CN115902071A

CN115902071A - Method for detecting fat-soluble vitamins in sample

Info

Publication number: CN115902071A
Application number: CN202310189805.4A
Authority: CN
Inventors: 章申燕; 李红娥; 李宁; 张伟; 王晓禄; 佟西海
Original assignee: Beijing Gbi Biotechnology Co ltd
Current assignee: Beijing Gbi Biotechnology Co ltd
Priority date: 2023-02-23
Filing date: 2023-02-23
Publication date: 2023-04-04
Also published as: CN116754677A

Abstract

The present invention provides a method for the detection of a fat-soluble vitamin in a sample, said method comprising the step of contacting a polymer, in particular a styrene-based polymer coated magnetic bead, with the sample. The method can be used in sample preparation and pretreatment of mass spectrometry and liquid chromatography. The invention also provides application of the magnetic beads in detection of fat-soluble vitamins in a sample and a kit for detecting the fat-soluble vitamins in the sample.

Description

Method for detecting fat-soluble vitamins in sample

Technical Field

The invention belongs to the field of biology, and particularly relates to a method for detecting fat-soluble vitamins in a sample.

Background

Fat-soluble vitamins meanVitamins which are soluble in water and soluble in fatty organic solvents include, for example, vitamin A, vitamin D, vitamin E and vitamin K, where vitamin D includes, for example, vitamin D ₂ And vitamin D ₃ . Fat-soluble vitamins have diverse effects, and besides being directly involved in and influencing specific metabolic processes, they also bind to intracellular nuclear receptors to influence the expression of specific genes. The fat-soluble vitamins play an important role in the growth, metabolism and development process of human bodies. The reasonable vitamin level is very important for maintaining the health of human body, the content of fat-soluble vitamin in the human body is accurately measured, people can be guided to scientifically and reasonably supplement vitamin, diseases are prevented, the life quality is improved, and meanwhile, the method has auxiliary diagnosis significance for clinical judgment, treatment management and physiological evaluation of deficiency or excess of fat-soluble vitamin. The determination of fat-soluble vitamins is an important task in clinical samples.

At present, when fat-soluble vitamins in a sample are clinically measured, because the components of the clinical sample are complex and cannot be directly detected, the sample needs to be pretreated firstly, so that the sample can possibly enter a subsequent analysis procedure after being properly separated and purified. The pretreatment methods of fat-soluble vitamins adopted in the prior art comprise Protein Precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE) and the like. However, the pretreatment method in the prior art has the defect that automation cannot be realized and/or effective detection of the fat-soluble vitamin A, D, E, K cannot be realized at the same time.

CN113376270B discloses a pretreatment method for detecting protein precipitation of a sample of fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry, which comprises the step of treatment by adopting a negative pressure device. The method is simple to operate, but because the protein precipitation is actually used for diluting the sample, the concentration of the substance to be detected in the on-machine sample is low, and the requirement on the sensitivity of a measuring instrument is high; moreover, the method requires negative pressure operation, which is not favorable for realizing full automation of the operation process. CN110779780A discloses a sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry, which uses ethanol for protein precipitation, and uses n-hexane for liquid-liquid extraction, nitrogen for drying extraction liquid, and sample redissolution to carry out sample injection analysis. The method involves the steps of extraction, nitrogen blowing and the like in the experimental process, needs a large amount of manual operation and is not beneficial to the automation of treatment; and the operation process is complicated, and the requirement on experimenters is high. CN111198238A discloses a method for extracting and detecting vitamin D in serum, which comprises the steps of firstly carrying out protein precipitation on a sample, then loading a precipitated mixed solution onto an active carbon 96-well plate, eluting by using strong volatile reagents such as n-hexane and the like, carrying out re-dissolution on the sample after the eluent is dried by nitrogen, and then selectively adsorbing a target substance by using a solid-phase extraction method. However, in the experiment, the solid phase extraction filler can generate swelling phenomenon after contacting with the organic reagent, the flow rate of the solution passing through the solid phase extraction filler can be influenced, so that the problem of poor uniformity among pores is generated, and the processing of drying the nitrogen by blowing is not beneficial to the processing automation. CN114487209a discloses a method for detecting liposoluble vitamin a, vitamin D and vitamin E in serum by using a magnetic solid-phase extraction material, which utilizes an external magnetic field and a magnetic solid-phase extraction filler (hydrophilic-lipophilic balance type magnetic solid-phase extraction magnetic beads, HLB magnetic beads), however, the method has low sensitivity for detecting vitamin a and cannot detect vitamin K.

Therefore, the existing methods can not realize sensitive, effective and/or automatic detection of the fat-soluble vitamin A, D, E, K in the sample. Clinical testing objectively requires providing a method that overcomes at least one of the problems of the prior art described above, and in particular is effective in detecting fat-soluble vitamins; and preferably further automation of the detection can be achieved.

Disclosure of Invention

The inventor of the invention finds that the detection of a plurality of fat-soluble vitamins can be realized by contacting a sample with polymer-coated magnetic beads (especially hydrophobic polymer magnetic beads), the high detection sensitivity is realized, and the simultaneous detection of a plurality of fat-soluble vitamins including vitamin K can be further realized.

Based on this finding, in a first aspect of the present invention, a method for detecting a target analyte in a sample is provided, comprising the step of contacting polymer-coated magnetic beads to the sample, wherein the target analyte is preferably a fat-soluble vitamin.

The magnetic bead of the invention is a magnetic particle, which has a magnetic core and a coating material coated on the surface of the magnetic core, wherein the coating material is a polymer material, especially a hydrophobic polymer, such as alkyl modified silica gel or a styrene-based polymer, the alkyl modified silica gel is, for example, C18 or C8 alkyl modified silica gel, and the styrene-based polymer is, for example, styrene-divinylbenzene copolymer.

The magnetic beads preferably have an appropriate particle size, for example, an average particle size of 1 to 200. Mu.m, for example, 10 to 100. Mu.m, or 30 to 50. Mu.m.

In the present invention, the "sample" means a product form from which the component to be detected is derived. Herein, the sample may refer to a biological sample, e.g. a clinical sample, which is preferably selected from the group consisting of whole blood, plasma, serum, urine, saliva, tears, bile, gastric fluid, interstitial fluid and lymphatic fluid.

In the invention, the magnetic bead has a magnetic core and a polymer coated on the surface of the magnetic core. The polymer may be a styrene-based polymer. The styrene-based polymer refers to a polymer formed by polymerizing styrene as a main monomer, or referred to as a styrene-based polymer. The styrene-based polymer herein may be polystyrene formed with styrene as the only monomer, or may be a polymer formed by adding other crosslinking agents (e.g., divinylbenzene), such as styrene-divinylbenzene copolymer. The polymers may be formed by different processes (e.g., fluidized bed processes, reaction kettle processes) and may have different degrees of crosslinking.

In the present invention, the magnetic core of the magnetic bead comprises magnetic particles, which may consist of any substance having magnetic properties, preferably selected from oxides of iron, cobalt, nickel, more preferably ferroferric oxide. As known in the art, the magnetic core may be prepared using any suitable method.

The inventors have further found in their research that the movement and/or separation of the sample can be further achieved by the action of the applied magnetic field. For example, the magnetic field is applied to realize rapid and sufficient transfer of the sample, particularly liquid (including solution or suspension), and simultaneously, the problems of blocking a chromatographic column caused by residues in the transfer process of the magnetic material can be avoided. Based on this finding, automated manipulation of moving and/or separating a sample under an applied magnetic field can be achieved.

Accordingly, in the method of the present invention, a step of moving and/or separating the sample under application of a magnetic field is further included. More specifically, the step of applying the magnetic field to move and/or separate the sample is performed by placing a magnet on the outside of the wall of the container containing the sample such that the sample is attracted to the inside of the wall of the container when the magnetic field is applied, or by placing a magnet on the bottom of the container, thereby transferring the sample on the machine, and achieving the problem of no magnetic beads remaining in the sample on the machine.

In some embodiments, the moving and/or separating the sample is performed by removing the sample from a container holding the sample and the magnetic beads, or by removing the magnetic beads from a container holding the sample and the magnetic beads.

In some embodiments, the step of moving and/or separating the sample under application of the magnetic field may be accomplished by placing a magnet outside the sidewall of the container containing the sample such that the sample is drawn onto the container sidewall when the magnetic field is applied, or by inserting a magnet into the container containing the sample and removing the magnet.

The magnet may be a permanent magnet or an electromagnet, preferably an electromagnet, which may be conveniently switched on and off to control the presence and absence of the magnetic field. In the case of an electromagnet, the on-off of the electromagnet can be controlled by a Programmable Logic Controller (PLC) so as to meet the full-automatic requirement of sample processing.

In a particular operation, the magnet may be placed or inserted into a solution or suspension containing the sample.

In one embodiment, the step of moving and/or separating the sample under the application of the magnetic field is achieved by placing a magnet outside the sidewall of the container containing the sample, such that the sample is attracted to the container sidewall when the magnetic field is applied. In a more specific embodiment, the magnet may be present in two positions: a side suction position and a bottom suction position. For magnets in a side-draw position, the magnet is located outside the sidewall of the container holding the solution or suspension of the sample, such that when a magnetic field is applied (e.g., by turning on the power supply of an electromagnet or moving a permanent magnet) the sample is drawn onto the container sidewall; for a magnet in a bottom-attractive position, the magnet is positioned below the bottom wall of a container holding a solution or suspension of the sample, such that when a magnetic field is applied (e.g., by turning on the power supply of an electromagnet or moving a permanent magnet) the sample is attracted to the bottom wall of the container. In some embodiments, there may be only magnets in side attraction; in other embodiments, only magnets in the bottom attraction position may be present; in other embodiments, both magnets in the side-attracting and bottom-attracting positions may be present.

The position and/or the on-off state of the magnet are switched to be matched with the operation of transferring liquid (solution or suspension), so that the automatic operation of transferring liquid between different containers can be effectively realized.

In addition, the present invention may better achieve adequate transfer of liquid by a "side-attracting" magnet for moving and/or separating a sample under an applied magnetic field by placing the magnet outside the sidewall of a container holding a solution or suspension of the sample such that the sample is attracted to the container sidewall when the magnetic field is applied. Compared with the bottom suction position design that the magnet is positioned below the bottom wall of the container, the side suction magnet can effectively avoid the defects of liquid loss (insufficient bottom liquid suction) caused by a bottom suction position magnetic field and the defects that the magnetic material is remained and blocks a chromatographic column and the like caused by the fact that the magnetic material is sucked away, and has convenient transfer operation and automation realization.

Based on the above findings, the method of the present invention further comprises the step of moving and/or separating the sample under the applied magnetic field. Further, the step of moving and/or separating the sample under the applied magnetic field is achieved by placing a magnet outside the sidewall of the container holding the solution or suspension of the sample, such that the sample is attracted to the container sidewall when the magnetic field is applied, or by inserting the magnet into the solution or suspension holding the sample and removing the magnet.

In the method of the invention, the container may be a multi-well plate such as a 2-well plate, a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate, or a separate tube.

In the method of the present invention, the following steps may be included:

a) Contacting the magnetic beads with the sample to adsorb target analytes in the sample;

b) Applying a magnetic field to separate the magnetic beads having the target analyte adsorbed thereon from the non-adsorbed sample components;

c) The magnetic beads having the target analyte adsorbed thereon are brought into contact with an eluent, so that the target analyte is allowed to pass from the magnetic beads into the eluent.

In some embodiments of the invention, there is further included a step of further activating and equilibrating the magnetic beads prior to step a, and/or a step of washing the magnetic beads having the target analyte adsorbed thereon after step b) and prior to step c), wherein the washing allows removal of non-specifically adsorbed components other than the target analyte from the magnetic beads while retaining or substantially retaining the target analyte still adsorbed on the magnetic beads.

In some embodiments of the invention, the moving and/or separating of the sample in the method is achieved by aspirating a liquid (or fluid) in one container and releasing the liquid (or fluid) in another container.

In some embodiments of the invention, the container is a multi-well plate or a separate tube; for example, 2-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, etc.; the container may have a U-shaped or V-shaped bottom shape.

In some embodiments of the present invention, the organic solvent for activation is selected from the group consisting of methanol, ethanol, acetonitrile, isopropanol, and a combination thereof, the organic solvent for equilibration is a mixed solvent of water and an organic solvent selected from the group consisting of methanol, ethanol, acetonitrile, and isopropanol, and the solvent for rinsing is water or a mixed solvent of water and an organic solvent selected from the group consisting of methanol, ethanol, acetonitrile, and isopropanol; the solvent for elution is selected from acetonitrile, methanol, ethanol, isopropanol and any combination thereof, or any combination of water and acetonitrile, methanol, ethanol or isopropanol. Preferably, the washing reagents used for washing include a weak washing reagent which may contain 80% to 100% water and a strong washing reagent which may contain 60% to 90% water.

In the method of the present invention, the transfer of the sample between different containers can be achieved by moving the pipetting needle up and down in the container. Preferably, the base of the pipetting needle is provided with an elastic element, so that the pipetting needle can elastically retract when moving downwards to contact the bottom of the container; preferably, the pipetting needle base is provided with an adjustment structure such that the stop position of the pipetting needle tip can be adjusted as close as possible to the bottom of the container without damaging the pipetting needle and/or the container.

In the method of the present invention, the method further comprises the steps of:

the polymer-treated sample is analyzed using an analytical method selected from the group consisting of mass spectrometry and liquid chromatography, including combinations thereof, such as liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS).

In another aspect of the present invention, there is provided a use of polymer-coated magnetic beads (e.g., styrene-based polymer-coated magnetic beads) for detecting a lipid-soluble vitamin in a sample. More specifically, the fat-soluble vitamins are detected using mass spectrometry or liquid chromatography.

In another aspect of the present invention, there is further provided a fat-soluble vitamin detection kit or kit comprising polymer-coated magnetic beads (e.g., styrene-based polymer-coated magnetic beads) and optionally a fat-soluble vitamin as a reference substance. Specifically, the kit is used for detecting the fat-soluble vitamin by mass spectrometry or liquid chromatography. More specifically, the kit further comprises an eluent and optionally a eluent. In some embodiments, the kit or kit is for performing a method according to the invention.

The present inventors have found that the magnetic beads (including PS magnetic beads, HLB magnetic beads, C18 magnetic beads, and C8 magnetic beads) used in the present invention can be effectively used for detecting fat-soluble vitamins. Especially hydrophobic polymer magnetic beads (such as styrene-based polymer magnetic beads and C8 or C18 magnetic beads), and 5 common fat-soluble vitamins (vitamin A and vitamin D) ₂ Vitamin D ₃ Vitamin E and vitamin K ₁ ) Has good grasping and adsorbing effects, especially on vitamin K (such as vitamin K) ₁ ) The detection of (2) is also significantly superior to other types of magnetic beads.

Description of the terms

The terms "magnetic bead", "magnetic particle" and "magnetic particle" are used interchangeably herein to refer to a particle that is magnetically charged.

As used herein, "polymer" refers to a macromolecular compound that may have a relative molecular mass ranging from several thousand to several million and may be a mixture of many homologs with different relative molecular masses.

Herein, the "hydrophobic polymer" refers to a polymer in which the surface (including the inner pore surface) of a magnetic bead formed of the polymer is hydrophobic, such as a styrene-based polymer and an alkyl-modified silica gel polymer, and more specifically, the alkyl-modified silica gel polymer is, for example, a C8 or C18 alkyl-modified silica gel polymer. Magnetic beads coated with a hydrophobic polymer are referred to herein as hydrophobic polymer magnetic beads, such as styrene-based polymer magnetic beads, alkyl-modified silica gel polymer magnetic beads (particularly C18 or C8 alkyl-modified silica gel polymer magnetic beads, also referred to herein as C18 or C8 silica gel magnetic beads, or C18 or C8 magnetic beads).

Herein, "styrene-based polymer" means a polymer formed by polymerizing styrene as a main monomer, and may also be referred to as "styrenic polymer". The styrene-based polymer herein may be polystyrene formed with styrene as the only monomer, or a polymer formed by adding other crosslinking agents (e.g., divinylbenzene), such as styrene-divinylbenzene copolymer. The polymers may be formed by different processes (e.g., fluidized bed processes, reaction vessel processes) and may have different degrees of crosslinking. In this context, the styrene-divinylbenzene copolymer refers to a polymer formed by polymerizing styrene and divinylbenzene as monomers, and particularly includes a copolymer formed by using styrene as a monomer and divinylbenzene as a crosslinking agent.

Herein, "styrene-based polymer magnetic beads" means magnetic beads coated with a styrene-based polymer, for example, polystyrene magnetic beads (PS magnetic beads) means magnetic beads coated with a polystyrene polymer, particularly, a polymer formed by crosslinking styrene and divinylbenzene. In addition, when a magnetic bead of a styrene-based polymer is mentioned herein, it means that the benzene ring of the polymer has no modification group (particularly, hydrophilic modification group).

As used herein, "HLB magnetic beads" are a specific type of polymer-coated magnetic beads, also known as hydrophilic-lipophilic balance magnetic beads. The polymer may further contain a hydrophilic group on the basis of the lipophilic styrene-based skeleton, and a typical hydrophilic group may have an N-vinylpyrrolidone structure, and may also have a structure such as a carboxyl group, a sulfonic acid group, an amine group, and the like. By a suitable ratio of lipophilic structures to hydrophilic groups, a balance of hydrophilic and lipophilic properties within a certain range is achieved.

Herein, "C18 magnetic beads" and "C8 magnetic beads" as specific types of polymer-coated magnetic beads refer to magnetic beads having a silica gel material as a framework and a coated magnetic core modified by a C18 or C8 alkyl group, for example, octadecyl silica gel polymeric magnetic beads, i.e., C18 magnetic beads, can be obtained by octadecyl trichlorosilane bonding, and octyl silane polymeric magnetic beads, i.e., C8 magnetic beads, can be obtained by octane trichlorosilane bonding.

Herein, "contacting a magnetic bead with a sample" means exposing the sample to the magnetic bead such that an interaction occurs.

Herein, fat-soluble vitamins refer to a class of vitamins insoluble in water and soluble in fats and non-polar organic solvents (e.g., benzene, ether, chloroform, etc.), including vitamin A, D, E, K, etc. Vitamin D is cyclopentane polyhydrophenanthrene compound belonging to sterols, including vitamin D ₂ And vitamin D ₃ Also includes its active metabolismForms such as 25-hydroxyvitamin D ₃ 25-hydroxy vitamin D ₂ 1, 25-dihydroxyvitamin D ₃ 24, 25-dihydroxyvitamin D ₃ And so on. The vitamin K includes naturally occurring vitamin K ₁ And K ₂ And the like.

Herein, when referring to the detection of a fat-soluble vitamin, one or more of said fat-soluble vitamins may be detected as target analyte simultaneously, said fat-soluble vitamins being selected from vitamin a, vitamin D ₂ Vitamin D ₃ Vitamin E, vitamin K ₁ And vitamin K ₂ Preferably two or more of said fat-soluble vitamins are detected simultaneously. In a specific embodiment wherein only one of said fat-soluble vitamins is detected, said fat-soluble vitamin is preferably vitamin K (e.g. vitamin K) ₁ And/or K ₂ ). In a specific embodiment wherein two or more of the fat-soluble vitamins are detected simultaneously, the fat-soluble vitamins preferably comprise at least vitamin K (e.g. vitamin K) ₁ And/or K ₂ ) In particular the fat-soluble vitamins may also include vitamin a, vitamin D ₂ Vitamin D ₃ Vitamin E, vitamin K ₁ And vitamin K ₂ Or any combination thereof.

Herein, the particle size means the average particle size of the particle population. The particle size measurement method includes a sieving method, a microscopic method, a sedimentation method, a resistance method, and the like.

As used herein, "liquid chromatography" refers to chromatography using a liquid as the mobile phase, which may have a stationary phase in a variety of forms. "mass spectrometry" refers to a method of detecting ions (charged atoms, molecules or molecular fragments, molecular ions, isotopic ions, fragment ions, rearranged ions, multiply charged ions, metastable ions, negative ions, ions generated by ion-molecule interactions, etc.) that move by separating them according to their mass-to-charge ratios using an electric field and a magnetic field. Liquid chromatography or mass spectrometry herein also includes combinations or combinations thereof, such as liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS), and the like.

In this context, the term "selected from" means that it may be an option listed thereafter, or a combination of one or more of them.

In this context, the purpose of "activation" is to create an environment compatible with the sample solvent and to remove impurities from the container; the purpose of "equilibration" is on the one hand to remove the organic solvent remaining upon activation and on the other hand to facilitate the acceptance of a sample with water as the main solvent, corresponding to a switch of the solvent system; "washing" means an operation for washing away interfering components adsorbed on the magnetic beads but retaining target molecules. Accordingly, "activator," "equilibrating agent," and "rinse" refer to the agents, particularly solvents and solvent combinations, required to accomplish the above-described activation, equilibration, and rinsing operations.

Herein, "retaining the target analyte still adsorbed on the magnetic bead" means that a majority of the target analyte still remains adsorbed on the magnetic bead, e.g. at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even at least 98% of the target analyte still is adsorbed on the magnetic bead.

Herein, "sample" refers to an object to be tested, and may be a biological sample, such as a clinical sample, including whole blood, plasma, serum, urine, saliva, tears, bile, gastric juice, interstitial fluid, lymph fluid, etc., and may be a biological material selected from the group consisting of whole blood, plasma, serum, urine, saliva, tears, bile, gastric juice, interstitial fluid, and lymph fluid, and the sample may be any combination of the above samples. The sample may be in liquid or fluid form, or even in solid form. Samples in solid form may optionally be tested after treatment such as homogenization.

Drawings

Figure 1 is a schematic view of the magnet of the present invention in a side-attracted position,

fig. 2 is a schematic view of the magnet of the present invention in a bottom attracted position.

In fig. 1, the magnet 4 is located at the outer side of the side wall of the container 1 (plate well), the sample solution or suspension 2 is in the container 1, and the magnetic beads 3 are gathered and fixed at the side wall of the container 1 under the action of the external magnetic field of the magnet 4 in the side attraction position, so that the transfer operation of the solution or suspension 2 is not affected, and the liquid in the container 1 can be ensured to be sufficiently sucked out.

In fig. 2, the magnet 4 is positioned below the bottom wall of the container 1 (well) and the sample solution or suspension 2 is present in the container 1. The sample solution or suspension 2 in the container 1 can be agitated or shaken while the magnet 4 is in the bottom attraction position.

Detailed Description

In the following examples, unless otherwise specified, the reagents and instruments used are those commonly used in the art and are available from chemical or biological products/preparations; the procedures used in the following examples are conventional in the art, and the procedures of these experiments can be unambiguously known and the corresponding results obtained by those skilled in the art according to the prior art or the operation manual provided by the manufacturer.

Examples

Example 1.

The standard substances, main reagent sources and instrument models used in the present invention are shown in the following table.

TABLE 1 details of sources of standard substances

Name of standard substance	Brand
		Vitamin A-d5	Toronto Research Chemicals
25-hydroxy vitamin D ₂ -d6	Toronto Research Chemicals
		25-hydroxy vitamin D ₃ -d6	Medical Isotopes
Vitamin E-d6	Sigma-Aldrich
		Vitamin K-d7	Sigma-Aldrich
Vitamin A	Sigma Alrdich
		25-hydroxy vitamin D ₂	Toronto research chemistry canada
25-hydroxy vitamin D ₃	Toronto research chemistry TRC of canada
		Vitamin E	Sigma Alrdich
Vitamin K ₁	Sigma-Aldrich

TABLE 2 details of the sources of the main reagents

Name of reagent	Brand
		Bovine serum albumin	Thermo Fisher
High purity water	Watsons water
		Formic acid	Fisher
Methanol	Fisher chemical
		Acetonitrile	Fisher chemical

TABLE 3 Instrument make and model

Name of instrument	Brand	Model number
			Liquid chromatography tandem mass spectrometer	Aiboceisi Limited	AB SCIEX Triple Quad ^TM 4500MD

The PS beads (polystyrene beads) and HLB beads (hydrophilic lipophilic balance beads) used in the examples herein were prepared according to the method described in CN111250067 a. Specifically, PS beads were prepared according to CN111250067a example 2, HLB beads were prepared according to CN111250067a example 3, and C18 beads/C8 beads (octadecyl polymeric beads/n-octyl polymeric beads) were prepared according to CN 113559830A example 1.

1. Preparation of test solutions

a. Preparation of mixed standard working solution

5 kinds of vitamin A and 25-hydroxy vitamin D are prepared ₂ 25-hydroxy vitamin D ₃ Vitamin E and vitamin K ₁ The mixed standard working solution is ready for use. The stock solution concentrations used and the formulated mixed standard working solution concentrations are shown in table 4 below.

TABLE 4 preparation of the Mixed Standard working solutions

Compound (I)	Stock solution concentration (μ g/mL)	Volume removal (mu L)	Mixed standard working solution (mu g/mL)
				Vitamin A	100	200	20
25-hydroxy vitamin D ₂	10	50	0.5
				25-hydroxy vitamin D ₃	10	200	2
Vitamin E	1000	300	300
				Vitamin K ₁	1	200	0.2
Methanol	-	50

b. Preparation of spiked serum samples

Taking a 9 mL human serum sample, adding 1 mL standard-mixing working solution, mixing for 2min to obtain standard-adding serum sample (vitamin A:2.0 μ g/mL; 25-hydroxy vitamin D) ₂ 0.05 mu g/mL; 25-hydroxy vitamin D ₃ 0.2 mu g/mL; vitamin E is 30 mug/mL; vitamin K ₁ :0.02 μg/mL)。

c. Preparation of working solution with 5 kinds of vitamin internal standards

5 vitamins, namely vitamin A and 25-hydroxy vitamin D, are taken out of a refrigerator at 20 ℃ below zero ₂ 25-hydroxy vitamin D ₃ Vitamin E and vitamin K ₁ The single standard internal standard stock solution is unfrozen at room temperature, and internal standard working solution is prepared according to the table 5 after uniform mixing for later use.

Table 5 internal standard working liquid preparing table

Compound (I)	Stock solution concentration (μ g/mL)	Volume removal (mu L)	Internal standard working solution (mu g/mL)
				Vitamin A-d5	100	200	2
25-hydroxy vitamin D ₂ -d6	10	50	0.05
				25-hydroxy vitamin D ₃ -d6	10	200	0.2
Vitamin E-d6	1000	300	30
				Vitamin K _1- d7	1	200	0.02
80% methanol	-	9050	-

d. Sample pretreatment

Magnetic bead activation: adding 2 mg magnetic solid phase extraction filler (PS magnetic beads, HLB magnetic beads, C18 magnetic beads or C8 magnetic beads) into 400 μ L methanol for activation, shaking at the bottom suction position for 0.5 min, transferring the 96-well plate to the side suction position, standing for 0.5 min, and transferring the activation reagent in the 96-well plate to a waste liquid tank by a liquid transfer system.

Magnetic bead balancing: transferring 400 mu L of water by a liquid transfer system to perform magnetic solid phase extraction and filler balance; oscillating for 0.5 min at the bottom suction position, transferring the 96-well plate to the side suction position, and standing for 0.5 min; the pipetting system transfers the equilibration reagents in the 96-well plate to a waste reservoir.

Internal standard and sample addition: transferring 400 mu L of internal standard working solution by a liquid transfer system, then adding 400 mu L of sample, and oscillating for 5 min at a bottom suction position; the side-draw position was stationary for 1 min and the pipetting system transferred the sample reagents in the 96-well plate to the waste reservoir.

Leaching 1: transferring 400 mu L of pure water by a liquid transferring system, and oscillating for 1 min at a bottom suction position; the side-draw station was allowed to stand for 1 min and the pipetting system transferred the rinse 1 reagents in the 96-well plate to the waste reservoir.

Leaching 2: transferring 400 mu L of 20% acetonitrile by a liquid transferring system, and oscillating for 1 min at a bottom suction position; the side-draw position was stationary for 1 min and the pipetting system transferred the reagents for elution 2 from the 96-well plate to the waste reservoir.

Elution is carried out: transferring 100 mu L of acetonitrile by a liquid transferring system, and oscillating for 2min at a bottom suction position; the side draw station was allowed to stand for 2min and the pipetting system transferred the eluted reagents from the 96 well plate to a 96 well V plate. Waiting for the detection on the computer.

e. HPLC-MS analysis

1) Liquid chromatography detection conditions: a chromatographic column: an octadecyl packed chromatography column (2.1 mm X50 mm,1.7 μm, 130A);

mobile phase: phase A is 0.1% formic acid water solution, and phase B is 0.1% formic acid methanol solution;

gradient elution procedure: 0-0.2min, 80% by weight B; 0.2-2.0min, 98% of (B); 2.0-4.8min,98% b; 4.8-4.9 min,80% by weight B; 4.9-6.0min, 80% by weight of (B);

flow rate: 0.4 mL/min;

sample introduction amount: 10. mu L;

column temperature: 45. is at one hundred percent

2) Mass spectrum conditions:

an ion source: atmospheric Pressure Chemical Ionization (APCI); the detection mode is as follows: multiple Reaction Monitoring (MRM); air curtain gas (CUR): 35 psi, atomizing gas (GS 1): 40 psi, temperature (TEM): 400. c, collision gas (CAD): 8 psi. Mass spectral parameters of parent ion, daughter ion, residence time, cone hole voltage, collision energy, etc. for each compound are shown in table 6 below.

TABLE 6 Mass Spectrometry parameters for the Compounds

Compound (I)	Q1	Q3	DP	EP	CE	CXP
							Vitamin A	269.1	93.1	140	8	50	8
Vitamin A-d5	274.1	93.1	40	8	21	8
							25-hydroxy vitamin D ₂	395.3	269.4	78	9	24	20
25-hydroxy vitamin D ₂ -d6	401.2	269.3	130	10	24	5
							25-hydroxy vitamin D ₃	383.4	229.2	60	10	28	20
25-hydroxy vitamin D ₃ -d6	389.4	229.2	81	13	35	20
							Vitamin E	431	165.2	180	7	35	12
Vitamin E-d6	437.3	171.2	140	10	35	26
							Vitamin K ₁	451.4	187.1	60	8	36	10
Vitamin K-d7	458.3	194.2	95	13	36	19

f. The result of the detection

After being eluted, styryl polymer magnetic beads, hydrophilic-lipophilic balance magnetic beads, C18 magnetic beads and C8 magnetic beads are connected with a mass spectrometer for mass spectrometry, and the results are shown in Table 7.

TABLE 7 response results of various types of magnetic bead signals

Detection object	HLB magnetic bead Signal	PS bead signal	C18 magnetic bead signal	C8 magnetic bead signal
					Vitamin A	3.05×10 ⁶	2.91×10 ⁶	1.45×10 ⁶	1.03×10 ⁶
25-hydroxy vitamin D ₂	3.21×10 ⁵	1.04×10 ⁶	6.93×10 ⁵	8.89×10 ⁵
					25-hydroxy vitamin D ₃	3.34×10 ⁵	1.23×10 ⁶	7.11×10 ⁵	1.76×10 ⁵
Vitamin E	2.21×10 ⁶	5.58×10 ⁶	1.40×10 ⁶	5.59×10 ⁵
					Vitamin K ₁	1.52×10 ³	2.93×10 ⁶	7.33×10 ⁵	1.24×10 ⁶

As can be seen from the above experimental results, the magnetic beads of the present invention can be used for detecting fat-soluble vitamins. Especially PS magnetic beads, C18 magnetic beads and C8 magnetic beads, for 5 fat-soluble vitamins (vitamin A, 25-hydroxyvitamin D) ₂ 25-hydroxy vitamin D ₃ Vitamin E and vitamin K ₁ ) Has good grabbing and adsorbing effects, especially on vitamin K ₁ The detection of (2) is also significantly better than other types of magnetic beads, of which PS beads perform the most excellent.

Example 2: styrene based Polymer magnetic bead Performance inspection

The standard, primary reagent, and magnetic bead sources were the same as in example 1.

The embodiment provides a liquid chromatography tandem mass spectrometry detection method of fat-soluble vitamins based on magnetic solid phase extraction, which comprises the following steps:

1. sample preparation

1.1 The internal standard working solution was prepared as in example 1.

1.2 Repetitive sample preparation

The method comprises the steps of taking 10 real serum samples, taking 1 mL from each real serum sample, mixing the samples together to prepare a 10 mL real serum sample, repeatedly measuring the sample 15 times, and inspecting the repeatability of the method.

1.3 Accuracy sample preparation

Taking out the sample of the reference substance from the refrigerator, thawing for standby, and ensuring that the measured value is within the required range of the quality control product.

2. Sample pretreatment and HPLC-MS were the same as in example 1.

3. Results of the experiment

3.1 Repeatability of

Samples of known concentration 2-3 were tested in parallel at least 10 times and the Coefficient of Variation (CV) should be no greater than 15.0%.

The experimental result shows that: in the determination method for capturing and enriching the fat-soluble vitamins in the serum by the styryl polymer magnetic beads, the repeatability meets the requirement of clinical detection.

3.2 Accuracy of

The detection is carried out using as a sample a reference substance (standard substance) which can be used for evaluating a conventional method, and the measurement result thereof should be within a concentration range allowed for the standard substance.

The experimental result shows that: in the determination method for capturing and enriching the fat-soluble vitamins in the serum by the styryl polymer magnetic beads, the accuracy meets the requirement of clinical detection.

Claims

1. A method of detecting a target analyte in a sample, comprising the step of contacting polymer-coated magnetic beads to the sample, wherein the target analyte is a lipid-soluble vitamin.

2. The method of claim 1, wherein the polymer is a hydrophobic polymer.

3. The method of claim 1 or 2, wherein the polymer is an alkyl modified silica gel polymer or a styrene based polymer.

4. The method according to claim 3, wherein the alkyl modified silica gel polymer is a C18 or C8 alkyl modified silica gel polymer and/or the styrene based polymer is a styrene-divinylbenzene copolymer.

5. The method according to claim 1 or 2, wherein the magnetic beads have an average particle diameter of 1 to 200 μm, 10 to 100 μm, or 30 to 50 μm.

6. The method of claim 1 or 2, wherein the sample is or comprises a biological material selected from the group consisting of whole blood, plasma, serum, urine, saliva, tears, bile, gastric fluid, interstitial fluid and lymphatic fluid.

7. The method of claim 1 or 2, further comprising the step of applying a magnetic field to move and/or separate the sample.

8. The method of claim 7, wherein the step of applying the magnetic field to move and/or separate the sample is accomplished by placing a magnet on the outside of the wall of the vessel containing the sample such that the sample is attracted to the inside of the wall of the vessel when the magnetic field is applied, or by inserting a magnet into the vessel containing the sample and removing the magnet.

9. The method of claim 8, wherein the applied magnetic field is generated by a permanent magnet or an electromagnet.

10. The method of claim 7, wherein the moving and/or separating of the sample is performed by removing the sample from a container containing the sample and the magnetic beads, or by removing the magnetic beads from a container containing the sample and the magnetic beads.

11. The method of claim 8, wherein the container is a multi-well plate or a separate tube.

12. The method according to claim 1 or 2, comprising the steps of:

13. The method of claim 12, further comprising the step of activating and equilibrating the magnetic beads prior to step a), and/or the step of washing the magnetic beads having target analytes adsorbed thereon after step b) and prior to step c), wherein the washing allows removal of non-specific adsorbed components other than the target analytes from the magnetic beads while leaving the target analytes adsorbed on the magnetic beads.

14. The method of claim 12, wherein the elution uses an eluent selected from the group consisting of: acetonitrile, methanol, ethanol, isopropanol, and any combination thereof, or any combination of water and acetonitrile, methanol, ethanol, or isopropanol.

15. The method of claim 13, wherein the activation uses an activating agent selected from the group consisting of: methanol, ethanol, acetonitrile, isopropanol, and combinations thereof, the rinsing using a rinsing agent selected from the group consisting of: water or a mixed solvent of water and an organic solvent selected from the group consisting of methanol, ethanol, acetonitrile and isopropanol.

16. The method according to claim 1 or 2, further comprising the steps of:

analyzing the polymer-treated sample using an analytical method selected from the group consisting of mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

17. The method of claim 1 or 2, wherein the fat soluble vitamin is or comprises vitamin K.

18. The method of claim 17, wherein the fat soluble vitamins further comprise a vitamin selected from the group consisting of vitamin a, vitamin D ₂ Vitamin D ₃ And vitamin E, or any combination thereof.

19. A fat-soluble vitamin detection kit comprises polymer-coated magnetic beads.

20. The kit of claim 19, wherein the polymer-coated magnetic beads are hydrophobic polymer-coated magnetic beads.

21. The kit of claim 19, wherein the polymer-coated magnetic beads are styrene-based polymer-coated magnetic beads or alkyl-modified silica gel polymer magnetic beads.

22. The kit of claim 19, further comprising a fat soluble vitamin as a reference substance.

23. The kit of claim 19, wherein the kit is for carrying out the method of claim 1 or 2.

24. Use of polymer-coated magnetic beads for the detection of fat-soluble vitamins.

25. The use of claim 24, wherein the polymer-coated magnetic beads are styrene-based polymer-coated magnetic beads.

26. Use according to claim 24, wherein the fat-soluble vitamin is detected using the method according to claim 1 or 2.