Kit and method for detecting vitamin K1 and K2 by magnetic bead method

Abstract

The present invention discloses a kit and method for detecting vitamin K1 and K2 by magnetic bead method, the kit comprising an internal standard solution, a leaching solution, an eluent, a calibration solution and magnetic beads, the magnetic beads being composed of a magnetic carrier core and a shell layer wrapped in an outer layer modification; wherein the internal standard is vitamin K1-D7 and vitamin K2-D7, and the internal standard solution comprises 30-60% methanol, 0.1-0.5% sodium dodecyl sulfate and 1-5% polyethylene glycol. The kit provided by the present invention is applied to determine the content of vitamin K1K2, which simplifies the pre-treatment process of the sample, simplifies the operation of the test, and improves the throughput of clinical detection compared with the existing method. The present invention uses a magnetic bead method to determine vitamin K1K2, instead of protein precipitation, liquid-liquid extraction method, to reduce the interference of the matrix on vitamin K1K2 when the traditional method is used to extract vitamin K.

Description

Technical Field

[0001] The invention belongs to the field of analytical technology and relates to the detection of vitamins, in particular to a kit and a method for detecting vitamins K1 and K2 using a magnetic bead method. Background Art

[0002] Vitamin K is a derivative of 2-methyl-1,4-naphthoquinone. Vitamin K widely found in nature includes vitamin K1 and vitamin K2. Vitamin K1, also known as plant menaquinone or phylloquinone, is mainly found in dark green vegetables (such as cabbage, spinach, lettuce, etc.) and vegetable oils. Vitamin K2 is a product of intestinal bacteria. Vitamin K3 is a synthetic water-soluble menaquinone that can be taken orally and injected. The most important ones are vitamin K1 and K2.

[0003] Vitamin K controls coagulation factor II (prothrombin), coagulation factor VII (conversion accelerating factor precursor), and factor IX (cnvisma5), which is a component of plasma prothrombin. The coagulation factors that depend on vitamin K are protein C, protein S, and protein Z. Protein C and protein S are procoagulants. The two bone matrix proteins required for normal bone metabolism are vitamin K dependent. The two vitamin K proteins are osteocalcin (BGP) and matrix protein (MGP). Calcium-binding protein plays a very important role in regulating bone and body calcium. It can increase bone density and prevent osteoporosis. It has a cleaning effect on bone spurs, arteriosclerotic calcium plaques, and tissue calcium deposition and hardening, which deserves further research and discussion. Carboxylation reaction and vitamin K circulation are the utilization pathways of vitamin K. Large doses of vitamin K (1-10 mg) can overcome the blockade of coumarin. It regenerates vitamin K hydroquinone through liver reductase. Vitamin K dissolves in the lipids of the mitochondrial membrane and plays an electron transfer role. The semihydroquinone free radicals produced by the reduction of vitamin K by body enzymes prevent cell proliferation and increase sensitivity to radiation. It can be used as a sensitizer for tumor radiotherapy. Vitamin K selectively and directly relaxes smooth muscle, and has a significant inhibitory effect on smooth muscle contraction caused by acetylcholine, and has analgesic and sedative effects, and can enhance the sedative and analgesic effects of salicylic acid and morphine.

[0004] The human body needs very little vitamin K, but it maintains normal blood coagulation, reduces heavy bleeding during menstruation, and prevents internal bleeding and hemorrhoids. People who often have nosebleeds should get more vitamin K from natural foods. Vitamin K is mainly absorbed in the small intestine and metabolized with chylomicrons. The body's storage of vitamin K is limited, and the first fat-soluble vitamin deficiency caused by lipid absorption disorder is vitamin K deficiency. Vitamin K is a commonly used hemostatic drug in clinical practice. Since 1974, Chinese scholars have found through a series of experimental studies and clinical observations that vitamin K also has obvious antispasmodic and analgesic effects. In recent years, a large number of medical workers have found in clinical observations that vitamin K also has obvious antiasthmatic effects on bronchiolitis, bronchial asthma, asthmatic tracheitis, and tracheitis; it has a certain therapeutic effect on pediatric diarrhea; and it also has a certain inhibitory effect on certain bacteria. Accurately quantifying the levels of vitamin K1 and K2 in the human body can help guide doctors to rationally supplement multiple vitamins and minerals, improve the nutritional status of human nutrients, ensure the normal growth and development of the people, bone development, intellectual development, and improve immune function.

[0005] Common methods for detecting vitamin K1 and K2 in the human body mainly include protein precipitation, liquid-liquid extraction, and solid phase extraction. The protein precipitation method usually uses organic solvents to precipitate proteins and take the supernatant for analysis. The sample purification efficiency is low, which is easy to cause blockage of the chromatographic column and contamination of the mass spectrometer. The liquid-liquid extraction method usually uses normal phase organic solvents (n-hexane, methyl tert-butyl ether), and the sample needs nitrogen blowing. The organic solvent is easy to volatilize, which is not friendly to the laboratory environment and personnel. The solid phase extraction method uses SPE plates to purify the sample at the same time. The sample purification effect is better, but the SPE solid phase filler is easily swollen by organic solvents, and the uniformity between batches is poor. The SPE solid phase extraction method has high consumables cost and is not suitable for large-scale clinical sample testing. For example, the "method for simultaneous detection of vitamin K1, MK4 and MK7" disclosed in patent CN113933410B uses a highly volatile organic solvent of n-hexane. The method requires protein precipitation, extraction, nitrogen blowing, and re-dissolution. The operation is complicated and has a certain impact on the body of the experimenter. For example, Yoshitomo Suhara, Maya Kamao, Naoko Tsugawa, et al published an article titled "Method for the Determination of Vitamin K Homologues in Human Plasma Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry". This method uses Sep Pak silica SPE plates for purification and enrichment, transfers to 96-well plates, and tests on a machine. The amount of solvent used in the SPE plate is relatively large, which can easily cause the SPE plate to swell, resulting in uneven results.

[0006] Currently, there is no test kit for the joint detection of vitamin K1 and K2 in human serum. The detection is time-consuming, cumbersome, requires high technical personnel, and cannot be used in conjunction with automation. Summary of the invention

[0007] In order to solve the above problems, the present invention first provides a kit for detecting vitamin K1K2 by automatic sample pretreatment based on magnetic method, which uses magnetic bead method to determine K1K2 instead of protein precipitation and liquid-liquid extraction to reduce the interference of matrix on vitamin K1K2 when extracting vitamin K by traditional method.

[0008] Secondly, the present invention provides a liquid chromatography-tandem mass spectrometry method for automated quantitative determination of vitamins K1 and K2 using magnetic beads.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A kit for detecting vitamin K1 and K2 by a magnetic bead method, the kit comprising an internal standard solution, a leaching solution, an eluent, a calibration solution and magnetic beads, wherein the magnetic beads are composed of a magnetic carrier core and a shell layer wrapped in an outer modified layer; wherein the internal standards are vitamin K1-D7 and vitamin K2-D7, and the internal standard solution comprises 30-60% methanol, 0.1-0.5% sodium dodecyl sulfate and 1-5% polyethylene glycol.

[0011] As a preferred embodiment of the present invention, the eluent is a 40-70% methanol aqueous solution.

[0012] As a preferred embodiment of the present invention, the eluent is one or more of methanol, ethanol, acetonitrile and isopropanol.

[0013] As a preferred embodiment of the present invention, the calibrator solution includes a mixed solution of vitamin K1 and vitamin K2 with a series of concentrations.

[0014] As a preferred embodiment of the present invention, the calibration solution includes 20 ng / mL of vitamin K1 and 40 ng / mL of vitamin K2 with known series concentrations.

[0015] As a preferred embodiment of the present invention, the magnetic carrier core of the magnetic beads is Fe 3 O 4 , the outer layer is modified with 4-toluenesulfonyl chloride, and the particle size of the magnetic beads is 5μm.

[0016] The present invention also provides a method for detecting vitamin K1 and K2 using the magnetic bead method of the above kit, comprising the following steps:

[0017] 1) Add serum and internal standard solution to a centrifuge tube, vortex at 1100 rpm for 30 s, transfer the supernatant after centrifugation to a new centrifuge tube, add magnetic bead solution, vortex at 1100 rpm for 30 s, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube;

[0018] 2) Add the eluent to the centrifuge tube, mix well, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube; add the eluent again, mix well, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube;

[0019] 3) Add the eluent to the centrifuge tube, mix well, use a magnet to adsorb the magnetic beads to the tube wall, collect all the liquid in the centrifuge tube into a 96-well plate, and detect it on a liquid chromatography-tandem mass spectrometer.

[0020] As a preferred embodiment of the present invention, the amount of serum added is 200 μL, the amount of internal standard solution added is 50 μL, the amount of eluent added is 1000 μL, the amount of eluent added is 100 μL, the amount of calibration solution added is 200 μL, and the amount of magnetic beads added is 5 mg.

[0021] As a preferred embodiment of the present invention, in step 3), the chromatographic conditions are: chromatographic column: 2.1*50 mm, 1.7 μm Waters BEH C18 + online filter; flow rate: 0.4 mL / min; column temperature: 50° C.; injection: 20 μL;

[0022] The gradient elution program was: 0-0.8 min, 5% mobile phase A and 95% mobile phase B;

[0023] 0.8-3min, 5% mobile phase A and 95% mobile phase B;

[0024] 3-3.5 min, 0% mobile phase A and 100% mobile phase B;

[0025] 3.5-3.6 min, 0% mobile phase A and 100% mobile phase B;

[0026] 3.6-4min, 5% mobile phase A and 95% mobile phase B;

[0027] At 4 min, 5% mobile phase A and 95% mobile phase B.

[0028] As a preferred embodiment of the present invention, in step 3), the mass spectrometry conditions are: ESI+; capillary voltage: 3.0Kv; desolvation temperature: 500°C; desolvation flow rate: 1000L / h; cone voltage: 50V; scanning mode: multiple reaction monitoring mode.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1) The kit provided by the present invention is used to determine the content of vitamin K1K2. Compared with the existing methods, it simplifies the sample pretreatment process, simplifies the test operation, and improves the throughput of clinical testing.

[0031] 2) The present invention uses a magnetic bead method to determine vitamin K1K2, replacing protein precipitation and liquid-liquid extraction, thereby reducing the interference of the matrix on vitamin K1K2 when extracting vitamin K in traditional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a linear graph of Vitamin K1.

[0033] Figure 2 is a linear graph of Vitamin K2. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] In the present invention, the raw materials, reagents or equipment used can be purchased from the market.

[0036] The present invention first provides a kit for detecting vitamin K1 and K2 by a magnetic bead method, wherein the kit comprises an internal standard solution, a rinsing solution, an eluent, a calibration solution and magnetic beads, wherein the magnetic beads are composed of a magnetic carrier core and a shell layer wrapped in an outer modified layer; wherein the internal standards are vitamin K1-D7 and vitamin K2-D7, and the internal standard solution comprises 30-60% methanol, 0.1-0.5% sodium dodecyl sulfate and 1-5% polyethylene glycol.

[0037] The eluent is 40-70% methanol in water.

[0038] The eluent is one or more of methanol, ethanol, acetonitrile and isopropanol.

[0039] The calibrator solution includes a mixed solution of vitamin K1 and vitamin K2 with a series of concentrations.

[0040] The calibration solution includes 20 ng / mL of vitamin K1 and 40 ng / mL of vitamin K2 with known concentrations.

[0041] The magnetic carrier core of the magnetic beads is Fe 3 O 4 , the outer layer is modified with 4-toluenesulfonyl chloride, and the particle size of the magnetic beads is 5μm.

[0042] Example 1

[0043] This embodiment discloses a method for detecting vitamin K1 and K2 using the above kit, comprising the following steps:

[0044] 1) Add serum and internal standard solution to a centrifuge tube, vortex at 1100 rpm for 30 seconds, transfer the supernatant after centrifugation to a new centrifuge tube, add magnetic bead solution, vortex at 1100 rpm for 30 seconds, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube.

[0045] 2) Add the eluent to the centrifuge tube, mix well, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube; add the eluent again, mix well, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube;

[0046] 3) Add the eluent to the centrifuge tube, mix well, use a magnet to adsorb the magnetic beads to the tube wall, collect all the liquid in the centrifuge tube into a 96-well plate, and detect it on a liquid chromatography-tandem mass spectrometer.

[0047] The amount of serum added was 200 μL, the amount of internal standard solution added was 50 μL, the amount of eluent added was 1000 μL, the amount of eluent added was 100 μL, the amount of calibration solution added was 200 μL, and the amount of magnetic beads added was 5 mg.

[0048] Among them, the setting of chromatographic conditions:

[0049] Chromatographic column: Waters BEH C18 (2.1*50mm, 1.7μm) + online filter; flow rate: 0.4mL / min; column temperature: 50℃; injection: 20μL

[0050] The gradient elution program is shown in Table 1.

[0051] Table 1. Gradient elution program

[0052]

[0053]

[0054] Setting of mass spectrometry conditions:

[0055] The ion source parameters are shown in Table 2.

[0056] Table 2. Ion source parameters

[0057] Parameter Value Source ESI+ Capillary 3.0 Desolvation temp 500 Desolvation(L / Hr) 1000 Cone 50

[0058] Scanning mode: Multiple reaction monitoring (MRM) see Table 3.

[0059] Table 3. Multiple reaction monitoring

[0060]

[0061] See also Figure 1 and Figure 2 , Figure 1 is a linear graph of vitamin K1 obtained by the method of the present invention, Figure 2 is a linear graph of vitamin K2 obtained by the method of the present invention.

[0062] The method of the present invention was tested for the recovery rates of vitamin K1 and vitamin K2, and the results are shown in Table 4.

[0063] Table 4. Recovery rate of vitamin K1 and K2 (%)

[0064] project Low concentration (% recovery) Medium concentration (recovery rate%) High concentration (recovery %) Vitamin K1 102.12 98.86 98.08 Vitamin K2 98.34 99.74 102.00

[0065] The method of the present invention was subjected to intra-batch and inter-batch comparison at low concentrations, and the results are shown in Table 5.

[0066] Table 5. Intra-batch and inter-batch CV% of vitamin K1 and K2 (low concentration)

[0067]

[0068] The method of the present invention was subjected to high concentration intra-batch and inter-batch comparison, and the results are shown in Table 6.

[0069] Table 6. Intra-batch and inter-batch CV% of vitamin K1 and K2 (high concentration)

[0070]

[0071] It can be seen that both intra-assay and inter-assay CVs were less than 10%.

[0072] The kit provided by the present invention is used to determine the content of vitamin K1K2. Compared with the existing method, it simplifies the sample pretreatment process, simplifies the test operation, and improves the throughput of clinical testing. The present invention uses a magnetic bead method to determine vitamin K1K2, replacing protein precipitation and liquid-liquid extraction, reducing the interference of the matrix on vitamin K1K2 when extracting vitamin K in the traditional method.

[0073] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A kit for detecting vitamin K1 and K2 by magnetic bead method, It is characterized in that The kit comprises an internal standard solution, a rinse solution, an eluent, a calibration solution and magnetic beads, wherein the magnetic beads are composed of a magnetic carrier core and a shell layer wrapped in an outer modified layer; wherein the internal standards are vitamin K1-D7 and vitamin K2-D7, the internal standard solution comprises 30-60% methanol, 0.1-0.5% sodium dodecyl sulfate and 1-5% polyethylene glycol; the rinse solution is a 40-70% methanol aqueous solution; the eluent is one or more of methanol, ethanol, acetonitrile and isopropanol; The calibrator solution includes a mixed solution of vitamin K1 and vitamin K2 with a series of concentrations; The calibration solution includes 20 ng / mL vitamin K1 and 40 ng / mL vitamin K2 with known series concentrations; The magnetic carrier core of the magnetic beads is Fe 3 O 4 , the outer layer is modified with 4-toluenesulfonyl chloride, and the particle size of the magnetic beads is 5μm.

2. A method for detecting vitamin K1 and K2 using magnetic beads. It is characterized in that The method uses the kit according to claim 1, comprising the following steps: 1) Add serum and internal standard solution to a centrifuge tube, vortex at 1100 rpm for 30 s, transfer the supernatant after centrifugation to a new centrifuge tube, add magnetic bead solution, vortex at 1100 rpm for 30 s, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube; 2) Add the eluent to the centrifuge tube, mix well, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube; add the eluent again, mix well, use a magnet to adsorb the magnetic beads to the tube wall, and discard all the liquid in the centrifuge tube; 3) Add the eluent to the centrifuge tube, mix well, use a magnet to adsorb the magnetic beads to the tube wall, collect all the liquid in the centrifuge tube into a 96-well plate, and detect it on a liquid chromatography-tandem mass spectrometer.

3. The method for detecting vitamin K1 and K2 by magnetic bead method according to claim 2, It is characterized in that The amount of serum added is 200 μL, the amount of internal standard solution added is 50 μL, the amount of eluent added is 1000 μL, the amount of eluent added is 100 μL, the amount of calibration solution added is 200 μL, and the amount of magnetic beads added is 5 mg.

4. The method for detecting vitamin K1 and K2 by magnetic bead method according to claim 2, It is characterized in that In step 3), the chromatographic conditions are: chromatographic column: 2.1*50 mm, 1.7 μm Waters BEH C18 + online filter; flow rate: 0.4 mL / min; column temperature: 50° C.; injection: 20 μL; The gradient elution program was: 0-0.8 min, 5% mobile phase A and 95% mobile phase B; 0.8-3min, 5% mobile phase A and 95% mobile phase B; 3-3.5 min, 0% mobile phase A and 100% mobile phase B; 3.5-3.6 min, 0% mobile phase A and 100% mobile phase B; 3.6-4min, 5% mobile phase A and 95% mobile phase B; At 4 min, 5% mobile phase A and 95% mobile phase B.

5. The method for detecting vitamin K1 and K2 by magnetic bead method according to claim 2, It is characterized in that In step 3), the mass spectrometry conditions are: ESI+; capillary voltage: 3.0 Kv; desolvation temperature: 500°C; desolvation flow rate: 1000 L / h; cone voltage: 50 V; scanning mode: multiple reaction monitoring mode.

Images