CN112834681A

CN112834681A - Method for detecting content of vitamin K2(MK-7) in blood

Info

Publication number: CN112834681A
Application number: CN202110006256.3A
Authority: CN
Inventors: 邱天祎; 贾永娟; 许丽; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2021-01-05
Filing date: 2021-01-05
Publication date: 2021-05-25
Anticipated expiration: 2041-01-05
Also published as: CN112834681B

Abstract

The invention provides a method for detecting the content of vitamin K2(MK-7) in blood. Respectively detecting at least three standard solutions by using a high performance liquid tandem mass spectrometer under certain detection conditions to obtain chromatograms of the standard solutions, wherein each standard solution contains a vitamin K2(MK-7) standard product with known concentration and an internal standard substance; fitting according to the chromatogram of each standard solution to obtain a standard curve equation; mixing the internal standard working solution and a blood sample taken from at least 100 mu L of blood uniformly, adding absolute ethyl alcohol and mixing uniformly, adding normal hexane and mixing uniformly, centrifuging to obtain a supernatant, drying the supernatant with nitrogen, adding an acetonitrile solution containing 0-0.1% of formic acid and 5-20% of water and mixing uniformly, centrifuging to obtain a supernatant to obtain a sample to be detected; detecting the sample to be detected to obtain a chromatogram map; and calculating the vitamin content in the blood sample according to the chromatogram and a standard curve equation. The sample pretreatment can not involve SPE process, so the experiment cost can be reduced, and the blood consumption is less.

Description

Method for detecting content of vitamin K2(MK-7) in blood

Technical Field

The invention relates to the technical field of clinical chemistry, in particular to a method for detecting the content of vitamin K2(MK-7) in blood.

Background

Vitamin K is also called a blood coagulation vitamin,comprising K₁、K₂、K₃、K₄Etc. in several forms, wherein K₁、K₂Is naturally occurring and belongs to fat-soluble vitamins. All vitamin K is structurally similar, but the longer the side chain the better the absorption, the higher the bioactivity, the longer the time in the blood. Therefore, long-chain menaquinones (especially MK-7) are the most preferred because they are almost completely absorbed by the body and stay in the blood for the longest time.

The most common adult VitK (Vitamin K) deficient bleeding is known to occur in patients who ingest a low VitK diet and take antibiotics, and is seen in malabsorption syndrome and other gastrointestinal disorders such as cystic fibrosis, sprue, ulcerative colitis, and the like. In addition, the small amount of VitK transported by placenta, the low in vivo storage capacity of newborn infants and the sterile state of in vivo intestinal tracts prevent the VitK from being utilized, the low content of VitK in breast milk, the small amount of breast milk sucked by newborn infants and the fact that the immature liver of infants cannot synthesize normal amount of blood coagulation factors, so that the newborn infants and small infants commonly have the low prothrombin disease. However, administration of VitK2 in an amount exceeding the pharmacological dose may lead to hemolytic anemia in newborn, hyperbilirubinemia and hepatotoxicity, and may induce heart and lung diseases in adults.

Vitamin K2(MK-7), or Menaquinone-7, MK-7, with the molecular formula C₄₆H₆₄O₂Is a type of vitamin K2. In recent studies, papers published in journal of alzheimer's disease and scientific report investigated the effect of aortic sclerosis caused by calcification on dementia and retinal artery health, respectively. It has been found that the pathogenesis of both diseases is influenced by the active Matrix Gla Protein (MGP), and the currently most effective vascular calcification inhibitor is vitamin K2 (MK-7). In addition, research shows that MK-7 has the function of preventing and treating osteoporosis. In conclusion, the kit has important clinical significance for detecting the vitamin K2(MK-7) in blood.

At present, the content of vitamin K2(MK-7) in blood can be detected by adopting a liquid chromatography-mass spectrometry combined method, but most of pretreatment methods of blood samples comprise an SPE (Solid-Phase Extraction) process, so that the experiment cost is increased, and the blood consumption required by detection is large.

Disclosure of Invention

The invention provides a method for detecting the content of vitamin K2(MK-7) in blood, and the pretreatment method of a blood sample can not involve an SPE process, so that the experimental cost can be reduced, the dosage of the blood sample is reduced, and the method can be better applied to clinical examination.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention provides a method for detecting the content of vitamin K2(MK-7) in blood, which comprises the following steps:

respectively detecting at least three standard solutions by using a high performance liquid chromatography tandem mass spectrometer under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a vitamin K2(MK-7) standard substance and an internal standard substance with known concentrations, and the vitamin K2(MK-7) standard substances in different standard solutions have different concentrations;

fitting to obtain a standard curve equation of vitamin K2(MK-7) according to the chromatogram of each standard solution;

uniformly mixing a certain amount of internal standard working solution and a certain amount of blood sample, then adding a certain amount of absolute ethyl alcohol and uniformly mixing, then adding a certain amount of n-hexane and uniformly mixing, centrifuging to obtain supernatant, blow-drying the supernatant in a nitrogen blowing mode, adding a certain amount of diluent and uniformly mixing after blow-drying, centrifuging to obtain supernatant to obtain a sample to be detected, wherein the blood sample is serum or plasma obtained by processing at least 100 mu L of blood to be detected, the internal standard working solution contains an internal standard substance with known concentration, and the diluent is acetonitrile solution containing 0-0.1% formic acid and 5-20% water;

detecting a sample to be detected by using a high performance liquid chromatography tandem mass spectrometer under the same detection condition to obtain a chromatogram of the sample to be detected;

and calculating the content of vitamin K2(MK-7) in the blood sample according to the chromatogram of the sample to be detected and a standard curve equation of the vitamin K2 (MK-7).

Preferably, mixing a certain amount of internal standard working solution and a certain amount of blood sample, then adding a certain amount of absolute ethyl alcohol and mixing, then adding a certain amount of n-hexane and mixing, centrifuging to obtain a supernatant, blow-drying the supernatant by nitrogen blowing, adding a certain amount of diluent and mixing after blow-drying, centrifuging to obtain a supernatant to obtain a sample to be tested, including:

transferring 50-200 mu L of blood sample into a centrifuge tube by using a liquid transfer gun, then adding a certain amount of internal standard working solution, and mixing for 20s-2min by vortex oscillation at the rotating speed of 1000-;

then adding 0.3-1.0mL of absolute ethyl alcohol, mixing for 1-4min by vortex oscillation at the rotating speed of 1000 plus 2500rpm, adding 0.5-1.2mL of n-hexane, mixing for 4-10min by vortex oscillation at the rotating speed of 1000 plus 2500rpm, centrifuging for 5-12min at a high speed at the rotating speed of 10000 plus 12000rpm, and transferring the supernatant to another centrifuge tube;

blowing the transferred supernatant by using nitrogen, adding 50-400 mu L of diluent after blowing, mixing for 1-3min by vortex oscillation at the rotating speed of 1000-.

Preferably, the internal standard working solution contains 10-60ng/mL of vitamin K2(MK-7) -d7, and the addition amount is 10 mu L.

Preferably, the liquid phase condition of the detection conditions includes: a pentafluorophenyl chromatographic column, wherein the mobile phase A is an aqueous solution containing 0.5% formic acid, the mobile phase B is a mixed solution of methanol and acetonitrile, the volume ratio of the methanol to the acetonitrile in the mixed solution is 1:4, and a gradient elution mode is adopted in the elution process.

Preferably, the pentafluorophenyl chromatography column comprises: phenomenex Kinetex F5 column, length of column is 100mm, internal diameter is 2.1mm, and packing particle diameter is 1.7 μm.

Preferably, the flow rate is 0.80 mL/min;

the elution process comprises:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0.00	12	88
3.00	12	88
			3.01	0	100
4.00	0	100
			4.01	12	88
6.00	12	88

。

Preferably, the analysis time is 4-10min, the column temperature is 30-50 ℃, the sample injection amount is 5-50 mu L, and the flow rate is 0.50-1.00 mL/min.

Preferably, tandem mass spectrometry conditions among the detection conditions include: an APCI (+) detection mode, wherein the acquisition mode is MRM, the air pressure of an air curtain is 20-55psi, the pressure of collision air is 1-12psi, the atomization current value is 1-5 muA, the ionization temperature is 200-750 ℃, and the atomization air pressure is 10-90 psi; the assist gas pressure was 0 psi.

Preferably, before the separately detecting at least three standard solutions, further comprising:

preparing at least three standard working solutions, wherein the standard working solutions contain vitamin K2(MK-7) standard products with known concentrations, the concentration of the vitamin K2(MK-7) standard products in the standard working solutions is in the range of 0.3-153.6ng/mL, and the concentration of the vitamin K2(MK-7) standard products in different standard working solutions is different;

transferring 10 mu L of standard working solution and 10 mu L of internal standard working solution into a centrifuge tube by using a pipette, and then transferring and adding 80 mu L of diluent;

and (3) uniformly mixing the centrifugal tube in a vortex manner at the rotation speed of 1000-2500rpm for 0.5-1min to obtain a standard solution.

Preferably, the concentration of the vitamin K2(MK-7) standard in each standard working solution is 0.3ng/mL, 0.6ng/mL, 1.2ng/mL, 2.4ng/mL, 4.8ng/mL, 9.6ng/mL, 19.2ng/mL, 38.4ng/mL, 76.8ng/mL, 153.6ng/mL respectively.

The invention provides a method for detecting the content of vitamin K2(MK-7) in blood. Respectively detecting at least three standard solutions by using a high performance liquid tandem mass spectrometer under certain detection conditions to obtain chromatograms of the standard solutions, wherein each standard solution contains a vitamin K2(MK-7) standard product with known concentration and an internal standard substance; fitting according to the chromatogram of each standard solution to obtain a standard curve equation; mixing the internal standard working solution and a blood sample taken from at least 100 mu L of blood uniformly, adding absolute ethyl alcohol and mixing uniformly, adding normal hexane and mixing uniformly, centrifuging to obtain a supernatant, drying the supernatant with nitrogen, adding an acetonitrile solution containing 0-0.1% of formic acid and 5-20% of water and mixing uniformly, centrifuging to obtain a supernatant to obtain a sample to be detected; detecting the sample to be detected to obtain a chromatogram map; and calculating the content of vitamin K2(MK-7) in the blood sample according to the chromatogram and the standard curve equation of the sample to be detected. Because the sample pretreatment can not involve the SPE process, the experiment cost can be reduced, and the blood sample dosage is reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a flowchart of a method for detecting the vitamin K2(MK-7) content in blood according to an embodiment of the invention;

FIG. 2 is a chemical structural formula of vitamin K2(MK-7) provided by an embodiment of the invention;

FIG. 3 is a chromatogram of a vitamin K2(MK-7) standard in a standard solution provided by an embodiment of the invention;

FIG. 4 is a chromatogram of vitamin K2(MK-7) -d7 in a standard solution provided by an embodiment of the invention;

FIG. 5 is a mass spectrum of vitamin K2(MK-7) standard substance and vitamin K2(MK-7) -d7 in a standard solution provided by an embodiment of the invention;

FIG. 6 is a chromatogram of vitamin K2(MK-7) in a test sample according to an embodiment of the invention;

FIG. 7 is a chromatogram of vitamin K2(MK-7) -d7 in a test sample, according to an embodiment of the invention;

FIG. 8 is a mass spectrum of vitamin K2(MK-7) standard and vitamin K2(MK-7) -d7 in a test sample according to an embodiment of the invention;

FIG. 9 is a graph showing the linear relationship between vitamin K2(MK-7) according to an embodiment of the present invention.

In the drawings, 1: vitamin K2(MK-7), 2: vitamin K2(MK-7) -d 7.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.

As shown in figure 1, the embodiment of the invention relates to the detection of endogenous substances of vitamins, and particularly provides a method for detecting the content of vitamin K2(MK-7) in blood, which is not used for the diagnosis and treatment of diseases and can comprise the following steps:

step 101: and respectively detecting at least three standard solutions by using a high performance liquid chromatography tandem mass spectrometer under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a vitamin K2(MK-7) standard substance and an internal standard substance with known concentrations, and the vitamin K2(MK-7) standard substances in different standard solutions have different concentrations.

Referring to FIG. 2, FIG. 2 shows the chemical structural formula of vitamin K2 (MK-7).

Step 102: and fitting to obtain a standard curve equation of the vitamin K2(MK-7) according to the chromatogram of each standard solution.

Step 103: uniformly mixing a certain amount of internal standard working solution and a certain amount of blood sample, then adding a certain amount of absolute ethyl alcohol and uniformly mixing, then adding a certain amount of n-hexane and uniformly mixing, centrifuging to obtain supernatant, blow-drying the supernatant by means of nitrogen blowing, then adding a certain amount of diluent and uniformly mixing, centrifuging to obtain supernatant to obtain a sample to be detected, wherein the blood sample is serum or plasma obtained by processing at least 100 mu L of blood to be detected, the internal standard working solution contains an internal standard substance with known concentration, and the diluent is acetonitrile solution containing 0-0.1% formic acid and 5-20% water.

In detail, the absolute ethyl alcohol is used as a precipitator, can precipitate proteins in a blood sample, and enables vitamin K2(MK-7) combined with the proteins to be in a free state, the normal hexane is used as an extractant, can extract vitamin K2(MK-7) in the blood sample, and the diluent is used for redissolving substances after drying.

In the embodiment of the invention, based on the matching use of absolute ethyl alcohol and n-hexane, the vitamin K2(MK-7) can be retained to the maximum extent, and meanwhile, the interferent is removed, so that the detection accuracy is improved.

In the embodiment of the invention, the internal standard and the blood sample are mixed uniformly, then absolute ethyl alcohol is added and mixed uniformly, then normal hexane is added and mixed uniformly, the supernatant is dried by nitrogen after centrifugation, the diluent is added for redissolution and mixing uniformly after drying, and finally the supernatant is centrifuged again to obtain the sample to be detected. The pretreatment only uses precipitated protein and a liquid-liquid extraction mode to treat the blood sample, not only can remove the interferent while keeping vitamin K2(MK-7) to the maximum extent, but also is simple and easy to operate, consumes short time, and can obtain the sample to be detected without SPE, thereby detecting the content of vitamin K2(MK-7) in the blood, obviously reducing the experimental cost, avoiding the loss of the vitamin K2(MK-7) to be detected in the SPE process, and reducing the dosage of the blood sample.

In detail, the blood sample can be obtained by processing at least 100. mu.L of blood to be tested. The dosage of blood to be detected is not high and can be as low as 100 mu L, the blood sampling quantity is less, and the blood sampling experience of a detected person is good. Typically, blood samples are stored at 4 ℃ until ready for analysis. After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded.

Step 104: and detecting the sample to be detected by using a high performance liquid chromatography tandem mass spectrometer under the same detection condition to obtain a chromatogram of the sample to be detected.

Step 105: and calculating the content of vitamin K2(MK-7) in the blood sample according to the chromatogram of the sample to be detected and a standard curve equation of the vitamin K2 (MK-7).

In the embodiment of the invention, based on the blood sample pretreatment process, the sample pretreatment can be simplified while the detection effect is ensured, the operation is simple, and the error caused by the operation can be reduced or avoided, so that the quantification can be more accurate, and the experiment cost can be obviously reduced. In addition, the pretreatment method is simple, the experiment cost is low, and the blood consumption is small, so that the application and popularization of actual detection are facilitated.

In an embodiment of the present invention, preferably, in the step 103, mixing a certain amount of internal standard working solution and a certain amount of blood sample uniformly, then adding a certain amount of absolute ethanol and mixing uniformly, then adding a certain amount of n-hexane and mixing uniformly, centrifuging to obtain a supernatant, blow-drying the supernatant by a nitrogen blowing method, adding a certain amount of diluent and mixing uniformly after blow-drying, and centrifuging to obtain the supernatant to obtain the sample to be tested, includes:

using a pipette to pipette 50-200 μ L (such as 50, 100, 150 or 200 μ L) of blood sample into a centrifuge tube, adding a certain amount (such as 10 μ L) of internal standard working solution, and mixing by vortex shaking at the rotation speed of 1000-2500rpm (such as 1000, 1500, 2000 or 2500rpm) for 20s-2min (such as 20s, 30s, 1min, 1.5min or 2 min);

adding 0.3-1.0mL (such as 0.3, 0.5, 0.7, 0.9 or 1.0mL) of anhydrous ethanol, mixing by vortex shaking at 2500rpm (such as 1000, 1500, 2000 or 2500rpm) for 1-4min (such as 1, 2, 3 or 4min), adding 0.5-1.2mL (such as 0.5, 0.7, 0.9, 1.0 or 1.2mL) of n-hexane, mixing by vortex shaking at 2500rpm (such as 1000, 1500, 2000 or 2500rpm) for 4-10min (such as 4, 5, 7, 9 or 10min), centrifuging at 12000rpm (such as 10000, 10500, 11000, 11500 or 12000rpm) for 5-12min (such as 5, 7, 9, 10 or 12min), and transferring the supernatant to another centrifuge tube;

blowing the supernatant with nitrogen, adding 50-200 μ L (such as 50, 100, 150 or 200 μ L) of diluent after blowing, mixing for 1-3min (such as 1, 1.5, 2.0, 2.5 or 3min) by vortex oscillation at the rotation speed of 1000-2500rpm (such as 1000, 1500, 2000 or 2500rpm), and then centrifuging for 5-12min (such as 5, 7, 9, 10 or 12min) at the rotation speed of 10000-14000rpm (such as 10000, 11000, 12000, 13000 or 14000rpm), and taking the supernatant to obtain the sample to be tested.

In the embodiment of the invention, the type, the dosage and the matching use of the reagents used for the pretreatment of the blood sample are optimized, and the pretreatment mode is used for treating the blood sample, so that the target object can be effectively extracted, and meanwhile, the impurity interference in the matrix is reduced, and the lower detection limit (the detection limit can be as low as 0.00606ng/mL) in the biological sample is realized, so as to meet the requirement of clinical medical examination.

Based on the above, in one embodiment of the present invention, preferably, the internal standard working solution contains vitamin K2(MK-7) -d7 in an amount of 10. mu.L to 60ng/mL (e.g., 10, 20, 30, 40, 50 or 60 ng/mL).

In detail, an isotope of vitamin K2(MK-7) is preferably used as an internal standard in the examples of the present invention. Wherein d7 represents 7H atoms substituted by deuterium atoms. The vitamin K2(MK-7) -d7 isotope label is used as an internal standard substance, so that the detection of a blood sample is not easily influenced by matrix and the like, the identification of the vitamin K2(MK-7) is more accurate, the analysis time is short, the interference is small, the internal standard is appropriate in quantification, and the specificity is strong, and the accuracy and the sensitivity are high.

In detail, the vitamin K2(MK-7) -d7 standard may be dissolved with an acetonitrile solution containing isopropanol in an amount of 15-30% (e.g., 15, 20, 25, or 30%) to obtain an internal standard stock solution, and the internal standard stock solution may be diluted with a diluent to obtain an internal standard working solution.

In one embodiment of the present invention, the liquid phase condition in the detection condition includes: a pentafluorophenyl chromatographic column, wherein the mobile phase A is an aqueous solution containing 0.5% formic acid, the mobile phase B is a mixed solution of methanol and acetonitrile, the volume ratio of the methanol to the acetonitrile in the mixed solution is 1:4, and a gradient elution mode is adopted in the elution process.

In the embodiment of the invention, when the blood sample is detected based on the matching use of the pentafluorophenyl chromatographic column and the specific mobile phase, a target object and impurities can be better separated, a lower serum quantitative limit is realized on the premise of not carrying out SPE pretreatment, the blood sample dosage is reduced, the analysis time is shortened, and the personnel cost and the instrument use cost are further reduced.

In detail, when the liquid phase condition is used for detecting the content of vitamin K2(MK-7) in blood, the retention time of vitamin K2(MK-7) can be as low as 2.31min, the analysis time can be 6min or even lower, the analysis time is short, the rapid and accurate detection of the content of vitamin K2(MK-7) in a blood sample can be realized, and the characteristics of stable retention time among batches, good peak appearance effect of a target peak, good repeatability of a separation effect and the like can be realized.

Based on the above, in one embodiment of the present invention, preferably, the pentafluorophenyl chromatography column comprises: phenomenex Kinetex F5 column, length of column is 100mm, internal diameter is 2.1mm, and packing particle diameter is 1.7 μm.

Based on the above, in one embodiment of the present invention, the flow rate is preferably 0.80 mL/min; the elution process is shown in table 1 below.

TABLE 1

In table 1, mobile phase a is an aqueous solution containing 0.5% formic acid, mobile phase B is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1: 4.

Based on the above, in one embodiment of the present invention, it is preferable that the analysis time is 4 to 10min, the column temperature is 30 to 50 ℃, the sample amount is 5 to 50. mu.L, and the flow rate is 0.50 to 1.00 mL/min.

For example, the analysis duration may be 4, 5, 6, 7, 8, 9, or 10 min. In detail, the length of the analysis period is influenced by the flow rate of the mobile phase, for example, when the flow rate is 1ml/min, the analysis period can be shortened to 4 min.

For example, the column temperature may be 30, 35, 40, 45, or 50 ℃, the sample size may be 5, 10, 20, 30, 40, or 50 μ L, and the flow rate may be 0.50, 0.60, 0.70, 0.80, 0.90, or 1.00 mL/min.

In one embodiment of the present invention, the tandem mass spectrometry condition among the detection conditions includes: an APCI (+) detection mode, wherein the acquisition mode is MRM, the air pressure of an air curtain is 20-55psi, the pressure of collision air is 1-12psi, the atomization current value is 1-5 muA, the ionization temperature is 200-750 ℃, and the atomization air pressure is 10-90 psi; the assist gas pressure was 0 psi.

For example, the curtain gas pressure may be 20, 30, 40, 50, or 55psi, the impinging gas pressure may be 1, 4, 7, 10, or 12psi, the atomization current value may be 1, 2, 3, 4, or 5 μ Α, the ionization temperature may be 200, 350, 500, 600, or 750 ℃, and the atomization gas pressure may be 10, 30, 50, 70, or 90 psi.

Usually, at least three coordinate points are needed for establishing the standard curve equation so as to ensure the accuracy of the established equation, so at least three standard solutions need to be prepared in advance, and the standard curve equation of the vitamin K2(MK-7) can be fitted according to the chromatogram obtained by detecting each standard solution.

In detail, a linear range can be set by combining the detected population, the dosage of the blood to be detected, the dilution degree of the blood sample, the dilution degree of the standard working solution, the approximate content range of vitamin K2(MK-7) in the human body and the like, so as to ensure that most of the detection results of the clinical samples fall within a reportable range.

In one embodiment of the present invention, before the separately detecting at least three standard solutions, further comprising: preparing at least three standard working solutions, wherein the standard working solutions contain vitamin K2(MK-7) standard products with known concentrations, the concentration of the vitamin K2(MK-7) standard products in the standard working solutions is in the range of 0.3-153.6ng/mL, and the concentration of the vitamin K2(MK-7) standard products in different standard working solutions is different; transferring 10 mu L of standard working solution and 10 mu L of internal standard working solution into a centrifuge tube by using a pipette, and then transferring and adding 80 mu L of diluent; the centrifuge tube is vortexed and mixed at the rotation speed of 1000-.

In one embodiment of the present invention, preferably, the concentration of the vitamin K2(MK-7) standard in each standard working fluid is 0.3ng/mL, 0.6ng/mL, 1.2ng/mL, 2.4ng/mL, 4.8ng/mL, 9.6ng/mL, 19.2ng/mL, 38.4ng/mL, 76.8ng/mL, 153.6ng/mL, respectively.

In detail, the standard curve equation of vitamin K2(MK-7) obtained by fitting may be generally y ═ kxx + b. Wherein, the two variables x and y can be the peak area ratio of the vitamin K2(MK-7) standard substance to the corresponding internal standard substance in the chromatogram of each standard solution, and the concentration ratio of the vitamin K2(MK-7) standard substance to the corresponding internal standard substance in each standard solution. Therefore, according to the peak area ratio of the vitamin K2(MK-7) and the internal standard substance in the chromatogram of the sample to be detected and the concentration of the internal standard substance in the sample to be detected, the concentration of the vitamin K2(MK-7) in the sample to be detected can be calculated by substituting the peak area ratio into a standard curve equation.

In general, the standard curve equation needs to be re-determined before each test, and the standard curve equation obtained each time is usually only applied to one time of the current test, such as one time of the on-off period of the high performance liquid tandem mass spectrometer.

In conclusion, the embodiment of the invention provides a method for detecting the content of vitamin K2(MK-7) in blood, and an internal standard method is combined with a high performance liquid chromatography tandem mass spectrometry method, so that the accuracy of a quantitative result is improved, and the system error is eliminated; the sample pretreatment is simple, the sample pretreatment time is greatly saved, and the experiment cost is saved; the sample detection is carried out by combining specific detection conditions, so that the detection process is simple, convenient and quick, the sample analysis time is shortened, and the content of vitamin K2(MK-7) in a patient body is more favorably monitored in clinical treatment, thereby providing an experimental basis for personalized administration of related medicaments, reducing or avoiding the occurrence of vitamin K2(MK-7) deficiency symptoms, and being more favorable for guiding the administration of patients.

The present invention will be described in detail below by way of examples, but the present invention is not limited to the following examples.

Example 1

The embodiment of the invention is used for obtaining the standard curve equation.

1.1 preparation of Standard stock solutions

Precisely weighing 2.33mg of a vitamin K2(MK-7) standard substance, placing the vitamin K2 standard substance into a 2mL volumetric flask, dissolving the vitamin K2 standard substance with absolute ethyl alcohol, and fixing the volume to 2mL to obtain a standard stock solution. Wherein, the standard quality test report indicates that the purity is 95%, so that the concentration of the vitamin K2(MK-7) standard substance in the obtained standard stock solution is 1.10675mg/mL, the standard stock solution is stored at the temperature of minus 80 ℃, and the effective period is 6 months.

1.2 preparation of stock solutions for internal standards

Unsealing 0.25mg of vitamin K2(MK-7) -d7 standard substance, adding 1mL of acetonitrile and 200 mu L of isopropanol to dissolve to obtain an internal standard stock solution, wherein the concentration of the vitamin K2(MK-7) -d7 standard substance is 187.5 mu g/mL, and the internal standard stock solution is stored at the temperature of minus 80 ℃ and has the effective period of 1 year.

1.3 Instrument for detection

High performance liquid tandem mass spectrometer: sciex 6500 plus.

1.4 Mass Spectrometry conditions

An APCI (+) detection mode, wherein the acquisition mode is MRM, the pressure of air curtain gas is 20psi, the pressure of collision gas is 9psi, the atomization current value is 3 muA, the ionization temperature is 400 ℃, and the pressure of atomization gas is 65 psi; the assist gas pressure was 0 psi.

For other parameters of the mass spectrometry conditions, see table 2 below.

TABLE 2

Categories	Name of substance	Retention time	Parent ion	Daughter ions	DP	EP	CE	CXP
									Qualitative and quantitative	MK-7	2.31min	649.5	187.1	110	8	52	10
Qualitative and quantitative	IS/MK-7-d7	2.31min	656.5	194.1	110	8	52	10

In Table 2, MK-7 represents vitamin K2(MK-7), IS/MK-7-d7 represents vitamin K2(MK-7) -d7, DP represents the optimized declustering voltage for positive ion mode, EP represents the focusing voltage, CE represents the collision voltage, and CXP represents the collision cell ejection voltage.

1.5 liquid phase conditions

1.5.1 chromatography columns

Phenomenex Kinetex F5 column (2.1 x 100mm,1.7 μm).

1.5.2 Mobile phase

Mobile phase A: an aqueous solution containing 0.5% formic acid;

mobile phase B: the mixed solution of methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 1: 4.

1.5.3 elution mode

Gradient elution was used, with the mobile phase ratios and flow rates as shown in table 1 above.

1.5.4 others

The online filter is SSI COL PRE-FILTER WATER 1/160.5M; the analysis time is 6.0 min; the column temperature was 40 ℃; the sample injection amount is 20 mu L; the flow rate was 0.80 mL/min.

1.6 preparation of Standard working solution

Taking a proper amount of standard stock solution, diluting with diluent (acetonitrile solution containing 0.1% formic acid and 5% water, the same below) to prepare ten standard working solutions of which the concentration of the vitamin K2(MK-7) standard product is 0.3-153.6ng/mL, and storing at-80 ℃.

Wherein, in the ten standard working solutions, the concentration of the vitamin K2(MK-7) standard substance is respectively 0.3ng/mL, 0.6ng/mL, 1.2ng/mL, 2.4ng/mL, 4.8ng/mL, 9.6ng/mL, 19.2ng/mL, 38.4ng/mL, 76.8ng/mL and 153.6 ng/mL.

1.7 preparation of internal standard working solution

Sucking 10 mu L of internal standard stock solution into a 1.5mL centrifuge tube, adding 615 mu L of diluent, and uniformly mixing for 1min in a vortex manner at the rotating speed of 2500r/min to obtain internal standard intermediate solution; and (3) sucking 100 mu L of internal standard intermediate solution into a 10mL volumetric flask, adding diluent to reach a constant volume of 10mL, uniformly oscillating to obtain internal standard working solution with the concentration of 30ng/mL of the vitamin K2(MK-7) -d7 standard product, and storing at-80 ℃.

1.8 preparation of Standard solution

And for each standard working solution, respectively transferring 10 mu L of standard working solution, 10 mu L of internal standard working solution and 80 mu L of diluent into a 1.5mL centrifuge tube by using a pipette, then, uniformly mixing for 1min in a vortex mode at the rotating speed of 2500rpm to obtain a mixed solution, and transferring the mixed solution to serve as the standard solution to be detected.

Thus, ten standard solutions were obtained for ten standard working solutions.

1.9 detecting the standard solution to generate a standard curve equation

After obtaining each standard solution, the ten standard solutions can be respectively detected by using a high performance liquid tandem mass spectrometer, and a chromatogram and a mass spectrogram of each standard solution are correspondingly obtained.

Referring to FIGS. 3 to 5, FIG. 3 shows chromatograms of vitamin K2(MK-7) standard in a standard solution, FIG. 4 shows a chromatogram of vitamin K2(MK-7) -d7 in a standard solution, and FIG. 5 shows mass spectra of vitamin K2(MK-7) standard and vitamin K2(MK-7) -d7 in a standard solution.

As can be seen from the chromatograms shown in FIGS. 3-5, the detection method provided by the embodiment of the invention has the advantages of accurate identification of the target compound, short analysis time, small interference and strong specificity.

From the chromatogram of the standard solution, the chromatographic peak area of the vitamin K2(MK-7) standard substance and the chromatographic peak area of the vitamin K2(MK-7) -d7 can be obtained, and then the standard curve equation of the vitamin K2(MK-7) can be obtained by combining the vitamin K2(MK-7) standard substance and the known concentration of the vitamin K2(MK-7) -d7 in each standard solution.

Referring to FIG. 9, FIG. 9 shows the linear relationship of vitamin K2(MK-7) obtained. From the linear relationship graph, a standard curve equation of vitamin K2(MK-7) can be obtained: y is 0.35X-0.0024, R is 0.9995, and the correlation coefficient R is²Is more than 0.999, Y is the peak area ratio of vitamin K2(MK-7) to vitamin K2(MK-7) -d7, and X is the concentration ratio of vitamin K2(MK-7) to vitamin K2(MK-7) -d 7.

It can be seen that the correlation coefficient R²> 0.9900, indicating good linearity. When the content of the vitamin K2(MK-7) in the blood is calculated based on the standard curve equation, the accuracy is high, and the error is small.

After the standard curve equation is obtained, the blood sample can be pretreated to obtain a sample to be detected, the sample to be detected is detected under the same detection condition, and the content of vitamin K2(MK-7) in the blood sample can be obtained by combining the obtained standard curve equation.

Example 2

The embodiment of the invention is used for detecting the content of vitamin K2(MK-7) in blood.

2.1 obtaining blood samples

Collecting at least 100 μ L of blood to be detected, centrifuging at 3500rpm for 10min, collecting supernatant to obtain serum, and storing the serum as blood sample at 4 deg.C under refrigeration until it is ready for analysis.

After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded.

2.2 blood sample pretreatment

Transferring 100 mu L of serum into a 2mL centrifuge tube by using a pipette gun, then adding 10 mu L of internal standard working solution, and mixing for 1min by vortex oscillation at the rotating speed of 2500 rpm; then adding 700 mu L of absolute ethyl alcohol, and mixing for 3min by vortex oscillation at the rotating speed of 2500 rpm; then adding 800 mu L of n-hexane, mixing for 5min by vortex oscillation at the rotating speed of 2500rpm, then centrifuging for 5min at the rotating speed of 12000rpm, and transferring the supernatant to a 1.5mL centrifuge tube; blowing the transferred supernatant by using nitrogen, adding 100 mu L of diluent for redissolution, mixing for 1min by vortex oscillation at the rotating speed of 2500rpm, then centrifuging at the rotating speed of 14000rpm for 10min at a high speed, and transferring 90 mu L of the supernatant obtained by high-speed centrifugation to obtain the sample to be detected.

2.3 detection of samples to be tested

Under the detection conditions of the embodiment 1, the same high performance liquid tandem mass spectrometer is used for detecting the sample to be detected, and the chromatogram of the sample to be detected is obtained.

Referring to FIGS. 6 to 8, FIG. 6 shows chromatograms of vitamin K2(MK-7) in a test sample, FIG. 7 shows chromatograms of vitamin K2(MK-7) -d7 in a test sample, and FIG. 8 shows mass spectrograms of vitamin K2(MK-7) and vitamin K2(MK-7) -d7 in a test sample.

Referring to fig. 3 to 8, the retention time of vitamin K2(MK-7) in the sample to be tested is consistent with the retention time of vitamin K2(MK-7) -d7, and is slightly delayed but the delay time is consistent with the retention time of vitamin K2(MK-7) and the retention time of vitamin K2(MK-7) -d7 in the standard solution, respectively.

2.4 calculation of vitamin K2(MK-7) content in the sample to be tested

And correspondingly substituting the chromatographic peak areas of vitamin K2(MK-7) and vitamin K2(MK-7) -d7 and the known concentration of vitamin K2(MK-7) -d7 in the sample to be detected in the chromatogram of the sample to be detected into the standard curve equation, so as to calculate the content of vitamin K2(MK-7) in the sample to be detected.

The concentration range of the vitamin K2(MK-7) standard in the standard working solution prepared in step 1.6 was 0.3-153.6ng/mL, and it was found that there was a 10-fold conversion between standard dilution and sample treatment by combining the standard solution preparation process described in step 1.8 and the blood sample pretreatment process described in step 2.2, and therefore, the linear range (clinically reportable range) of vitamin K2(MK-7) was 0.03-15.36ng/mL, and the linearity of vitamin K2(MK-7) was good in the range of 0.03-15.36 ng/mL.

Example 3

The embodiment of the invention is used for detecting the content of vitamin K2(MK-7) in blood plasma.

In step 2.1, blood to be tested is treated to obtain serum. In contrast, the present embodiment treats blood to be tested to obtain plasma, and the obtained plasma is used as a blood sample, while the other treatments are consistent with the above embodiment 2.

By measuring the content of vitamin K2(MK-7) in the plasma, the obtained detection result is consistent with the detection result obtained in example 2 within an allowable error range.

Example 4

The embodiment of the invention is used for determining the quantitative limit and the detection limit.

The control samples with low concentration were selected and diluted with physiological saline to different degrees to prepare blood sample dilutions with different concentrations, and these blood sample dilutions were measured in the blood sample pretreatment method and measurement conditions in example 2. The detection limit and the quantitative limit of the vitamin K2(MK-7) are shown as follows:

(1) limit of detection (LOD): 0.00606 ng/mL.

(2) Limit of quantitation (LOQ): 0.0202 ng/mL.

In this example, the detection limit of vitamin K2(MK-7) can be as low as 0.00606ng/mL, and the quantification limit can be as low as 0.0202 ng/mL. Therefore, the detection method provided by the embodiment of the invention has high sensitivity, can improve the parallelism of low-concentration samples, enhances the accuracy and can meet clinical requirements.

Due to the high sensitivity, the requirement of the sample volume of the blood to be detected in the embodiment of the invention can be wider, so that the overall accuracy of sample detection is improved. In addition, the embodiment of the invention can accurately quantify the biological sample with low vitamin K2(MK-7) content, and ensures the high accuracy and wide applicability of the detection method.

In addition, the detection limit and the quantification limit of the detection method provided by the embodiment of the invention are both lower, the dosage of the blood to be detected is less, and the dosage of the blood can be as low as 100 mu L. Therefore, the detection method provided by the embodiment of the invention can effectively reduce the dosage of the blood sample on the premise of realizing normal detection of the content of vitamin K2(MK-7) in human serum/plasma.

Example 5

The embodiment of the invention is used for measuring the recovery rate and the precision.

The standard working solution containing vitamin K2(MK-7) standard substance was prepared into high, medium and low concentrations, and subjected to sample recovery rate test and precision test according to the test method in example 2, and the recovery rate and precision of vitamin K2(MK-7) were determined by repeated analysis for 3 batches as shown in Table 3.

TABLE 3

Adding quantity of scalar	0.06ng/mL	0.48ng/mL	1.92ng/mL
				Average recovery rate	102％	100％	95％
Precision (RSD)	3.94％	3.50％	1.60％

It can be seen that vitamin K2(MK-7) has an average recovery rate of 95-102% in the range of 3 low, medium and high addition levels, has good reproducibility and good sample addition recovery rate, has a relative standard deviation of 1.60-3.94%, has high accuracy of detection results, and can eliminate system errors.

Based on the above, the detection method described in the above embodiment 2 has the advantages that the technical indexes such as detection limit, quantitative limit, recovery rate and precision meet the requirements, the reproducibility is good, the sample recovery rate is high, and the accuracy of the detection result is improved.

In summary, the method for detecting the blood concentration content of vitamin K2(MK-7) in blood provided by the embodiment of the invention combines an internal standard method with a high performance liquid chromatography-mass spectrometry combined method, so that interference factors are greatly reduced, sample pretreatment is simple, labeled koji does not need pretreatment, the sample pretreatment time is greatly saved, the quantification is accurate, the reproducibility is good, the specificity is strong, the sensitivity is high, the detection result is more accurate, the cost is low, the analysis time is short, the method meets clinical requirements, is suitable for detection of clinical large-batch blood samples, and can provide experimental basis for personalized administration of related drugs and reduction or avoidance of the occurrence of vitamin K2(MK-7) deficiency symptoms.

Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for detecting the content of vitamin K2(MK-7) in blood, which comprises the following steps:

2. The method of claim 1,

mixing a certain amount of interior label working solution and a certain amount of blood sample, then adding a certain amount of absolute ethyl alcohol and mixing, add a certain amount of n-hexane and mixing again, the supernatant is got in the centrifugation, weathers the supernatant through the mode that nitrogen blows, adds a certain amount of diluent and mixing after weathering, and the centrifugation is got the supernatant and is obtained the sample that awaits measuring, include:

3. The method of claim 2,

the internal standard working solution contains 10-60ng/mL vitamin K2(MK-7) -d7, and the addition amount is 10 mu L.

4. The method of claim 1,

the liquid phase conditions in the detection conditions include: a pentafluorophenyl chromatographic column, wherein the mobile phase A is an aqueous solution containing 0.5% formic acid, the mobile phase B is a mixed solution of methanol and acetonitrile, the volume ratio of the methanol to the acetonitrile in the mixed solution is 1:4, and a gradient elution mode is adopted in the elution process.

5. The method of claim 4,

the pentafluorophenyl chromatographic column comprises: phenomenex Kinetex F5 column, length of column is 100mm, internal diameter is 2.1mm, and packing particle diameter is 1.7 μm.

6. The method of claim 4,

the flow rate is 0.80 mL/min;

the elution process comprises:

。

7. The method of claim 4,

the analysis time is 4-10min, the column temperature is 30-50 ℃, the sample injection amount is 5-50 mu L, and the flow rate is 0.50-1.00 mL/min.

8. The method of claim 1,

tandem mass spectrometry conditions among the detection conditions include: an APCI (+) detection mode, wherein the acquisition mode is MRM, the air pressure of an air curtain is 20-55psi, the pressure of collision air is 1-12psi, the atomization current value is 1-5 muA, the ionization temperature is 200-750 ℃, and the atomization air pressure is 10-90 psi; the assist gas pressure was 0 psi.

9. The method of claim 1,

before the separately detecting at least three standard solutions, further comprising:

10. The method of claim 9,

the concentration of vitamin K2(MK-7) standard in each standard working solution is 0.3ng/mL, 0.6ng/mL, 1.2ng/mL, 2.4ng/mL, 4.8ng/mL, 9.6ng/mL, 19.2ng/mL, 38.4ng/mL, 76.8ng/mL and 153.6ng/mL respectively.