CN115078559B - Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application - Google Patents

Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application Download PDF

Info

Publication number
CN115078559B
CN115078559B CN202210295641.9A CN202210295641A CN115078559B CN 115078559 B CN115078559 B CN 115078559B CN 202210295641 A CN202210295641 A CN 202210295641A CN 115078559 B CN115078559 B CN 115078559B
Authority
CN
China
Prior art keywords
magnetic particles
vitamin
kit
antibody
quadrupole mass
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210295641.9A
Other languages
Chinese (zh)
Other versions
CN115078559A (en
Inventor
吴超超
薛建有
高强
杨利娜
李鸣晖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Baichen Medical Instrument Co ltd
Hangzhou Baichen Medical Laboratory Co ltd
Original Assignee
Hangzhou Baichen Medical Instrument Co ltd
Hangzhou Baichen Medical Laboratory Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Baichen Medical Instrument Co ltd, Hangzhou Baichen Medical Laboratory Co ltd filed Critical Hangzhou Baichen Medical Instrument Co ltd
Priority to CN202210295641.9A priority Critical patent/CN115078559B/en
Publication of CN115078559A publication Critical patent/CN115078559A/en
Application granted granted Critical
Publication of CN115078559B publication Critical patent/CN115078559B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/08—Preparation using an enricher
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a vitamin D rapid detection method based on single quadrupole mass spectrometry, a kit and application, wherein 25-OH-D antibody coupled magnetic beads are used as carriers, and 25-OH-D 2 and 25-OH-D 3 are rapidly and selectively detected by utilizing liquid chromatography and single quadrupole mass spectrometry; meanwhile, a kit for rapid detection based on the method and application thereof are provided. Compared with the traditional LC-MS/MS detection method, the invention saves 80% of operation time, and greatly reduces the purchase cost, the debugging difficulty and the requirements of operators of detection equipment; and has excellent performances in detection accuracy, quantitative lower limit and dilution reliability.

Description

Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application
Technical Field
The invention relates to the field of analytical chemistry and immunology mass spectrometry detection, in particular to a vitamin D rapid detection method based on single quadrupole mass spectrometry, a kit and application.
Background
Vitamin D is a fat-soluble ring-opening steroid compound capable of maintaining physical health, and mainly comprises vitamin D 3 of animal origin and vitamin D 2 of plant origin, wherein the vitamin D is catalyzed by 25-hydroxylase of a living body to form 25-hydroxy vitamin D (25-OH-D), which is mainly represented by 25-hydroxy vitamin D 2(25-OH-D2 as shown in a formula I) and 25-hydroxy vitamin D 3(25-OH-D3 as shown in a formula II).
The existing vitamin D detection methods include an enzyme-linked immunosorbent assay, liquid chromatography (HPLC method) and liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS method). The ELISA method has the advantages of automatic detection and labor saving, and has high clinical laboratory acceptance, but low specificity, and can not distinguish 25-OH-D 2 from 25-OH-D 3. The HPLC method can separate 25-OH-D 2、25-OH-D3 from other vitamin D metabolites, and has higher specificity than the ELISA method, and has the defects of long analysis time, low detection flux, low sensitivity and large required sample amount, and can not be applied to a rapid detection scene. LC-MS/MS can detect 25-OH-D 2 and 25-OH-D 3 simultaneously, has very high specificity, and has been increasingly used in component detection and separation.
The working principle of triple quadrupole mass spectrometry used by the LC-MS/MS method is that a liquid chromatograph is used for separating a sample, then an ion source is used for entering the mass spectrum, a first quadrupole selects parent ions, a second quadrupole is used for entering the mass spectrum, inert gases such as nitrogen or argon are cracked into sub-ions, a third quadrupole is used for entering the mass spectrum, then the sub-ions are selected, and then a detector is used for detecting signals. Compared with Shan Siji rods, the triple quadrupole rod has the advantages of increased length, more ion vibration times and better sensitivity in theory. The LC-MS/MS method has very complex sample pretreatment, needs manual operation and is easy to make mistakes; adding internal standard precipitated protein prepared by methanol into a sample, adding ethyl acetate, n-butanol or methyl tertiary butyl ether to extract 25-OH-D in the sample, centrifuging, taking supernatant nitrogen for blowing, adding methanol or acetonitrile for redissolution, and detecting by using LC-MS/MS. Special protective equipment is also required, with the use of toxic and environmentally hazardous agents. The LC-MS/MS instrument has high cost, large occupied test bed space and ground space, high energy consumption, complex operation and maintenance, and needs to be operated by a trained technician, and collision gas needs to be additionally provided. After liquid chromatographic separation, detection of parent and daughter ions is required by a Multiple Reaction Monitoring (MRM) scan mode.
In vitamin D detection, the prior art mainly adopts a technology of coating magnetic beads with biotin-labeled antibodies. Chinese patent application "25-hydroxyvitamin D analysis method based on immunopurification combined mass spectrometry" (publication No. CN 111366646A) adopts biotin labeled antibody to coat magnetic beads to enrich 25-OH-D in a sample, and then LC-MS/MS detection analysis is carried out. The Chinese patent application "a rapid sensitive analysis method of 25 hydroxyvitamin D based on the immune mass spectrometry" (publication No. CN 111879876A) also adopts biotin labeled antibody to coat 25-OH-D in a magnetic bead enrichment sample, 4-phenyl-1, 2, 4-triazolin-3, 5-dione (PTAD) is added for derivatization, and then LC-MS/MS detection analysis is carried out. The prior art adopts biotin labeled antibody to coat magnetic beads for at least 50 hours, has complex operation and long time consumption, and is difficult to apply to clinical detection and popularization. The sample amounts were 100- μl and 50- μl, respectively, and pretreatment required manual handling, failing to achieve automation.
Disclosure of Invention
The invention aims to: the invention aims to provide a method for rapidly and selectively detecting 25-OH-D 2 and 25-OH-D 3 by using 25-OH-D antibody coupled magnetic beads as a carrier and utilizing liquid chromatography and single quadrupole mass spectrometry; it is a further object of the present invention to provide a method for rapid, selective detection of 25-OH-D 2 and 25-OH-D 3 after preparation of the 25-OH-D antibody-conjugated magnetic beads in the foregoing method as a kit product; the invention also aims to provide a rapid detection kit containing 25-OH-D antibody coupled magnetic particles and vitamin D3 isotope internal standard solution; the invention also aims to provide the application of the kit in LC-MS single quadrupole mass spectrometry for rapidly detecting vitamin D.
The technical scheme is as follows: in order to achieve the above object, the method for detecting vitamin D based on single quadrupole mass spectrometry of the present invention comprises the following steps:
(1) Preparing 25-OH-D antibody coupled magnetic particles by using the magnetic particles; mixing a sample, the antibody coupled magnetic particles and an internal standard solution, enriching and washing to obtain an eluent;
(2) Separating the eluent by liquid chromatography to obtain vitamin D isolate containing 25-OH-D;
(3) Subjecting the vitamin D isolate to single-quadrupole mass spectrometry, selecting positive ion mode by adopting ESI ion source,
Wherein the internal standard solution comprises 25-OH-D 2 -D3 and 25-OH-D 3 -D6, and the magnetic particles comprise carboxylated magnetic particles or tosyl magnetic particles.
Further, the preparation of the 25-OH-D antibody-coupled magnetic particles in the step (1) may adopt a one-step method or a two-step method, preferably a one-step method, according to the mixing timing of the antibody buffer, and specifically includes:
(11) Preparing MES buffer solution of the 25-OH-D antibody;
(12) Mixing with MES buffer solution of carboxylated magnetic particles, adding carbodiimide solution for coupling for 0.5-1 hour, and adding quenching buffer solution to seal unreacted carboxyl on the magnetic particles;
(13) Washing with high-concentration alcohol solvent for several times, and discarding supernatant;
(14) And washing for several times by using alcohol solvents with gradually reduced gradient concentration to recover the activity of the antibody, thus obtaining the 25-OH-D antibody coupled magnetic particles.
The invention adopts carboxylated magnetic particles or tosyl magnetic particles to carry out antibody coupling for detecting vitamin D, and the coupling time only needs 0.5-1 hour; in the prior art, biotin is adopted for coupling magnetic beads, streptavidin is required to react with antibodies and then be coupled with biotinylated magnetic beads, the steps are more, longer reaction time is required, and at least 50 hours are required to realize coupling.
As a preferable technical scheme, the carboxylated magnetic particles with the diameter of 0.5-3 mu m are preferably carboxylated magnetic particles with the diameter of 1 mu m, have the characteristics of high carboxyl bonding specific surface area and quick magnetic response, can couple more antibodies, use less magnetic particles during enrichment, and have the magnetic attraction time of about 3-5 seconds, thus being more suitable for automatic separation and operation. Whereas more magnetic particles, longer magnet times and more washing steps are required.
As a preferred scheme of the invention, after the 25-OH-D antibody coupled magnetic particles are prepared, the 25-OH-D antibody coupled magnetic particles, an internal standard solution, a diluent and an eluent are respectively packaged in a kit product.
The sample according to the invention is selected from human or animal ex vivo blood, saliva, tissue cells, for vitamin D detection for non-disease diagnosis or treatment for direct purposes, e.g. for exploring metabolites of vitamins under the action of 25-OH-D enzymes; or can be selected from non-living tissue samples for experimental study or teaching; or can be selected from plant or microorganism tissue samples for detecting vitamin D content and constitution.
Further, in the step (2), a C18 reverse column is adopted for liquid chromatography, a mobile phase A is an aqueous solution containing 2mM ammonium formate and 0.1% formic acid, a mobile phase B is a methanol solution containing 2mM ammonium formate and 0.1% formic acid, gradient elution is carried out, the flow rate is 0.4mL/min, the column temperature is 50 ℃, and the sample injection is 20 mu L.
As a preferred embodiment of the invention, the C18 reverse column is Waters BEH C18, 2.1X10 mm,2.7 μm.
Further, in the step (2), the single quadrupole mass spectrometry is selected from H-CLASS WATERS QDA, positive ion mode, SIR scanning, 25-OH-D 2 ion 395m/z, 25-OH-D 2 -D3 ion 398m/z, 25-OH-D 3 ion 383m/z, 25-OH-D 3 -D6 ion 389m/z, and cone voltage of 22V.
The invention provides a kit, which at least comprises: coupling magnetic particles with 25-OH-D antibodies prepared from carboxylated magnetic particles or tosyl magnetic particles; isotopic internal standard solutions comprising 25-OH-D 2 -D3 and 25-OH-D 3 -D6.
The preparation method of the magnetic microparticles has been described above, and the magnetic microparticles are generally stored in an organic reagent, washed with a gradient concentration before use, and the antibody activity is recovered. The content of the kit is generally 5-100. Mu.L of magnetic particles per 100. Mu.L of sample.
Further, the kit may further comprise at least the following reagents:
washing solution, PBS 100-400. Mu.L;
An internal standard solution comprising 20-60ng/mL of 25-OH-D 2 -D3 and 25-OH-D 3 -D6, and 30-100. Mu.L of 30-70% methanol solution;
sample dilution, 50-300. Mu.L sodium acetate-acetic acid buffer, pH 3-5;
50-400 mu L of 80-100% methanol as eluent;
a 25-OH-D mobile phase comprising 0.2M ammonium formate, 10% formic acid, 90% water.
The kit can be used for detecting 25-OH-D 2 and 25-OH-D 3 in vitamin D by LC-MS or LC-MS/MS, is particularly suitable for LC-MS single quadrupole mass spectrometry detection, and achieves the optimal solution in terms of cost, detection sensitivity/specificity and detection time. Specifically, a sample is mixed with 25-OH-D antibody coupled magnetic particles and an internal standard solution by using a kit, and an eluent is obtained through enrichment and washing; subjecting the eluate to Waters BEH C18 liquid chromatography to obtain vitamin D isolate containing 25-OH-D; and detecting the vitamin D separation product through a single quadrupole mass spectrum H-CLASS WATERS QDA, scanning positive ion mode and SIR, and analyzing chromatographic peak indexes.
The method and the kit for detecting 25-OH-D 2 and 25-OH-D 3 based on 25-OH-D antibody coupled magnetic particles are used for detection, and the whole detection process is not more than 17 minutes.
According to the method, a one-step method antibody coupling magnetic bead method is selected, firstly, a carrier capable of enriching 25-OH-D is prepared by coupling the carboxyl magnetic bead with the antibody, the antibody coupling process can be completed in about 2 hours, and the coating time of the antibody coupling magnetic particles is greatly shortened. The serum sample dissociates 25-OH-D from the binding protein through dissociation liquid, stable isotope internal standard is added, the antibody coupling magnetic particles enrich 25-OH-D, non-specific impurities are removed through washing, then high concentration organic reagent is used for eluting, 25-OH-D is further separated through liquid chromatography, and single quadrupole mass spectrometry detection is carried out. 25-OH-D 2 has a methyl group attached to the carbon atom at position 24 (see FIGS. I and II), which is 12Da different from the molecular weight of 25-OH-D 3, and 25-OH-D antibody-coupled magnetic particles can recognize and enrich 25-OH-D 2 and 25-OH-D 3 and their respective stable isotope internal standards in the sample.
The invention combines the advantages of an immunological method and a mass spectrometry method, prepares the required reagent for pretreatment into a kit, and a user only needs to add the sample into a single separation and purification reagent strip in the kit, wherein the pretreatment step can be completed manually or automatically by an iMS-1000 device, the sample is treated for 14 minutes at the highest speed, and the mass spectrometry detection needs 3 minutes. In the pretreatment operation step, dissociation, internal standard adding and enrichment are simultaneously carried out, and when the internal standard is calibrated in real time, the treatment time is effectively saved, and a sample can be finished from pretreatment to detection in 17 minutes.
According to the invention, by utilizing the characteristic that 25-OH-D is unstable in heat and easy to dehydrate in a mass spectrum ion source, WATERS QDA single quadrupole mass spectrum uses SIR detection and positive ion mode, 383m/z and 395m/z are respectively selected as mass spectrum detection ions of 25-OH-D 3 (400.7 Da) and 25-OH-D 2 (412.7 Da), and the purpose of high detection specificity of triple quadrupole mass spectrum MRM is achieved. Single quadrupole mass spectrometers are cheaper than triple quadrupole mass spectrometers; the device has small volume, is easy to move, can be directly placed above the liquid chromatograph, and effectively saves an experiment table top; the energy consumption is lower, the instrument operation is simple, and excessive conventional maintenance is not needed; no additional collision gas is provided. According to the invention, 25-OH-D in serum samples is enriched through the specificity of the antibody in the pretreatment, and through liquid phase separation and selection, 25-OH-D 2 and 25-OH-D 3 are detected by utilizing the selectivity of single quadrupole mass spectrometry, so that the detection effect same as that of the traditional LC-MS/MS method can be realized, the automation can be realized in the pretreatment, the detection time is short, the sample consumption is low, the single quadrupole mass spectrometry cost is lower, and the method is beneficial to clinical detection and application.
Drawings
FIG. 1 is a schematic diagram of the structure of a reagent strip in example 1;
FIG. 2 is a calibration curve of 25-OH-D 2 in example 1;
FIG. 3 is a calibration curve of 25-OH-D 3 in example 1;
FIG. 4 is a mass spectrum of 25-OH-D 3 in example 1;
FIG. 5 is a mass spectrum of 25-OH-D 3 -D6 in example 1;
FIG. 6 is a mass spectrum of 25-OH-D 2 in example 1;
FIG. 7 is a mass spectrum of 25-OH-D 2 -D3 in example 1.
Detailed Description
The invention will be further described with reference to the drawings and specific examples. It should be noted that the reagents used in the present invention are all in volume percent concentrations unless specifically stated.
Example 1
Preparation of 1.25-OH-D antibody-coupled magnetic particles
Using a 10kD ultrafiltration tube, 1-10mg of 25-OH-D antibody was centrifuged at 12000g for 10min at 4℃and 400. Mu.L of MES (1-200 mM, pH 3-7) was added thereto, and 12000g was centrifuged for 10min to replace the antibody preservation solution with the MES reaction buffer. 1000. Mu.L (1-20 mg) of carboxylated magnetic particles were added to a 2mL EP tube, the supernatant was removed by magnetic separation, MES was added to resuspend the magnetic particles, the antibody was removed from the ultrafiltration tube and added to the magnetic particles, and the mixture was mixed at room temperature for 10min. Adding 5-100 mu L of 1-100mg/mL carbodiimide solution, and coupling for 0.5-1 hour at 37 ℃. 200. Mu.L of quenching buffer (5-100 mM Tris-HCl, pH 5-8) was added and mixed at 37℃for 10min, and unreacted carboxyl groups on the beads were blocked. Removing supernatant by magnetic separation, adding 80% -100% methanol to wash the antibody coupled magnetic particles for 1-5 times, removing endogenous antigen bound on the antibody, and discarding supernatant. The antibody-coupled magnetic particles are washed with 60-80% methanol, 40-60% methanol and 20-40% methanol sequentially, the antibody activity is recovered by gradient, and the antibody-coupled magnetic particles are stored in 1-20% methanol.
Preparation of 25-OH-D purification reagent strips
Preparation of 25-OH-D purification strips, the wells of which may comprise:
(1) 25-OH-D antibody coupled magnetic particles
(2) Multiple sets of 100-400 mu L PBS washing solution
(3) An internal standard solution comprising 20-60ng/mL of 25-OH-D 2 -D3 and 25-OH-D 3 -D6, and 30-100. Mu.L of 30-70% methanol solution;
(4) Sample diluent, 50-300 mu L of sodium acetate-acetic acid buffer, 0.05-2mol/L of sodium acetate, and acetic acid to adjust the pH to 3-5;
(5) 50-400 mu L of 80-100% methanol as eluent;
Sealing with sealing aluminum film to obtain 25-OH-D purified reagent strip, and preserving at 2-8deg.C. Fig. 1 shows a reagent strip a of one embodiment, which at least comprises a sample addition well 1, a washing solution reagent well 2, an internal standard solution reagent well 3, an antibody-coupled magnetic particle reagent well 4, a sample diluent reagent well 5, an eluent reagent well 6, and a sample elution well 7. The reagent strip is suitable for automatic sampling, enrichment, washing and elution of single-person automatic equipment, and can be modified in adaptability by a person skilled in the art without departing from the principle of the invention, and the reagent strip is within the protection scope of the invention.
3.25 Preparation of the OH-D calibration Curve
Calibration curves were prepared with 30% -70% methanol, 70% -30% PBS solution, as shown in Table 1, in ng/mL units, 80. Mu.L per tube for packaging, and storage at-80 ℃.
TABLE 1 concentration of 25-OH-D calibration curve
Name of the name Marking 1 Marking 2 Marking 3 Marking 4 Marking 5 Marking 6
25-OH-D2 5 10 25 50 75 100
25-OH-D3 5 10 25 50 75 100
FIGS. 2 and 3 show that the scaling curves of 25-OH-D 2 and 25-OH-D 3 are 5-100 ng/mL, and the correlation coefficients are 0.9993 and 0.9991, respectively, which meet the requirements (R2 is more than or equal to 0.99).
Preparation of 4.25-OH-D quality control product
Mixing the serum with 10-300mM sodium acetate buffer solution with pH of 3-5 at a ratio of 1:0.5-1:5, dialyzing with dialysis bag for several times, removing 25-OH-D in the serum, adding 25-OH-D standard substance, preparing low-concentration and high-concentration quality control product with low quality control concentration of 15ng/mL and high quality control concentration of 70ng/mL, packaging with 80 μl of each tube, and preserving at-80deg.C.
5.25-OH-D flow phase preparation
The 25-OH-D mobile phase comprises 0.2M ammonium formate, 10% formic acid and 90% water, and is prepared by packaging with 8mL ampoule bottles, packaging with 5.0mL of each bottle, and storing at 2-8deg.C.
6.25-OH-D kit preparation
And (3) the prepared 25-OH-D separation and purification reagent strip, a 25-OH-D calibration curve, a 25-OH-D low-high quality control and mobile phase are combined into a 25-OH-D kit.
7. Sample processing
50 Mu L of serum is added into a 25-OH-D separation and purification reagent strip hole 1, a reagent strip and a sample are placed into an iMS-1000 instrument, a corresponding number of suction heads are placed, a 25-OH-D treatment program is set, and the post-treatment is completed after about 12 minutes. 100. Mu.L of the treated 25-OH-D reticle 1-6, low, high quality control or sample to be tested was removed from the 14 wells and detected by WATERS QDA mass spectrometer in 96 well plates.
8. Chromatographic parameters
(1) Chromatographic column: waters BEH C18 (2.1X10 mm,2.7 μm).
(2) Mobile phase: a bottle of 5mL of 25-OH-D mobile phase was added to 500mL of water (mobile phase A) or methanol (mobile phase B), respectively, and mixed well.
(3) Liquid chromatography elution gradient: 0-1min 80% B,1-2.3min 90% B,2.3-2.5min 90% B,2.5-2.6min 80% B,2.6-3min 80% B. Flow rate: 0.4mL/min; column temperature: 50 ℃; and (3) sample injection: 20. Mu.L.
9. Mass spectral parameters
The mass spectrum model is WATERS QDA, the positive ion mode, SIR scanning, 25-OH-D 2 ion 395m/z, 25-OH-D 2 -D3 ion 398m/z, 25-OH-D 3 ion 383m/z, 25-OH-D 3 -D6 ion 389m/z and the cone voltage is 22V.
As shown in Table 2 and FIGS. 5 to 7, the peak at 1.7min was 25-OH-D3, the peak at 1.67 was 25-OH-D 3 -D6, the peak at 1.88min was 25-OH-D 2, the peak at 1.86 was 25-OH-D2-D3, none of the interfering peaks,
TABLE 2 detection signals of 25-OH-D 3 and 25-OH-D 2 enriched in 25-OH-D antibody coupled magnetic particles
Detection index 25-OH-D3 25-OH-D3-d6 25-OH-D2 25-OH-D2-d3
Peak area 20629.3 8738.5 16938.2 6643.9
Example 2
The present example uses conventional triple quadrupole mass spectrometry detection and does not use carboxylated magnetic particle pretreatment for evaluation of subsequent test examples.
1. Sample processing
Precisely sucking 100 mu L of calibrator or quality control material solution or serum sample into a 1.5mL centrifuge tube, adding 200 mu L of methanol to prepare an internal standard solution, and mixing and rotating for 5min; adding 500 mu L of n-hexane, and mixing for 5min; centrifuging at 4deg.C at high speed (14000 g) for 10min, sucking 300 μl of supernatant, placing into a clean 1.5mL centrifuge tube, blow-drying with nitrogen at room temperature, adding 140 μl of aqueous solution containing 0.1% formic acid-75% methanol, re-dissolving, vortex shaking for 5min, centrifuging at 4deg.C at high speed (14000 g) for 10min, and collecting 80 μl of supernatant for mass spectrometry sample injection analysis.
2. Liquid chromatography separation of 25-OH-D
(1) Chromatographic column: waters BEH C18 (2.1X10 mm,2.7 μm).
(2) Mobile phase: mobile phase A is 2mmol/L ammonium formate and 0.1% formic acid aqueous solution; mobile phase B was 2mmol/L ammonium formate and 0.1% methanolic formate.
(3) Liquid chromatography elution gradient: 0-1min 80% B,1-2.3min 90% B,2.3-2.5min 90% B,2.5-2.6min 80% B,2.6-3min 80% B. Flow rate: 0.4mL/min; column temperature: 50 ℃; and (3) sample injection: 20. Mu.L. Flow rate: 0.4mL/min; column temperature: 50 ℃; and (3) sample injection: 10 mu L.
3. Triple quadrupole mass spectrometry detection
(1) Ion source: electrospray ion source, positive ion mode;
(2) Capillary voltage: 3.0kV; desolventizing temperature: 350 ℃; desolventizing gas: 950L/hr; taper hole gas: 0L/hr;
(3) Scanning mode: multiple Reaction Monitoring (MRM) conditions are shown in Table 3
TABLE 3 MRM conditions for each compound
Test example 1 precision verification
The sample concentrations of the low and high 2 concentration levels were measured by the method described in example 1, and repeated 3 times a day for 5 days, and the results are shown in tables 4 and 5.
TABLE 4 detection of the precision of 25-OH-D 2 at different concentrations (5 days precision)
TABLE 5 detection of the precision of 25-OH-D 3 at different concentrations (5 days precision)
Test example 2 verification of correctness
The 25-OH-D level differentiated serum was selected as the recovery experimental base sample, and mixed with 900ul of base samples according to 100ul of low, medium and high standard samples, respectively, as shown in Table 6, requiring: the low concentration is 3 times the lower limit of the linear range; the medium concentration is the middle concentration in the linear range; the high concentration is 70% of the upper limit of the linear range. The above "base sample" and "experimental sample" were repeated for 6 times in parallel, the experimental results were recorded and statistical analysis was performed for the case of labeled recovery at 3 concentration levels of low, medium, and high, recovery = (detection value-base sample value)/theoretical value x 100%,85% < recovery < 115%.
TABLE 6 recovery formulation table with labels
The accuracy of the method was verified by standard addition recovery, and the results are shown in Table 7 and Table 8, wherein the average recovery rate of 25-OH-D 3 is 98.21%, the average recovery rate of 25-OH-D 2 is 100.28%, and the requirements (85% < average recovery rate < 115%) are met.
TABLE 7 25-OH-D 3 correctness verification results
Table 8 results of verification of correctness of 25-OH-D 2
Further, 25-OH-D 3 in 5 samples of the second endocrine chamber interstitial evaluations 202121 to 202125 in 2021 were examined according to the standards of the health department interstitial evaluation, and the results are shown in Table 9, and the detection values were close to the target values, and were within the allowable range.
Table 9 results of the second ventricular septum evaluation sample from the 2021 Ministry of health
Detection result of room interstitial evaluation sample 25-OH-D 3 Detection value Target value Allowed range
Sample 202121 6.6 6.6 5-8.3
Sample 202122 24.5 28.7 21.5-35.8
Sample 202123 14.9 15.2 11.4-19.0
Sample 202124 27.8 33.7 25.3-42.2
Sample 202125 21.3 22.3 16.7-31.8
Finally, the results of the vitamin D serum toxicology control commercial quality control of UTAK company at low, medium, and high 3 levels of 25-OH-D 3 and 25-OH-D 2 were tested to verify the accuracy of the method, and the results are shown in Table 10, with a bias of less than 15% for the detection values and target values. The method provided by the invention has the advantages of comprehensive labeling recovery, compartment quality evaluation of the health department and commercial quality control detection result judgment, and accurate detection result.
Table 10 UTAK commercial quality control test results
Test example 3 lower limit of quantitation
The lower limit of quantification is the lowest value of the target analyte detected by the LC-MS/MS method under the premise of meeting the laboratory requirements for accuracy and precision. The lower limit of quantification is to select 3 samples with concentration close to the detection limit, each concentration sample is divided into 5 samples to be treated, each sample is repeatedly measured for 3 times, 3 batches are continuously measured, the total precision (CV) of each concentration sample and the deviation between the measured concentration mean value and the theoretical concentration of each concentration sample are respectively evaluated, and the measured mean value of the lowest concentration sample with CV less than or equal to 20% and bias less than 15% is taken as the lower limit of quantification of the method. The results are shown in tables 11 and 12:
TABLE 11 quantitative lower limit results for 25-OH-D 3
Table 12 quantitative lower limit results for 25-OH-D 2
Test example 4 dilution reliability
Plasma samples near the upper limit of the linear range were collected, diluted 2, 4 and 8 times with water, respectively, and the dilution was judged to pass the verification, requiring a recovery rate (dilution detection value/theoretical value) of 80% or less than 120%. The results are shown in tables 13 and 14:
TABLE 13 dilution reliability results for 25-OH-D 3
TABLE 14 dilution reliability results for 25-OH-D 2
Test example 5 comparison of operating time and cost
Compared with the traditional LC-MS/MS method of the embodiment 2, the automatic method has obvious advantages, can effectively save laboratory space, instruments, consumables, time and other costs while ensuring accuracy, and the results are shown in tables 15 and 16.
TABLE 15 comparison of the advantages and disadvantages of the automated method according to the invention and the conventional LC-MS/MS method
TABLE 16 step and time comparison required for the automated method of the present invention and the conventional LC-MS/MS method
/>

Claims (5)

1. A vitamin D rapid detection method based on single quadrupole mass spectrometry is characterized by comprising the following steps:
(1) Preparing 25-OH-D antibody coupled magnetic particles by using carboxylated magnetic particles; mixing a sample, the antibody coupled magnetic particles and an internal standard solution, enriching and washing to obtain an eluent;
the internal standard solution comprises 25-OH-D 2 -D3 and 25-OH-D 3 -D6, and the preparation method of the 25-OH-D antibody coupled magnetic particles comprises the following steps:
(11) Preparing MES buffer solution of the 25-OH-D antibody;
(12) Mixing the mixture with MES buffer solution of carboxylated magnetic particles, adding carbodiimide solution for coupling for 0.5-1 hour, and then adding quenching buffer solution to seal unreacted carboxyl on the carboxylated magnetic particles;
(13) Washing with high-concentration alcohol solvent for several times, and discarding supernatant;
(14) Washing for several times by using alcohol solvent with gradually reduced gradient concentration to recover antibody activity, so as to obtain 25-OH-D antibody coupled magnetic particles;
(2) Separating the eluent by liquid chromatography to obtain vitamin D separation product containing 25-OH-D, wherein the liquid chromatography separation adopts a Waters BEH C18 reverse column, the mobile phase A is aqueous solution containing 2mM ammonium formate and 0.1% formic acid, the mobile phase B is methanol solution containing 2mM ammonium formate and 0.1% formic acid, the gradient elution is carried out, the flow rate is 0.4 mL/min, the column temperature is 50 ℃, and the sample injection is 20 mu L; wherein the liquid chromatography elution gradient: 80% B for 0-1 min, 90% B for 1-2.3 min, 90% B for 2.3-2.5 min, 80% B for 2.5-2.6 min, 80% B for 2.6-3 min;
(3) Detecting the vitamin D isolate by a single-quadrupole mass spectrum H-CLASS WATERS QDA, adopting an ESI ion source, selecting a positive ion mode, performing SIR scanning, and performing cone hole voltage of 22V on 25-OH-D 2 ion 395 m/z, 25-OH-D 2 -D3 ion 398 m/z, 25-OH-D 3 ion 383 m/z and 25-OH-D 3 -D6 ion 3839 m/z;
Wherein, the peak time of 25-OH-D 3 is 1.7min, the peak time of 25-OH-D 3 -D6 is 1.67min, the peak time of 25-OH-D 2 is 1.88 min, and the peak time of 25-OH-D 2 -D3 is 1.86min.
2. The method for rapidly detecting vitamin D based on single quadrupole mass spectrometry according to claim 1, wherein the method comprises the following steps: after the 25-OH-D antibody coupled magnetic particles are prepared, the magnetic particles, an internal standard solution, a diluent and an eluent are respectively packaged in a kit product.
3. A kit for use in a rapid vitamin D detection method based on single quadrupole mass spectrometry as defined in claim 1, wherein:
The kit at least comprises the following reagents:
The 25-OH-D antibody-coupled magnetic particles prepared by carboxylated magnetic particles comprise isotopic internal standard solutions of 25-OH-D 2 -D3 and 25-OH-D 3 -D6.
4. A kit according to claim 3, characterized in that it comprises the following reagents:
washing solution, PBS 100-400. Mu.L;
An internal standard solution comprising 20-60ng/mL of 25-OH-D 2 -D3 and 25-OH-D 3 -D6, and 30-100. Mu.L of 30-70% methanol solution;
Sample dilution, 50-300. Mu.L sodium acetate-acetic acid buffer, pH 3-5;
50-400 mu L of 80-100% methanol as eluent;
a 25-OH-D mobile phase comprising 0.2M ammonium formate, 10% formic acid, 90% water.
5. The kit of claim 4, wherein: the whole detection time is not more than 17 minutes.
CN202210295641.9A 2022-03-24 2022-03-24 Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application Active CN115078559B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210295641.9A CN115078559B (en) 2022-03-24 2022-03-24 Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210295641.9A CN115078559B (en) 2022-03-24 2022-03-24 Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application

Publications (2)

Publication Number Publication Date
CN115078559A CN115078559A (en) 2022-09-20
CN115078559B true CN115078559B (en) 2024-05-28

Family

ID=83247783

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210295641.9A Active CN115078559B (en) 2022-03-24 2022-03-24 Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application

Country Status (1)

Country Link
CN (1) CN115078559B (en)

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012058619A1 (en) * 2010-10-29 2012-05-03 Cohesive Technologies Inc. Lc-ms configuration for purification and detection of analytes having a broad range of hydrophobicities
CN103391920A (en) * 2010-11-26 2013-11-13 利奥制药有限公司 Calcium-sensing receptor-active compounds
CN105527364A (en) * 2015-08-26 2016-04-27 袁洪 Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry
CN106199016A (en) * 2016-06-30 2016-12-07 深圳市亚辉龙生物科技股份有限公司 25 hydroxy-vitamine D chemiluminescence immune detection reagent kits and preparation method thereof
CN109188001A (en) * 2018-08-13 2019-01-11 深圳天辰医疗科技有限公司 A kind of detection reagent of 25-hydroxy-vitamin D and preparation method thereof and kit
CN110988245A (en) * 2019-12-25 2020-04-10 中储粮镇江粮油质量检测中心有限公司 Method for rapidly detecting content of vitamin E in vegetable oil and fat and analogues thereof by ultra-high performance combined phase chromatography-mass spectrometry
CN111122881A (en) * 2019-12-26 2020-05-08 宁波奥丞生物科技有限公司 Detection kit for chemiluminescence of 25-hydroxy vitamin D magnetic particles
CN111208250A (en) * 2020-01-14 2020-05-29 南京品生医学检验实验室有限公司 Kit for detecting 5 fat-soluble vitamins in serum by ultra-high performance liquid chromatography tandem mass spectrometry
CN111208248A (en) * 2020-01-14 2020-05-29 南京品生医学检验实验室有限公司 Method for detecting 5 fat-soluble vitamins in serum by ultra-high performance liquid chromatography tandem mass spectrometry
CN111366646A (en) * 2020-02-27 2020-07-03 苏州新波生物技术有限公司 25-hydroxyvitamin D analysis method based on immune purification and mass spectrometry detection
CN111718377A (en) * 2019-03-19 2020-09-29 海南长安国际制药有限公司 Platinum substance with structural formulas H1 and H2, preparation and application thereof
CN111936851A (en) * 2018-01-29 2020-11-13 Dh科技发展私人贸易有限公司 Methods and systems for detecting vitamin D metabolites
CN113009017A (en) * 2021-02-25 2021-06-22 杭州佰辰医学检验所有限公司 Hormone mass spectrometry detection method based on antibody coupling magnetic bead enrichment technology
CN113063871A (en) * 2021-03-24 2021-07-02 杭州佰勤医疗器械有限公司 Metabolic small molecule detection method and detection system

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5449202B2 (en) * 2008-02-25 2014-03-19 ウオーターズ・テクノロジーズ・コーポレイシヨン Methods and kits for determining the presence and amount of vitamin D analogs in a sample
WO2012037365A1 (en) * 2010-09-16 2012-03-22 Quest Diagnostics Investments Incorporated Mass spectrometric determination of eicosapentaenoic acid and docosahexaenoic acid
EP2633325B1 (en) * 2010-10-29 2016-12-28 Cohesive Technologies Inc. Lc-ms separation and detection of vitamin d metabolites
JP6739080B2 (en) * 2016-11-17 2020-08-12 学校法人東京理科大学 Vitamin D quantification method, mass spectrometer and vitamin D quantification reagent kit

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012058619A1 (en) * 2010-10-29 2012-05-03 Cohesive Technologies Inc. Lc-ms configuration for purification and detection of analytes having a broad range of hydrophobicities
CN103391920A (en) * 2010-11-26 2013-11-13 利奥制药有限公司 Calcium-sensing receptor-active compounds
CN105527364A (en) * 2015-08-26 2016-04-27 袁洪 Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry
CN106199016A (en) * 2016-06-30 2016-12-07 深圳市亚辉龙生物科技股份有限公司 25 hydroxy-vitamine D chemiluminescence immune detection reagent kits and preparation method thereof
CN111936851A (en) * 2018-01-29 2020-11-13 Dh科技发展私人贸易有限公司 Methods and systems for detecting vitamin D metabolites
CN109188001A (en) * 2018-08-13 2019-01-11 深圳天辰医疗科技有限公司 A kind of detection reagent of 25-hydroxy-vitamin D and preparation method thereof and kit
CN111718377A (en) * 2019-03-19 2020-09-29 海南长安国际制药有限公司 Platinum substance with structural formulas H1 and H2, preparation and application thereof
CN110988245A (en) * 2019-12-25 2020-04-10 中储粮镇江粮油质量检测中心有限公司 Method for rapidly detecting content of vitamin E in vegetable oil and fat and analogues thereof by ultra-high performance combined phase chromatography-mass spectrometry
CN111122881A (en) * 2019-12-26 2020-05-08 宁波奥丞生物科技有限公司 Detection kit for chemiluminescence of 25-hydroxy vitamin D magnetic particles
CN111208248A (en) * 2020-01-14 2020-05-29 南京品生医学检验实验室有限公司 Method for detecting 5 fat-soluble vitamins in serum by ultra-high performance liquid chromatography tandem mass spectrometry
CN111208250A (en) * 2020-01-14 2020-05-29 南京品生医学检验实验室有限公司 Kit for detecting 5 fat-soluble vitamins in serum by ultra-high performance liquid chromatography tandem mass spectrometry
CN111366646A (en) * 2020-02-27 2020-07-03 苏州新波生物技术有限公司 25-hydroxyvitamin D analysis method based on immune purification and mass spectrometry detection
CN113009017A (en) * 2021-02-25 2021-06-22 杭州佰辰医学检验所有限公司 Hormone mass spectrometry detection method based on antibody coupling magnetic bead enrichment technology
CN113063871A (en) * 2021-03-24 2021-07-02 杭州佰勤医疗器械有限公司 Metabolic small molecule detection method and detection system

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Analysis of Vitamin D and its Metabolites using Thermospray Liquid Chromatography/Mass Spectrometry;David Watson etal.;《BIOMEDICAL CHROMATOGRAPHY》;第5卷(第4期);第153-159页 *
Development and comparison of three liquid chromatography–atmospheric pressure chemical ionization/mass spectrometry methods for determining vitamin D metabolites in human serum;Mary Bedner etal.;Journal of Chromatography A;第1240卷;正文第1-4节 *
UPLC-MS/MS法测定人血浆中25羟基维生素D的浓度及其应用;张叶 等;《中国药房》;第27卷(第14期);全文 *
液相色谱-串联质谱法检测血清25-羟基维生素D的方法建立和性能评价;宋斌斌 等;《检验医学》;第30卷(第05期);第417、418页 *
超高效液相色谱-串联质谱法测定血清中25-羟基维生素D和维生素K_1;尹丹阳 等;《卫生研究》;第49卷(第04期);全文 *
高效液相色谱-串联质谱法测定食品及保健品中维生素D的含量;李涵博 等;《甘肃中医药大学学报》;第37卷(第02期);全文 *

Also Published As

Publication number Publication date
CN115078559A (en) 2022-09-20

Similar Documents

Publication Publication Date Title
US11650216B2 (en) Methods for detecting dihydroxyvitamin D metabolites by mass spectrometry
US11333639B2 (en) LC-MS configuration for purification and detection of analytes having a broad range of hydrophobicities
CN106855545B (en) Method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed
EP3278100A1 (en) Solid phase extraction, derivatization with crown ethers, and mass spectrometry, methods, reagents and kits
EP1866633A2 (en) Methods for detecting vitamin d metabolites by mass spectrometry
CN113533555B (en) Detection kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry and detection method thereof
CN113063871B (en) Metabolic small molecule detection method and detection system
CN113125601B (en) Method for simultaneously detecting concentrations of 4 fat-soluble vitamins in serum
CN114674961A (en) Kit for synchronously detecting 17 steroid hormones in serum without derivatization and application thereof
CN114019069A (en) Pretreatment reagent for detecting small molecular substances in biological sample
CN112748198A (en) Method and device for detecting antifungal drugs in serum by liquid chromatography tandem mass spectrometry technology
CN112014509A (en) Method for synchronously determining angiotensin I and aldosterone in sample
CN115876933A (en) Method for detecting steroid hormone in serum
CN113607854A (en) Method and detection kit for simultaneously detecting multiple vitamins
CN115078559B (en) Vitamin D rapid detection method based on single quadrupole mass spectrometry, kit and application
CN113311080B (en) Joint detection method for multiple vitamin D metabolites, detection kit and application thereof
CN114755321A (en) Method for detecting fat-soluble vitamins in serum
CN113302499A (en) High speed sample workflow for LC-MS based HbA1c measurement
CN110806447B (en) Screening method and content determination method for bordetella pertussis tracheal cytotoxin
CN105699575A (en) Method and kit for testing cortisol in saliva by efficient liquid chromatogram and tandem mass spectrometry combination technology
CN115112810B (en) Sample pretreatment method for 25-hydroxy vitamin D detection and application
CN114674943B (en) Detection of 25 (OH) D in serum2And 25 (OH) D3Liquid chromatography mass spectrometry tandem detection method of (C)
KR20060123270A (en) Method for the determination of 25-hydroxycholecalciferol in feed
CN112697898B (en) Method for rapidly determining content of ustilagin A in urine or cell culture medium by liquid chromatography-mass spectrometry
CN117630263A (en) Method for detecting various free amino acids in plasma based on liquid chromatography-tandem mass spectrometry

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant