CN113419017A - Pretreatment reagent for detecting small molecular substances in biological sample - Google Patents
Pretreatment reagent for detecting small molecular substances in biological sample Download PDFInfo
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a pretreatment reagent for detecting small molecular substances in a biological sample. The reagent provided by the invention comprises a magnetic bead suspension, an activation solution and an eluent. The reagent has high extraction efficiency and high extraction recovery rate of the to-be-detected object in the biological sample, requires a small amount of samples, and can efficiently and accurately enrich the to-be-detected object in the biological sample. Compared with an immune biological magnetic bead kit, the unsealed biological magnetic bead kit has long effective period and is more stable. In addition, the matched instrument has low cost and is more beneficial to the automation of the pretreatment process.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a pretreatment reagent for detecting small molecular substances in a biological sample.
Background
Biological samples generally refer to flowers, leaves, stems, roots, seeds, etc. of plants, body fluids (e.g., urine, blood, saliva, bile, gastric juice, lymph fluid and other secretions of organisms, etc.) of animals (including humans), hair, muscle and some tissue organs (e.g., thymus, pancreas, liver, lung, brain, stomach, kidney, etc.), and various microorganisms. Because the composition of the biological sample is complex and is easy to interfere with the subsequent analysis and detection, the pretreatment of the sample is required before the analysis and detection.
Biological sample pretreatment, also known as sample preparation, sample purification, sample extraction, is an important component in the biological analysis process. For liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for clinical testing applications, it is essential that the biological sample be processed prior to analytical detection of the sample. The primary purpose of pre-sample processing is to separate one or more target analytes from other components in the sample matrix. Biological sample pre-treatment is a critical loop in the development and optimization of LC-MS/MS methods, since sample pre-treatment is a bottleneck restriction for high-throughput detection and a major source of analytical errors. Failure of LC-MS/MS methods is often due to improper sample pretreatment, resulting in suppression of target analyte signals or interference by co-presence in the biological matrix.
The biological sample has the following characteristics: firstly, the composition of a biological sample matrix is very complex, and the sample not only contains macromolecular protein and nucleic acid, but also contains micromolecular components such as fat, sugar, uric acid, urea and the like, and also contains salts such as sodium salt and the like, which can interfere the analysis and detection; secondly, the concentration of part of target analytes in the biological sample is too low, and if necessary enrichment and concentration are not carried out, the sensitivity of the instrument can not be achieved; and the biological sample is incompatible with an LC-MS/MS analysis system, and if the analysis system is not damaged by pretreatment, adverse effects such as chromatographic column blockage, ion source pollution, matrix effect generation and the like are caused. The purpose of the pretreatment of the biological sample is therefore: purifying a sample to remove interfering components (protein, phospholipid, salt and the like); matrix effect is reduced, sensitivity is improved, samples are concentrated, and mass spectrum detection signals are improved; protecting the sample introduction system and the chromatographic column; and fourthly, simplifying the analysis process and improving the detection flux. In short, performing effective sample pretreatment is a prerequisite for developing sensitive, accurate, high-throughput LC-MS/MS analysis.
At present, the pretreatment method before LC-MS/MS detection of a biological sample mainly comprises the following steps:
1. protein precipitation method (PPT): PPT is the sample pretreatment method which is earliest in application, least in time consumption, widely applied to biological analysis, has the characteristics of simplicity, rapidness and economy, and is a common or even the first-choice sample pretreatment method. Blood samples must first be deproteinized prior to LC-MS/MS analytical assays. Protein is denatured by adding protein precipitant, then mixed and centrifuged to form protein precipitate and supernatant, which is directly injected or diluted for sample detection. Common protein precipitants are: mixing organic solvent miscible with water, such as acetonitrile, methanol, ethanol, acetone, etc. in 2-3 times volume of the sample to change the hydrogen bond between and inside protein molecule for coagulation, and centrifuging to eliminate protein; neutral salt, saturated ammonium sulfate, sodium sulfate, etc to dehydrate and precipitate protein; ③ strong acid: such as 10% trichloroacetic acid, 6% perchloric acid, etc., so that the pH is lower than the isoelectric point of the protein, and insoluble salts are formed and precipitated; zinc salt, copper salt, zinc sulfate and the like, wherein the metal salt ions and protein carboxyl form insoluble salt to precipitate; enzyme, surfactin, etc. and some matters with unstable acid and firm protein combination are measured. The method is time-consuming and low in efficiency when a large number of samples are processed; PPT treated samples have low purification degree, and if proper chromatographic separation is not carried out, strong matrix effect is caused; the concentration of the sample is diluted in the treatment process, so that the method is not suitable for pretreatment of the sample with less content of the substance to be detected; the high-speed centrifuge is needed in the treatment process, the cost is high, and the automation of the whole process is not facilitated.
2. Liquid-liquid extraction (LLE): LLE uses an organic solvent immiscible with water to extract the target analyte from the aqueous phase to the organic phase. The LLE treatment by hand comprises the basic steps of adding an organic solvent, vortex mixing, centrifuging, blow-drying and recombining and the like, and the influence of the characteristics of the organic solvent, the volume of the solvent and the pH on the extraction effect is considered according to the properties of an analyte during operation, so that the solvent which has high solubility to the analyte, low boiling point, is easy to concentrate, is insoluble in water, is non-toxic, stable and is not easy to emulsify is preferably selected. Emulsification can lead to reduced extraction recovery, and is avoided by increased solvent volume, mild mixing, deproteinization, and the like. LLE has the advantages of high flexibility, relatively high selectivity, relatively low phospholipid residue, capability of concentrating analytes and the like. However, the method has complex operation steps and is time-consuming; the extraction recovery rate of the analyte with large polarity is low; the required sample amount is large; in the treatment process, a high-speed centrifuge, a nitrogen blowing device, a nitrogen generator/nitrogen steel cylinder and other devices are needed, so that the cost is high and the automation of the whole process is not facilitated.
3. Solid Phase Extraction (SPE) SPE retains and elutes target analytes based on different partition coefficients of various components of a sample matrix, and selectively removes impurities (interferents) from the sample matrix to concentrate the analytes. The SPE utilizes extraction columns or micro-porous plates to fill different types of fillers, realizes different separation modes (reverse phase, normal phase, ion exchange and the like), selectively retains analytes and enables interferents to flow out, or vice versa, and therefore the purpose of purifying samples is achieved. SPE operations typically go through activation, equilibration, loading, washing, elution steps. Wetting filler holes by adding an organic solvent; then adding a balance solvent which is miscible with the organic phase solvent to replace the organic solvent; adding the biological sample after proper treatment to an SPE small column; then adding a solvent for cleaning, and removing impurities and interferents; and finally, adding a solvent to elute the target analyte from the SPE filler, and collecting the target analyte for sample injection analysis. SPE has the characteristics of clean sample, small amount of required sample, suitability for mass spectrometry and the like. The method has complex and time-consuming operation steps and low manual operation flux; the consumable cost is high, the microporous plate is filled with 2mg, and the extraction column is filled with 30 mg; in the treatment process, equipment such as a mechanical pump or a positive pressure device (nitrogen-containing gas generator/nitrogen steel cylinder) is required, the cost is high, and the automation is not facilitated.
4. Immune biomagnetic bead method: usually, a carboxyl group, a tosyl group, or streptavidin is modified on the surface of a magnetic bead, and covalent or non-covalent coupling of an antibody is performed through the surface-modified groups, so that a magnetic bead coated with a specific antibody is formed, and separation and detection of a specific analyte antigen are realized. The principle of the magnetic particle chemiluminescence immunoassay is as follows: fluorescein Isothiocyanate (FITC) -labeled antibodies, the antigen to be tested and Alkaline Phosphatase (AP) -labeled antibodies form a complex of a "sandwich" structure. And then adding magnetic particles connected with an anti-fluorescein antibody, connecting the antigen-antibody complex to the magnetic particles through the specific binding of the anti-fluorescein antibody and fluorescein, directly precipitating in an external magnetic field, and separating the complex formed by immunoreaction from other unbound substances. After removal of the supernatant, the precipitated complex is washed and an enzymatic chemiluminescent substrate is added. The substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, and when the excited state intermediate returns to the ground state, photons are emitted to form a luminescence reaction, namely, a luminometer can be used for detecting the luminescence intensity of the reaction. The luminous intensity is in direct proportion to the content of the antigen to be detected, and the concentration of the antigen to be detected in the sample can be calculated by using a corresponding calculation method. In the application of the immunomagnetic bead method, an antibody with strong specificity to an object to be detected needs to be designed; the method is suitable for detecting macromolecular compounds, and cross interference reaction is easy to generate for small molecular substances to be detected by adopting an immunomagnetic bead method; furthermore, since antibodies are involved, the reagent kit is once unsealed, and the time for storage is very limited.
5. Molecular imprinting magnetic beads: the method comprises the steps of carrying out cross-linking polymerization on a functional monomer in the presence of a template molecule (target molecule, also called imprinting molecule) and a magnetic bead, and then eluting to remove the template molecule, thus preparing the magnetic bead coated by a polymer, wherein the polymer on the surface of the magnetic bead has a complementary structure with the target molecule on the arrangement of a three-dimensional hole and a functional group, thereby realizing the selection and separation of the target molecule. For example, the patent publication No. CN111474282A discloses a molecularly imprinted magnetic bead for extracting phosphorus-containing compounds such as organophosphorus pesticides in food samples. The preparation method of the molecularly imprinted polymer comprises the steps of mixing a proper amount of template molecules, functional monomers, magnetic beads, a cross-linking agent and an initiator according to a certain proportion, reacting the template molecules under a proper initiation condition to generate a polymer, and finally removing the template molecules by using a proper solvent. The method can generate large nonspecific adsorption when extracting a target object, is mainly used for food samples or environmental samples with simple components, and is not suitable for biological samples such as serum or plasma with complex matrix composition. In addition, when polymer particles produced by polymerization reaction are treated by a solvent, template molecules in the polymer particles are difficult to completely clean, and serious errors can be caused when a sample with low concentration is extracted by the molecular template polymer.
Therefore, the above methods have certain disadvantages in enriching small molecule targets in biological samples, and therefore, there is still a need to further develop reagents capable of efficiently and accurately enriching small molecule analytes from biological samples.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a reagent capable of efficiently and accurately enriching small molecular substances in a biological sample.
The invention relates to a pretreatment reagent for detecting small molecular substances in a biological sample, which comprises the following components: magnetic bead suspension, activating solution, resuspension, cleaning solution and eluent;
the magnetic beads in the magnetic bead suspension are magnetic beads with surface modified octadecyl groups;
the activating solution is trichloroacetic acid aqueous solution;
the eluent is a mixed solution of formic acid, methanol and water;
the heavy suspension is water, and the weight percentage of the water,
the cleaning liquid is water.
In the reagent of the present invention, each component is present independently of each other. Wherein, the magnetic particle suspension: the ferromagnetic particle suspension with the surface modified by hydrophilic or hydrophobic groups can realize the separation of magnetic particles and solution under the external magnetic field. Activating solution: a solution containing a liquid modifier. Eluent: aqueous phase or organic phase or a solution containing liquid modifier in a certain ratio of aqueous phase to organic phase. Resuspending: aqueous phase or organic phase or a solution of a certain ratio of aqueous phase to organic phase. Internal standard solution: a solution containing an internal stable isotope label or structural analog of the test agent. Cleaning solution: aqueous phase or organic phase or a solution of a certain ratio of aqueous phase and organic phase. In the present example, the blood sample is treated to detect 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin A, vitamin E and/or an immunosuppressant.
In some embodiments, the solution in the suspension of magnetic beads is purified water, wherein the density of the magnetic beads is 10 mg/mL.
In some embodiments, the volume fraction of trichloroacetic acid in the activation solution is 0.1%.
In some embodiments, the volume ratio of formic acid, methanol, and water in the eluent is 0.1:90: 10.
The pretreatment reagent further comprises a dissociation agent, wherein the dissociation agent comprises: 1.5g/L sodium dodecyl sulfate, 100g/L dimethyl sulfoxide, 50g/L ethanol, 0.05M/LTris-HCl.
In some embodiments, the pretreatment reagent of the present invention further comprises an internal standard solution. The internal standard solution is a solution of small molecular substances, and the small molecular substances comprise posaconazole, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin A, vitamin E and/or an immunosuppressant; the immunosuppressant is tacrolimus, sirolimus, everolimus, cyclosporine A, ascomycin, and sirolimus-d3Everolimus-d4And/or cyclosporins A-d12。
The invention also provides a pretreatment method for detecting small molecular substances in a biological sample, which treats the biological sample by using the pretreatment reagent.
In the embodiment of the invention, the biological sample is blood, and the small molecular substance is posaconazole, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin A, vitamin E and/or an immunosuppressive agent; the immunosuppressant is tacrolimus, sirolimus, everolimus, cyclosporine A, ascomycin, and sirolimus-d3Everolimus-d4And/or cyclosporins A-d12。
In some embodiments, the pre-processing method comprises:
activating the magnetic beads by the activating solution, and then resuspending the magnetic beads by ultrapure water to obtain a magnetic bead working solution;
mixing blood with the magnetic bead working solution, incubating and separating the magnetic beads;
after the magnetic beads are washed with ultrapure water, the magnetic beads are eluted with an eluent, and then a supernatant is taken.
In the magnetic bead working solution, the density of the magnetic beads is 10 mg/mL;
the volume ratio of the blood to the magnetic bead working solution is (10-100): 100;
the incubation is carried out at room temperature for 2-5 min;
the mass-volume ratio of the magnetic beads to the eluent is 1mg:100 mu L.
Taking the pretreatment of posaconazole in a blood sample as an example, in the pretreatment method, the density of magnetic beads in the magnetic bead working solution is 10 mg/ml; the volume ratio of the blood to the magnetic bead working solution is 1: 10; the incubation is carried out for 5min at room temperature; the mass-volume ratio of the magnetic beads to the eluent is 1mg:100 mu L.
The invention also provides a method for detecting posaconazole in blood, which comprises the following steps:
after the blood is treated by the pretreatment method, the liquid chromatogram of LC-MS/MS is used for detection;
the LC-MS/MS liquid chromatography conditions include:
the chromatographic column is a C18 column, the column temperature is 50 ℃, and the flow rate of the mobile phase is 0.4 mL/min;
the mobile phase A is an aqueous solution containing 2mM ammonium acetate and 0.1 wt% formic acid;
the mobile phase B is a methanol solution containing 2mM ammonium acetate and 0.1 wt% formic acid;
the elution procedure included:
0-1.2 min, the volume fraction of the mobile phase A is from 95% to 10%,
1.2-2.0 min, the volume fraction of the mobile phase A is 10%,
2.0-2.1 min, the volume fraction of the mobile phase A is from 10% to 95%,
2.1-3.0 min, wherein the volume fraction of the mobile phase A is 95%;
the mass spectrum detection conditions of the LC-MS/MS comprise:
the ion source is an electrospray ionization ion source and is in a positive ion mode; capillary voltage 0.8 kV; the temperature of desolventizing gas is 500 ℃; the desolventizing air flow rate is 1000L/h; the reverse blowing airflow speed is 50L/h; the desolventizing gas and the back blowing gas are nitrogen with the purity of 99.9 percent; the collision gas is argon with the purity of 99.999 percent; the scanning mode is a multi-reaction monitoring mode, the ion pair of the posaconazole is 701.4/127.0, the cone hole voltage is 70V, and the collision energy is 56 eV.
Taking the pretreatment of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in a blood sample as an example, the pretreatment method comprises the following steps:
mixing the blood sample with the analysis solution, vortexing for 1min, and dissociating at 37 deg.C for 30 min; the volume ratio of the blood sample to the analysis solution is 1: 2;
mixing the dissociated sample with the magnetic bead suspension, and incubating for 2 min; the volume ratio of the magnetic bead suspension to the blood sample is 1: 1;
the precipitate was collected by centrifugation, washed with a washing solution, and then eluted with an eluent.
In the magnetic bead working solution, the density of the magnetic beads is 1 mg/ml; the volume ratio of the blood to the magnetic bead working solution is 1: 10; the incubation is carried out for 5min at room temperature; the mass-volume ratio of the magnetic beads to the eluent is 1mg:100 mu L.
The invention also provides a detection method of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, which comprises the following steps: after the blood is treated by the pretreatment method of the invention, the liquid chromatogram of LC-MS/MS is used for detection.
The LC-MS/MS liquid chromatography conditions include:
the chromatographic column is a C18 column, the column temperature is 45 ℃, and the flow rate of the mobile phase is 0.4 mL/min;
the mobile phase A is an aqueous solution containing 0.1 weight percent of formic acid;
the mobile phase B is a methanol solution containing 0.1 wt% of formic acid;
the elution procedure included:
0-1.0 min, the volume fraction of the mobile phase A is 20 percent,
1.0-1.8 min, the volume fraction of the mobile phase A is from 20% to 10%,
1.8-2.6 min, the volume fraction of the mobile phase A is from 10% to 2%,
2.6-3.0 min, the volume fraction of the mobile phase A is 2%,
3.0-4.0 min, the volume fraction of the mobile phase A is from 2% to 20%;
the mass spectrum detection conditions of the LC-MS/MS comprise:
the ion source is an electrospray ionization ion source and is in a positive ion mode; capillary voltage 2.0 kV; the temperature of the desolventizing agent is 400 ℃; the desolventizing air flow rate is 800L/h; the reverse blowing airflow speed is 50L/h; the desolventizing gas and the back blowing gas are nitrogen with the purity of 99.9 percent; the collision gas is argon with the purity of 99.999 percent; the scanning mode is a multi-reaction monitoring mode.
Taking pretreatment of vitamin A and vitamin E in a blood sample as an example, in the pretreatment method,
in the pretreatment method, the density of magnetic beads in the magnetic bead working solution is 10 mg/ml; the volume ratio of the blood to the magnetic bead working solution is 1: 1; the incubation is carried out for 2min at room temperature; the mass-volume ratio of the magnetic beads to the elution liquid is 1mg:100 mu L.
The invention also provides a detection method of vitamin A and vitamin E, which comprises the following steps: after the blood is treated by the pretreatment method, the detection is carried out by LC-MS/MS liquid chromatography.
The LC-MS/MS liquid chromatography conditions include:
the chromatographic column is a C18 column, the column temperature is 45 ℃, the sample injection temperature is 8 ℃, and the flow rate of the mobile phase is 0.4 mL/min;
the mobile phase A is an aqueous solution containing 0.1 weight percent of formic acid;
the mobile phase B is a methanol solution containing 0.1 wt% of formic acid;
the elution procedure included:
0-0.4 min, the volume fraction of the mobile phase A is from 15% to 5%,
0.4-2 min, the volume fraction of the mobile phase A is from 5% to 0%,
2-2.7 min, the volume fraction of the mobile phase A is 0 percent,
2.7-3.3 min, wherein the volume fraction of the mobile phase A is from 0% to 15%;
the mass spectrum detection conditions of the LC-MS/MS comprise:
the ion source is an electrospray ionization ion source and is in a positive ion mode; capillary voltage 3.0 kV; the temperature of the desolventizing agent is 600 ℃; the desolventizing gas flow rate is 1100L/h; the reverse blowing airflow speed is 50L/h; the desolventizing gas and the back flushing gas are nitrogen with the purity of 99.9 percent; the collision gas is argon with the purity of 99.999 percent; the scanning mode is a multi-reaction monitoring mode.
Taking the pretreatment of an immunosuppressant in a blood sample as an example, in the pretreatment method,
the immunosuppressant is tacrolimus, sirolimus, everolimus, cyclosporine A, ascomycin, and sirolimus-d3Everolimus-d4And/or cyclosporins A-d12。
The invention also provides a detection method of the immunosuppressant, which comprises the following steps: after the blood is treated by the pretreatment method of the invention, the liquid chromatogram of LC-MS/MS is used for detection.
The LC-MS/MS liquid chromatography conditions include:
the chromatographic column is a C18 column, the column temperature is 50 ℃, the sample injection temperature is 2-8 ℃, and the flow rate of the mobile phase is 0.5 mL/min;
the mobile phase A is an aqueous solution containing 2mM ammonium acetate and 0.1 wt% formic acid;
the mobile phase B is a methanol solution containing 2mM ammonium acetate and 0.1 wt% formic acid;
the elution procedure included:
0-0.2 min, the volume fraction of the mobile phase A is 50%,
0.2-1 min, the volume fraction of the mobile phase A is from 50% to 0%,
1-2 min, the volume fraction of the mobile phase A is 0 percent,
2-2.1 min, the volume fraction of the mobile phase A is from 0% to 50%,
2.1-3 min, wherein the volume fraction of the mobile phase A is 50%;
the mass spectrum detection conditions of the LC-MS/MS comprise:
the ion source is an electrospray ionization ion source and a positive ion mode, and the scanning mode is a multi-reaction monitoring mode; the ion source parameters include: ionization voltage is 5.5kV, temperature is 550 ℃, gas curtain gas is 35psi, collision gas is 7, spraying gas is 50psi, auxiliary heating gas is 50psi, and an MRM ion channel adopts ammonia adduct ions.
The reagent provided by the invention comprises a magnetic bead suspension, an activation solution and an eluent. The reagent has high extraction efficiency and high extraction recovery rate of the to-be-detected object in the biological sample, requires a small amount of samples, and can efficiently and accurately enrich the to-be-detected object in the biological sample. Compared with an immune biological magnetic bead kit, the effective period after unsealing is long and the stability is higher. In addition, the matched instrument has low cost and is more beneficial to the automation of the pretreatment process.
Drawings
FIG. 1 shows a linear regression equation for the detection of posaconazole in 4 batches;
FIG. 2 shows the 25-hydroxyvitamin D2 working curve and the 25-hydroxyvitamin D3 working curve;
fig. 3 shows the working curves for vitamin a and vitamin E.
Detailed Description
The invention provides a pretreatment reagent for detecting small molecular substances in a biological sample, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein may be made and utilized without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1
1. Pretreatment kit for extracting posaconazole in serum/plasma
Magnetic microparticle suspension (Dynabeads RPC18, particle size 1 micron): the surface of the suspension of ferromagnetic particles modified by octadecyl has the concentration of 10mg/mL (1mL), and the separation of the magnetic particles and the solution can be realized under the external magnetic field.
Activation solution (0.1% trichloroacetic acid): to 2mL of ultrapure water, 2. mu.L of trichloroacetic acid was added to prepare a 0.1% trichloroacetic acid solution.
Resuspending: ultrapure water, 3 mL.
Internal standard solution: 10 mu L of posaconazole-d 4 standard liquid (1mg/mL) is transferred, 4.92mL of 50% methanol water is added, and internal standard liquid is obtained after uniform mixing.
Cleaning solution: ultrapure water, 30 mL.
Eluent (90% methanol water with 0.1% formic acid): 9000. mu.L acetonitrile was added to 1000. mu.L ultrapure water, 10. mu.L formic acid was added thereto, and the mixture was mixed well.
2. Use steps and recovery rate of posaconazole pretreatment kit
1mL of magnetic particle suspension was taken in an EP tube and placed in a magnetic particle separator containing a magnet to hold the magnetic particles on the tube wall. The supernatant liquid was removed with a pipette and the EP tube was removed from the separator. 1mL of the activation solution was then added to the EP tube and the magnetic microparticles were resuspended by multiple pipetting. The magnet was again introduced and the activating solution was removed. Then the magnetic beads are resuspended in 2.5mL of resuspension liquid to obtain magnetic particle working solution.
Another 1.5mL EP tube was taken for sample preparation: add 10. mu.L of internal standard solution and 10. mu.L of serum sample (concentration of posaconazole 1 mg/L). After mixing evenly, 100 mul of prepared magnetic particle working solution is added. After mixing, incubation was carried out for 5 minutes at room temperature, the magnet was reintroduced, and the sample matrix in the supernatant was removed. The magnet was removed and 500. mu.L of washing solution was added to wash the beads. The magnetic beads were again immobilized under magnetic field and the supernatant removed. Finally, 100. mu.L of eluent was added to resuspend the beads. After the magnetic beads are fixed, taking the supernatant to carry out detection in an LC-MS/MS MRM mode, and recording the peak area of posaconazole. For comparison, pure solvents of posaconazole at the same concentration (no extraction required) were also analyzed and the peak areas were recorded.
Liquid chromatography detection conditions of LC-MS/MS: the chromatographic column is a C18 column, the column temperature is 50 ℃, and the flow rate of the mobile phase is 0.4 mL/min.
Mobile phase A: 0.1% formic acid solution containing 2mM ammonium acetate,
mobile phase B: 0.1% formic acid methanol with 2mM ammonium acetate;
0-1.2 min, the volume fraction of the mobile phase A is from 95% to 10%,
1.2-2.0 min, the volume fraction of the mobile phase A is 10%,
2.0-2.1 min, the volume fraction of the mobile phase A is from 10% to 95%,
2.1-3.0 min, wherein the volume fraction of the mobile phase A is 95%;
the liquid chromatograph is ACQUITY UPLC I-Class (Watt).
The mass spectrum detection conditions of the LC-MS/MS are as follows: the ion source is an electrospray ionization ion source (ESI, positive ion mode); capillary voltage 0.8 kV; the temperature of desolventizing gas is 500 ℃; the desolventizing air flow rate is 1000L/h; the reverse blowing airflow speed is 50L/h; the desolventizing gas and the back blowing gas are nitrogen with the purity of 99.9 percent; the collision gas is argon with the purity of 99.999 percent; the scanning mode is a multi-reaction monitoring mode, the ion pair of the posaconazole is 701.4/127.0, the cone hole voltage is 70V, and the collision energy is 56 eV. The liquid chromatograph is ACQUITY UPLC I-Class (Watt).
The result shows that the peak area difference between the pure posaconazole solution and the serum extracting solution is less than 5%, which indicates that the extraction recovery rate is high based on the magnetic bead pretreatment method. In addition, it can be shown that 40 μ g of magnetic particles is sufficient to completely extract the analyte from 1 μ L of serum, whereas the column-based solid phase extraction protocol typically extracts 1 μ L of sample with 1mg of packing, and therefore the extraction efficiency of the magnetic bead-based pretreatment method proposed by the present invention is higher.
3. Linear verification of posaconazole pretreatment kit
A set of posaconazole serum calibrators (6 concentration levels) were prepared at concentrations of 0.2. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 5. mu.g/mL, and 10. mu.g/mL, respectively. Four batches of calibrator were processed in parallel using the pretreatment kit of example 1 (pretreatment procedure same as above), and the extracted solution was subjected to LC-MS/MS detection (detection conditions same as above). In the concentration of posaconazoleAnd (3) performing linear regression on the abscissa by taking the ratio of the peak area of the posaconazole to the peak area of an internal standard (posaconazole-d 4) thereof as the ordinate to obtain a linear regression equation (namely a standard curve) of the posaconazole, wherein the linear regression equations of 4 batches are shown in the following table, and the fitted standard curve is shown in attached figures 1-3. Linear correlation coefficient R in all batches2All are more than 0.999, and the linearity is good.
| Batches of | Linear regression equation | Coefficient of correlation (R)2) |
| 1 | y＝1.3093×x+0.00508921 | 0.999203 |
| 2 | y＝1.41915×x+0.0111105 | 0.999542 |
| 3 | y＝1.34366×x+0.0127576 | 0.999640 |
| 4 | y＝1.21213×x+0.0134247 | 0.999809 |
4. Accuracy evaluation of pretreatment kit for antifungal drugs
And detecting the posaconazole serum quality control substance according to the detection method, wherein the concentration of the low-concentration posaconazole serum quality control substance is 0.85 mu g/mL, and the concentration of the high-concentration posaconazole serum quality control substance is 4.4 mu g/mL. The pretreatment kit is used for carrying out parallel three-batch treatment according to the method and then carrying out LC-MS/MS detection. And calculating the concentration and accuracy of each compound in the quality control product according to the ratio of the area of the posaconazole peak in the quality control product to the area of the isotope internal standard substance peak and the linear regression equation of posaconazole, wherein the specific data are shown in the following table.
As can be seen from the table, the accuracy of the low-concentration and high-concentration quality control products of the three batches is between 97.9% and 99.1%, which shows that the accuracy and the repeatability of the pretreatment method are high.
5. Influence of different magnetic bead working solution concentrations on extraction rate of posaconazole
Respectively preparing magnetic bead working solutions of 0.5mg/ml, 0.8mg/ml, 1.2mg/ml, 2mg/ml, 4mg/ml and 5mg/ml from 10mg/ml magnetic bead suspensions, and extracting posaconazole in serum by using the prepared magnetic bead working solutions with different concentrations, wherein the extraction rates and CVs of the magnetic bead working solutions with different concentrations are shown in the following table. As can be seen from the table, the magnetic bead working solution concentration of 4mg/ml has the highest extraction rate and the best effect.
| Concentration of magnetic beads (mg/ml) | 0.5 | 0.8 | 1.2 | 2 | 4 | 5 |
| Extraction ratio/% | 57.4 | 58.6 | 56.5 | 66.8 | 95.4 | 80.3 |
| CV/％ | 2.8 | 2.6 | 5.0 | 2.3 | 1.8 | 3.9 |
6. Comparison of extraction rates of posaconazole by different group-modified magnetic beads
Respectively using magnetic beads modified by amino, carboxyl, tosyl and octadecyl (the particle diameter is 1 micron, the concentration of the working solution of the magnetic beads is 4mg/ml) to extract the posaconazole in the serum, wherein the magnetic bead treatment step and the pretreatment step are the same as above, and the results are shown in the following table
| Magnetic bead modifying group | Amino group | Carboxyl group | Tosyl radical | Octadecyl radical |
| Extraction ratio/% | 1.4 | 0.5 | 0.4 | 95.0 |
| CV/％ | 106 | 137 | 145 | 2.3 |
As can be seen from the table, the extraction rate of the magnetic beads modified by octadecyl groups is the highest, while the extraction rate of the magnetic beads modified by other groups is almost zero.
7. Comparison of extraction rates of posaconazole by eluents of different proportions
When posaconazole samples are extracted, 10% -100% methanol water (containing 0.1% formic acid) eluent is respectively used for eluting octadecyl magnetic beads, and the influence of different proportions of the eluents on the extraction rate is calculated, and the results are shown in the following table.
| Methanol ratio | 10％ | 20％ | 30％ | 40％ | 50％ | 60％ | 70％ | 80％ | 90％ |
| The extraction rate% | 6.3 | 10.2 | 12.6 | 32.5 | 43.8 | 70.6 | 73.2 | 89.7 | 95.8 |
As is clear from the table, the highest extraction rate was obtained by elution with 90% methanol water (containing 0.1% formic acid).
Example 2
1. Pretreatment kit for extracting 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 from serum
Magnetic microparticle suspension (Dynabeads RPC18, particle size 1 micron): the surface of the suspension of ferromagnetic particles modified by octadecyl has the concentration of 10mg/mL (1mL), and the separation of the magnetic particles and the solution can be realized under the external magnetic field.
A dissociating agent: a buffer containing dimethyl sulfoxide; weighing 1.5g of sodium dodecyl sulfate, adding a small amount of 0.05M Tris-HCl buffer solution, dissolving, weighing 100g of dimethyl sulfoxide and 50g of ethanol, placing in a 1L volumetric flask, and fixing the volume with 0.05M Tris-HCl buffer solution.
Activation solution (0.1% trichloroacetic acid): to 2mL of ultrapure water, 2. mu.L of trichloroacetic acid was added to prepare a 0.1% trichloroacetic acid solution.
Resuspending: ultrapure water, 3 mL.
Internal standard solution: 500ng/mL of 25-hydroxyvitamin D2-D3 and 500ng/mL of 25-hydroxyvitamin D3-D3.
Cleaning solution: ultrapure water, 30 mL.
Eluent (90% methanol water with 0.1% formic acid): 9000. mu.L acetonitrile was added to 1000. mu.L ultrapure water, 10. mu.L formic acid was added thereto, and the mixture was mixed well.
2. Use procedure of pretreatment kit for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
Step one, dissociation of 25-hydroxy vitamin D in a sample
1) Taking 100 mu L of blood sample/calibrator/quality control product, 10 mu L of internal standard solution and 200 mu L of dissociation agent, and uniformly mixing in a sample tube by vortex for 1 min;
2) and (3) putting the mixed solution into an environment with the temperature of 37 ℃ for dissociation for 30 min.
Step two, 25-hydroxy vitamin D enrichment purification
1) After dissociation, adding 100 mu L of magnetic particle suspension into the mixed solution, uniformly mixing and then incubating for 2 min;
2) after removing the solution, 500. mu.L of washing solution was added, mixed well and washed.
Step three, eluting and redissolving 25-hydroxy vitamin D
1) After washing, the solution was removed, 100. mu.L of eluent was added and mixed well.
Step four, transfer detection
And transferring the solution into a microporous plate, and detecting by using a liquid chromatography-tandem mass spectrometer.
Step five, result processing
1) And fitting a calibration curve according to the corresponding relation between the peak area ratio of 25-hydroxyvitamin D2 to 25-hydroxyvitamin D2-D3, the peak area ratio of 25-hydroxyvitamin D3 to 25-hydroxyvitamin D3-D3 in the calibrator and the concentration of 25-hydroxyvitamin D2 and the concentration of 25-hydroxyvitamin D3 in the calibrator.
And obtaining the contents of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the blood sample to be detected according to the peak area ratio of 25-hydroxyvitamin D2 to 25-hydroxyvitamin D2-D3 and the peak area ratio of 25-hydroxyvitamin D3 to 25-hydroxyvitamin D3-D3 in the blood sample to be detected.
Wherein, the chromatographic conditions for the liquid chromatography-tandem mass spectrometry (Waters I-Class TQD) detection are as follows: a chromatographic column: octadecyl (C18) 2.1X 50mm, 2.6 μm; the mobile phase A is an aqueous solution containing 0.1 wt% of formic acid; the mobile phase B is a methanol solution containing 0.1 wt% of formic acid; column temperature: 45 ℃; the sample amount is 20 mu L, the detection time is 4min, a gradient elution program is adopted, and the gradient elution parameters are as follows:
| time (min) | Flow rate (mL/min) | Mobile phase A volume ratio% | Mobile phase B volume ratio% |
| 0 | 0.4 | 20 | 80 |
| 1.00 | 0.4 | 20 | 80 |
| 1.80 | 0.4 | 10 | 90 |
| 2.60 | 0.4 | 2 | 98 |
| 3.00 | 0.4 | 2 | 98 |
| 4.00 | 0.4 | 20 | 80 |
Wherein, the mass spectrum conditions in the liquid chromatogram-tandem mass spectrometry (Waters I-Class TQD) detection are as follows: ionization mode: electrospray ionization source (ESI), positive ion mode; capillary voltage 2.0 Kv; desolventizing agent temperature: 400 ℃; desolventizing agent gas flow: 800L/h; an acquisition mode: multiple Reaction Monitoring (MRM) mode. The specific ion parameters are as follows:
3. linearity of pretreatment kits for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
As shown in FIG. 2, the working curve of the test substance is shown, the abscissa represents the concentration of the test substance, and the ordinate represents the peak area ratio of the test substance to the peak area of the corresponding internal standard, so that a good linear relationship can be seen, wherein, the 25-hydroxy groupThe linear relation of the vitamin D2 is that y is 0.0107x +0.00294, the linear coefficient r is 0.9995, and r is20.9990; the linear relation of the 25-hydroxy vitamin D3 is that y is 0.0260x-0.000193, the linear coefficient r is 0.9995, r2＝0.9990。
| Compound (I) | Linear relation | Coefficient of linearity r | Linear coefficient r2 |
| 25-hydroxy vitamin D2 | y＝0.0107x+0.00294 | r＝0.9995 | r2＝0.9990 |
| 25-hydroxy vitamin D3 | y＝0.0260x-0.000193 | r＝0.9995 | r2＝0.9990 |
4. Accuracy evaluation of pretreatment kit for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
Detecting corresponding serum quality control products according to the detection method, and performing LC-MS/MS detection after the pretreatment kit performs parallel three-batch treatment according to the method. And calculating the concentration and accuracy of each compound in the quality control product according to the ratio of the peak area of the substance to be detected in the quality control product to the peak area of the isotope internal standard substance and a linear regression equation of the substance to be detected, wherein specific data are shown in the following table.
5. Influence of different magnetic bead working solution concentrations on extraction rates of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
0.5mg/ml, 1.0/ml, 2.0/ml, 4.0ml, 5.0ml and 6.0ml of magnetic bead working solution are respectively prepared from 10mg/ml magnetic bead suspension, and the prepared magnetic bead working solution with different concentrations is used for extracting 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in serum, wherein the extraction rates of the magnetic bead working solution with different concentrations are shown in the following table. As can be seen from the table, the magnetic bead working solution concentration of 4mg/ml has the highest extraction rate and the best effect.
6. Comparison of extraction rates of different groups of modified magnetic beads on 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum were extracted using magnetic beads modified with amino groups, carboxyl groups, tosyl groups, and octadecyl groups (particle size 1 μm, concentration of magnetic bead working solution 4mg/ml), respectively, and the magnetic bead treatment step and pretreatment step were as described above, and the results are shown in the following table. As can be seen from the table, the extraction rate of the magnetic beads modified by octadecyl groups is the highest, while the extraction rate of the magnetic beads modified by other groups is almost zero.
Example 3
1. Pretreatment kit for extracting vitamin A and vitamin E in serum
Magnetic microparticle suspension (Dynabeads RPC18, particle size 1 micron): the surface of the suspension of ferromagnetic particles modified by octadecyl has the concentration of 10mg/mL (1mL), and the separation of the magnetic particles and the solution can be realized under the external magnetic field.
Activation solution (0.1% trichloroacetic acid): to 2mL of ultrapure water, 2. mu.L of trichloroacetic acid was added to prepare a 0.1% trichloroacetic acid solution.
Resuspending: ultrapure water, 3 mL.
Internal standard solution: mu.L vitamin A-D6 (50. mu.g/mL, 50% methanol in water (v/v)) and 50. mu.L vitamin E-D6 (50. mu.g/mL, 50% methanol in water (v/v)) were mixed.
Cleaning solution: ultrapure water, 30 mL.
Eluent (90% methanol water with 0.1% formic acid): 9000. mu.L acetonitrile was added to 1000. mu.L ultrapure water, 10. mu.L formic acid was added thereto, and the mixture was mixed well.
2. Use steps of pretreatment kit for vitamin A and vitamin E
Enrichment and purification: putting 100 mu L of sample into a 1.5mL centrifuge tube, adding 10 mu L of internal standard working solution, mixing uniformly, then adding 100 mu L of magnetic bead solution, mixing uniformly, incubating for 2min, putting the centrifuge tube on a magnetic frame, standing for 1min, and removing the solution by a pipettor;
washing: adding 500 μ L of distilled water, mixing, placing the centrifuge tube on the magnetic frame again, standing for 1min, removing distilled water with a pipettor, adding 500 μ L of distilled water again, mixing, placing the centrifuge tube on the magnetic frame again, standing for 1min, removing distilled water with a pipettor;
and (3) elution: adding 100 μ L90% methanol water (containing 0.1% formic acid), mixing, placing the centrifuge tube on magnetic frame, standing for 1min, sucking eluate with pipette into 96-well plate, and performing detection on the machine.
The instrument comprises the following steps: liquid phase: waters ACQUITY UPLC I-Class; mass spectrum: waters
TQD。
Mass spectrum conditions: ion source mode: ESI +; and (3) data acquisition mode: multiple Reaction Monitoring (MRM); capillary voltage: 3.0 kV; desolventizing temperature: 600 ℃; desolventizing gas flow: 1100L/Hr. The ion pair parameters for vitamin A/E are shown in the table below.
| Compound (I) | Parent ion | Daughter ions | Taper hole energy (V) | Collision energy (V) |
| Vitamin A | 269.2 | 92.9 | 24 | 20 |
| Vitamin A-D6 | 275.2 | 96.0 | 26 | 23 |
| Vitamin E | 431.4 | 165.1 | 30 | 15 |
| Vitamin E-D6 | 437.4 | 171.1 | 44 | 20 |
Liquid phase conditions: a chromatographic column: kinetex C18(2.6 μm, 2.1X 50mm), mobile phase A being an aqueous solution containing 0.1% by weight of formic acid; the mobile phase B is a methanol solution containing 0.1 wt% of formic acid;
column temperature: 45 ℃, sample introduction amount of 10 μ L, sample introduction temperature: 8 ℃, flow rate: 0.4 mL/min; a gradient elution procedure was used, as shown in the table below.
| Time (min) | A(V，％) | B(V，％) |
| 0 | 15 | 85 |
| 0.4 | 5 | 95 |
| 2.0 | 0 | 100 |
| 2.7 | 0 | 100 |
| 3.3 | 15 | 85 |
Data processing: and fitting a calibration curve by taking the concentration of the calibrator as the abscissa and the area of the calibrator/the area of the internal standard as the ordinate, and substituting the area of the substance in the serum to be detected/the area of the internal standard into the calibration curve so as to obtain the content of the vitamin A/E in the substance to be detected.
3. Linearity of vitamin a and E pretreatment kit
As shown in the figure, the graph is a working curve of the detection object, the abscissa represents the concentration of the detection object, and the ordinate represents the peak area ratio of the detection object to the peak area of the corresponding internal standard, so that a good linear relation can be seen.
| Detecting substance | Linear range | Calibration curve | Correlation coefficient R2 |
| Vitamin A | 0.01-5μg/ml | y＝39.6346*X+0.0796237 | 0.999641 |
| Vitamin E | 0.1-50μg/mL | y＝1.07878*X+0.0193714 | 0.995599 |
4. Accuracy evaluation of vitamin A and vitamin E pretreatment kit
Detecting corresponding serum quality control products according to the detection method, and performing LC-MS/MS detection after the pretreatment kit performs parallel three-batch treatment according to the method. And calculating the concentration and accuracy of each compound in the quality control product according to the ratio of the peak area of the substance to be detected in the quality control product to the peak area of the isotope internal standard substance and a linear regression equation of the substance to be detected, wherein specific data are shown in the following table.
5. Influence of concentration of magnetic bead working solution on extraction rate of vitamin A and vitamin E
Respectively preparing magnetic bead working solutions of 0.5mg/ml, 0.8mg/ml, 1.2mg/ml, 2mg/ml, 4mg/ml and 5mg/ml from 10mg/ml magnetic bead suspensions, and extracting the sample in the serum by using the prepared magnetic bead working solutions with different concentrations, wherein the extraction rates and CVs of the magnetic bead working solutions with different concentrations are shown in the following table. As can be seen from the table, the magnetic bead working solution concentration of 4mg/ml has the highest extraction rate and the best effect.
6. Comparison of extraction rates of different modifying group magnetic beads on vitamin A and vitamin E
The method comprises the following steps of respectively extracting vitamin A and vitamin E in serum by using magnetic beads modified by different groups (the particle size is 1 micron, the concentration of a magnetic bead working solution is 4mg/ml), and performing magnetic bead treatment and pretreatment in the same way, wherein the results are shown in the following table. As can be seen from the table, the extraction rate of the magnetic beads modified with octadecyl groups was the highest, while the extraction of the magnetic beads modified with other groups was almost none.
Example 4
1. Use procedure of immunosuppressant pretreatment kit
Respectively adding an internal standard solution and a treated hydrophobic base magnetic bead solution into a whole blood sample to be detected, oscillating and incubating, then carrying out magnetic separation, then washing by using a magnetic bead cleaning solution, carrying out magnetic separation again after washing, then adding an eluent for elution, obtaining a supernatant after magnetic separation, and finishing the sample treatment process. And finally, the supernatant enters a liquid chromatography tandem mass spectrum to finish the final detection.
The first step is as follows: putting 100 mu L of calibrator/quality control sample/whole blood sample to be tested into a 1.5mL centrifuge tube/glass bottle;
the second step is that: adding 10 mu L of internal standard solution, mixing at 1500rpm by an oscillator for 1min in a vortex manner to fully mix the internal standards;
the third step: adding 100 mu L of magnetic bead solution, mixing at 1500rpm with an oscillator in a vortex manner for 1min, and incubating for 2min to ensure that the magnetic beads are fully combined to extract a target substance;
the fourth step: placing on a magnetic frame, performing magnetic separation, and sucking and discarding liquid by a pipette;
the fifth step: adding 500 mu L of magnetic bead cleaning solution into a 1.5mL centrifuge tube/glass bottle, and mixing for 1min by a vortex of an oscillator;
and a sixth step: placing on a magnetic frame, performing magnetic separation, and sucking and discarding liquid by a pipette;
the seventh step: repeating the fifth step and the sixth step to fully clean and remove impurities in the sample;
eighth step: adding 100 μ L of magnetic bead eluent, and mixing with vortex for 1 min;
the ninth step: and placing the sample on a magnetic frame for magnetic separation, and sucking liquid into a sample injection vial by a pipette gun for liquid chromatography tandem mass spectrometry detection.
Liquid phase system: AB SCIEX
HPLC
And (3) analyzing the column:
c18 (2.1X 50mm, 2.6 μm) column
Mobile phase: a: 2mM ammonium acetate + 0.1% formic acid + water B: 2mM ammonium acetate + 0.1% formic acid + methanol
Flow rate: 0.5ml/min
Column temperature: 50 deg.C
Sample chamber temperature: 2-8 deg.C
Sample introduction volume: 10 μ l
The gradient elution procedure is shown in the table:
| time (min) | Flow rate (mL/min) | Mobile phase A | Mobile phase B |
| 0.00 | 0.5 | 50 | 50 |
| 0.20 | 0.5 | 50 | 50 |
| 1.00 | 0.5 | 0 | 100 |
| 2.00 | 0.5 | 0 | 100 |
| 2.10 | 0.5 | 50 | 50 |
| 3.00 | 0.5 | 50 | 50 |
The mass spectrometry method comprises the following steps: positive ion MRM scan analysis, using electrospray ion source (ESI), includes ion source parameters and MRM ion channels. The ion source parameters comprise ionization voltage and temperature, air curtain gas, collision gas, spray gas and auxiliary heating gas, and the specific information is shown in the table:
| ionization voltage (V) | 5500 |
| Temperature (. degree.C.) | 550 |
| Air curtain gas (psi) | 35 |
| Air for collision | 7 |
| Spray mist (psi) | 50 |
| Auxiliary heating gas (psi) | 50 |
MRM ion channels: ammonia adduct ions are adopted, and the specific ion pair information is shown as the table:
and (4) calculating a result: and (3) adopting liquid chromatogram tandem mass spectrometer data analysis software, taking the ratio of the area of the calibrator peak to the area of the internal standard peak as a vertical coordinate, taking the corresponding calibrator concentration as a horizontal coordinate, establishing linear regression, and automatically calculating the four immunosuppressive agents in the whole blood sample to be detected according to the method set by the data analysis software.
2. Linearity of immunosuppressant pretreatment kit
As shown in the figure, the graph is a working curve of the detection object, the abscissa represents the concentration of the detection object, and the ordinate represents the peak area ratio of the detection object to the peak area of the corresponding internal standard, so that a good linear relation can be seen.
3. Accuracy evaluation of immunosuppressant pretreatment kit
Detecting corresponding quality control products according to the detection method, and performing LC-MS/MS detection after the treatment of three parallel batches by the pretreatment kit according to the method. And calculating the concentration and accuracy of each compound in the quality control product according to the ratio of the peak area of the substance to be detected in the quality control product to the peak area of the internal standard substance of the syngen and a linear regression equation of the substance to be detected, wherein specific data are shown in the following table.
4. Influence of concentration of magnetic bead working solution on extraction rate of immunosuppressant
Magnetic bead working solutions with different concentrations are respectively prepared from 10mg/ml magnetic bead suspensions, the prepared magnetic bead working solutions with different concentrations are used for extracting the immunosuppressant in the whole blood, and the extraction rates of the magnetic bead working solutions with different concentrations are shown in the following table. As can be seen from the table, the magnetic bead working solution concentration of 4mg/ml has the highest extraction rate and the best effect.
5. Influence of different modifying group magnetic beads on extraction rate of immunosuppressant
The immunosuppressants in the whole blood were extracted by using different group-modified magnetic beads (particle size 1 μm, concentration of magnetic bead working solution 4mg/ml), and the magnetic bead treatment step and the pretreatment step were the same as above, and the results are shown in the following table. As can be seen from the table, the extraction rate of the magnetic beads modified with octadecyl groups was the highest.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (15)
1. A pretreatment reagent for detecting small molecular substances in a biological sample, which comprises: magnetic bead suspension, activation liquid, resuspension, cleaning liquid and eluent;
the magnetic beads in the magnetic bead suspension are magnetic beads with surface modified octadecyl groups;
the activating solution is trichloroacetic acid aqueous solution;
the eluent is a mixed solution of formic acid, methanol and water;
the heavy suspension is water, and the weight percentage of the water,
the cleaning liquid is water.
2. The pretreatment reagent of claim 1, wherein the solution of the suspension of magnetic beads is purified water, and the density of the magnetic beads is 10 mg/mL.
3. The pretreatment reagent according to claim 1, wherein the volume fraction of trichloroacetic acid in the activation solution is 0.1%.
4. The pretreatment reagent according to claim 1, wherein the volume ratio of formic acid, methanol and water in the eluate is 0.1:90: 10.
5. The pretreatment reagent according to any one of claims 1 to 4, further comprising a dissociation agent comprising: 1.5g/L sodium dodecyl sulfate, 100g/L dimethyl sulfoxide, 50g/L ethanol and 0.05M/L Tris-HCl.
6. The pretreatment reagent according to any one of claims 1 to 4, further comprising an internal standard solution, wherein the internal standard solution is a solution of a small molecule substance, and the small molecule substance comprises posaconazole, 25-hydroxyvitamin D2 and 25-hydroxyVitamin D3, vitamin a, vitamin E, and/or an immunosuppressive agent; the immunosuppressant is tacrolimus, sirolimus, everolimus, cyclosporine A, ascomycin, and sirolimus-d3Everolimus-d4And/or cyclosporins A-d12。
7. A pretreatment method for detecting a small molecular substance in a biological sample, characterized in that the biological sample is treated with the pretreatment reagent according to any one of claims 1 to 6.
8. The pretreatment method according to claim 7, wherein the biological sample is blood; the small molecular substance is posaconazole, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin A, vitamin E and/or an immunosuppressant; the immunosuppressant is tacrolimus, sirolimus, everolimus, cyclosporine A, ascomycin, and sirolimus-d3Everolimus-d4And/or cyclosporins A-d12。
9. The pretreatment method according to claim 8, comprising:
activating the magnetic beads by the activating solution, and then resuspending the magnetic beads by ultrapure water to obtain a magnetic bead working solution;
mixing blood with the magnetic bead working solution, incubating and separating the magnetic beads;
washing the magnetic beads with ultrapure water, eluting with an eluent, and then taking a supernatant;
in the magnetic bead working solution, 0, the density of the magnetic beads is 10 mg/mL;
the volume ratio of the blood to the magnetic bead working solution is (10-100): 100;
the incubation is carried out at room temperature for 2-5 min;
the mass-volume ratio of the magnetic beads to the eluent is 1mg:100 mu L.
10. The pretreatment method according to claim 9, further comprising
Analyzing the blood with the analyzing agent before mixing the blood with the magnetic beads;
the volume ratio of the blood to the resolving agent is 1: 2; the analysis was carried out at 37 ℃ for 30 min.
11. A method for detecting small molecular substances in blood, which is characterized by comprising the following steps:
the pretreatment method according to any one of claims 6 to 9, wherein the blood is treated and then detected by LC-MS/MS liquid chromatography.
12. The detection method according to claim 11, wherein the small molecule substance is posaconazole:
the LC-MS/MS liquid chromatography conditions include:
the chromatographic column is a C18 column, the column temperature is 50 ℃, and the flow rate of the mobile phase is 0.4 mL/min;
the mobile phase A is an aqueous solution containing 2mM ammonium acetate and 0.1 wt% formic acid;
the mobile phase B is a methanol solution containing 2mM ammonium acetate and 0.1 wt% formic acid;
the elution procedure included:
0-1.2 min, the volume fraction of the mobile phase A is from 95% to 10%,
1.2-2.0 min, the volume fraction of the mobile phase A is 10%,
2.0-2.1 min, the volume fraction of the mobile phase A is from 10% to 95%,
2.1-3.0 min, wherein the volume fraction of the mobile phase A is 95%;
the mass spectrum detection conditions of the LC-MS/MS comprise:
the ion source is an electrospray ionization ion source and is in a positive ion mode; capillary voltage 0.8 kV; the temperature of desolventizing gas is 500 ℃; the desolventizing air flow rate is 1000L/h; the reverse blowing airflow speed is 50L/h; the desolventizing gas and the back blowing gas are nitrogen with the purity of 99.9 percent; the collision gas is argon with the purity of 99.999 percent; the scanning mode is a multi-reaction monitoring mode.
13. The detection method according to claim 11, wherein the small molecule substance is 25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3,
the LC-MS/MS liquid chromatography conditions include:
the chromatographic column is a C18 column, the column temperature is 45 ℃, and the flow rate of the mobile phase is 0.4 mL/min;
the mobile phase A is an aqueous solution containing 0.1 weight percent of formic acid;
the mobile phase B is a methanol solution containing 0.1 wt% of formic acid;
the elution procedure included:
0-1.0 min, the volume fraction of the mobile phase A is 20 percent,
1.0-1.8 min, the volume fraction of the mobile phase A is from 20% to 10%,
1.8-2.6 min, the volume fraction of the mobile phase A is from 10% to 2%,
2.6-3.0 min, the volume fraction of the mobile phase A is 2%,
3.0-4.0 min, the volume fraction of the mobile phase A is from 2% to 20%;
the mass spectrum detection conditions of the LC-MS/MS comprise:
the ion source is an electrospray ionization ion source and is in a positive ion mode; capillary voltage 2.0 kV; the temperature of desolventizing gas is 400 ℃; the desolventizing air flow rate is 800L/h; the reverse blowing airflow speed is 50L/h; the desolventizing gas and the back blowing gas are nitrogen with the purity of 99.9 percent; the collision gas is argon with the purity of 99.999 percent; the scanning mode is a multi-reaction monitoring mode.
14. The detection method according to claim 11, wherein the small molecule substance is vitamin A and/or vitamin E,
the LC-MS/MS liquid chromatography conditions include:
the chromatographic column is a C18 column, the column temperature is 45 ℃, the sample injection temperature is 8 ℃, and the flow rate of the mobile phase is 0.4 mL/min;
the mobile phase A is an aqueous solution containing 0.1 weight percent of formic acid;
the mobile phase B is a methanol solution containing 0.1 wt% of formic acid;
the elution procedure included:
0-0.4 min, the volume fraction of the mobile phase A is from 15% to 5%,
0.4-2 min, the volume fraction of the mobile phase A is from 5% to 0%,
2-2.7 min, the volume fraction of the mobile phase A is 0 percent,
2.7-3.3 min, wherein the volume fraction of the mobile phase A is from 0% to 15%;
the mass spectrum detection conditions of the LC-MS/MS comprise:
the ion source is an electrospray ionization ion source and is in a positive ion mode; capillary voltage 3.0 kV; the temperature of desolventizing gas is 600 ℃; the desolventizing gas flow rate is 1100L/h; the reverse blowing airflow speed is 50L/h; the desolventizing gas and the back blowing gas are nitrogen with the purity of 99.9 percent; the collision gas is argon with the purity of 99.999 percent; the scanning mode is a multi-reaction monitoring mode.
15. The detection method according to claim 11, wherein the small molecule substance is an immunosuppressant; the immunosuppressant is tacrolimus, sirolimus, everolimus, cyclosporine A, ascomycin, and sirolimus-d3Everolimus-d4And/or cyclosporins A-d12；
The LC-MS/MS liquid chromatography conditions include:
the chromatographic column is a C18 column, the column temperature is 50 ℃, the sample injection temperature is 2-8 ℃, and the flow rate of the mobile phase is 0.5 mL/min;
the mobile phase A is an aqueous solution containing 2mM ammonium acetate and 0.1 wt% formic acid;
the mobile phase B is a methanol solution containing 2mM ammonium acetate and 0.1 wt% formic acid;
the elution procedure included:
0-0.2 min, the volume fraction of the mobile phase A is 50%,
0.2-1 min, the volume fraction of the mobile phase A is from 50% to 0%,
1-2 min, the volume fraction of the mobile phase A is 0 percent,
2-2.1 min, the volume fraction of the mobile phase A is from 0% to 50%,
2.1-3 min, wherein the volume fraction of the mobile phase A is 50%;
the mass spectrum detection conditions of the LC-MS/MS comprise:
the ion source is an electrospray ionization ion source and a positive ion mode, and the scanning mode is a multi-reaction monitoring mode; the ion source parameters include: the ionization voltage is 5.5kV, the temperature is 550 ℃, the air curtain gas pressure is 35psi, the collision gas pressure is 7, the spray gas pressure is 50psi, the auxiliary heating gas pressure is 50psi, and the MRM ion channel adopts ammonia adduct ions.
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| CN114371065A (en) * | 2021-12-28 | 2022-04-19 | 上海固容生物科技有限公司 | Method for processing liquid biopsy sample of biological sample (magnetic bead separation method) and application thereof |
| CN114487209A (en) * | 2022-03-08 | 2022-05-13 | 天津国科医工科技发展有限公司 | Liquid chromatography tandem mass spectrometry detection method of fat-soluble vitamins based on magnetic solid phase extraction |
| CN114509526A (en) * | 2021-12-31 | 2022-05-17 | 福建医科大学孟超肝胆医院(福州市传染病医院) | LC-MS/MS-based pure solvent type whole blood immunosuppressant drug concentration joint inspection kit and detection method |
| CN114544836A (en) * | 2022-03-11 | 2022-05-27 | 天津国科医工科技发展有限公司 | Pretreatment method and detection method for detecting trace estrogen, 17-hydroxypregnanolone, aldosterone and dehydroepiandrosterone sulfate |
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| EP2015074A1 (en) * | 2007-06-14 | 2009-01-14 | Roche Diagnostics GmbH | Preparation of samples for LC-MS/MS using magnetic particles |
| DE102014005993A1 (en) * | 2014-04-28 | 2015-10-29 | Magnamedics Gmbh | Method for enriching trace components from a liquid biological sample |
| EP3548892B1 (en) * | 2016-12-02 | 2024-02-21 | Magtivio B.V. | Method to analyze compounds in biological samples |
| CN109406650A (en) * | 2018-10-25 | 2019-03-01 | 美康生物科技股份有限公司 | Kit and detection method for four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
| CN110470753A (en) * | 2019-07-24 | 2019-11-19 | 天津国科医工科技发展有限公司 | Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake |
| CN111398480A (en) * | 2020-04-21 | 2020-07-10 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting triazole antifungal drugs and glycopeptide antibiotics and detection method thereof |
| CN111879876A (en) * | 2020-08-07 | 2020-11-03 | 苏州新波生物技术有限公司 | Rapid and sensitive analysis method for 25 hydroxyvitamin D based on immune mass spectrometry technology |
| CN112067388A (en) * | 2020-09-07 | 2020-12-11 | 苏州英芮诚生化科技有限公司 | High-throughput detection pretreatment method and kit for fat-soluble vitamins in blood plasma |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114371065A (en) * | 2021-12-28 | 2022-04-19 | 上海固容生物科技有限公司 | Method for processing liquid biopsy sample of biological sample (magnetic bead separation method) and application thereof |
| CN114509526A (en) * | 2021-12-31 | 2022-05-17 | 福建医科大学孟超肝胆医院(福州市传染病医院) | LC-MS/MS-based pure solvent type whole blood immunosuppressant drug concentration joint inspection kit and detection method |
| CN114487209A (en) * | 2022-03-08 | 2022-05-13 | 天津国科医工科技发展有限公司 | Liquid chromatography tandem mass spectrometry detection method of fat-soluble vitamins based on magnetic solid phase extraction |
| CN114544836A (en) * | 2022-03-11 | 2022-05-27 | 天津国科医工科技发展有限公司 | Pretreatment method and detection method for detecting trace estrogen, 17-hydroxypregnanolone, aldosterone and dehydroepiandrosterone sulfate |
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