CN116026971A

CN116026971A - Kit and detection method for detecting full-spectrum fat-soluble vitamins and metabolites thereof in human serum and plasma

Info

Publication number: CN116026971A
Application number: CN202310326425.0A
Authority: CN
Inventors: 赵妍; 陈怡昭; 王春燕; 孙美珊; 冯涛; 邹浩
Original assignee: Suzhou Yikun Biotechnology Co ltd
Current assignee: Suzhou Yikun Biotechnology Co ltd
Priority date: 2023-03-30
Filing date: 2023-03-30
Publication date: 2023-04-28
Anticipated expiration: 2043-03-30
Also published as: CN116026971B

Abstract

The invention provides a kit for detecting full spectrum fat-soluble vitamins and metabolites thereof in human serum and plasma, which comprises a sample pretreatment reagent, wherein the pretreatment reagent comprises the following components: water, aqueous methanol at a concentration of 60% to 80% (V/V) and methyl tert-butyl ether. The invention also provides a method for detecting the blood sample by using the kit. By verifying serum and plasma matrix, the parameters in all aspects are ensured to meet the requirements. The kit has good precision and high accuracy among test result batches. The kit is used for detecting fat-soluble vitamins in serum and plasma, accurately quantifies clinical projects, and can meet clinical requirements.

Description

Kit and detection method for detecting full-spectrum fat-soluble vitamins and metabolites thereof in human serum and plasma

Technical Field

The invention belongs to the field of disease diagnosis kits, and particularly relates to a kit for detecting full-spectrum fat-soluble vitamins and metabolites thereof in human serum and plasma and a detection method.

Background

Vitamins are important substances that maintain normal physiological functions in humans and animals, and such substances must be obtained from foods and play an important role in the growth and metabolism of humans. Vitamins are classified into two major classes, water-soluble vitamins and fat-soluble vitamins, wherein fat-soluble vitamins mainly include Vitamin A (VA), vitamin D (VD), vitamin E (VE), vitamin K (VK), and the like.

Vitamin A is related to vision, growth and development and immune system, and deficiency of vitamin A can cause night blindness, xerophthalmia, dermatoses, hypoimmunity, growth and development retardation of children, etc.; vitamin D is a fat-soluble vitamin, mainly comprises two active ingredients of vitamin D2 and vitamin D3, wherein the vitamin D is converted into 25-hydroxy vitamin D in the liver, and the 25-hydroxy vitamin D is clinically used as an index for evaluating the nutrition level of the vitamin D in the body; vitamin E is an important biological oxidant, and deficiency of vitamin E can cause anemia, hypoimmunity, and nervous system degenerative disease. Vitamin K is involved in the synthesis of coagulation factors and in calcium and phosphorus metabolism in bone. Thus, the detection of fat-soluble vitamins can assess the nutritional status of the fat-soluble vitamins in a patient. Has diagnostic assistance significance for clinical judgment, treatment management and physiological assessment of fat-soluble vitamin deficiency or excess.

The traditional detection method of fat-soluble vitamins comprises a colorimetry method, an ultraviolet deactivated spectrophotometry method, a fluorescence method, a microbiological method and the like. These methods can only measure the content of a certain vitamin, and have the disadvantages of complex operation steps, large sample size of blood sample, unstable result and many interference factors, so that a sensitive, accurate and rapid analysis method needs to be established.

Chinese patent application number CN202110241142.7 discloses: a sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry. The invention provides a sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry, which adopts a specific protein precipitator and is mixed with an internal standard working solution to form an internal standard solution, so that sample pretreatment is simply and efficiently completed, any additional operations such as sample enrichment are not needed, the recovery rate of the fat-soluble vitamins A, E, K and K2 in a serum sample can be remarkably improved, the cost of sample pretreatment is reduced, the sensitivity of detection of the fat-soluble vitamins in serum is greatly improved, and the accuracy and stability of detection results can be ensured. However, the pretreatment method is too simple, the concentration of K1 and K2 in serum is low, the quantitative lower limit of each item is not shown in the patent, the data is not complete, and the credibility is lacking.

Chinese patent application number CN202011615598.7 discloses: a kit and a detection method for detecting a fat-soluble vitamin, the kit comprising: at least three bottles of fat-soluble vitamin calibrator, at least three bottles of fat-soluble vitamin quality control product, mixed internal standard protein precipitant, mobile phase additive and extract; fat-soluble vitamins include vitamin A, vitamin E, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin K1 and vitamin K2; the mixed internal standard protein precipitant comprises a mixed solution containing a protein precipitant, a vitamin A-D3 internal standard, a vitamin E-D6 internal standard, a 25-hydroxy vitamin D2-D3 internal standard, a 25-hydroxy vitamin D3-D6 internal standard, a vitamin K1-D7 internal standard and a vitamin K2-D7 internal standard; the mobile phase additive comprises a methanol solution containing formic acid and an acetonitrile solution containing formic acid. The invention can detect multiple fat-soluble vitamins at the same time, but the standard curve needs to be combined with a quality control product to determine the accuracy problem, and whether the slope and the intercept of the standard curve equation are respectively in a preset range or not needs to be determined, and the supernatant is removed for experiment after the standard curve equation is feasible.

The development and operation of the fat-soluble vitamin detection kit and the detection method are simpler, the efficiency is higher, the result is more accurate, and the sensitivity is higher.

Disclosure of Invention

In order to solve the problems, the invention provides a pretreatment kit for effectively extracting 5 fat-soluble vitamins from serum/plasma samples. The kit is matched with a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to measure the fat-soluble vitamins in serum/plasma samples: vitamin a (vitamin a, VA) vitamin E (vitamin E, V E), 25-hydroxyvitamin D2 (25 (OH) VD2, VD 2) and 25-hydroxyvitamin D3 (25 (OH) VD3, VD 3), vitamin K1 (vitamin K1, VK 1), serum/plasma samples were sampled after treatment with the pretreatment kit. In the electrospray ionization mode, a multi-reaction monitoring mode (MRM) is adopted for detection, and finally an internal standard method is used for accurate quantitative analysis. The invention detects fat-soluble vitamin by liquid chromatography tandem mass spectrometry technology, and the pretreatment adopts an SPE plate purification method, which is simple, has extremely high efficiency and outstanding advantages. Specifically, the kit can effectively extract fat-soluble vitamins, can provide high-resolution, high-specificity and high-sensitivity detection by using a liquid chromatography-mass spectrometry method, avoids a freeze-drying process by a pretreatment method, avoids derivatization, has a simple pretreatment method and short time consumption, and can effectively improve detection flux.

In the present invention, "MTBE" refers to tert-butyl ether, an organic compound of the formula C ₅ H ₁₂ O is colorless transparent liquid, insoluble in water and soluble in ethanol and diethyl ether.

In the invention, the SPE plate refers to a solid phase extraction plate, and is a sample pretreatment device for extraction, separation and concentration, which is developed from a chromatographic column. The SPE technology is based on a liquid-solid phase chromatography theory, adopts a mode of selective adsorption and selective elution to enrich, separate and purify a sample, and is a physical extraction process comprising liquid phase and solid phase; it can also be regarded approximately as a simple chromatographic process.

In the present invention, when the ratio of the amounts is referred to, "V" represents the volume in liters (L); "m" represents mass in grams (g); "n" represents the amount of a substance in moles (mol).

In the invention, the effect of the eluent is to clean impurities remained on the solid phase extraction plate after sample loading, so as to ensure cleaner elution process.

In the present invention, the "eluent" is used to elute the target from the solid phase extraction plate.

In the present invention, "sample" and "specimen" have the same meaning.

In one aspect, the present invention provides a method of pretreatment of a blood sample.

Specifically, the pretreatment method comprises the following steps: sequentially eluting the sample on the solid phase extraction plate by using water and 60% -80% (V/V) methanol aqueous solution; drying the column bed, and eluting the sample by using methyl tertiary butyl ether; and (5) re-dissolving the eluent by using methanol after drying.

Preferably, the aqueous methanol solution is a 70% (V/V) aqueous methanol solution.

Specifically, the dosage ratio of water to methanol is 1:1 (V/V).

Preferably, the specific operation of the drying bed is as follows: blowing for 1-3min at a vacuum degree of 10-15 mmHg; further preferably 2min.

Preferably, the eluent is dried by a nitrogen blower, the specific condition is that the eluent is dried at room temperature, and the drying time is 10-20min, generally 15min, based on the drying state of the sample.

Preferably, the methanol is redissolved and then comprises a mixing step, which can be high-speed vortex mixing; the step of homogenizing may further comprise a centrifugation step, wherein the centrifugation is a high-speed centrifugation at 10000-14000rpm for 3-8min, and in some embodiments 5min.

In practical application, in the pretreatment method, before the sample enters the solid-phase extraction plate, the sample can also comprise other treatment processes, such as adding internal standard working solution, vortex mixing, high-speed centrifugation, acetonitrile addition and the like; the solid phase extraction plate can also comprise a methanol and ultrapure water pretreatment step, and the dosage ratio of the methanol to the ultrapure water can be 1:1 (V/V).

In particular, the above reagents need to be kept drop-by-drop for 3-5 seconds and not too fast when passing through the solid phase extraction plate, which is a concern in the conventional operation of solid phase extraction plates.

Preferably, the pretreatment method comprises the following steps:

(1) Sucking 300 mu L of blood sample into the EP tube;

(2) Adding 60-80 mu L of mixed internal standard working solution; vortex mixing for 1min, and high-speed centrifuging for 20s;

(3) Then respectively adding 750-900 mu L of acetonitrile into the sample in the step (2), and swirling for 5min; centrifuging at high speed for 5min;

(4) Sequentially using methanol and ultrapure water to pretreat the solid-phase extraction plate;

(5) Taking 0.8-1.0 mL supernatant from the centrifuged sample in (3) and adding the supernatant to the pretreated solid phase extraction plate in (4);

(6) Sequentially using ultrapure water and 60-80% methanol aqueous solution to clean the solid phase extraction plate; blowing for about 2min at a vacuum degree of 10-15 mmHg;

(7) Finally, eluting the target substance from the solid phase extraction plate into a centrifuge tube by using 0.9-1.1mL MTBE;

(8) And (3) drying the eluent by using a nitrogen blowing instrument, adding 70-90 mu L of methanol into a nitrogen-dried sample for re-dissolution, uniformly mixing, and centrifuging at a high speed for 5min.

Further preferably, the conditions of the high-speed centrifugation may be 10000 to 14000rpm.

Further preferably, the blood sample of step (1) is plasma or serum.

Further preferably, the ratio of the amount of the mixed internal standard working solution to the amount of the blood sample in the step (2) may be 1:4 or others.

Further preferably, the ratio of acetonitrile to blood sample in step (3) may be 11:4.

further preferably, in the step (4), the amounts of methanol and ultrapure water used are 1mL (V/V), respectively.

Further preferably, the ratio of the amount of methanol to the amount of ultrapure water in the step (6) may be 1:1 (V/V).

Further preferably, the supernatant from the centrifuged sample obtained in step (8) is used for liquid chromatography tandem mass spectrometry in a sample bottle or a 96-well plate.

According to the method, the invention simultaneously provides application of methanol and methyl tertiary butyl ether in preparing a kit for detecting full spectrum fat-soluble vitamins and metabolites thereof in human serum and plasma.

In another aspect, the invention provides a kit for the detection of a full spectrum of fat-soluble vitamins and their metabolites in human serum and plasma.

The kit is a kit for liquid chromatography tandem mass spectrometry.

The fat-soluble vitamins at least comprise: VA, VE, VD2, VD3, VK 1.

The kit comprises a pretreatment reagent, wherein the pretreatment reagent comprises: water, aqueous methanol at a concentration of 60% to 80% (V/V) and methyl tert-butyl ether.

The pretreatment reagent can also comprise mixed internal standard working solution and acetonitrile.

The kit also comprises a chromatographic mobile phase, wherein the chromatographic mobile phase comprises a mobile phase A and a mobile phase B.

Specifically, the mobile phase A is an aqueous solution comprising ammonium fluoride and formic acid.

Preferably, the ratio of ammonium fluoride to ultrapure water in the mobile phase A is 1:1600-1:2400 (n/V); the preparation method of the mobile phase A comprises the following steps: sequentially adding ultrapure water and ammonium fluoride into the reagent bottle according to the proportion; mixing well. The mixing can be carried out by adopting ultrasonic waves.

Specifically, the mobile phase B is ammonium fluoride methanol solution.

Preferably, the ratio of the ammonium fluoride to the methanol in the mobile phase B is 1:2500-1:7500 (n/V).

Preferably, the kit also comprises other reagents for chromatography or mass spectrometry.

Other reagents for chromatography or mass spectrometry include, but are not limited to: standard, quality control product and needle washing liquid.

In yet another aspect, the invention provides methods of using the aforementioned kits.

The using method comprises a sample pretreatment step and a chromatographic tandem mass spectrometry step.

The sample pretreatment step refers to the blood sample pretreatment method.

Preferably, in the chromatographic tandem mass spectrometry step, the chromatographic mobile phase conditions are as follows:

in some embodiments, the liquid phase gradient elution parameters may be:

in some embodiments, the mass spectrometry source parameters may be:

the invention has the beneficial effects that:

the matrix for the test of the kit comprises serum and plasma, and the sample matrix has a plurality of types. By verifying serum and plasma matrix, the parameters in all aspects are ensured to meet the requirements. The kit has good precision and high accuracy among test result batches. The kit is used for detecting fat-soluble vitamins in serum and plasma, accurately quantifies clinical projects, and can meet clinical requirements.

Drawings

FIG. 1 shows the VA test standard curve.

FIG. 2 is a VA chromatogram.

FIG. 3 is a VE assay standard curve.

FIG. 4 is a VE chromatogram.

Fig. 5 is a VD2 detection standard curve.

FIG. 6 is a VD2 chromatogram.

FIG. 7 shows a VD3 detection standard curve.

FIG. 8 is a VD3 chromatogram.

Fig. 9 is a VK detection standard curve.

FIG. 10 is a VK chromatogram.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

The instruments used in the following examples were as follows:

triple quadrupole mass spectrometry detection system (QSight 420MD, a bioscience limited company of Yiku, su); thermo Scientific Fresco21 high speed refrigerated centrifuge (U.S.); multitube Vortex mixer (Vortex Genie2, usa); positive pressure device (Biotage, sweden); an adjustable pipette (Eppendorf 0.5-10. Mu.L, 10-100. Mu.L, 100-1000. Mu.L); glassware, measuring cylinders, etc.

The reagent consumables used in the following examples are as follows:

HLB-SPE plate (Waters, U.S.), ultra pure water (Milli-Q IQ7000, U.S.); methanol (Merk, usa); acetonitrile (Fisher, usa); isopropanol (Fisher, usa); 2, 6-di-tert-butyl-4-methylphenol (Sigma, germany); bovine serum albumin (Sigma, germany); proClin ™ (Sigma, germany); ascorbic acid (Sigma, germany).

In the following examples, the standards used are as follows:

vitamin A (VA) (TRC, canada); vitamin a internal standard (VA-d 5) (TRC, canada); 25-hydroxyvitamin D2 (VD 2) (TRC, canada); 25-hydroxyvitamin D2 internal standard (VD 2-D3) (IsoScience, USA); 25-hydroxyvitamin D3 (VD 3) (TRC, canada); 25-hydroxyvitamin D3 internal standard (VD 3-D6) (TRC, canada); vitamin E (VE) (TRC, canada); vitamin E internal standard (VE-d 6) (TRC, canada); vitamin K1 (TRC, canada); vitamin K1 internal standard (VK 1-d 7) (TRC, canada).

The preparation method of part of the reagents in the following examples is as follows, unless otherwise indicated:

mobile phase a was 0.5. 0.5 mM ammonium fluoride aqueous solution, preparation method: sequentially adding 500 mL ultrapure water and 0.5 mL 500 mM ammonium fluoride into a reagent bottle; uniformly mixing and carrying out ultrasonic treatment for 10 minutes. The label was FV mobile phase A-and stored at room temperature (expiration date: 7 days).

Mobile phase B is 0.2 mM ammonium fluoride methanol solution, and the preparation method comprises the following steps: sequentially adding 500 mL methanol and 0.2 mL 500 mM ammonium fluoride into a reagent bottle; uniformly mixing and carrying out ultrasonic treatment for 10 minutes. The label was FV mobile phase B-and stored at room temperature (expiration date: 30 days).

The perfusion injector 1/perfusion sample injection needle 1 is acetonitrile/ultrapure water, 1/1 (V/V), and the preparation method comprises the following steps: sequentially adding 250 mL acetonitrile and 250 mL ultrapure water into a reagent bottle; uniformly mixing and carrying out ultrasonic treatment for 10 minutes. The label was FV perfusion syringe 1-and stored at room temperature (expiration date: 1 month).

The perfusion injector 2/perfusion sampling needle 2 is isopropanol, and the preparation method comprises the following steps: adding 500 mL isopropanol to the reagent bottle; the label is a perfusion syringe 2-and stored at room temperature (expiration date: 1 month).

The filling plunger rod is sealed and cleaned to isopropanol/ultrapure water, 1/4 (V/V), and the preparation method comprises the following steps: sequentially adding 100 mL isopropanol and 400 mL ultrapure water into a reagent bottle; uniformly mixing and carrying out ultrasonic treatment for 10 minutes. The label was a pouring plunger rod, sealed and rinsed-and stored at room temperature (expiration date: 1 month).

The transfer fluid is methanol/ultrapure water, 7/3 (V/V), and the preparation method comprises the following steps: sequentially adding 350 mL methanol and 150 mL ultrapure water into the reagent bottle; uniformly mixing and carrying out ultrasonic treatment for 10 minutes. The label is a transfusion-and stored at room temperature (expiration date: 1 month).

In the following examples, the following conditions of Table 1 were used for chromatography unless otherwise specified:

TABLE 1

In the following examples, the liquid phase gradient elution parameters are shown in table 2 below:

TABLE 2

In the following examples, a list of mass spectrometry source parameters in the mass spectrometry method is shown in table 3 below:

TABLE 3 Table 3

In the following examples, the list of target compounds and internal standard collection parameters is shown in table 4 below:

TABLE 4 Table 4

Remarks: the two ends of the time window can be finely adjusted according to the actual situation. VA-d5, VD2-d3 VD3-d3, VE-d6 and VK1-d7 are internal standards.

In the present invention, "LOD" refers to the detection limit, also known as the detection limit, which refers to the corresponding amount of 3 times the value of the instrument background signal generated by the matrix blank, or the average value of the background signal generated by the matrix blank plus 3 times the mean standard deviation. Is one of important indexes of sensitivity.

In the invention, the lower limit of the designated amount of the LLMI refers to the lowest concentration or content of the detected substance can be accurately and quantitatively measured by a specific method on the premise that the limit error can meet the preset requirement.

In the present invention, "CV" means a coefficient of variation, which is a ratio of standard deviation to average value, expressed as a percentage (%).

In the present invention, "LQC" refers to a low concentration quality control, and in an embodiment, low concentration refers to a relatively low concentration within a certain range, which has no fixed range, and which refers to a range different for different detection objects.

In the present invention, "HQC" refers to a high concentration quality control, and in an embodiment, high concentration refers to a relatively high concentration within a certain range, which has no fixed range, and which refers to a range different for different detection objects.

Example 1A method for pretreatment of plasma/serum samples

The method comprises the following steps:

(1) Draw 300 μl of plasma/serum sample into a 1.5mL EP tube; plasma sample: blood is collected by a blood collection tube (anticoagulation tube), whole blood is centrifugally separated (3000 rpm,10 min), processed into plasma, and the plasma is immediately placed in a refrigerator for light-shielding and refrigeration at 2-8 ℃ after sample collection and processing, and is sent to a laboratory for detection as soon as possible, and can not be stored for a long time at room temperature. If the sample cannot be immediately detected, the sample is kept at-20 ℃ or-80 ℃ in a dark place. Serum samples: venous blood is collected by a vacuum negative pressure blood collection tube (coagulation tube), whole blood is centrifugally separated (3000 rpm,10 min), processed into serum, and the serum is immediately placed in a refrigerator for light-shielding and refrigeration at 2-8 ℃ after sample collection and processing, and is sent to a laboratory for detection as soon as possible, and can not be stored for a long time at room temperature. If the sample cannot be immediately detected, the sample is kept at-20 ℃ or-80 ℃ in a dark place.

(2) Adding 75 mu L of mixed internal standard working solution (refer to Table 4, wherein the diluent is methanol); vortex mixing for 1min, and high speed centrifuging (10000-14000 rpm,20 s);

(3) Then 825 mu L acetonitrile is added into the sample in the step (2), and vortex is carried out for 5min; centrifuging at high speed (10000-14000 rpm) for 5min;

(4) The SPE plates were pretreated with 1mL of methanol followed by 1mL of ultrapure water (SPE plates were kept wet);

(5) Taking 0.9 mL supernatant from the centrifuged sample in (3) and adding the supernatant to the pretreated SPE plate in (4);

(6) The SPE vials were rinsed with 1mL of ultrapure water followed by 1mL of 70% methanol water; blowing for 2min at 10-15mmHg (no water can be left in the bed at last, and drying the bed;

(7) Finally eluting the target from the SPE vial into a 1.5mL centrifuge tube using 1mL MTBE;

(8) Drying the eluent by a nitrogen blowing instrument (room temperature, about 15 min), adding 70-90 mu L of methanol into a nitrogen-dried sample for re-dissolution, uniformly mixing by high-speed vortex, vibrating for 5min, and centrifuging at high speed (10000-14000 rpm) for 5min. Taking the supernatant to finish the sample pretreatment step.

Remarks: the drops are guaranteed to be dropped for 3-5 seconds and cannot be too fast when passing through SPE.

Example 2 method for detecting fat-soluble vitamins in plasma/serum

The following steps are added on the basis of example 1:

(9) Taking supernatant (60-70 mu L) from the centrifuged sample in the step (8) and placing the supernatant in a sample injection bottle (with a lining pipe) or a 96-well plate for liquid chromatography tandem mass spectrometry sample loading analysis.

Example 3A blood sample pretreatment kit

Including all of the reagents used in example 1.

Example 4A kit for the detection of full spectrum fat-soluble vitamins and their metabolites in human serum and plasma

Including all of the reagents used in example 2.

The kit also comprises standard substances S1-S5, which are standard substances of 5 detection targets, specifically VA, VE, VD2, VD3 and VK 1.

Also comprises a high-concentration quality control product HQC and a low-concentration quality control product LQC.

The method comprises the following steps:

example 5

Example 5 was set up with reference to example 4, with the difference that: methanol was 80% methanol. The actual detection of methanol is described in step (6) of example 1.

Example 6

Example 5 was set up with reference to example 4, with the difference that: the methanol was 60% methanol. The actual detection of methanol is described in step (6) of example 1.

Experimental example 1 detection of the Performance of the kit

The performance of the kit of example 4 was tested as follows:

1. detection Limit (LOD)

Evaluation criteria: the signal to noise ratio of each sample can be obtained in the Simplicity software. Wherein the sample concentration with the average signal to noise ratio being more than or equal to 3:1 is LOD. While the average signal-to-noise ratio of the LOD should be higher than the background signal-to-noise ratio of the blank samples.

(1) Screening low concentration serum/plasma samples, selecting suitable samples;

(2) The experimental method comprises the following steps: each sample was tested in duplicate 1 time. Sample injection sequence of sample to be measured: random sampling is carried out.

The LOD measured in this experiment was as follows, and all satisfied the evaluation requirements.

The signal to noise ratio of the single detection result of the LOD sample is calculated as follows:

2. lower limit of quantification (LLMI)

Evaluation criteria: the average value and theoretical value of the detection concentration of LLMI of the kit are biased to be less than or equal to +/-20 percent and CV is less than or equal to 15 percent. Serum/plasma samples were screened for low concentration, no bias data, and only CV was detected.

Theoretical results of the lower limit of quantification are summarized as follows:

the experimental method comprises the following steps: each sample was tested in duplicate 5 times. Sample injection sequence of sample to be measured: random sampling is carried out.

The 5 assays for analyte VA were as follows:

the LLMI concentration of VA meeting the evaluation criteria was 6.616 ng/mL.

The 5 detection results for analyte VD3 are as follows:

the LLMI concentration of VD3 meeting the evaluation criteria was 1.416ng/mL.

The 5 detection results for analyte VD2 are as follows:

the LLMI concentration of VD2 meeting the evaluation criteria was 1.170 ng/mL.

The 5 detection results for analyte VE are as follows:

the LLMI concentration of VE meeting the evaluation criteria was 0.337. Mu.g/mL.

The 5 detection results for analyte VK1 are as follows:

the LLMI concentration of VK1 meeting the evaluation criteria was 0.098 ng/mL.

3. Precision in batch

Evaluation criteria: CV is less than or equal to 15 percent.

The theoretical concentration of serum quality control is as follows:

the experimental method comprises the following steps: each sample was tested in duplicate 5 times. Sample injection sequence of sample to be measured: from low to high, the low concentration sample is in front and the high concentration sample is in back.

Experimental results:

the precision CV in all the serum sample batches is less than or equal to 15 percent, and all the serum sample batches meet the evaluation standard.

4. Precision between batches

Evaluation criteria: CV is less than or equal to 15 percent.

The theoretical concentration of serum quality control is as follows:

the experimental method comprises the following steps: three batches of standard curves were analyzed for serum LQC and HQC samples (1 st:2 replicates; 2) ^nd :4 replicate samples; 3 ^rd :5 duplicate samples). Sample injection sequence of sample to be measured: from low to high, the low concentration sample is in front and the high concentration sample is in back.

Experimental results:

/>

all the serum samples meet the evaluation standard in the precision CV less than or equal to 15% among batches.

5. Accuracy of

Evaluation criteria: the recovery rate is 85-115%.

The theoretical labeling concentration of serum samples is as follows:

the experimental method comprises the following steps: serum samples were tested by standard curve analysis. Sample injection sequence of sample to be measured: from low to high, the low concentration sample is in front and the high concentration sample is in back.

Standard curves and chromatograms of VA, VE, VD2, VD3, VK1 are shown in fig. 1-10.

Experimental results:

/>

the recovery rate of serum samples of all projects is within 85-115%, and all the projects meet the evaluation standard.

6. Anti-interference capability

The anti-interference capability of the detection kit shows that when a clinical sample is hemolyzed, the hemoglobin: not more than 50 mg/dL does not affect the accuracy of the detection result, and when patients have bilirubinemia, bilirubin: less than or equal to 20 mg/dL does not affect the accuracy of the detection result, when the clinical sample is triglyceride: the accuracy of the detection result is not affected by less than or equal to 10 mg/dL, and the specific result is as follows:

hemoglobin:

/>

bilirubin:

/>

triglycerides:

/>

experimental example 2 crowd sample detection Effect

Blood samples of 15 patients with diseases were selected for detection, and the detection results are shown in the following table:

the results show that the kit can be used for diagnosing diseases to a certain extent for the detection results of VA, VE, VD2, VD3 and VK1 in blood.

Experimental example 3

Referring to the method in example 1, the detection procedure of the kit of example 4 was adjusted, specifically: the acetonitrile addition amount in the step (3) was 880. Mu.L.

The in-batch precision of HQC was measured and the results were as follows:

experimental example 4

Referring to the method in example 1, the detection procedure of the kit of example 4 was adjusted, specifically: the supernatant in the step (5) was added in an amount of 1mL.

The in-batch precision of HQC was measured and the results were as follows:

experimental example 5

Referring to the method in example 1, the detection procedure of the kit of example 4 was adjusted, specifically: the amount of redissolved methanol in step (8) is 70 μl.

The in-batch precision of HQC was measured and the results were as follows:

/>

Claims

1. a method of pretreatment of a blood sample, comprising: sequentially eluting the sample on the SPE plate by using water and 60% -80% V/V methanol aqueous solution; drying the column bed, and eluting the sample by using methyl tertiary butyl ether; and (5) re-dissolving the eluent by using methanol after drying.

2. The method for pretreatment of blood samples according to claim 1, wherein the ratio of water to methanol is 1:1.

3. the method for pretreatment of blood samples according to claim 1, wherein the specific operation of drying the bed is as follows: blowing for 1-3min at vacuum degree of 10-15 mmHg.

4. The method for pretreatment of blood samples according to claim 1, wherein said solid phase extraction plate comprises a pretreatment step of methanol and ultrapure water in a volume ratio of 1:1.

5. the method for pretreatment of a blood sample according to any one of claims 1 to 4, comprising the steps of:

(1) Sucking 300 mu L of blood sample into the EP tube;

6. The method of claim 5, wherein the blood sample in step (1) is plasma or serum; the dosage ratio of the mixed internal standard working solution to the blood sample in the step (2) is 1:4, a step of; the dosage ratio of acetonitrile to blood sample in the step (3) is 11:4.

7. the application of methanol and methyl tertiary butyl ether as sample pretreatment reagent in preparing kit for detecting full spectrum fat-soluble vitamin and its metabolite in human serum and plasma.

8. A kit for the detection of a full spectrum of fat-soluble vitamins and their metabolites in human serum and plasma, comprising a sample pretreatment reagent, characterized in that the pretreatment reagent comprises: water, aqueous methanol at a concentration of 60-80% V/V and methyl tert-butyl ether.

9. The kit of claim 8, further comprising a chromatographic mobile phase a; the mobile phase A is an aqueous solution comprising ammonium fluoride; the ratio of the ammonium fluoride to the ultrapure water in the mobile phase A is 1:1600-1:2400n/V.

10. The kit of any one of claims 8-9, further comprising a standard or quality control.