说明。
实施例1
一种同时检测苯并[a]芘及其代谢产物的方法，包括以下步骤：
取血液样品(血站提供)置于4℃，10000×g离心10min，取上层血清100μL，加入10μL苯并[a]芘、3-羟基苯并[a]芘，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮和苯并[a]芘-7,8-二氢二醇-9,10-环氧化物的混标(10ppm，溶剂为超纯水)作为内标，涡旋后加入500μL的甲基叔丁基醚溶液进行液液萃取10min，收集上层液，在下层液中加入500μL的甲基叔丁基醚溶液继续进行液液萃取10min，如此重复三次，将三次所得的上层萃取液混合用氮吹仪浓缩至近干，复溶于1.0mL纯水中，得到预处理后的待测样品；
对固相萃取小柱(Supelclean LC-C18,500mg,6mL)，依次用5.0mL二氯甲烷、5.0mL甲醇、5.0mL超纯水对C18固相萃取柱进行活化，活化过程中固相萃取小 柱保持湿润不干状态。当固相萃取小柱中超纯水液面距上层筛板1mm左右时，关闭固相萃取装置，并将固相萃取小柱装满超纯水，待用。将预处理后的待测样品加入已活化的固相萃取小柱中，进行加标血样的富集，富集结束后用10.0mL超纯水冲洗样品瓶及固相萃取小柱，真空泵抽干10min。然后将富集好的固相萃取小柱进行洗脱，洗脱条件为先加入5.0mL甲醇，5.0mL二氯甲烷，分别收集洗脱液，40℃水浴氮吹，氮气流速以洗脱液液面轻微下凹为宜浓缩至近干时，加入500μL甲醇定容，混匀后采用GHP注射器过滤器(GHP Syringe Filter)(0.2μm，13mm)进行过滤，得到处理后的待测样品，待仪器分析；
处理后的待测样品通过超高效液相色谱-串联质谱联用仪(UPLC-MS/MS，沃特世(Waters)Xevo TQD)进行分析，获取多反应监测(MRM)数据，并利用保留时间锁定和特征离子对锁定的方式定性筛查样品中可能苯并[a]芘及其代谢产物；采用超高效液相色谱柱(ACQUITY UPLC)BEH C18(规格为2.1×100mm，1.7μm)，流动相：A为水，B为甲醇。梯度洗脱程序：起始比例A为50％，B为50％；梯度洗脱程序：起始比例A为50％，B为50％，维持0.5min；0.5～1.0min，A降至20％，B升至80％，维持至7.0min；7.0～7.5min，A降至0％，B升至100％，维持至12.0min；12.2min时恢复至起始梯度平衡系统至18.0min。流速为200μL/min；柱温为室温；进样体积为10.0μL；洗针液为乙腈和水混合溶液(1:1,v/v)。串联质谱条件：电离模式为电喷雾电离(ESI)，正离子和负离子模式混合；检测方式为选择反应监测(MRM)；离子源温度为120℃，毛细管电压为3.5kV，锥孔电压为30V，脱溶剂温度为380℃，脱溶剂气流速为600L/h，锥孔气流速为50L/h。然后，记录苯并[a]芘及其代谢产物的监测离子对、碰撞能等参数数据。
效果实施例
(1)苯并[a]芘及其代谢产物的监测离子对、碰撞能参数
按实施例1所述方法，获得苯并[a]芘及其代谢产物的监测离子对、碰撞能参数数据见下表1。苯并[a]芘及其代谢产物对应的提取离子流色谱图，进一步参见图2所示。
表1.苯并[a]芘及其代谢产物监测特征离子对及碰撞能参数
Figure PCTCN2020128800-appb-000001
配制不同溶度的苯并[a]芘、3-羟基苯并[a]芘，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮和苯并[a]芘-7,8-二氢二醇-9,10-环氧化物(BPDE)的标准溶液，根据定量离子对的色谱峰面积绘制标准曲线；在此基础上，对洗脱液中目标物的含量进行定量。最终获得不同目标物的回收率，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮回收率略低，约40％，其余目标物回收率均高于75％。
结果显示，本发明实施例所述方法，可以有效固相萃取苯并[a]芘及其代谢产物，获得干净的洗脱液并去除杂质的干扰；然后同时提取并检测出血清样品中含有的多种苯并[a]芘本体及其代谢产物。同时，本发明实施例所述采用保留时间锁定与特征离子对锁定的方法，可以高效、快速对样品中可能存在的苯并[a]芘本体及其代谢产物进行定性筛查和定量分析。
相比于传统的检测方法，本发明实施例所述方法在对待测样品中苯并[a]芘本体及其代谢产物的初步筛查和定量分析方面具有潜在的应用价值；特别是针对哺乳动物(包括人)体液样品，由于苯并[a]芘在进入体内会存在部分代谢的情况，单独检测苯并[a]芘，或单独针对其代谢产物的进行检测，均不能全面、准确地反映出体液样品内的苯并[a]芘的暴露水平，而本发明实施例所述方法就能很好地改善上述问题，且该方法更加高效，更加灵敏。进一步地，本发明实施例所述方法可为苯并[a]芘及其代谢产物对人体健康的影响及相关毒理学研究提供重要的参考信息。
例如，相对于气质联用方法，本发明实施例所述液质联用方法测定苯并[a]芘的代谢物不需要衍生化。另外，针对液体样品，在一定条件下(例如灵敏度足 够高)，本发明所述方法甚至可以直接进样，无需做任何前处理，而气质联用的方法则必须进行相应溶剂转换方可进样。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种同时检测苯并[a]芘及其代谢产物的方法，其特征在于，包括以下步骤：
    (1)取待测样品，对所述待测样品进行预处理，所述待测样品含有苯并[a]芘和所述苯并[a]芘的代谢产物，所述代谢产物包括3-羟基苯并[a]芘，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮和苯并[a]芘-7,8-二氢二醇-9,10-环氧化物中的一种或多种；
    (2)用活化后的C18固相萃取柱对所述预处理后的待测样品进行富集处理，然后对所述C18固相萃取柱进行洗脱，收集洗脱液；然后对所述洗脱液浓缩至近干后，定容和经过膜处理后，得到处理后的待测样品；
    (3)采用超高效液相色谱-串联质谱联用检测方式，所述处理后的待测样品经液相色谱分离和多反应监测模式检测后，利用保留时间锁定和特征离子对信息锁定，检测出所述处理后的待测样品中含有的所述苯并[a]芘和所述苯并[a]芘的代谢产物，所述特征离子对信息包括母离子和子离子。
  2. 如权利要求1所述的方法，其特征在于，所述待测样品包括哺乳动物体液样品，所述体液样品包括血液样品、尿液样品、脑脊液、组织提取液中的至少一种。
  3. 如权利要求1所述的方法，其特征在于，所述步骤(3)中，所述超高效液相色谱条件为：BEH C18色谱柱，规格为2.1mm×100mm×1.7μm，流动相为水和甲醇的混合溶液，按梯度洗脱程序洗脱；进样体积为5.0～20.0μL；洗针液为体积比1:1的乙腈和水的混合溶液。
  4. 如权利要求3所述的方法，其特征在于，所述梯度洗脱程序为：起始梯度按水：甲醇＝50：50比例洗脱，维持0.5min，然后在0.5～1.0min内，变化为水：甲醇＝20：80比例洗脱，维持至7.0min；然后在7.0～7.5min内，变化为水：甲醇＝0：100比例洗脱，维持至12.0min；然后在12.2min时恢复至所述起始梯度平衡系统至18.0min；流速为200μL/min。
  5. 如权利要求1所述的方法，其特征在于，所述步骤(3)中，所述串联质谱条件为：电离模式为电喷雾电离，正离子和负离子模式混合；检测方式为选择反应监测；离子源温度为80～150℃，毛细管电压为3.0～4.0kV，锥孔电压为20～40V，脱溶剂温度为300～500℃，脱溶剂气流速为500～700L/h，锥孔气流速为30～80L/h。
  6. 如权利要求1所述的方法，其特征在于，所述步骤(2)中，所述C18固相萃取柱的活化过程包括为：依次用5.0～10.0mL的二氯甲烷、甲醇和超纯水进行活化，所述活化过程中，所述C18固相萃取柱保持不干状态。
  7. 如权利要求1所述的方法，其特征在于，所述步骤(2)中，然后对所述C18固相萃取柱进行洗脱的具体过程包括：用5.0～20.0mL超纯水冲洗盛放所述预处理后的待测样品的容器及所述富集处理后的C18固相萃取柱，然后抽干所述富集处理后的C18固相萃取柱中残留水分；用5.0～10.0mL的甲醇和二氯甲烷进行洗脱。
  8. 如权利要求1所述的方法，其特征在于，所述步骤(2)之后，所述步骤(3)之前还包括配制多个浓度梯度的标准溶液，然后对所述标准溶液进行定量测定并制作标准曲线；所述标准溶液包括苯并[a]芘、3-羟基苯并[a]芘，苯并[a]芘-1,6-二酮、苯并[a]芘-3,6-二酮和苯并[a]芘-7,8-二氢二醇-9,10-环氧化物。
  9. 如权利要求8所述的方法，其特征在于，将步骤(3)中对所述处理后的待测样本测定获得的数据，代入所述标准曲线，定量分析所述待测样本中的所述苯并[a]芘和所述苯并[a]芘的代谢产物。
  10. 一种包含如权利要求1-9任意一项所述同时检测苯并[a]芘及其代谢产物的方法在生化分析检测或生命科学领域的应用。
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