CN110907572B - A device and method for measuring 8-isomeric prostaglandin F2α in urine - Google Patents

Abstract

The invention relates to a device and a method for measuring 8-isomeric prostaglandin F2 alpha in urine, belonging to the field of analytical chemistry. The method comprises the following steps: and after the prepared sample is injected, conveying the sample by using an enrichment pump and enriching the sample by using a graphene oxide bonded silica gel solid phase extraction column. And after the sample is enriched, switching through a six-way valve, conveying a mobile phase by using an analysis pump, eluting the solid-phase extraction column from a flow direction opposite to the sample enrichment, separating the eluted components through the analysis column, and detecting and determining the content of the components to be analyzed in the sample through a detector. The online solid phase extraction method realizes the high multiple enrichment of the sample on the enrichment column, improves the sample introduction amount by nearly three orders of magnitude under the condition that the chromatographic peak is not obviously widened, greatly improves the analysis sensitivity of the method, and can accurately determine the content of 8-isoprostane F2 alpha in pg/mL order of magnitude in the urine sample. Provides a sensitive, accurate and reliable new method for researching the correlation between smoking and oxidative stress reaction.

Description

A device and method for measuring 8-isomeric prostaglandin F2α in urine

technical field

The invention relates to a measuring device, especially a device for measuring 8-isomeric prostaglandin F2α in urine, and also relates to a measuring method, which belongs to the field of analytical instruments.

Background technique

Oxidative stress refers to the imbalance between oxidation and anti-oxidation in the body. Free radicals produce a negative effect in the body that tends to oxidize, which will lead to inflammatory infiltration of neutrophils, increased secretion of proteases, and the production of a large number of oxidized intermediates. In living organisms, free radicals and other highly reactive chemicals are difficult to measure directly due to their low concentrations and short lifetimes. Therefore, detection methods such as free radicals, active substances, oxidative stress, spin trapping, DNA markers, lipid and protein oxidative stress, cellular fluorescent probes, etc. may be possible in the qualitative detection of active substances and oxidative damage in vivo and in vitro. Errors and human influences do occur.

8-Hydroxyisomeric prostaglandin F2α is a representative important biomarker of oxidative stress, and its content plays an important role in evaluating the degree of oxidative damage in vivo and studying the pathological process of diseases.

Currently, 8-isomeric prostaglandin F2α can be analyzed by ELISA, GC-MS and HPLC/MS/MS. The HPLC-MS/MS method has good sensitivity and selectivity, and is suitable for the analysis of 8-isomeric prostaglandin F2α. Due to the low content of this compound in urine samples and the presence of other compounds with similar structures, the sample usually requires solid-phase extraction or a combination of liquid-liquid extraction-solid-phase extraction for purification, concentration and quantification; sample pretreatment The process requires multiple transfers, which is not only cumbersome to operate, but also introduces many factors of analysis errors. On the other hand, the matrix background of urine samples is complex. When HPLC/MS/MS is used for analysis, the matrix effect of the sample will also affect the analysis results.

Therefore, it is necessary to find an effective, reliable and convenient method to measure the content of 8-hydroxyisomeric prostaglandin F2α to evaluate the effect of cigarette smoke exposure on oxidative stress.

Contents of the invention

The purpose of the present invention is to provide a device and method for measuring 8-isomeric prostaglandin F2α in the urine of smokers in order to solve the deficiencies in the prior art. , Accurate measurement requirements, providing a scientific and simple new method for the evaluation of the impact of cigarette smoke exposure on oxidative stress.

Unless otherwise specified, the percentages used in the present invention are all mass percentages.

To achieve the above object, the technical scheme adopted in the present invention is as follows:

A device for measuring 8-hydroxyisomeric prostaglandin F2α in urine, the device includes a six-way valve with a sample inlet, a sampler, an enrichment pump, an analysis pump, a detector, and an online solid-phase extraction column , analytical column and chromatographic workstation; a six-way valve with an inlet is used for switching between the solid phase extraction column and the analytical column; the solid phase extraction material of the solid phase extraction is graphene oxide bonded silica gel;

When the sample is enriched, the sample to be tested enters the analysis system from the injector, is transported by the enrichment pump, enters from the seventh inlet with a six-way valve of the inlet, and flows out from the fourth outlet through the solid phase extraction column. The components to be tested are enriched on the solid-phase extraction column, and then discharged into the waste liquid through the first and sixth ports; Mouth flow;

During sample analysis, after the sample is enriched, switch through the six-way valve, the mobile phase of the enrichment pump is discharged from the fifth port of the six-way valve, and the mobile phase of the analysis pump enters from the second port of the six-way valve. Outflow through the solid-phase extraction column, and the flow direction opposite to the sample enrichment elutes the components to be analyzed enriched on the solid-phase extraction column, and flows out from the fourth and third ports, and flows through the analysis column for separation. Then, the content of the components to be analyzed in the sample is determined by detection with a detector.

A method for assaying 8-isomeric prostaglandin F2α in urine that the present invention relates to comprises the following steps:

Step (1), after the prepared sample is injected, use the enrichment pump to transport the solid phase extraction column through the graphene oxide bonded silica gel for enrichment, and continue to wash the solid phase extraction column with 2.0mL of mobile phase after enrichment;

Step (2), after the sample is enriched, switch through the six-way valve, use the analytical pump to transport the mobile phase, and elute the solid phase extraction column from the flow direction opposite to the sample enrichment, and the eluted components flow through the analytical column for separation. The content of the components to be analyzed in the sample is determined by detector detection; the sample enrichment and sample analysis of the online solid-phase extraction device are independent pipelines, which do not cross each other, and the chromatographic analysis and solid-phase extraction enrichment are carried out at the same time.

Further, in step (1), the sample preparation process is as follows:

Take 7.5mL of smoker urine sample collected, add 200μL of internal standard working solution, and dilute to 10mL with methanol; shake well, bring back to the laboratory within 20h, and centrifuge at 20000r/min for 10min at high speed; take the supernatant for about 5.0mL, filtered with a 0.25μm syringe filter, used for online solid-phase extraction enrichment.

Further, the internal standard is deuterated 8-isomeric prostaglandin F2α.

Further, the column medium is 25% methanol, after the enrichment is completed, continue to wash the sample with 1.5-4.0mL mobile phase, the flow rate of the enrichment pump is 1.0mL/min; the injection volume is 0.5-2.0mL.

Further, the ultra-high performance liquid chromatography mass spectrometry analysis conditions are as follows:

Chromatographic column: Waters ACQUITY UPLC BEH-C ₁₈ , 2.1×30mm, 1.7μm; mobile phase: 45% methanol containing 0.1% ammonium acetate, flow rate 0.6mL/min;

The mass spectrometry conditions are as follows:

The gas is high-purity nitrogen; electrospray ion source negative ion scanning; multiple reaction detection; electrospray voltage is 4500V, ion source temperature: 500°C; auxiliary gas Gas1 pressure is 60psi, auxiliary gas Gas2 pressure is 80psi, curtain gas pressure is 20psi, Collision pressure is 8psi; 8-isomeric prostaglandin F2α detection ion pair is 353.30/193.10, deuterated internal standard detection ion pair is 357.30/197.10, ion residence monitoring time is 100ms; declustering voltage: 80V, entrance voltage: 10V , Collision energy: 30V, collision cell outlet voltage: 15V.

The injector of the present invention is a liquid chromatography large-volume injector, and the injection volume is 0.01-5.0mL;

The enrichment pump is an ordinary liquid chromatography pump, which can withstand the pressure of 400bar;

The analytical pump is an ultra-high performance liquid chromatography binary pump; it can perform gradient elution; it can withstand a pressure of 1200bar;

The detector is a triple quadrupole tandem mass spectrometer, and the ionization source of the mass spectrometer is an electrospray ionization source;

The specification of the solid phase extraction column is 2.1×8mm, and the solid phase extraction material packed in the column is graphene oxide bonded silica gel (GO@SiO ₂ ), with a particle size of 5.0 μm;

The chromatographic column is Waters ACQUITY UPLC BEH-C18 (2.1×30mm, 1.7μm);

The chromatographic workstation can control the connected components at the same time, realize the online switching between the solid phase extraction column and the analytical column, and the collection of sample analysis data.

The specific analysis process is as follows:

A. Preparation of standard working solution

The standard 8-isomeric prostaglandin F2α and the internal standard d ₄ -8-isomeric prostaglandin F2α in the present invention were purchased from the United States (Cayman Chemical Co.), with a purity of ≥98%.

Firstly, the standard substance and the internal standard substance were mixed with methanol to form a standard stock solution and an internal standard stock solution with a concentration of 1.0 μg/mL, respectively.

Then the standard stock solution and the internal standard stock solution were diluted step by step with actual urine containing 25% methanol, and the concentration of the standard product was prepared as 5, 20, 100, 200, 500, 1000, 2000 pg/mL, and the concentration of the internal standard All are 200pg/mL series of standard working solutions for the working curve.

Finally, dilute the internal standard stock solution with methanol step by step to prepare an internal standard working solution with a concentration of 1.0 ng/mL.

B. Analytical sample preparation

Take 7.5mL of smoker urine sample collected, add 200μL of internal standard working solution, and dilute to 10mL with methanol; shake well, bring back to the laboratory within 20h, and centrifuge at 20000r/min for 10min at high speed; take the supernatant for about 5.0mL, filtered with a 0.25μm syringe filter, used for online solid-phase extraction enrichment. In the present invention, the deuterated 8-isomeric prostaglandin F2α is used as the internal standard, and the internal standard is added when the sample is sampled. The introduced error is brought, and the obtained analysis result is more reliable.

C. Selection of solid phase extraction column material and preparation of solid phase extraction column

The solid-phase extraction and purification of 8-isoprostane F2α generally adopts reversed-phase solid-phase extraction. In the present invention, C-18, polystyrene reversed-phase resin, graphitized carbon black balls, HILIC, graphene oxide bonded silica gel, etc. were compared According to the extraction effect of solid phase extraction materials, it is found that graphene oxide bonded silica gel has a good enrichment effect on 8-hydroxyisomeric prostaglandin F2α of polar molecules, and can selectively adsorb 8-hydroxyisomeric prostaglandin F2α. It has high dereversibility, and has a good separation and purification effect on the components with large content of carbohydrates, proteins, uric acid, urea, inorganic salts, etc. in urine samples, and can effectively avoid urine sample protein in the conventional solid phase extraction purification process. The problem of clogging the solid phase extraction column and chromatographic column due to residual impurities. In the present invention, graphene oxide bonded silica gel is selected as the solid-phase extraction material for 8-hydroxyisomeric prostaglandin F2α.

The preparation method of graphene oxide bonded silica gel: Accurately weigh 50g each of graphene and sodium nitrate, grind them thoroughly and add them into a 10L round bottom flask. Place the round-bottomed flask in an ice bath, slowly add 500 mL of 98% concentrated sulfuric acid under stirring conditions, and stir well until uniform. Then slowly add well-ground potassium permanganate powder until the color turns dark green. Then place the round bottom flask in a 40°C water bath and react for 2-3h under stirring. When the mixture becomes viscous, add 400mL of ultrapure water slowly, raise the temperature of the water bath to 90°C, and continue the reaction for 40min. Then 5 L of ultrapure water was added to the mixture, and then 300 mL of 30% hydrogen peroxide was added dropwise to continue the reaction for 2 h. After the reaction was completed, the reaction solution was poured into 10L of pure water, the precipitate was collected by centrifugation, washed with pure water until neutral, and dried in vacuum to obtain graphene oxide.

Using N,N-dicyclohexylcarbodiimide as a coupling agent, the graphene oxide-bonded silica gel was synthesized by reacting the carboxyl groups on the surface of graphene oxide with the amino groups on the surface of aminated silicon spheres. Disperse 6.0g of graphene oxide into 750mL DMF solution and ultrasonically disperse for 30min, then add 6.0g of N,N-dicyclohexylcarbodiimide and 100g of silicon amide spheres (particle size 5μm), and place in a water bath at 60°C React under rapid stirring for 24h. After the reaction, the precipitate was collected by centrifugation at 10,000 rpm for 10 min, washed with pure water and methanol several times to remove impurities, and dried at 80°C. Graphene oxide bonded silica gel was obtained.

Solid-phase extraction column packing: the obtained graphene oxide-bonded silica gel filler is packed into a stainless steel solid-phase extraction column sleeve (2.1×8mm) by a homogenate column packing method, and the solid-phase extraction column of the present invention can be obtained. The solid phase extraction column is stable under the mobile phase conditions of the method of the present invention, the adsorption and elution are reversible, and it can be used repeatedly.

D. On-line solid phase extraction enrichment conditions

Graphene oxide bonded silica gel is a reversed-phase solid-phase extraction material, generally with water-polar organic solvent (methanol, acetonitrile, ethanol, etc.) % methanol is used as the medium to pass through the column, not only the enrichment effect of 8-isomeric prostaglandin F2α is good, but also the sample purification effect is good. Therefore, the present invention selects 25% methanol medium to pass through the column, and the mobile phase of the enrichment pump to transport the sample through the solid phase extraction column is also 25% methanol. Experiments show that after the enrichment of the solid phase extraction column is completed, continuing to wash with the mobile phase can improve the purification effect of the sample, and the volume of the washing liquid in the range of 1.5-4.0mL has no effect on the recovery rate of the solid phase extraction. The present invention selects enrichment to complete Then continue to wash the sample with 2.0 mL of mobile phase.

The flow rate of the enrichment pump (that is, the flow rate at which the sample is transported through the solid-phase extraction column) has no significant impact on the analysis results, but if the flow rate is too slow, it will take a long time for sample enrichment; if the flow rate is too high, the back pressure of the chromatographic system will be large, and the solid phase The service life of the extraction material will be shortened; therefore, considering the comprehensive effect and time, the flow rate of the enrichment pump is selected to be 1.0mL/min in the present invention.

In the present invention, the method of passing the sample of actual urine standard addition (the standard addition concentration is 2.0 μg/mL) has been tested, and the extraction capacity of the solid phase extraction column has been tested. Both the internal standard and the internal standard will not leak through, and the recovery rate of the standard-added sample is above 96.7%. In actual sample analysis, the greater the sample injection volume, the higher the analysis sensitivity, but in the present invention, the sensitivity within the range of 0.5-2.0 mL can meet the requirements of actual sample analysis. Therefore, the present invention selects the sample injection volume as 0.5-2.0 mL (determined according to the actual sample situation). Since the injection volume and concentration of the actual sample analysis are much smaller than the injection volume and concentration of the extraction capacity test, in the actual sample analysis process, the injection volume of 0.5-2.0mL will not exceed the extraction capacity of the solid phase extraction column.

E. Selection of chromatographic and mass spectrometric analysis conditions

After the online solid-phase extraction enrichment is completed, the mobile phase of the analytical pump can elute the 8-isomeric prostaglandin F2α enriched on the solid-phase extraction column by switching the six-way valve, and separate it through the analytical column. Since the component 8-isomeric prostaglandin F2α to be tested in the present invention is a polar compound, Waters ACQUITY UPLC BEH-C ₁₈ (2.1×30mm, 1.7μm) ultra-high performance liquid chromatography column was selected as the analytical column. In the case of a selected chromatographic column, the mobile phase conditions of the analysis pump have been further optimized, and the results show that: using 45% methanol (containing 0.1% ammonium acetate) as the mobile phase, and a flow rate of 0.6mL/min, can The 8-isomeric prostaglandin F2α is eluted from the solid-phase extraction column, and can be well separated from the interfering components on the analytical column, and the sample analysis time is short, and the analysis cycle of each sample only needs 5.0min. Therefore, The present invention selects this mobile phase condition for use.

In the present invention, the mass spectrometry detector is API-4000 from Applied Biotechnology Corporation of the United States, and the mass spectrometry conditions are optimized through needle pump sample injection. The mass spectrometry conditions selected after optimization are: the gas is high-purity nitrogen, and the negative ion scanning of the electrospray ion source is selected for use. Multiple reaction detection; electrospray voltage is 4500V, ion source temperature: 500°C; auxiliary gas Gas1 pressure is 60psi, auxiliary gas Gas2 pressure is 80psi, curtain gas pressure is 20psi, and collision air pressure is 8psi. The detection ion pair of 8-isomeric prostaglandin F2α is 353.30/193.10, the detection ion pair of deuterated internal standard is 357.30/197.10, and the ion dwell monitoring time is 100ms. The declustering voltage (DP) was 80V, the entry voltage (EP) was 10V, the collision energy (CE) was 30V, and the collision cell exit voltage (CXP) was 15V.

Under the selected chromatographic and mass spectrometric conditions, the typical selected ion chromatograms of urine samples and internal standards are shown in Figure-2.

F. Determination of sample analysis process

On the basis of the optimization of the above conditions, the analysis process of the samples was determined. Inject 0.5-2.0 mL of the prepared sample, use the enrichment pump to use 25% methanol as the mobile phase, and transport it through the solid-phase extraction column at a flow rate of 1.0 mL/min for 2.5-4.0 min (including 2.0 mL of mobile phase washing). After the sample is enriched, switch through the six-way valve, use the analytical pump to use 45% methanol (containing 0.1% ammonium acetate) as the mobile phase, and a flow rate of 0.6mL/min to elute the solid from the flow direction opposite to the sample enrichment. Phase extraction column, the eluted components flow through the analytical column for separation (Waters ACQUITY UPLC BEH-C ₁₈ ), and then detect and determine the content of the components to be analyzed in the sample through detector detection. The chromatographic analysis time is 6.0min.

The sample enrichment and sample analysis of the online solid-phase extraction device of the present invention are independent pipelines that do not cross each other, and the chromatographic analysis and solid-phase extraction enrichment can be performed simultaneously. That is, during the chromatographic analysis of the sample, the enrichment of the next sample can be carried out. After the chromatographic analysis is completed, the solid phase extraction enrichment of the next sample is also completed at the same time, and the chromatographic analysis can be performed immediately. Compared with the conventional online solid phase extraction analysis method, the analysis time is effectively shortened.

G. Working curve, limit of detection and limit of quantitation

In order to further eliminate the interference of the matrix effect, in addition to using the isotope internal standard, the method of adding standard in the actual urine sample was also used to make the working curve. That is to add different concentration gradients of the components to be tested in the actual urine sample, and make a working curve by regression with the corresponding concentration by "subtracting the peak area of the spiked sample minus the peak area of the blank, and then dividing by the peak area of the internal standard"; During the curve process, the matrix of the working curve is completely consistent with the actual sample matrix, effectively avoiding the interference of the sample matrix. Under the selected experimental conditions, the linear regression equation is A=1.028C+0.0102, the correlation coefficient r=0.9996, and the linear relationship is good. After the standard working solution of minimum concentration is diluted again, when with signal-to-noise ratio (S/N)=3 and S/N=10, corresponding sample concentration calculates detection limit and quantitative limit, the detection limit of method among the present invention and quantification limit (injection volume 1.0mL) are respectively 0.82pg/mL and 2.4pg/mL, and the method of the present invention has very high sensitivity.

H. Recovery and precision of the method

In order to verify the precision and recovery rate of the method, add a known amount of reference substance stock solution in the actual urine sample according to the concentration range of the standard curve, and the addition amount is set to high (500pg/mL), medium (200pg/mL), low ( 80pg/mL) at 3 concentration levels, each concentration was injected and measured 7 times, and the measurement was continued for 7 days, and the recovery rate, intra-day relative standard deviation, and inter-day relative standard deviation were calculated. The intraday recovery rate of the inventive method is between 92.2%～101.5%, the intraday relative standard deviation is between 2.8～3.2%; the interday recovery rate is between 91.4%～97.5%, and the interday relative standard deviation is between 3.1～3.8% %between. The method has good recovery and precision.

Compared with prior art, the present invention has following advantage:

A. In view of the low content of 8-isoprostane F2α in urine samples and the complex matrix of urine samples, the present invention adopts on-line solid phase extraction to enrich and measure 8-isoprostane F2α, which realizes that the samples are collected on the enrichment column High-fold enrichment increases the sample injection volume by nearly three orders of magnitude without obvious broadening of the chromatographic peaks, and greatly improves the analytical sensitivity of the method, which can accurately determine pg/mL 8-isoprostane in urine samples F2α.

B. The sample enrichment and sample analysis of the online solid-phase extraction device of the present invention are independent pipelines, which do not cross each other, and the chromatographic analysis and solid-phase extraction enrichment can be carried out at the same time. That is, during the chromatographic analysis of the sample, the enrichment of the next sample can be carried out. After the chromatographic analysis is completed, the solid phase extraction enrichment of the next sample is also completed at the same time, and the chromatographic analysis can be performed immediately. Compared with the conventional online solid phase extraction analysis method, the analysis time is effectively shortened.

C. The present invention uses graphene oxide bonded silica gel for the first time to enrich the polar molecule 8-hydroxyisomeric prostaglandin F2α by solid phase extraction. The material has a good enrichment effect on 8-hydroxyisomeric prostaglandin F2α, can selectively adsorb 8-hydroxyisomeric prostaglandin F2α, has high elution reversibility, and is effective for carbohydrates, proteins, and uric acid in urine samples Components with high content such as urea, urea, and inorganic salts have good separation and purification effects, which can effectively avoid the problem of clogging solid-phase extraction columns and chromatographic columns due to residual impurities such as protein in urine samples during the conventional solid-phase extraction purification process.

D. The present invention uses deuterated 8-isomeric prostaglandin F2α as the internal standard, and the internal standard is added when the sample is sampled, which can effectively deduct the errors introduced by the processes of sample storage, transportation, pretreatment, etc., and the obtained analysis The result is more reliable.

E. The present invention uses an elution solid-phase extraction column opposite to the flow direction of sample enrichment to analyze 8-isomeric prostaglandin F2α by ultra-high performance liquid chromatography. Using the flow direction opposite to sample enrichment for elution can effectively shorten the elution path and avoid broadening of chromatographic peaks; using ultra-high performance liquid chromatography analysis can effectively shorten the analysis time of samples and improve analysis sensitivity.

F. The present invention adopts the method of adding standard in the actual urine sample to make the working curve. That is to add standard substances of the components to be tested with different concentration gradients in the actual urine sample, and make a working curve by regression with the corresponding concentration by "subtracting the peak area of the spiked sample minus the peak area of the blank, and then dividing by the peak area of the internal standard". In the process of making the working curve, the matrix of the working curve is completely consistent with the actual sample matrix, which effectively avoids the influence of the sample matrix on the ionization efficiency of the mass spectrometer, and can obtain more accurate and reliable analysis results.

Description of drawings

Fig. 1 is the schematic structural diagram of the device in the sample enrichment state of the present invention;

Fig. 2 is the structural representation of the device under the sample analysis state of the present invention;

Fig. 3 is the typical selected ion chromatogram of the urine sample and internal standard of embodiment 1, wherein: a, sample; b, internal standard.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them.

Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

As shown in Figure 1, the device of 8-hydroxy isomerism prostaglandin F2α in the measurement urine of the present embodiment, described device comprises the six-way valve 7 of band sample inlet, injector 6, enrichment pump 5, Analysis pump 2, detector 4, online solid phase extraction column 1, analysis column 3 and chromatographic workstation. The six-way valve 7 with the sample inlet is provided with seven ports, namely the first port to the seventh port.

The detector 4 is connected to one end of the analytical column 3, the other end of the analytical column 3 is connected to the third port, the analytical pump 2 is connected to the second port, the enrichment pump 5 is connected to the sampler 6, and the sampler 6 is connected to the seventh port .

The six-way valve 7 with a sample inlet is used for switching between the solid phase extraction column 1 and the analysis column 3; the solid phase extraction material of the solid phase extraction is graphene oxide bonded silica gel.

When the sample is enriched, the sample to be tested enters the device from the injector 6, is transported by the enrichment pump 5, enters from the seventh sampling port with a six-way valve of the sampling port, and flows out from the fourth port through solid phase extraction. Column 1, the components to be measured are enriched on the solid phase extraction column 1, and then discharged into the waste liquid through the first port and the sixth port; in this state, the mobile phase of the analysis pump 2 is passed by the second It enters through the fourth mouth and exits through the fourth mouth.

As shown in Figure 2, during sample analysis, after the sample is enriched, switch through the six-way valve, the mobile phase of the enrichment pump 5 is discharged from the fifth port of the six-way valve, and the mobile phase of the analysis pump 2 is discharged by the six-way valve. The second port enters, the first port flows out, flows through the solid phase extraction column 3, and the flow direction opposite to the sample enrichment elutes the components to be analyzed enriched on the solid phase extraction column 3, and flows from the fourth port and the second port. The three outlets flow out, flow through the analysis column 3 for separation, and then detect and determine the content of the components to be analyzed in the sample through the detector.

The present embodiment is based on the assay method of 8-isomer prostaglandin F2α in the urine of above-mentioned device, carries out as follows:

The test samples were urine samples from non-smokers. Take 7.5mL of smoker urine sample collected, add 200μL of internal standard working solution, and dilute to 10mL with methanol; shake well, bring back to the laboratory within 20h, and centrifuge at 20000r/min for 10min at high speed; take the supernatant for about 5.0mL, filtered with a 0.25μm syringe filter, used for online solid-phase extraction enrichment.

The sample injection volume was 2.0 mL, and after the sample injection, the enrichment pump used 25% methanol as the mobile phase at a flow rate of 1.0 mL/min to transport through the solid phase extraction column for enrichment. After the sample has completely passed through the solid phase extraction column, continue to send 2.0mL of mobile phase to wash the solid phase extraction column.

After the online solid-phase extraction enrichment and washing are completed, switch through the six-way valve, and use the analytical pump to use 45% methanol (containing 0.1% ammonium acetate) as the mobile phase at a flow rate of 0.6mL/min, from the opposite flow direction of the sample enrichment , to elute the component 8-isomeric prostaglandin F2α to be tested, and separate it through the analytical column [Waters ACQUITY UPLC BEH-C ₁₈ (2.1×30mm, 1.7μm) ultra-high performance liquid chromatography column], and through the selected mass spectrometry Condition detection. The sample analysis period is 6.0min. After the analysis, the content of 8-hydroxyisomeric prostaglandin F2α is calculated from the working curve according to the sample (peak area of the component to be tested/peak area of the internal standard). The content of 8-hydroxyisomeric prostaglandin F2α in this sample was 119.2 pg/mL.

Typical selected ion chromatograms of urine samples and internal standards are shown in Figure 3, where: a, sample; b, internal standard.

Example 2

The device of this embodiment is the same as that of Embodiment 1.

The present embodiment is based on the assay method of 8-isomer prostaglandin F2α in the urine of above-mentioned device, carries out as follows:

The test samples were urine samples from light smokers. Take 7.5mL of smoker urine sample collected, add 200μL of internal standard working solution, and dilute to 10mL with methanol; shake well, bring back to the laboratory within 20h, and centrifuge at 20000r/min for 10min at high speed; take the supernatant for about 5.0mL, filtered with a 0.25μm syringe filter, used for online solid-phase extraction enrichment.

The sample injection volume was 2.0 mL, and after the sample injection, the enrichment pump used 25% methanol as the mobile phase at a flow rate of 1.0 mL/min to transport through the solid phase extraction column for enrichment. After the sample has completely passed through the solid phase extraction column, continue to send 2.0mL of mobile phase to wash the solid phase extraction column.

After the online solid-phase extraction enrichment and washing are completed, switch through the six-way valve, and use the analytical pump to use 45% methanol (containing 0.1% ammonium acetate) as the mobile phase at a flow rate of 0.6mL/min, from the opposite flow direction of the sample enrichment , to elute the component 8-isomeric prostaglandin F2α to be tested, and separate it through the analytical column [Waters ACQUITY UPLC BEH-C ₁₈ (2.1×30mm, 1.7μm) ultra-high performance liquid chromatography column], and through the selected mass spectrometry Condition detection. The sample analysis period is 6.0min. After analysis, calculate the content of 8-hydroxyisomeric prostaglandin F2α from the working curve according to the sample (peak area of the component to be tested/peak area of the internal standard). The content of 8-hydroxyisomeric prostaglandin F2α in this sample was 164.6 pg/mL.

Example 3

The device of this embodiment is the same as that of Embodiment 1.

The present embodiment is based on the assay method of 8-isomer prostaglandin F2α in the urine of above-mentioned device, carries out as follows:

The samples tested were urine samples from moderate smokers. Take 7.5mL of smoker urine sample collected, add 200μL of internal standard working solution, and dilute to 10mL with methanol; shake well, bring back to the laboratory within 20h, and centrifuge at 20000r/min for 10min at high speed; take the supernatant for about 5.0mL, filtered with a 0.25μm syringe filter, used for online solid-phase extraction enrichment.

The sample injection volume was 1.5 mL, and after the sample injection, the enrichment pump used 25% methanol as the mobile phase at a flow rate of 1.0 mL/min to transport through the solid phase extraction column for enrichment. After the sample has completely passed through the solid phase extraction column, continue to send 2.0mL of mobile phase to wash the solid phase extraction column.

After the online solid-phase extraction enrichment and washing are completed, switch through the six-way valve, and use the analytical pump to use 45% methanol (containing 0.1% ammonium acetate) as the mobile phase at a flow rate of 0.6mL/min, from the opposite flow direction of the sample enrichment , to elute the component 8-isomeric prostaglandin F2α to be tested, and separate it through the analytical column [Waters ACQUITY UPLC BEH-C ₁₈ (2.1×30mm, 1.7μm) ultra-high performance liquid chromatography column], and through the selected mass spectrometry Condition detection. The sample analysis period is 6.0min. After the analysis, the content of 8-hydroxyisomeric prostaglandin F2α is calculated from the working curve according to the sample (peak area of the component to be tested/peak area of the internal standard). The content of 8-hydroxyisomeric prostaglandin F2α in this sample was 186.6 pg/mL.

Example 4

The device of this embodiment is the same as that of Embodiment 1.

The present embodiment is based on the assay method of 8-isomer prostaglandin F2α in the urine of above-mentioned device, carries out as follows:

The samples tested were urine samples from heavy smokers. Take 7.5mL of smoker urine sample collected, add 200μL of internal standard working solution, and dilute to 10mL with methanol; shake well, bring back to the laboratory within 20h, and centrifuge at 20000r/min for 10min at high speed; take the supernatant for about 5.0mL, filtered with a 0.25μm syringe filter, used for online solid-phase extraction enrichment.

The sample injection volume was 1.0 mL, and after the sample injection, the enrichment pump used 25% methanol as the mobile phase at a flow rate of 1.0 mL/min to transport through the solid phase extraction column for enrichment. After the sample has completely passed through the solid phase extraction column, continue to send 2.0mL of mobile phase to wash the solid phase extraction column.

After the online solid-phase extraction enrichment and washing are completed, switch through the six-way valve, and use the analytical pump to use 45% methanol (containing 0.1% ammonium acetate) as the mobile phase at a flow rate of 0.6mL/min, from the opposite flow direction of the sample enrichment , to elute the component 8-isomeric prostaglandin F2α to be tested, and separate it through the analytical column [Waters ACQUITY UPLC BEH-C ₁₈ (2.1×30mm, 1.7μm) ultra-high performance liquid chromatography column], and through the selected mass spectrometry Condition detection. The sample analysis period is 6.0min. After analysis, calculate the content of 8-hydroxyisomeric prostaglandin F2α from the working curve according to the sample (peak area of the component to be tested/peak area of the internal standard). The content of 8-hydroxyisomeric prostaglandin F2α in this sample was 240.3 pg/mL.

Summary of actual sample analysis

In addition to the situation of Examples 1-4, we analyzed 12 smoking volunteers (smoking every day: heavy 25-50 sticks, moderate 15-25 sticks, mild 5-15 sticks) 8-heterogeneous prostate according to the established method The content of F2α, and non-smokers as the control, all the results are shown in Table-1.

It can be seen from the results in Table 1 that there is a certain correlation between the 8-isomeric prostaglandin F2α in the urine of smokers and the amount of smoking. The order is: heavy smokers>moderate smokers>light smokers>non-smokers. The level of exposure to human cigarette smoke can be preliminarily judged by the determination of 8-isomeric prostaglandin F2α.

Table 1. Determination results of 8-isomeric prostaglandin F2α in the urine of smokers

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the present invention. within the scope of protection.

Claims (5)

1. An assay method for 8-isomeric prostaglandin F2α in urine, characterized in that: it is applicable to a device for measuring 8-hydroxyisomeric prostaglandin F2α in urine, including a six-way valve with a sample inlet , injector, enrichment pump, analysis pump, detector, on-line solid phase extraction column, analysis column and chromatographic workstation; six-way valve with injection port is used for switching between solid phase extraction column and analysis column; Including the following steps: Step (1), after the prepared sample is injected, use the enrichment pump to transport it through the graphene oxide bonded silica gel solid phase extraction column for enrichment, and continue to wash the solid phase extraction column with 2.0mL of mobile phase after enrichment; After sample injection, the enrichment pump uses 25% methanol as the mobile phase; Among them, when the sample is enriched, the sample to be tested enters the analysis system from the injector, is transported by the enrichment pump, enters the seventh inlet with a six-way valve for the inlet, and flows out from the fourth outlet through solid phase extraction. In this state, the mobile phase of the analysis pump enters through the second port of the six-way valve, from The fourth outflow; Step (2), after the sample is enriched, switch through the six-way valve, use the analytical pump to transport the mobile phase, and elute the solid-phase extraction column from the flow direction opposite to the sample enrichment, and the eluted components flow through the analytical column for separation. The content of the components to be analyzed in the sample is determined by detector detection; the sample enrichment and sample analysis of the online solid phase extraction device are independent pipelines, which do not cross each other, and the chromatographic analysis and solid phase extraction enrichment are carried out at the same time; the analysis pump uses 0.1 The 45% methyl alcohol of % ammonium acetate is mobile phase; Among them, when the sample is analyzed, after the sample is enriched, switch through the six-way valve, the mobile phase of the enrichment pump is discharged from the fifth port of the six-way valve, and the mobile phase of the analysis pump is entered through the second port of the six-way valve. The flow out of the first port flows through the solid phase extraction column, and the flow direction opposite to the sample enrichment elutes the enriched components to be analyzed on the solid phase extraction column, and flows out from the fourth and third ports, and flows through the analysis column After separation, the content of the components to be analyzed in the sample is determined by detector detection. 2. The assay method according to claim 1, characterized in that: in step (1), the sample preparation process is as follows: Take 7.5 mL of smoker urine sample collected, add 200 µL of internal standard working solution, and dilute to 10 mL with methanol; shake well, bring back to the laboratory within 20 h, and centrifuge at 20,000 r/min for 10 min at high speed; About 5.0 mL of the supernatant was filtered with a 0.25 µm syringe filter for online solid-phase extraction enrichment. 3. The assay method according to claim 2, characterized in that: the internal standard is deuterated 8-isomeric prostaglandin F2α. 4. The assay method according to claim 1, characterized in that: after the enrichment is completed, continue to wash the sample with 1.5-4.0mL of mobile phase, the flow rate of the enrichment pump is 1.0mL/min; the injection volume is 0.5-2.0mL mL. 5. assay method according to claim 1, is characterized in that: ultra-high performance liquid chromatography mass spectrometry analysis condition is as follows: Chromatographic column: Waters ACQUITY UPLC BEH-C ₁₈ , 2.1×30mm, 1.7µm; mobile phase: 45% methanol containing 0.1% ammonium acetate, flow rate 0.6mL/min; The mass spectrometry conditions are as follows: The gas is high-purity nitrogen; electrospray ion source negative ion scanning; multiple reaction detection; electrospray voltage is 4500 V, ion source temperature: 500 ℃; auxiliary gas Gas1 pressure is 60 psi, auxiliary gas Gas2 pressure is 80 psi, curtain gas pressure 20psi, collision gas pressure 8 psi; 8-isomeric prostaglandin F2α detection transition is 353.30/193.10, deuterated internal standard detection transition is 357.30/197.10, ion dwell monitoring time is 100 ms; declustering voltage: 80 V, inlet voltage: 10 V, collision energy: 30V, collision cell outlet voltage: 15V.

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