CN115078559A - Rapid vitamin D detection method based on single quadrupole mass spectrometry, kit and application - Google Patents

Rapid vitamin D detection method based on single quadrupole mass spectrometry, kit and application Download PDF

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CN115078559A
CN115078559A CN202210295641.9A CN202210295641A CN115078559A CN 115078559 A CN115078559 A CN 115078559A CN 202210295641 A CN202210295641 A CN 202210295641A CN 115078559 A CN115078559 A CN 115078559A
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magnetic particles
vitamin
mass spectrometry
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吴超超
薛建有
高强
杨利娜
李鸣晖
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Hangzhou Baichen Medical Instrument Co ltd
Hangzhou Baichen Medical Laboratory Co ltd
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Abstract

The invention discloses a vitamin D rapid detection method based on single quadrupole mass spectrometry, a kit and application thereof, wherein a 25-OH-D antibody coupled magnetic bead is used as a carrier, and liquid chromatography and single quadrupole mass spectrometry are utilized to rapidly and selectively detect vitamin DDetection of 25-OH-D 2 And 25-OH-D 3 (ii) a Meanwhile, a kit based on the method for rapid detection and application thereof are provided. Compared with the traditional LC-MS/MS detection method, the method saves 80% of the operation time, and greatly reduces the purchase cost of detection equipment, the debugging difficulty and the requirements of operators; and has excellent performance in detection accuracy, lower limit of quantitation and dilution reliability.

Description

Rapid vitamin D detection method based on single quadrupole mass spectrometry, kit and application
Technical Field
The invention relates to the field of analytical chemistry and immunological mass spectrometry detection, in particular to a rapid vitamin D detection method based on single quadrupole mass spectrometry, a kit and application.
Background
Vitamin D is a fat-soluble open-ring sterol compound for maintaining health, and mainly comprises animal-derived vitamin D 3 And vitamin D of plant origin 2 Vitamin D is catalyzed by the 25-hydroxylase of the organism to form 25-hydroxyvitamin D (25-OH-D), which is predominantly expressed as 25-hydroxyvitamin D 2 (25-OH-D 2 Compounds of formula I) and 25-hydroxyvitamin D 3 (25-OH-D 3 A compound of formula II).
At present, vitamin D detection methods comprise an enzyme-linked immunosorbent assay, a liquid chromatography (HPLC method) and a liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS method). The enzyme linked immunosorbent assay has the advantages of automatic detection and labor saving, has high clinical laboratory acceptance, but has low specificity and can not distinguish 25-OH-D 2 And 25-OH-D 3 . The HPLC method can convert 25-OH-D 2 、25-OH-D 3 The method has the advantages of high specificity compared with an enzyme-linked immunosorbent assay when being separated from other vitamin D metabolites, and has the defects of long analysis time, low detection flux, low sensitivity and large required sample amount, and cannot be applied to a rapid detection scene. LC-MS/MS can simultaneously detect 25-OH-D 2 And 25-OH-D 3 Is very highHas been increasingly used in component detection and separation.
Figure RE-GDA0003711921740000011
The working principle of the triple quadrupole mass spectrometry used by the LC-MS/MS method is that a sample is separated by liquid chromatography, then enters the mass spectrometry through an ion source, a first quadrupole selects parent ions, enters a second quadrupole, is broken into daughter ions through inert gases such as nitrogen or argon, enters a third quadrupole, selects the daughter ions, and then enters a detector to detect signals. Compared with a single quadrupole, the length of the triple quadrupole is increased, the ion vibration times are more, and theoretically, the sensitivity is better. The sample pretreatment of the LC-MS/MS method is very complex, manual operation is needed, and errors are easy to occur; adding internal standard precipitate protein prepared by methanol into a sample, adding ethyl acetate, n-butyl alcohol or methyl tert-butyl ether to extract 25-OH-D in the sample, centrifuging, taking supernatant, blowing nitrogen, adding methanol or acetonitrile to redissolve, and detecting by LC-MS/MS. Toxic and environmentally hazardous agents are used, and special protective equipment is also required. The LC-MS/MS instrument has high cost, large occupied test bed space and ground space, high energy consumption, complex operation and maintenance, needs to be operated by a professional trained technician, and needs to additionally provide collision gas. After liquid chromatography separation, detection of parent and daughter ions is required to be achieved by Multiple Reaction Monitoring (MRM) scan mode.
In the detection of vitamin D, the prior art mainly adopts a technology that biotin labeled antibodies coat magnetic beads. Chinese patent application "a 25-hydroxyvitamin D analysis method based on immune purification and mass spectrometry detection" (publication No. CN111366646A) adopts biotin-labeled antibody to coat 25-OH-D in magnetic bead enriched samples, and then the samples are detected and analyzed by LC-MS/MS. Chinese patent application "a 25 hydroxyvitamin D rapid sensitive analysis method based on immune mass spectrometry" (publication No. CN111879876A) also adopts biotin-labeled antibody to coat 25-OH-D in magnetic bead enriched sample, adds 4-phenyl-1, 2, 4-triazoline-3, 5-diketone (PTAD) for derivatization, and then carries out LC-MS/MS detection and analysis. In the prior art, the biotin labeled antibody is adopted to coat the magnetic beads for at least 50 hours, the operation is complex, the time is long, and the application to clinical detection and popularization is difficult. The sample dosage is 100-muL and 50-muL respectively, and the pretreatment needs manual operation and can not be automated.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for rapidly and selectively detecting 25-OH-D by using 25-OH-D antibody coupled magnetic beads as carriers and utilizing liquid chromatography and single quadrupole mass spectrometry 2 And 25-OH-D 3 The method of (1); the invention also aims to provide a method for rapidly and selectively detecting 25-OH-D after 25-OH-D antibody coupled magnetic beads are prepared into a kit product in the method 2 And 25-OH-D 3 The method of (1); still another object of the present invention is to provide a rapid assay kit comprising 25-OH-D antibody-coupled magnetic particles and vitamin D3 isotope internal standard solution; the invention also aims to provide 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 invention provides a method for detecting vitamin D based on single quadrupole mass spectrometry, comprising the following steps:
(1) preparing 25-OH-D antibody coupling magnetic particles by using the magnetic particles; mixing the sample, the antibody coupling magnetic particles and the internal standard solution, enriching and washing to obtain an eluent;
(2) separating the eluate by liquid chromatography to obtain a vitamin D isolate containing 25-OH-D;
(3) subjecting the vitamin D isolate to single quadrupole mass spectrometry, selecting a positive ion mode by using an ESI ion source,
wherein the internal standard solution comprises 25-OH-D 2 -D3 and 25-OH-D 3 -d6, said magnetic particles comprising carboxylated magnetic particles or tosyl magnetic particles.
Further, the preparation of the 25-OH-D antibody-coupled magnetic particles in step (1) may adopt a one-step method or a two-step method according to the mixing time of the antibody buffer, preferably adopts a one-step method, and specifically includes:
(11) preparing MES buffer solution of the 25-OH-D antibody;
(12) mixing with MES buffer solution of the 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 for several times by using a high-concentration alcohol solvent, and discarding the supernatant;
(14) and washing the magnetic particles for several times by using an alcohol solvent with gradually reduced gradient concentration to recover the activity of the antibody to obtain 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 is only 0.5-1 hour; the existing detection technology adopts biotin coupling magnetic beads, streptavidin needs to react with an antibody, and then the streptavidin is coupled with biotinylated magnetic beads, so that the coupling can be realized through multiple steps, long reaction time is needed, and at least 50 hours are needed.
As a preferred technical scheme, the carboxylated magnetic particles have the diameter of 0.5-3 microns, preferably the carboxylated magnetic particles with the diameter of 1 micron are adopted, the carboxylated magnetic particles have the characteristics of high carboxyl bonding specific surface area and quick magnetic response, the magnetic particles with the same mass can be coupled with more antibodies, the magnetic particles used in enrichment are few, and meanwhile, the magnetic attraction time is about 3-5 seconds, so that the magnetic particle magnetic carrier is more suitable for automatic separation and operation. Otherwise, more magnetic particles, longer magnetic attraction time and more washing steps are required.
In a preferred embodiment of the present invention, the 25-OH-D antibody-coupled magnetic particles are prepared and then packaged in a kit product together with an internal standard solution, a diluent, and an eluent.
The sample is selected from blood, saliva and tissue cells of human or animal bodies, and is used for vitamin D detection with non-disease diagnosis or treatment as direct purposes, such as exploring metabolites of vitamins under the action of 25-OH-D enzyme; or non-living tissue samples for experimental research or teaching; or a tissue sample selected from plants or microorganisms for detecting the content and composition of vitamin D therein.
Further, the liquid chromatography in the step (2) adopts a C18 reverse-phase column, wherein the mobile phase A is an aqueous solution containing 2mM ammonium formate and 0.1% formic acid, the mobile phase B is a methanol solution containing 2mM ammonium formate and 0.1% formic acid, the gradient elution is carried out, the flow rate is 0.4mL/min, the column temperature is 50 ℃, and 20 mu L of sample injection is carried out.
As a preferred embodiment of the present invention, the C18 reverse column is Waters BEH C18, 2.1X 50mm, 2.7 μm.
Further, the single quadrupole mass spectrometry in the step (2) adopts H-class Waters QDa, positive ion mode, SIR scanning, 25-OH-D 2 Ion 395m/z, 25-OH-D 2 398m/z, 25-OH-D for the D3 ion 3 Ion 383m/z, 25-OH-D 3 D6 ions 389m/z, the cone hole voltage is 22V.
The invention provides a kit, at least comprising: coupling 25-OH-D antibody prepared by using carboxylated magnetic particles or tosyl magnetic particles with magnetic particles; and comprises 25-OH-D 2 -D3 and 25-OH-D 3 -isotopic internal standard solutions of d 6.
The method for producing magnetic microparticles has been described above, and generally, the magnetic microparticles are stored in an organic reagent, washed with a gradient concentration before use, and the antibody activity is recovered. The kit is typically included in an amount of 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-;
an internal standard solution comprising 20-60ng/mL of 25-OH-D 2 -D3 and 25-OH-D 3 -d6, and 30-100 μ L of 30-70% methanol solution;
sample diluent, 50-300 mul sodium acetate-acetic acid buffer solution, pH 3-5;
eluent, 50-400 μ L of 80-100% methanol;
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 in vitamin D by LC-MS or LC-MS/MS 2 And 25-OH-D 3 The method 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. In particular to a kitMixing the sample with 25-OH-D antibody coupling magnetic particles and an internal standard solution, and obtaining an eluent through enrichment and washing; subjecting the eluate to Waters BEH C18 liquid chromatography to obtain a vitamin D isolate containing 25-OH-D; and detecting the vitamin D isolate by a single quadrupole mass spectrometry H-class Waters QDa, and analyzing a chromatographic peak index by a positive ion mode and SIR scanning.
25-OH-D antibody-coupled magnetic particle-based 25-OH-D detection method 2 And 25-OH-D 3 The method and the kit thereof carry out detection, and the whole process does not exceed 17 minutes.
According to the invention, a one-step method of coupling the magnetic beads with the antibodies is selected, firstly, the carboxyl magnetic beads are coupled with the antibodies to prepare a carrier capable of enriching 25-OH-D, the coupling process of the antibodies can be completed in about 2 hours, and the coating time of the antibody coupling magnetic particles is greatly shortened. Dissociating 25-OH-D and binding protein of a serum sample through a dissociation solution, adding a stable isotope internal standard, enriching 25-OH-D through antibody coupling magnetic particles, washing to remove non-specific impurities, eluting with a high-concentration organic reagent, further separating 25-OH-D through liquid chromatography, and performing single quadrupole mass spectrometry. 25-OH-D 2 Mainly a methyl group (shown in figures I and II) is connected to the 24-position carbon atom, and is connected with 25-OH-D 3 The molecular weight of the sample is 12Da, and the 25-OH-D antibody coupled magnetic particles can identify and enrich the 25-OH-D in the sample 2 And 25-OH-D 3 And their respective stable isotope internal standards.
The invention combines the advantages of an immunological method and a mass spectrometry, the required reagent for pretreatment is prepared into the kit, a user only needs to add a sample into a single reagent strip for separation and purification in the kit, the pretreatment step can be manually completed, the pretreatment step can also be automatically completed through equipment such as iMS-1000, the sample is treated once only within 14 minutes at the fastest time, and the mass spectrometry detection needs 3 minutes. In the pretreatment operation step, the dissociation, the internal standard addition and the enrichment are carried out simultaneously, the processing time is effectively saved while the internal standard is calibrated in real time, and the detection result from the pretreatment to the detection of one sample can be completed within 17 minutes.
The invention utilizes the characteristic that 25-OH-D is thermally unstable and easily dehydrated in a mass spectrum ion source, namely a Waters QDa single quadrupole rodMass Spectrometry Using SIR detection and Positive ion mode, 383m/z and 395m/z were chosen as 25-OH-D, respectively 3 (400.7 Da) and 25-OH-D 2 And (412.7Da) to achieve the purpose of high detection specificity of the triple quadrupole mass spectrum MRM. Compared with a triple quadrupole mass spectrometer, the single quadrupole mass spectrometer is lower in price; the volume is small, the carrying is easy, the liquid chromatography can be directly placed above the liquid chromatography, and the experiment table is effectively saved; the energy consumption is lower, the instrument is simple to operate, and excessive conventional maintenance is not needed; no additional supply of collision gas is required. The pretreatment of the invention enriches 25-OH-D in a serum sample through the specificity of an antibody, and finally, the invention selectively detects the 25-OH-D by utilizing a single quadrupole mass spectrum 2 And 25-OH-D 3 The method can realize the detection effect same as that of the traditional LC-MS/MS method, and the pretreatment can realize automation, the detection time is short, the sample dosage is low, the cost of the single quadrupole mass spectrum is lower, and the method is beneficial to clinical detection and application.
Drawings
FIG. 1 is a schematic view showing the structure of a reagent strip in example 1;
FIG. 2 shows 25-OH-D in example 1 2 Calibrating a curve;
FIG. 3 shows 25-OH-D in example 1 3 Calibrating a curve;
FIG. 4 shows 25-OH-D in example 1 3 Mass spectrogram;
FIG. 5 shows 25-OH-D in example 1 3 -d6 mass spectrum;
FIG. 6 shows 25-OH-D in example 1 2 Mass spectrogram;
FIG. 7 shows 25-OH-D in example 1 2 -d3 mass spectrum.
Detailed Description
The invention is further described below with reference to the figures and the specific embodiments. It is to be noted that, unless otherwise specified, all reagents used in the present invention are in volume percent concentration.
Example 1
1. Preparation of 25-OH-D antibody coupled magnetic particles
1-10mg of 25-OH-D antibody was centrifuged at 12000g for 10min at 4 ℃ using a 10kD ultrafilter tube, and 400. mu.L of MES (1-200mM, pH 3-7) was added thereto and centrifuged at 12000g for 10min to replace the antibody stock solution with the MES reaction buffer. Add 1000. mu.L (1-20mg) of carboxylated magnetic microparticles into a 2mL EP tube, remove the supernatant by magnetic separation, add MES to resuspend the magnetic microparticles, remove the antibody from the ultrafiltration tube and add to the magnetic microparticles, mix for 10min at room temperature. 5-100 mu L of the ready-prepared 1-100mg/mL carbodiimide solution is added, and coupling is carried out for 0.5-1 hour at 37 ℃. Add 200. mu.L of quenching buffer (5-100mM Tris-HCl, pH5-8) and mix for 10min at 37 ℃ to block unreacted carboxyl groups on the beads. And (3) magnetically separating to remove supernatant, adding 80-100% methanol to wash the antibody coupled magnetic particles for 1-5 times, removing endogenous antigens combined on the antibody, and discarding the supernatant. Washing the antibody-coupled magnetic particles with 60-80% methanol, 40-60% methanol and 20-40% methanol respectively, recovering antibody activity by gradient, and storing the antibody-coupled magnetic particles in 1-20% methanol.
2. Preparation of 25-OH-D purification reagent strips
Preparing a 25-OH-D purification reagent strip, wherein the reagent strip hole can contain:
(1)25-OH-D antibody coupled magnetic particles
(2) Multi-group 100-400 μ L PBS wash 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 μ L of 30-70% methanol solution;
(4) sample diluent, 50-300 mu L sodium acetate-acetic acid buffer solution, 0.05-2mol/L sodium acetate, and acetic acid to adjust the pH value to 3-5;
(5) eluent, 50-400 μ L of 80-100% methanol;
sealing with aluminum sealing film to obtain 25-OH-D purified reagent strip, and storing at 2-8 deg.C. Fig. 1 shows a reagent strip a of one embodiment, which at least comprises a sample application 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, enriching, washing and eluting of single-person automatic equipment, and can be modified by persons skilled in the art without departing from the principle of the invention, and the reagent strip is within the protection scope of the invention.
Preparation of 3.25-OH-D calibration Curve
A calibration curve was prepared using 30% -70% methanol, 70% -30% PBS solution, as shown in Table 1, in ng/mL units, 80. mu.L/tube was packaged and stored at-80 ℃.
TABLE 125-OH-D calibration Curve concentration
Name (R) Marking 1 Marking 2 Marking 3 Marking 4 Marking 5 Marking 6
25-OH-D 2 5 10 25 50 75 100
25-OH-D 3 5 10 25 50 75 100
FIGS. 2 and 3 show 25-OH-D 2 And 25-OH-D 3 The calibration curve ranges are 5-100 ng/mL, the correlation coefficients are 0.9993 and 0.9991 respectively, and the requirements are met (the requirement that R2 is more than or equal to 0.99).
4.25-OH-D quality control Material preparation
Mixing the serum with 10-300mM pH3-5 sodium acetate buffer solution according to the proportion of 1: 0.5-1: 5, dialyzing for several times by using a dialysis bag, removing 25-OH-D in the serum, adding 25-OH-D standard substance, preparing a low-concentration and high-concentration quality control product, wherein the low-concentration is 15ng/mL, the high-concentration is 70ng/mL, each tube is packaged by 80 mu L, and storing at-80 ℃.
5.25-OH-D flow phase formulation
The 25-OH-D mobile phase comprises 0.2M ammonium formate, 10% formic acid and 90% water, and is packaged in 8mL ampoule bottles, 5.0mL each bottle, and stored at 2-8 deg.C.
Preparation of 6.25-OH-D kit
And (3) forming a 25-OH-D kit by using the prepared 25-OH-D separation and purification reagent strip, a 25-OH-D calibration curve, a 25-OH-D low-quality control and mobile phase.
7. Sample processing
Adding 50 mu L of serum into a 25-OH-D separation and purification reagent strip hole 1, placing the reagent strip and the sample into an iMS-1000 instrument, placing a corresponding number of suction heads, setting a 25-OH-D treatment program, and finishing the treatment after about 12 min. And taking 100 mu L of the processed 25-OH-D marked line 1-6, low and high quality control or sample to be detected out of the 14-hole, putting the sample into a 96-hole plate, and detecting by a Waters QDa mass spectrometer.
8. Chromatographic parameters
(1) A chromatographic column: waters BEH C18 (2.1X 50mm, 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) and mixed.
(3) Liquid chromatography elution gradient: 80% B at 0-1min, 90% B at 1-2.3min, 90% B at 2.3-2.5min, 80% B at 2.5-2.6 min, and 80% B at 2.6-3 min. Flow rate: 0.4 mL/min; column temperature: 50 ℃; sample introduction: 20 μ L.
9. Parameters of mass spectrum
Mass spectrum model Waters QDa, positive ion mode, SIR scan, 25-OH-D 2 Ion 395m/z, 25-OH-D 2 398m/z, 25-OH-D for the D3 ion 3 Ion 383m/z, 25-OH-D 3 D6 ions 389m/z, the cone hole voltage is 22V.
As shown in Table 2 and FIGS. 5 to 7, the peak at 1.7min was 25-OH-D3, and the peak at 1.67 was 25-OH-D 3 D6, peak at 1.88min 25-OH-D 2 The peak at 1.86 is 25-OH-D2-D3, and no interference peak exists,
TABLE 225-OH-D antibody coupling to 25-OH-D enriched magnetic particles 3 And 25-OH-D 2 Detection signal of
Detecting the index 25-OH-D 3 25-OH-D 3 -d6 25-OH-D 2 25-OH-D 2 -d3
Peak area 20629.3 8738.5 16938.2 6643.9
Example 2
This example was tested using conventional triple quadrupole mass spectrometry without pretreatment with carboxylated magnetic particles and used for evaluation in subsequent experimental examples.
1. Sample processing
Precisely sucking 100 mu L of calibrator or quality control solution or serum sample into a 1.5mL centrifuge tube, adding 200 mu L of methanol to prepare an internal standard solution, and carrying out mixed spinning for 5 min; adding 500 μ L n-hexane, and performing mixed spinning for 5 min; centrifuging at 4 deg.C and high speed (14000g) for 10min, sucking 300 μ L of supernatant, placing in clean 1.5mL centrifuge tube, blowing dry with nitrogen at room temperature, adding 140 μ L of 0.1% formic acid-75% methanol-containing aqueous solution for redissolving, vortexing and shaking for 5min, centrifuging at 4 deg.C and high speed (14000g) for 10min, and collecting 80 μ L of supernatant for mass spectrometry sample injection analysis.
2. Liquid chromatography for separating 25-OH-D
(1) A chromatographic column: waters BEH C18 (2.1X 50mm, 2.7 μm).
(2) Mobile phase: the mobile phase A is 2mmol/L ammonium formate and 0.1% formic acid aqueous solution; the mobile phase B is 2mmol/L ammonium formate and 0.1% formic acid in methanol.
(3) Liquid chromatography elution gradient: 80% B at 0-1min, 90% B at 1-2.3min, 90% B at 2.3-2.5min, 80% B at 2.5-2.6 min, and 80% B at 2.6-3 min. Flow rate: 0.4 mL/min; column temperature: 50 ℃; sample introduction: 20 μ L. Flow rate: 0.4 mL/min; column temperature: 50 ℃; sample introduction: 10 μ L.
3. Triple quadrupole mass spectrometry detection
(1) An ion source: electrospray ion source, positive ion mode;
(2) capillary voltage: 3.0 kV; desolventizing temperature: 350 ℃; removing the solvent gas: 950L/hr; taper hole gas: 0L/hr;
(3) scanning mode: multiple Reaction Monitoring (MRM), conditions as shown in Table 3
TABLE 3 MRM Condition for the Compounds
Figure RE-GDA0003711921740000081
Test example 1 verification of precision
The concentrations of the samples at the low and high concentration levels were measured by the method described in example 1, and the measurements were repeated 3 times each day for 5 consecutive days, and the results are shown in tables 4 and 5.
TABLE 4 different concentrations for 25-OH-D 2 Detection of precision (5 days essence density)
Figure RE-GDA0003711921740000082
TABLE 5 different concentrations vs 25-OH-D 3 Detection of precision (5 days essence density)
Figure RE-GDA0003711921740000083
Test example 2 verification of accuracy
Serum with differentiated 25-OH-D levels was selected as the base sample for recovery experiments and mixed with 900ul of base sample according to 100ul of low, medium and high standard samples, as shown in Table 6, requiring: the low concentration is 3 times of the lower limit of the linear range; the medium concentration is the medium concentration in the linear range; the high concentration is 70% of the upper limit of the linear range. The above-mentioned "basic sample" and "experimental sample" were subjected to parallel measurement 6 times, the experimental results were recorded and statistical analysis was carried out for the spiked recovery conditions of the low, medium and high 3 concentration levels, with recovery rate ═ (detection value-basic sample value)/theoretical value × 100%, 85% < recovery rate < 115%.
TABLE 6 Tab-adding recovery preparation table
Figure RE-GDA0003711921740000091
The accuracy of the method was verified by first applying a standard, and the results are shown in tables 7 and 8, 25-OH-D 3 The average recovery rate is 98.21 percent, and the recovery rate is 25-OH-D 2 The average recovery rate is 100.28%, and the average recovery rate meets the requirements (the requirement is that the average recovery rate is more than 85% < 115%).
TABLE 725-OH-D 3 Accuracy verification result
Figure RE-GDA0003711921740000092
TABLE 825-OH-D 2 Accuracy verification result
Figure RE-GDA0003711921740000093
Figure RE-GDA0003711921740000101
Further, 25-OH-D in 5 samples of secondary evaluation of endocrine Chamber quality 202121 to 202125 in 2021 was determined according to evaluation criteria of Chamber quality of Ministry of health 3 The results are shown in Table 9, and the detection values were close to the target values and were within the allowable ranges.
Second time of laboratory test on laboratory test sample by Ministry of health in Table 92021
Sample for evaluation of Room quality 25-OH-D 3 The result of the detection Detection value Target value Allowable 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 commercial quality control of the vitamin D serum toxicology control of UTAK company is detected to be 25-OH-D with low, medium and high 3 levels 3 And 25-OH-D 2 The results of the verification of the accuracy of the method are shown in Table 10, and the deviation between the detection value and the target value is less than 15%. The method has the advantages of comprehensive bid adding recovery, health department indoor quality assessment and commercial quality control detection result judgment, and accurate detection result.
TABLE 10 UTAK commercial quality control test results
Figure RE-GDA0003711921740000102
Test example 3 lower limit of quantitation
The lower limit of the quantification is the lowest value of the target analyte detected by the LC-MS/MS method on the premise of meeting the requirements of laboratories on accuracy and precision. And selecting 3 samples with the concentration close to the detection limit as a lower limit of quantification, dividing each sample with the concentration into 5 parts for processing, repeatedly measuring each sample for 3 times, continuously measuring for 3 batches, respectively evaluating the total precision (CV) of each sample with the concentration and the deviation of the measured mean value of the concentration from the theoretical concentration, and taking the measured mean value of the sample with the lowest concentration, of which the CV is less than or equal to 20% and the bias is less than 15%, as the lower limit of quantification of the method. The results are shown in tables 11 and 12:
TABLE 1125-OH-D 3 Quantitative lower limit result
Figure RE-GDA0003711921740000111
TABLE 1225-OH-D 2 Quantitative lower limit result
Figure RE-GDA0003711921740000112
Test example 4 dilution reliability
And collecting plasma samples near the upper limit of the linear range, adding water to dilute the plasma samples by 2, 4 and 8 times respectively, wherein the recovery rate (dilution detection value/theoretical value) is required to be more than or equal to 80 percent and less than or equal to 120 percent, and judging that the dilution passes verification. The results are shown in tables 13 and 14:
TABLE 1325-OH-D 3 Dilution reliability results
Figure RE-GDA0003711921740000113
TABLE 1425-OH-D 2 Dilution reliability results
Figure RE-GDA0003711921740000114
Figure RE-GDA0003711921740000121
Test example 5 comparison of operating time and cost
Compared with the traditional LC-MS/MS method in the embodiment 2, the automatic method has obvious advantages, the accuracy is ensured, meanwhile, the cost of laboratory space, instruments, consumables, time and the like can be effectively saved, and the result is shown in tables 15 and 16.
TABLE 15 comparison of advantages and disadvantages of the automated method of the present invention and the conventional LC-MS/MS method
Figure RE-GDA0003711921740000122
TABLE 16 comparison of the steps and time required for the automated method of the invention and the conventional LC-MS/MS method
Figure RE-GDA0003711921740000123
Figure RE-GDA0003711921740000131

Claims (10)

1. A rapid vitamin D detection method based on single quadrupole mass spectrometry is characterized by comprising the following steps:
(1) preparing 25-OH-D antibody coupling magnetic particles by using the magnetic particles; mixing the sample, the antibody coupling magnetic particles and the internal standard solution, enriching and washing to obtain an eluent;
(2) separating the eluate by liquid chromatography to obtain a vitamin D isolate containing 25-OH-D;
(3) detecting the vitamin D isolate by single quadrupole mass spectrometry, adopting an ESI ion source, selecting a positive ion mode,
wherein the internal standard solution comprises 25-OH-D 2 -D3 and 25-OH-D 3 -d6, said magnetic particles comprising carboxylated magnetic particles or tosyl magnetic particles.
2. The method for rapidly detecting vitamin D based on single quadrupole mass spectrometry (MONOMMS) of claim 1, wherein the step of preparing 25-OH-D antibody coupled magnetic particles in step (1) comprises:
(11) preparing MES buffer solution of the 25-OH-D antibody;
(12) mixing with MES buffer solution of the 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 for several times by using a high-concentration alcohol solvent, and discarding the supernatant;
(14) and washing the magnetic particles for several times by using an alcohol solvent with gradually reduced gradient concentration to recover the activity of the antibody to obtain the 25-OH-D antibody coupled magnetic particles.
3. The rapid vitamin D detection method based on the single quadrupole mass spectrometry according to claim 1 or 2, wherein: after the 25-OH-D antibody coupling magnetic particles are prepared, the 25-OH-D antibody coupling magnetic particles, an internal standard solution, a diluent and an eluent are respectively packaged in a kit product.
4. The method for rapidly detecting vitamin D based on single quadrupole mass spectrometry according to claim 3, wherein the liquid chromatography in step (2) adopts a C18 reverse-phase column, the mobile phase A is an aqueous solution containing 2mM ammonium formate and 0.1% formic acid, the mobile phase B is a methanol solution containing 2mM ammonium formate and 0.1% formic acid, and the gradient elution is performed at a flow rate of 0.4mL/min, the column temperature is 50 ℃, and the sample is injected at 20 μ L.
5. The rapid vitamin D detection method based on the single quadrupole mass spectrometry as claimed in claim 4, wherein: the C18 reverse column was Waters BEH C18, 2.1X 50mm, 2.7 μm.
6. The rapid vitamin D detection method based on single quadrupole mass spectrometry of any one of claims 1-2 or 4-5, wherein the single quadrupole mass spectrometry in the step (2) is selected from H-class Waters QDa, positive ion mode, SIR scan, 25-OH-D 2 Ion 395m/z, 25-OH-D 2 398m/z, 25-OH-D for the D3 ion 3 Ion 383m/z, 25-OH-D 3 D6 ions 389m/z, the cone hole voltage is 22V.
7. A kit characterized by comprising at least the following reagents:
coupling 25-OH-D antibody prepared by using carboxylated magnetic particles or tosyl magnetic particles with magnetic particles;
comprising 25-OH-D 2 -D3 and 25-OH-D 3 -isotopic internal standard solutions of d 6.
8. A kit according to claim 7, characterized in that it comprises the following reagents:
washing solution, PBS 100-;
an internal standard solution comprising 20-60ng/mL of 25-OH-D 2 -D3 and 25-OH-D 3 -d6, and 30-100 μ L of 30-70% methanol solution;
sample diluent, 50-300 mul sodium acetate-acetic acid buffer solution, pH 3-5;
eluent, 50-400 μ L of 80-100% methanol;
25-OH-D mobile phase comprising 0.2M ammonium formate, 10% formic acid, 90% water.
9. The use of the kit of claim 8 in the rapid detection of vitamin D by LC-MS single quadrupole mass spectrometry, wherein:
mixing a sample with 25-OH-D antibody coupling magnetic particles and an internal standard solution by using a kit, and obtaining an eluent through enrichment and washing;
subjecting the eluate to Waters BEH C18 liquid chromatography to obtain a vitamin D isolate containing 25-OH-D;
and detecting the vitamin D isolate by single quadrupole mass spectrometry H-class Waters QDa, and analyzing a chromatographic peak index by positive ion mode and SIR scanning.
10. Use according to claim 9, characterized in that: the whole detection time is not more than 17 minutes.
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