阅读说明：本技术 一种同时检测苯并[a]芘及其代谢产物的方法及其应用 (Method for simultaneously detecting benzo [ a ] pyrene and metabolite thereof and application thereof ) 是由 罗茜 李芳� 向彬彬 于 2019-11-14 设计创作，主要内容包括：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：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；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；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,检测出待测样品中含有的所述苯并[a]芘及其代谢产物。该方法能高选择性检测苯并[a]芘及其代谢产物。本发明还提供了该方法的应用。(The invention provides a method for simultaneously detecting benzo [ a ] pyrene and metabolite thereof, which comprises the following steps: taking a sample to be detected, and pretreating the sample to be detected, wherein the sample to be detected contains benzo [ a ] pyrene and a metabolite of the benzo [ a ] pyrene; enriching the pretreated sample to be detected by using an activated C18 solid-phase extraction column, then eluting the C18 solid-phase extraction column, and collecting an eluent; concentrating to near dryness, fixing the volume and performing membrane treatment to obtain a treated sample to be detected; and detecting the benzo [ a ] pyrene and metabolites thereof contained in the sample to be detected by using retention time locking and characteristic ion pair information locking after the processed sample to be detected is subjected to liquid chromatography separation and multi-reaction monitoring mode detection in an ultra-high performance liquid chromatography-tandem mass spectrometry combined detection mode. The method can detect benzo [ a ] pyrene and its metabolite with high selectivity. The invention also provides the application of the method.)

1. A method for simultaneously detecting benzo [ a ] pyrene and metabolite thereof is characterized by comprising the following steps:

(1) taking a sample to be detected, and pretreating the sample to be detected, wherein the sample to be detected contains benzo [ a ] pyrene and metabolites of the benzo [ a ] pyrene, and the metabolites comprise one or more of 3-hydroxy benzo [ a ] pyrene, benzo [ a ] pyrene-1, 6-dione, benzo [ a ] pyrene-3, 6-dione and benzo [ a ] pyrene-7, 8-dihydrodiol-9, 10-epoxide;

(2) enriching the pretreated sample to be detected by using an activated C18 solid phase extraction column, then eluting the C18 solid phase extraction column, and collecting an eluent; concentrating the eluent to be nearly dry, performing constant volume and membrane treatment to obtain a treated sample to be detected;

(3) and detecting metabolites of benzo [ a ] pyrene and benzo [ a ] pyrene contained in the processed sample to be detected by using retention time locking and characteristic ion pair information locking after the processed sample to be detected is subjected to liquid chromatography separation and multi-reaction monitoring mode detection by adopting an ultra-performance liquid chromatography-tandem mass spectrometry combined detection mode, wherein the characteristic ion pair information comprises parent ions and daughter ions.

2. The method of claim 1, wherein the test sample comprises a mammalian bodily fluid sample comprising at least one of a blood sample, a urine sample, cerebrospinal fluid, a tissue extract.

3. The method of claim 1, wherein in step (3), the ultra high performance liquid chromatography conditions are: BEH C18 chromatographic column with specification of 2.1mm × 100mm × 1.7 μm, mixed solution of water and methanol as mobile phase, and gradient elution procedure; the sample injection volume is 5.0-20.0 mu L; the needle washing liquid is a mixed solution of acetonitrile and water in a volume ratio of 1: 1.

4. The method of claim 3, wherein the gradient elution procedure is: initial gradient as water: methanol 50: eluting at a ratio of 50, maintaining for 0.5min, and changing into water within 0.5-1.0 min: methanol 20: eluting at 80 proportion, and maintaining for 7.0 min; then changing into water within 7.0-7.5 min: methanol is 0: eluting at a ratio of 100, and maintaining for 12.0 min; then at 12.2min, the system was returned to the initial gradient equilibrium system to 18.0 min; the flow rate was 200. mu.L/min.

5. The method of claim 1, wherein in step (3), the tandem mass spectrometry conditions are: the ionization mode is electrospray ionization, and positive ions and negative ions are mixed; the detection mode is reaction monitoring; the ion source temperature is 80-150 ℃, the capillary voltage is 3.0-4.0 kV, the taper hole voltage is 20-40V, the desolventizing temperature is 300-500 ℃, the desolventizing air flow rate is 500-700L/h, and the taper hole air flow rate is 30-80L/h.

6. The method of claim 1, wherein in the step (2), the activation process of the C18 solid phase extraction column comprises: and (3) sequentially using 5.0-10.0 mL of dichloromethane, methanol and ultrapure water for activation, wherein the C18 solid-phase extraction column is kept in an undried state in the activation process.

7. The method of claim 1, wherein the specific process of eluting the C18 solid phase extraction column in step (2) comprises: washing the container containing the pretreated sample to be detected and the enriched C18 solid phase extraction column with 5.0-20.0 mL of ultrapure water, and then pumping out residual water in the enriched C18 solid phase extraction column; eluting with 5.0-10.0 mL of methanol and dichloromethane.

8. The method of claim 1, wherein after step (2) and before step (3), preparing a plurality of concentration gradient standard solutions, and then performing quantitative determination on the standard solutions and preparing a standard curve; the standard solution comprises benzo [ a ] pyrene, 3-hydroxy benzo [ a ] pyrene, benzo [ a ] pyrene-1, 6-dione, benzo [ a ] pyrene-3, 6-dione and benzo [ a ] pyrene-7, 8-dihydrodiol-9, 10-epoxide.

9. The method according to claim 8, wherein the data obtained by measuring the treated sample to be tested in the step (3) is substituted into the standard curve to quantitatively analyze the benzo [ a ] pyrene and the metabolite of the benzo [ a ] pyrene in the sample to be tested.

10. Use of a method for simultaneously detecting benzo [ a ] pyrene and its metabolites according to any one of claims 1 to 9 in biochemical analysis detection or in the field of life science.

Technical Field

The invention relates to the technical field of analysis and detection, and particularly relates to a method for simultaneously detecting benzo [ a ] pyrene and a metabolite thereof and application thereof.

Background

Benzo [ a ]]Pyrene (Benzo [ a ]]pyrene,B[a]P) of the formula C₂₀H₁₂The product is a strong carcinogen widely existing in food and environment, is derived from cooking processing, smoking, incomplete combustion of coal, petroleum and natural gas and the like, and can enter the body through food (such as fried smoke and roasted, cereals, vegetables, fat, oil and the like), breath (such as air, tobacco smoke, automobile exhaust and the like) and skin contact and other ways. B [ a ] into the body]P most of the cytochrome P450 in the body is oxidized to a series of epoxy compounds including phenols (for example, 3-hydroxybenzo [ a ]) except for a small part of the P which is excreted in the feces in the original form]Pyrene (3-OH-B [ a ]]P)), ketones (e.g. 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 (e.g. benzo [ a ])]Pyrene-7, 8-dihydrodiol (B [ a ]]P-7,8-dihydrodiol)), and further oxidations of the resulting benzo [ a ]]Pyrene-7, 8-dihydrodiol-9, 10-epoxide (B [ a ]]P-7,8-diol-9,10-epoxide, BPDE), which interact with biomolecules such as DNA, proteins and lipids to exhibit toxicity. Thus, pair B [ a]The qualitative and quantitative detection of P is significant.

At present, the human body exposure level evaluation method of BaP mainly takes 3-hydroxybenzo [ a ] pyrene in urine as a marker. Because the metabolic products in urine can only reflect the exposure condition in a short period, and the exposure of BaP is a long-term process, the prior detection means can not fully and effectively monitor the exposure level of BaP in a human body and the metabolic condition of BaP in the human body.

Disclosure of Invention

In view of the above, the invention provides a method for simultaneously detecting benzo [ a ] pyrene and its metabolite and an application thereof, and the method can be used for simultaneously detecting benzo [ a ] pyrene and its metabolite in a sample to be detected with high sensitivity and high selectivity, and accurately and comprehensively representing the exposure level of the benzo [ a ] pyrene.

In a first aspect, the invention provides a method for simultaneously detecting benzo [ a ] pyrene and its metabolite, comprising the following steps:

(1) taking a sample to be detected, and pretreating the sample to be detected, wherein the sample to be detected contains benzo [ a ] pyrene and metabolites of the benzo [ a ] pyrene, and the metabolites comprise one or more of 3-hydroxy benzo [ a ] pyrene, benzo [ a ] pyrene-1, 6-dione, benzo [ a ] pyrene-3, 6-dione and benzo [ a ] pyrene-7, 8-dihydrodiol-9, 10-epoxide;

(2) enriching the pretreated sample to be detected by using an activated C18 solid phase extraction column, then eluting the C18 solid phase extraction column, and collecting an eluent; concentrating the eluent to be nearly dry, performing constant volume and membrane treatment to obtain a treated sample to be detected;

(3) and detecting metabolites of benzo [ a ] pyrene and benzo [ a ] pyrene contained in the processed sample to be detected by using retention time locking and characteristic ion pair information locking after the processed sample to be detected is subjected to liquid chromatography separation and multi-reaction monitoring mode detection by adopting an ultra-performance liquid chromatography-tandem mass spectrometry combined detection mode, wherein the characteristic ion pair information comprises parent ions and daughter ions.

In an embodiment of the present invention, the step (3) of locking information by using retention time and characteristic ion pair means: and comparing the retention time and the characteristic ion pair information of the processed sample to be tested in a multi-reaction monitoring mode with the standard retention time and the characteristic ion pair information of the benzo [ a ] pyrene and the metabolite thereof respectively, and then determining the type of the benzo [ a ] pyrene and the metabolite thereof actually contained in the sample to be tested according to the comparison result.

Optionally, the standard retention time and characteristic ion pair information of the benzo [ a ] pyrene and its metabolites are based on measurement data of a standard solution containing benzo [ a ] pyrene and its metabolites under the same detection conditions.

In the embodiment of the invention, the processed sample to be detected can be detected by a college liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

Optionally, the sample to be tested comprises a mammalian body fluid sample comprising at least one of a blood sample, a urine sample, cerebrospinal fluid, a tissue extract. Wherein the mammal comprises a human.

Optionally, when the sample to be tested is a blood sample, the sample to be tested can be centrifuged at a high speed, and then the supernatant is taken and stored at a low temperature for later use. Wherein the pretreatment process when the blood sample is prepared comprises the following steps: and centrifuging the blood sample, collecting supernatant, extracting with an organic solvent for multiple times to obtain an extract, concentrating the extract to be nearly dry, and redissolving the extract into ultrapure water with the volume less than or equal to 1 mL.

Further, optionally, the organic solvent comprises one or more of methyl tert-butyl ether, ethyl acetate, acetonitrile HE methanol. In one embodiment, the organic solvent may be methyl tert-butyl ether.

Optionally, in the step (3), the ultra high performance liquid chromatography conditions are: BEH C18 chromatographic column with specification of 2.1mm × 100mm × 1.7 μm, mixed solution of water and methanol as mobile phase, and gradient elution procedure; the sample injection volume is 5.0-20.0 mu L; the needle washing liquid is a mixed solution of acetonitrile and water in a volume ratio of 1: 1.

Optionally, the gradient elution procedure is: initial gradient as water: methanol 50: eluting at a ratio of 50, maintaining for 0.5min, and changing into water within 0.5-1.0 min: methanol 20: eluting at 80 proportion, and maintaining for 7.0 min; then changing into water within 7.0-7.5 min: methanol is 0: eluting at a ratio of 100, and maintaining for 12.0 min; then at 12.2min, the system was returned to the initial gradient equilibrium system to 18.0 min; the flow rate was 200. mu.L/min.

Optionally, in the step (3), the tandem mass spectrometry conditions are: the ionization mode is electrospray ionization, and positive ions and negative ions are mixed; the detection mode is reaction monitoring; the ion source temperature is 120-150 ℃, the capillary voltage is 3.0-4.0 kV, the taper hole voltage is 20-40V, the desolventizing temperature is 300-500 ℃, the desolventizing air flow rate is 500-700L/h, and the taper hole air flow rate is 30-80L/h.

Optionally, in the step (2), the activation process of the C18 solid-phase extraction column includes: and (3) sequentially using 5.0-10.0 mL of dichloromethane, methanol and ultrapure water for activation, wherein the C18 solid-phase extraction column is kept in an undried state in the activation process.

Optionally, in the step (2), the specific process of eluting the C18 solid-phase extraction column comprises: washing the container containing the pretreated sample to be detected and the enriched C18 solid phase extraction column with 5.0-20.0 mL of ultrapure water, and then pumping out residual water in the enriched C18 solid phase extraction column; and eluting with 5.0-10.0 mL of methanol and dichloromethane in sequence. Wherein, the eluent can be concentrated to near dryness by high-purity nitrogen, and is directly analyzed by an instrument after redissolution.

Optionally, after the step (2), before the step (3), preparing a plurality of standard solutions with concentration gradients, and then performing quantitative determination on the standard solutions and making a standard curve; the standard solution comprises benzo [ a ] pyrene, 3-hydroxy benzo [ a ] pyrene, benzo [ a ] pyrene-1, 6-dione, benzo [ a ] pyrene-3, 6-dione and benzo [ a ] pyrene-7, 8-dihydrodiol-9, 10-epoxide.

Optionally, substituting the data obtained by measuring the processed sample to be detected in the step (3) into the standard curve, and quantitatively analyzing the benzo [ a ] pyrene and the metabolite of the benzo [ a ] pyrene in the sample to be detected.

According to the method for simultaneously detecting benzo [ a ] pyrene and metabolites thereof provided by the first aspect of the invention, an ultra-high performance liquid chromatography-tandem mass spectrometry method is adopted, so that benzo [ a ] pyrene and metabolites thereof in a sample to be detected can be detected with high sensitivity, high selectivity and high speed. Compared with the traditional detection method only aiming at benzo [ a ] pyrene alone or at the metabolite thereof alone, the method can detect the benzo [ a ] pyrene and the metabolite thereof in the sample to be detected more comprehensively, and reflect the exposure level of the benzo [ a ] pyrene of the sample to be detected more accurately. Meanwhile, the method is simple to operate and easy to develop into a standardized detection means for benzo [ a ] pyrene and metabolites thereof.

In a second aspect, the invention also provides an application of the method for simultaneously detecting benzo [ a ] pyrene and its metabolite in the fields of biochemical analysis and detection or life science.

For example, the method for simultaneously detecting benzo [ a ] pyrene and its metabolites can be used for detecting urine samples or blood samples of human bodies, evaluating the exposure level of benzo [ a ] pyrene in the samples, and the obtained detection data can serve in the fields of biochemical analysis detection or life science, or other fields.

Because benzo [ a ] pyrene is a strong carcinogen widely existing in food and environment, the benzo [ a ] pyrene seriously harms human health. Therefore, the method can comprehensively and accurately reflect the exposure level of the benzo [ a ] pyrene in the sample to be detected, has important significance for researching the exposure level of the benzo [ a ] pyrene in the human body and evaluating the human health, and can also provide important technical support for related researches of toxicology.

Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the invention.

Drawings

In order to more clearly illustrate the contents of the present invention, a detailed description thereof will be given below with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a process flow diagram of a method for simultaneously detecting benzo [ a ] pyrene and its metabolites in accordance with one embodiment of the present invention;

FIG. 2 is an ion flow chromatogram of benzo [ a ] pyrene and its metabolites provided by an embodiment of the present invention.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it should be noted that those skilled in the art can make various modifications and improvements without departing from the principle of the embodiments of the present invention, and such modifications and improvements are considered to be within the scope of the embodiments of the present invention.

The following examples are intended to illustrate the invention in more detail. The embodiments of the present invention are not limited to the following specific embodiments. The present invention can be modified and implemented as appropriate within the scope of the main claim.

Unless otherwise specified, the raw materials and other chemicals used in the examples of the present invention are commercially available.

Referring to fig. 1, an embodiment of the present invention further provides a method for simultaneously detecting benzo [ a ] pyrene and its metabolites, comprising the steps of:

s10, taking a sample to be detected, and pretreating the sample to be detected, wherein the sample to be detected contains benzo [ a ] pyrene and metabolites of the benzo [ a ] pyrene, and the metabolites comprise one or more of 3-hydroxy benzo [ a ] pyrene, benzo [ a ] pyrene-1, 6-dione, benzo [ a ] pyrene-3, 6-dione and benzo [ a ] pyrene-7, 8-dihydrodiol-9, 10-epoxide;

s20, enriching the pretreated sample to be detected by using an activated C18 solid phase extraction column, then eluting the C18 solid phase extraction column, and collecting eluent; concentrating the eluent to be nearly dry, performing constant volume and membrane treatment to obtain a treated sample to be detected;

s30, detecting metabolites of benzo [ a ] pyrene and benzo [ a ] pyrene contained in the processed sample to be detected by using retention time locking and characteristic ion pair information locking after the processed sample to be detected is detected in a liquid chromatography separation and multi-reaction monitoring mode by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry combined detection mode, wherein the characteristic ion pair information comprises parent ions and daughter ions.

Wherein in the step S10, the sample to be tested comprises a sample of a body fluid of a mammal, wherein the mammal comprises a human; the body fluid sample comprises at least one of a blood sample, a urine sample, a cerebrospinal fluid sample, and a tissue extract sample.

Optionally, when the sample to be detected is a blood sample, the pretreatment process includes: and centrifuging the blood sample, collecting supernatant, extracting for multiple times by using an organic solvent to obtain an extract, concentrating the extract to be nearly dry, and redissolving the extract into ultrapure water with the volume less than or equal to 1 mL. Wherein, the near-dry means that the volume of the extract liquid after the concentration process is ignored. For example, the volume of the extract after concentration treatment is less than 0.1 mL.

In one embodiment, the supernatant of the blood sample is extracted using methyl tert-butyl ether. In the embodiment of the invention, the benzo [ a ] pyrene and the metabolite thereof contained in the sample to be detected can be more fully extracted by adopting the methyl tert-butyl ether.

Optionally, the extract is concentrated to near dryness by nitrogen-blowing concentration. For example, the extract is concentrated using a nitrogen blower.

Optionally, in the step S20, the activation process of the C18 solid phase extraction column includes: and (3) sequentially using 5.0-10.0 mL of dichloromethane, methanol and ultrapure water for activation, wherein the C18 solid-phase extraction column is kept in an undried state in the activation process.

Optionally, in the step S20, the specific process of eluting the C18 solid phase extraction column includes: washing the container containing the pretreated sample to be detected and the enriched C18 solid phase extraction column with 5.0-20.0 mL of ultrapure water, and then pumping out residual water in the enriched C18 solid phase extraction column; eluting with 5.0-10.0 mL of methanol and dichloromethane.

Optionally, the residual water in the enriched C18 solid-phase extraction column is drained in a vacuum draining manner, and the draining time is 10-20 min.

Optionally, in S20, the eluent is concentrated to near dryness by nitrogen-blown concentration. For example, nitrogen blowing is performed by using a 40 ℃ water bath nitrogen blower, and the flow rate of nitrogen gas is always kept to make the liquid surface of the eluent in a concave state. Wherein, the near-dry means that the volume of the eluent after the concentration process is ignored. For example, the volume of the concentrated eluate is less than 0.1 mL.

Alternatively, in S20, the volume is determined by using methanol. After the volume fixing process, the membrane treatment is carried out through a filter membrane with the aperture of 0.2 mu m. Optionally, the constant volume can be but is not limited to 500-1000 μ L.

Optionally, in S30, the ultra high performance liquid chromatography conditions are: BEH C18 chromatographic column with specification of 2.1mm × 100mm × 1.7 μm, mixed solution of water and methanol as mobile phase, and gradient elution procedure; the sample injection volume is 5.0-20.0 mu L; the needle washing liquid is a mixed solution of acetonitrile and water in a volume ratio of 1: 1.

Optionally, the gradient elution procedure is: initial gradient as water: methanol 50: eluting at a ratio of 50, maintaining for 0.5min, and changing into water within 0.5-1.0 min: methanol 20: eluting at 80 proportion, and maintaining for 7.0 min; then changing into water within 7.0-7.5 min: methanol is 0: eluting at a ratio of 100, and maintaining for 12.0 min; then at 12.2min, the system was returned to the initial gradient equilibrium system to 18.0 min; the flow rate was 200. mu.L/min.

Optionally, in S20, the tandem mass spectrometry conditions are: the ionization mode is electrospray ionization, and positive ions and negative ions are mixed; the detection mode is reaction monitoring; the ion source temperature is 80-150 ℃, the capillary voltage is 3.0-4.0 kV, the taper hole voltage is 20-40V, the desolventizing temperature is 300-500 ℃, the desolventizing air flow rate is 500-700L/h, and the taper hole air flow rate is 30-80L/h.

Optionally, after the S20, the S30 further includes preparing a plurality of concentration gradient standard solutions, and then performing quantitative determination on the standard solutions and preparing a standard curve; the standard solution comprises benzo [ a ] pyrene, 3-hydroxy benzo [ a ] pyrene, benzo [ a ] pyrene-1, 6-dione, benzo [ a ] pyrene-3, 6-dione and benzo [ a ] pyrene-7, 8-dihydrodiol-9, 10-epoxide.

Optionally, substituting the data obtained by measuring the processed sample to be tested in the step S30 into the standard curve, and quantitatively analyzing the benzo [ a ] pyrene and the metabolite of the benzo [ a ] pyrene in the sample to be tested.

The following examples are intended to illustrate the invention in more detail.

Example 1

A method for simultaneously detecting benzo [ a ] pyrene and metabolite thereof comprises the following steps:

centrifuging blood sample (provided by blood station) at 4 deg.C and 10000 Xg for 10min, collecting upper layer serum 100 μ L, adding 10 μ L of mixed standard (10ppm, solvent is ultrapure water) of benzo [ a ] pyrene, 3-hydroxy benzo [ a ] pyrene, benzo [ a ] pyrene-1, 6-diketone, benzo [ a ] pyrene-3, 6-diketone and benzo [ a ] pyrene-7, 8-dihydrodiol-9, 10-epoxide as internal standard, vortexing, adding 500 μ L methyl tert-butyl ether solution for liquid-liquid extraction for 10min, collecting upper layer liquid, adding 500 μ L of methyl tert-butyl ether solution into the lower layer solution, performing liquid-liquid extraction for 10min, repeating the above steps for three times, mixing the upper layer extract obtained in the three times, concentrating the mixture by using a nitrogen blowing instrument until the mixture is nearly dry, and redissolving the mixture in 1.0mL of pure water to obtain a pretreated sample to be detected;

for the solid phase extraction cartridge (Supelclean LC-C18,500mg,6mL), the C18 solid phase extraction cartridge was activated with 5.0mL of dichloromethane, 5.0mL of methanol, and 5.0mL of ultrapure water in this order, and during the activation, the solid phase extraction cartridge was kept wet and dry. And when the surface of the ultrapure water in the solid-phase extraction small column is about 1mm away from the upper-layer sieve plate, closing the solid-phase extraction device, and filling the solid-phase extraction small column with ultrapure water for later use. And adding the pretreated sample to be detected into the activated solid phase extraction small column, enriching the labeled blood sample, washing the sample bottle and the solid phase extraction small column with 10.0mL of ultrapure water after the enrichment is finished, and pumping out for 10min by using a vacuum pump. Then eluting the enriched solid phase extraction column, wherein the elution conditions comprise that 5.0mL of methanol and 5.0mL of dichloromethane are added firstly, eluent is collected respectively, nitrogen blowing is carried out in water bath at 40 ℃, when the nitrogen flow rate is proper to be concentrated to be nearly dry because the liquid level of the eluent is slightly concave downwards, 500 mu L of methanol is added for constant volume, after uniform mixing, filtration is carried out by adopting a GHP Syringe Filter (GHP Syringe Filter) (0.2 mu m, 13mm), a processed sample to be detected is obtained, and the sample to be detected is analyzed by an instrument;

analyzing the processed sample to be detected by an ultra-high performance liquid chromatography-tandem mass spectrometry instrument (UPLC-MS/MS, Waters (Waters) Xevo TQD), acquiring multi-reaction monitoring (MRM) data, and qualitatively screening possible benzo [ a ] pyrene and metabolites thereof in the sample by using retention time locking and characteristic ion pair locking modes; using an ultra high performance liquid chromatography column (ACQUITY UPLC) BEH C18 (specification 2.1X 100mm, 1.7 μm), mobile phase: a is water and B is methanol. Gradient elution procedure: the initial proportion A is 50 percent, and the initial proportion B is 50 percent; gradient elution procedure: the initial ratio A is 50%, B is 50%, and the time is maintained for 0.5 min; 0.5-1.0 min, reducing A to 20%, increasing B to 80%, and maintaining for 7.0 min; 7.0-7.5 min, reducing A to 0%, increasing B to 100%, and maintaining for 12.0 min; at 12.2min, the system was returned to the initial gradient equilibrium system to 18.0 min. The flow rate is 200 muL/min; the column temperature is room temperature; the sample injection volume is 10.0 mu L; the needle washing solution is a mixed solution of acetonitrile and water (1:1, v/v). Tandem mass spectrometry conditions: the ionization mode is electrospray ionization (ESI), and the positive ion mode and the negative ion mode are mixed; the detection mode is selective reaction monitoring (MRM); the ion source temperature is 120 ℃, the capillary voltage is 3.5kV, the taper hole voltage is 30V, the desolventizing temperature is 380 ℃, the desolventizing airflow rate is 600L/h, and the taper hole airflow rate is 50L/h. And then recording parameter data such as monitoring ion pairs, collision energy and the like of benzo [ a ] pyrene and metabolites thereof.

Effects of the embodiment

(1) Monitoring ion pair and collision energy parameters of benzo [ a ] pyrene and metabolite thereof

The parameters of ion pairs and collision energy for monitoring benzo [ a ] pyrene and its metabolites were obtained as described in example 1 and are shown in Table 1 below. The chromatogram of the extracted ion current corresponding to benzo [ a ] pyrene and its metabolites is further shown in FIG. 2.

TABLE 1 monitoring characteristic ion-pair and collision energy parameters for benzo [ a ] pyrene and its metabolites

Preparing standard solutions of benzo [ a ] pyrene, 3-hydroxy benzo [ a ] pyrene, benzo [ a ] pyrene-1, 6-diketone, benzo [ a ] pyrene-3, 6-diketone and benzo [ a ] pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) with different solubilities, and drawing a standard curve according to the chromatographic peak area of a quantitative ion pair; on the basis, the content of the target substance in the eluate is quantified. Finally, the recovery rates of different target objects are obtained, the recovery rates of benzo [ a ] pyrene-1, 6-diketone and benzo [ a ] pyrene-3, 6-diketone are slightly lower and are about 40%, and the recovery rates of the other target objects are all higher than 75%.

The result shows that the method provided by the embodiment of the invention can effectively extract benzo [ a ] pyrene and metabolite thereof in a solid phase, obtain clean eluent and remove the interference of impurities; then simultaneously extracting and detecting a plurality of benzo [ a ] pyrene bodies and metabolites thereof contained in the serum sample. Meanwhile, the method adopting retention time locking and characteristic ion pair locking can be used for efficiently and quickly carrying out qualitative screening and quantitative analysis on benzo [ a ] pyrene bodies and metabolites thereof possibly existing in the sample.

Compared with the traditional detection method, the method disclosed by the embodiment of the invention has potential application value in the aspects of primary screening and quantitative analysis of the benzo [ a ] pyrene body and the metabolite thereof in the sample to be detected; particularly, for a body fluid sample of a mammal (including a human), because the benzo [ a ] pyrene is partially metabolized when entering the body, the exposure level of the benzo [ a ] pyrene in the body fluid sample cannot be completely and accurately reflected by singly detecting the benzo [ a ] pyrene or singly detecting metabolites of the benzo [ a ] pyrene. Furthermore, the method provided by the embodiment of the invention can provide important reference information for the influence of benzo [ a ] pyrene and its metabolites on human health and related toxicology research.

For example, the LC-MS method described in the examples herein does not require derivatization for determining metabolites of benzo [ a ] pyrene, as opposed to GC-MS. In addition, for liquid samples, under certain conditions (for example, the sensitivity is high enough), the method of the invention can even directly sample without any pretreatment, and the gas chromatography-mass spectrometry method needs to carry out corresponding solvent conversion to sample.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.