CN113391008A

CN113391008A - Method for performing pretreatment of fat-soluble vitamin detection sample based on automatic liquid transfer workstation

Info

Publication number: CN113391008A
Application number: CN202110151469.5A
Authority: CN
Inventors: 富士山; 卫晓乐; 曲亮; 马金飞; 刘华芬
Original assignee: Hangzhou Calibra Diagnostics Co ltd
Current assignee: Kailaipu Technology Co ltd; Zhejiang Diesel Diagnostic Technology Co ltd
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2021-09-14
Anticipated expiration: 2041-02-03
Also published as: CN113391008B

Abstract

The invention provides a method for carrying out pretreatment on a fat-soluble vitamin detection sample based on an automatic pipetting workstation, which is mainly used for detecting fat-soluble vitamins in a sample by high performance liquid chromatography tandem mass spectrometry, selects a proper operation mode and program parameters through brand-new application of the automatic pipetting workstation, realizes high flux, automation and low cost of liquid-liquid extraction of the fat-soluble vitamin sample, solves the problem that the liquid-liquid extraction needs a large amount of manual operation, and can perfectly replace an expensive SLE plate extraction method suitable for high flux detection; and a mixture of isooctane and isopropanol is used as an extracting agent, isopropanol is used as a dissolving solution, methanol and water are used as a double solvent, and meanwhile, the proportional relation is further optimized, so that the recovery rate of vitamin K1 in the sample is obviously improved, the subsequent high performance liquid chromatography tandem mass spectrometry detection result is more accurate, the sensitivity is higher, and the detection capability of K1 in the sample meets the actual requirement of a clinical reference interval.

Description

Method for performing pretreatment of fat-soluble vitamin detection sample based on automatic liquid transfer workstation

Technical Field

The invention belongs to the field of vitamin detection, particularly relates to a sample pretreatment method for fat-soluble vitamin detection, and particularly relates to a method for pretreating a serum sample needing fat-soluble vitamin detection through an automatic liquid transfer workstation.

Background

The fat-soluble vitamins have important effects on human health, especially on the growth and development of children, and the deficiency of vitamin A can cause xerophthalmia and nyctalopia, and researches show that the vitamin A level has obvious correlation with cancer risk; the main physiological role of vitamin D is to maintain the balance of calcium and phosphorus, thereby promoting bone health. In addition, studies have shown that vitamin D levels are clearly correlated with immune system function and the onset of a variety of diseases, such as the onset of tuberculosis, heart disease, and endocrine disorders. Vitamin E, also known as tocopherol, has a primary antioxidant function and can scavenge free radicals in the body. Vitamin E is associated with the risk of liver disease and cardiovascular disease. Vitamin K plays an important role in the blood coagulation process, has two natural forms (vitamin K1 and vitamin K2) in vivo, and is mainly detected by clinical assay.

The prior ADEK sample pretreatment method mainly adopts a liquid-liquid extraction method and a solid-liquid extraction method:

(1) the liquid-liquid extraction method is characterized in that a low-polarity organic extraction solvent is added into a blood sample, and a vortex oscillation mixing mode is adopted to extract fat-soluble vitamins, so that the obvious defects of low detection flux, low efficiency, large amount of manual operation in a pretreatment process, difficulty in conversion into a 96-pore plate high-flux method, difficulty in realization of high flux and automation and limitation of clinical application and popularization are achieved. CN106504947A provides a method for determining fat-soluble vitamins by a semi-automatic sample processing liquid chromatography technology, liquid-liquid extraction is carried out by adopting a semi-automatic liquid-liquid extractor in a traditional vortex oscillation blending mode, so that the number of channels for simultaneously extracting by the semi-automatic liquid-liquid extractor is very limited, seamless butt joint with a 96-pore plate cannot be realized, a plurality of fussy manual operation processes are brought to experimental operation, the operation time is increased along with the increase of the operation time, a large amount of labor is needed, and the method is not suitable for large-scale high-flux fat-soluble vitamin extraction.

(2) The solid-liquid extraction method, namely the solid-phase support liquid-liquid extraction method, realizes liquid-liquid distribution based on special diatomite filler particles, enriches and extracts fat-soluble vitamins in blood samples, and has the advantages of easy realization of high-throughput detection of a 96-pore plate, commercial products of consumables, high detection throughput, less manual operation, high consumable cost and the like. CN110763788A discloses a method for realizing high-throughput detection of fat-soluble vitamins in plasma by liquid chromatography-tandem mass spectrometry by adopting a solid-phase support liquid-liquid extraction method (SLE method), wherein although the sample pretreatment method is suitable for high-throughput detection of a 96-well plate, and the sample pretreatment method can be matched with an automatic pipetting workstation to realize automatic operation, the sample extraction process is mainly completed by the SLE plate, the automatic pipetting workstation can only replace the manual pipetting function and can not replace the extraction function of the SLE plate, and meanwhile, the problem of high cost of the SLE plate is necessarily caused.

Therefore, the method for pretreating the fat-soluble vitamin sample, which can really realize high-throughput and automatic operation, is urgently needed, can overcome the defects of the existing method, has the advantages of short sample treatment time, high treatment efficiency, high throughput, low material consumption cost, high automation degree and small manual workload, and is convenient for clinical popularization and use.

On the other hand, the content of vitamin K1 in a normal human body is low, and the clinical detection reference interval of vitamin K1 is only 0.13-1.39ng/mL, so that the requirements on the sample pretreatment recovery rate and the detection sensitivity of the detection method are very high, in the existing sample pretreatment method, although the selected extraction agent can simultaneously detect a plurality of fat-soluble vitamins in a sample, the extraction recovery rate of vitamin K1 is low, the detection sensitivity of vitamin K1 is limited, and whether K1 lacks clinical accurate judgment cannot be carried out.

Therefore, an improved pretreatment method of the fat-soluble vitamin sample is urgently needed for quantitatively analyzing the fat-soluble vitamin in serum by high performance liquid chromatography-tandem mass spectrometry, and on one hand, high throughput and automatic operation can be really realized, and high efficiency and convenience are realized; on the other hand, the material consumption cost can be greatly reduced; meanwhile, the pretreatment recovery rate of fat-soluble vitamins, especially vitamin K1, can be improved, so that the sensitivity of the subsequent high performance liquid chromatography tandem mass spectrometry detection result is improved, and the actual requirement of a clinical reference interval on the detection capability of vitamin K1 in blood plasma is met.

Disclosure of Invention

In order to solve the problems, the invention provides a method for pre-treating a sample needing fat-soluble vitamin detection by an automatic pipetting workstation, which realizes high flux, automation and low cost of liquid-liquid extraction of the fat-soluble vitamin sample which is difficult to realize in the past by brand-new application of the automatic pipetting workstation and selection of a proper operation mode and program parameters, solves the problem that the liquid-liquid extraction needs a large amount of manual operation, and can perfectly replace an expensive SLE plate extraction method suitable for high flux detection; meanwhile, by selecting more proper extracting agent, diluent and redissolving solution, the recovery rate of vitamin K1 with the lowest content in human serum is obviously improved, the subsequent high performance liquid chromatography tandem mass spectrometry detection result is more accurate, the sensitivity is higher, the detection capability of vitamin K1 in a blood sample meets the actual requirement of a clinical reference interval, and the method can be used for clinically and more accurately judging whether the vitamin K1 with the lower normal content in the human body is deficient or not.

The existing high-throughput pretreatment method of the fat-soluble vitamin detection sample is to carry out solid-liquid extraction by an SLE plate, so that the high-throughput detection of a 96-pore plate can be realized; liquid-liquid extraction is difficult to convert into a 96-pore plate high-flux method, high flux and automation are difficult to realize, although vortex mixed liquid-liquid extraction is performed by a semi-automatic liquid-liquid extractor, the number of channels is very limited, the operation is complicated, and the semi-automatic liquid-liquid extractor can not be butted with a 96-pore plate, because the structure of the 96-pore plate is not suitable for vortex mixing of large-volume solvents, the single pore of the 96-pore plate is generally a slender columnar structure, the inner diameter is very small, the inner volume is small, when two large-volume immiscible solvents are added, the purpose of effective mixing extraction can not be achieved through a traditional vortex oscillation mode, the semi-automatic liquid-liquid extraction method is not suitable for vortex mixing of the two immiscible solvents, and the semi-automatic liquid-liquid extraction method is difficult to be applied to detection of fat-soluble vitamins.

An automatic pipetting workstation is generally used for replacing manual work to perform high-throughput pipetting, wherein a 'mixing mode' is set, and the automatic pipetting workstation can be used for mixing two mutually dissolved solvents together, such as dilution, pipetting, liquid adding, mixing and the like, so that the pipetting workstation is conventionally used only as an auxiliary operation device and is widely used in sample pretreatment methods such as protein precipitation, liquid-liquid extraction, solid-phase extraction and the like. The research group surprisingly discovers that liquid-liquid extraction between two immiscible solvents can be carried out through an automatic liquid-transferring workstation, particularly through a 'mixing mode', liquid-liquid extraction of a fat-soluble vitamin detection sample can be smoothly completed, and high flux and automation of liquid-liquid extraction of the fat-soluble vitamin detection sample can be really realized through series settings of different operation modes and operation parameters thereof, so that a pretreatment process is more convenient, quicker and efficient, the treatment effect is good, the fat-soluble vitamin is high, and the SLE plate with high recovery rate can be completely replaced.

The principle of liquid-liquid extraction by using the 'mixing mode' of an automatic pipetting workstation is shown in figure 1, a biological sample and an extraction solvent are different in density and are not dissolved, the biological sample is distributed at the lower part of a hole, the extraction solvent is distributed at the upper part, the mixing mode is characterized in that a sample layer is sucked into a pipetting gun head by setting the liquid suction depth of the pipetting gun head, then the liquid discharge depth is adjusted, liquid is discharged at the upper part of a single hole, the biological sample is transferred to the upper part by the above mode, the extraction solvent is transferred to the lower part, then the initial state is re-distributed according to two phases of the density difference, the circulation process is carried out, so that the two phases are efficiently mixed, the fat-soluble vitamin extraction solvent in the biological sample is extracted, and the technical effect which cannot be realized in the 96-channel treatment process by the traditional vortex mixing mode is realized.

On one hand, the invention provides a pretreatment method for a high-throughput detection sample of fat-soluble vitamins based on an automatic pipetting workstation, which adopts a 'mixing mode' of the automatic pipetting workstation to carry out liquid-liquid extraction on the sample, wherein the adopted extractant is a mixture of isooctane and isopropanol.

The sample obtained by the method for pretreating the blood matrix sample based on the automatic pipetting workstation can be used for quantitatively analyzing fat-soluble vitamins in human serum, plasma and peripheral whole blood by a liquid chromatography-mass spectrometry combined technology.

The sample pretreatment method comprises the processes of sample dilution, sample extraction, blow-drying and redissolution.

The 'mixing mode' is equivalent to the operation of rapid circulating liquid suction and discharge by an automatic liquid transfer workstation. Through the blending mode of the automatic liquid-moving workstation, the object to be detected is extracted from the sample matrix into the organic solvent through the repeated liquid-absorbing and liquid-discharging process, so that the liquid-liquid extraction process realizes the effect of high flux, the defects of the existing liquid-liquid extraction method (high flux detection can not be realized) and the existing solid-liquid extraction method (high cost) are effectively overcome, and the method has the advantages of short sample processing time, high processing efficiency, high flux, low material consumption cost, high automation degree and small manual workload.

The fat-soluble vitamins comprise vitamin A, 25-hydroxy vitamin D2, 25-hydroxy vitamin D3, vitamin E, vitamin K1 and the like.

The automatic liquid-transfering workstation is suitable for the simultaneous liquid-transfering treatment of large-batch samples, usually has multiple types such as 96 channels, 384 channels and the like, has high liquid-transfering efficiency, can greatly improve the experimental efficiency, and can liberate experimenters immersed in high-flux repeated liquid-transfering work from experiments. In order to correspond to a 96-well plate in an experimental process, the invention preferably selects a 96-channel automatic pipetting workstation, but does not indicate that the automatic pipetting workstations of other channels are not applicable, and the method provided by the invention can be applied to the automatic pipetting workstations with any channel number.

When liquid-liquid extraction is carried out, the adopted extracting agent is a mixture of isooctane and isopropanol, and a large number of researches prove that the recovery rate of vitamin K1 in a serum sample is obviously improved when the extracting agent is the mixture of isooctane and isopropanol.

Furthermore, in the processes of sample dilution, extraction, blow-drying and redissolution, the automatic pipetting workstation can adopt a 'mixing mode'; in the extraction process of the sample, the program setting parameters of the 'blending mode' of the automatic pipetting workstation comprise: mixing for 50-100 times, and mixing volume of 0.3-0.9 ml.

In some implementations, the pretreatment method provided by the present invention, in addition to the extraction process, must be performed by a pipetting station, or other operations can be performed on a vortex shaker.

The existing sample extraction process usually needs to adopt an extracting agent for extracting for multiple times, the steps are complicated, and when the automatic liquid transfer workstation is adopted for extracting the sample, the extracting agent is only added once to complete the extraction. In the extraction process, quick liquid suction and liquid discharge are carried out through a 'mixing mode' of an automatic liquid-transferring workstation, liquid suction and liquid discharge are carried out according to one-time mixing, mixing times are required to be carried out for 50-100 times, the sample mixing volume of each channel is 0.3-0.9ml, the extraction effect is optimal at the moment, and the sample recovery rate is high.

Further, in the extraction process of the sample, the program setting parameters of the 'blending mode' of the automatic pipetting workstation further comprise: the liquid suction speed and the liquid discharge speed are 80-90%, the liquid suction depth is 90% and the liquid discharge depth is 50%.

Furthermore, the volume ratio of the isooctane to the isopropanol in the extracting agent is 9: 1.

Further, during the dilution of the sample, the program setting parameters of the "blending mode" of the automatic pipetting station include: the mixing times are 10-30 times, and the mixing volume is 100-.

When the automatic pipetting workstation is adopted for diluting the serum sample, the blending frequency is 10-30 times, and when the sample blending volume of each channel is 100-.

Further, in the dilution process of the sample, the program setting parameters of the 'blending mode' of the automatic pipetting workstation further comprise: the liquid suction speed and the liquid discharge speed are 80-90%, the liquid suction depth is 90% and the liquid discharge depth is 50%.

Further, in the sample dilution process, the dilution solution is 0.1-1 mg/mL BHT (2, 6-di-tert-butyl-4-methylphenol) solution, and the dissolving solvent is selected from one or more of methanol, ethanol, acetonitrile and isopropanol.

Further, the dissolving solvent is isopropyl alcohol.

Further, during the blow-drying and redissolving process of the sample, the redissolution solution is a mixture of methanol and water, wherein the volume ratio of the methanol to the water is 9: 1.

Further, in the sample pretreatment process, when liquid needs to be transferred, the 'liquid sucking/emptying mode' of the automatic liquid transferring workstation is adopted, and the program setting parameters are as follows: the liquid suction speed and the liquid discharge speed are 80-90%, the liquid suction depth is 90% and the liquid discharge depth is 50%.

During the pretreatment of the serum sample, including dilution, extraction and reconstitution processes, a "pipetting/emptying mode" is required.

Further, the sample is human serum, plasma, or peripheral whole blood.

Further, the method comprises the steps of:

1) diluting: taking 50-200ul of sample to a 96-well plate, adding 10-100 mu L of internal standard and 100-500 mu L of diluted solution, and uniformly mixing by adopting a 'uniformly mixing mode' of an automatic pipetting workstation;

2) and (3) extraction: adding 0.5-1.5mL of extracting agent, and performing liquid-liquid extraction by adopting a 'mixing mode' of an automatic liquid transfer workstation;

3) drying and redissolving: and (3) centrifuging, taking 0.5-1 mL of upper-layer extraction solvent, drying by using nitrogen, adding a redissolution, and redissolving by using a uniform mixing mode of an automatic liquid transfer workstation.

Furthermore, quality control substances can be added into the sample.

Further, the operation process of adding 10-100ul of internal standard in the step 1) comprises the following steps: adding a sample into a 96-hole plate and then placing the sample into a tray position 1 of a pipetting workstation, placing a 96-hole sample adding slot for placing internal standard working solution into a tray position 2 of the pipetting workstation, adopting a 'liquid suction/emptying' mode program in the pipetting workstation, sucking a proper amount of internal standard working solution from the sample adding slot in the tray position 2 and adding the internal standard working solution into the sample 96-hole plate in the tray position 1, and setting program parameters to be 80-90% of liquid suction speed and liquid discharge speed, 90% of liquid suction depth and 50% of liquid discharge depth.

Further, the operation process of taking the upper layer extraction solvent after centrifugation in the step 3) is as follows: the sample 96 well plate was placed in tray level 1 of the 96 channel pipetting station and another empty 96 well plate was placed in tray level 2 of the pipetting station, and the pipetting program selected "aspirate/empty mode" with the program set up parameters as follows: the liquid suction speed and the liquid discharge speed are 80-90%, the liquid suction depth is 90% and the liquid discharge depth is 50%.

Further, the adding of the redissolution in the step 3) and the redissolution in the 'mixing mode' of the automatic pipetting workstation comprise the following operation processes: placing a 96-well plate containing a sample in a tray position 1, placing a sample adding slot containing a redissolution in a tray position 2, and transferring the redissolution into the sample plate by adopting a liquid suction/emptying mode in a liquid transfer workstation; then, a 'mixing mode' program is operated to uniformly mix the residues.

Further, the operation of adding the internal standard working solution and the diluting solution in the step 1) can be completed by adopting any one of a manual multi-channel liquid shifter, a manual continuous liquid adder or a 96-channel liquid transferring workstation; or the step 1) can be performed by using any one of a 96-hole vortex oscillator or a 96-channel pipetting workstation; or the supernatant liquid is removed in the step 3) by selecting any one mode of a manual multi-channel liquid removing device or a 96-channel liquid removing work station; or the reconstitution process in the step (3) can be completed by selecting any one of a manual multi-channel pipettor or a 96-channel pipetting workstation.

Further, the sample obtained by the method is used for high-throughput detection of fat-soluble vitamins by liquid chromatography-tandem mass spectrometry; wherein the liquid chromatography adopts gradient elution, the chromatographic column is a C18 column, the mobile phase A is 0.05-0.2 volume percent formic acid aqueous solution, the mobile phase B is 0.05-0.2 volume percent formic acid methanol solution, and the volume ratio of the mobile phase A to the mobile phase B is 60-0%: 40-100%.

Further, the gradient elution procedure is as follows;

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	60	40
0.4	60	40
			1.2	25	75
3.0	0	100
			4.5	0	100
4.51	60	40
			5.00	60	40

Furthermore, the mass spectrum adopted by the liquid chromatography-tandem mass spectrometry method is a triple quadrupole mass spectrometer, and the mass spectrometry detection is carried out by adopting an atmospheric pressure chemical ionization source, an anode ion mode (APCI +) and a Multiple Reaction Monitoring (MRM) mode.

In another aspect, the present invention provides a use of a mixture of isooctane and isopropanol for preparing an extractant for liquid-liquid extraction of a sample for high-throughput detection of a fat-soluble vitamin by high performance liquid chromatography-tandem mass spectrometry, wherein the volume ratio of isooctane to isopropanol is 9: 1, and the fat-soluble vitamin is vitamin K1.

In another aspect, the invention provides a detection kit for determining human serum fat-soluble vitamins, which comprises a standard working solution, a quality control sample, an internal standard working solution, a diluent, an extracting agent, a redissolution solution and a liquid-phase elution solution; the extraction agent is a mixture of isooctane and isopropanol, and the volume ratio of the isooctane to the isopropanol is 9: 1.

Further, the diluent comprises a diluted solution and a dissolved solution, wherein the diluted solution is a BHT (2, 6-di-tert-butyl-4-methylphenol) solution of 0.1-1 mg/mL; the dissolving solvent is selected from one or more of methanol, ethanol, acetonitrile and isopropanol.

Further, the redissolution solution is a mixture of methanol and water, wherein the volume ratio of the methanol to the water is 9: 1.

Further, the standard working solution is: contains 5 fat-soluble vitamin solutions with standard concentration, including vitamin A1, 25 hydroxy vitamin D3, 25 hydroxy vitamin D2, vitamin E and vitamin K1); the quality control sample is as follows: serum matrix samples containing three different levels of low, medium and high; the internal standard working solution is as follows: isotope internal standard solutions with specific concentrations comprise VA1-d6, VD3-d6, VD2-d6, VK1-d4 and VE-d 6.

Further, the standard working solution has the following concentration: vitamin A1, 200-10000 ng/mL; 25-hydroxyvitamin D3, 40-2000 ng/mL; 25-hydroxyvitamin D2, 20-1000 ng/mL; 5000-250000 ng/mL of vitamin E; 1-50 ng/mL of vitamin K1.

Further, the quality control samples are serum matrix samples containing three different levels of concentration, namely low level, medium level and high level.

Further, the liquid chromatography elution solution comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.05-0.2 volume percent formic acid aqueous solution, and the mobile phase B is 0.05-0.2 volume percent formic acid methanol solution.

The invention has the following beneficial effects:

(1) through the automatic liquid transfer workstation, the high flux, automation and low cost of the liquid-liquid extraction of the fat-soluble vitamin sample which are difficult to realize in the past are realized, the problem that the liquid-liquid extraction needs a large amount of manual operation is solved, and the expensive SLE plate extraction method suitable for high-flux detection can be perfectly replaced.

(2) By selecting the optimal extraction agent and further optimizing the dilution and redissolution processes, the recovery rate of the fat-soluble vitamin K1 in the sample is obviously improved, the subsequent high performance liquid chromatography tandem mass spectrometry detection result is more accurate, the sensitivity is higher, the actual requirement of a clinical reference interval can be met, and whether the vitamin K1 with lower normal content in a human body is deficient or not can be accurately judged.

(3) The method is suitable for high-flux operation, simple and convenient to operate, short in sample processing time, high in processing efficiency, low in material consumption cost and high in automation degree.

Drawings

FIG. 1 is a schematic diagram of the liquid-liquid extraction principle in the "blending mode" of an automated pipetting station

FIGS. 2-1 to 2-5 are chromatograms of five fat-soluble vitamin labeled samples in example 1

FIG. 3 is a chromatogram of vitamin K1 detected from human serum sample in example 1

FIGS. 4-1 to 4-5 are standard curves for five fat-soluble vitamins prepared in example 1

Detailed Description

The invention will be described in further detail below with reference to the drawings and examples, which are intended to facilitate the understanding of the invention without limiting it in any way.

Example 1 sample preparation, pretreatment and testing

The following formulation is based on the use of solid standards, e.g., commercially available standard stock solutions, with adjustments to the formulation method based on actual use of standard stock solution concentrations.

First, sample preparation

1. Preparation of stock solution

0.1mg/mL BHT in methanolPreparation: 5mg of 2, 6-di-tert-butyl-p-cresol BHT was accurately weighed and dissolved in 50mL of methanol to obtain a 0.1mg/mL BHT methanol solution.

First-order stock solutionThe preparation of (1): precisely weighing Vitamin A (VA), 25-hydroxy vitamin D3(VD3), 25-hydroxy vitamin D2(VD2), Vitamin E (VE) and Vitamin K1(VK) standard substances, and adding a proper volume of BHT methanol solution to obtain a first-stage stock solution, wherein the details are shown in Table 1;

TABLE 1 preparation of first order stock solutions

Compound (I)	Weight mg	BHT methanol solution volume (mL)	First order stock solution concentration (mg/mL)
				Vitamin preparationA1	2	1	2
Vitamin D3	1	1	1
				Vitamin D2	1	1	1
Vitamin E	5	1	5
				Vitamin K	2	1	2

Second-stage stock solutionThe preparation of (1): and taking a proper amount of the first-stage stock solution, and then adding a BHT methanol solution to obtain second-stage stock solutions of the detected substances respectively, wherein the second-stage stock solutions are shown in a table 2.

TABLE 2 preparation of the second-stage stock solutions

Compound (I)	First order stock volume (μ L)	Add BHT methanol solution volume (μ L)	Concentration of second order stock solution (μ g/mL)
				Vitamin A1	100	900	200
Vitamin D3	50	950	50
				Vitamin D2	25	975	50
Vitamin K1	20	980	40

Three-stage stock solutionThe preparation of (1): taking a proper volume of the secondary stock solution, adding a BHT methanol solution to obtain a tertiary stock solution of the detected substance, and preparing the tertiary stock solution of vitamin K1 shown in Table 3.

TABLE 3 preparation of tertiary stock solutions

Note: the stock solutions prepared above were stored in a refrigerator at-80 ℃ in the dark.

2. Preparation of standard working solution

Mixed MIX stock solutionsThe preparation of (1): appropriate volumes of each standard stock solution were taken and mixed, and then diluted with BHT methanol solution to obtain 5 vitamin MIX stock solutions as shown in table 4.

Table 4 preparation of mixed MIX stock solutions

Standard working solutionThe configuration of (2): appropriate volumes of the mixed MIX stock solution were taken and diluted stepwise to obtain a series of standard working solutions as shown in table 5.

TABLE 5 preparation of Standard working solutions

Note: the prepared standard working solution is stored in a refrigerator at minus 80 ℃ in a dark place.

3. Preparation of internal standard working solution

Internal standard first-grade stock solutionThe preparation of (1): an appropriate amount of internal standard substance is precisely weighed, and an appropriate amount of 0.1mg/mL BHT methanol solution is added for dissolution to obtain a first-stage stock solution of the internal standard, which is detailed in Table 6.

TABLE 6 preparation of first order stock solutions

Compound (I)	Weight mg	Volume of BHT solution (mL)	Internal standard first order stock solution concentration (mg/mL)
				Vitamin A1-d6	10	1	10
Vitamin D3-D6	1	1	1
				Vitamin D2-D6	1	1	1
Vitamin E-d6	2	1	2
				Vitamin K1-d4	10	1	10

Internal standard secondary stock solutionThe preparation of (1): and taking an appropriate volume of the internal standard first-stage stock solution, and respectively adding an appropriate volume of 0.1mg/mL BHT methanol solution to obtain an internal standard second-stage stock solution, which is detailed in Table 7.

Method for preparing secondary stock solution of internal standard in Table 7

Compound (I)	First order stock volume (μ L)	Add volume of BHT solution (. mu.L)	Concentration of second order stock solution (μ g/mL)
				Vitamin A1-d6	10	990	100
Vitamin D3-D6	50	950	50
				Vitamin D2-D6	50	950	50
Vitamin K1-d4	10	990	100

Internal standard three-stage stock solutionThe preparation of (1): taking an appropriate volume of the internal standard secondary stock solution, and respectively adding an appropriate volume of 0.1mg/mL BHT methanol solution to obtain an internal standard tertiary stock solution, as detailed inTable 8.

Preparation method of table 8 internal standard three-stage stock solution

Compound (I)	Volume of second order stock solution (μ L)	Volume of BHT solution (μ L)	Third order stock solution concentration (μ g/mL)
				Vitamin K1-d4	100	900	10

Internal standard working fluidThe configuration of (2): and (3) taking each internal standard stock solution with a proper volume, mixing, and adding a proper amount of BHT methanol for dilution to obtain an internal standard working solution.

Preparation method of working solution in table 9

Second, sample pretreatment

(1) Adding a sample: taking 100 mu L of serum sample from the sample blood collection tube and adding the serum sample into a single hole of a 96-hole plate;

(2) adding an internal standard: transferring the 96-well plate subjected to sample adding in the step (1) into a tray position 1 of an S2-PIPETTE 96 channel semi-automatic pipetting workstation, simultaneously placing a 96-well sample adding groove for placing internal standard working solution into a tray position 2 of the automatic pipetting workstation, and absorbing 50 mu L of the internal standard working solution from the sample adding groove of the tray position 2 by adopting a 'liquid absorption/emptying mode' to transfer and add the internal standard working solution into the 96-well plate of the sample of the tray position 1, wherein program parameters are set to 80-90% of liquid absorption speed and liquid discharge speed, 90% of liquid absorption depth and 50% of liquid discharge depth; (ii) a

(3) Adding a diluting solvent: the step (2) is carried out, a liquid suction/emptying mode of a liquid suction workstation is adopted, and 200 mu L of 0.1mg/mL BHT methanol solution is sucked and transferred to be added into a 96-well sample plate;

(4) and (3) a uniform mixing process A: operating a program of a 'mixing mode' of a 96-channel automatic pipetting workstation, repeatedly absorbing liquid and emptying the sample solution in the tray position 1 to uniformly mix the sample and the added reagent, wherein the program parameters of the mixing process A are as follows: blending volume, 150 μ L; mixing for 10 times; liquid suction speed and liquid discharge speed, 90%; imbibition depth, 90%; the liquid discharge depth is 50%;

(5) adding an extraction solvent: placing a 96-hole sample adding slot for placing an extracting agent in a tray 2, sucking 1mL of mixed solution of isooctane and isopropanol (the volume ratio of the isooctane to the isopropanol is 9: 1) by adopting a liquid sucking/emptying mode, and adding the mixed solution into a sample 96-hole plate of the tray 1;

(6) and B, a uniform mixing process: operating a 'mixing mode' program of a 96-channel automatic pipetting workstation, repeatedly absorbing liquid and emptying the sample solution in the tray position 1 to uniformly mix the sample and the extraction solution, wherein program parameters of a mixing process B are as follows: mixing volume, 0.6 mL; mixing for 100 times; liquid suction speed and liquid discharge speed, 90%; imbibition depth, 90%; the liquid discharge depth is 50%;

(7) taking a supernatant: centrifuging a 96-pore sample plate at 4000rpm for 10min, placing the plate at a tray position 1 of a pipetting workstation, placing another empty 96-pore plate at a tray position 2 of the pipetting workstation, and sucking 0.9mL of upper-layer extraction solvent from the 96-pore sample plate by the 96-channel pipetting workstation in a liquid sucking/emptying mode to transfer the upper-layer extraction solvent into another 96-pore sample plate; the program set-up parameters are as follows: the liquid suction speed and the liquid discharge speed are 80-90%, the liquid suction depth is 90% and the liquid discharge depth is 50%;

(8) introducing nitrogen into the transferred supernatant for drying;

(9) and (3) redissolving: placing a 96-pore plate sample in a tray position 1, placing a sample adding slot of 90% methanol of a redissolution solution in a tray position 2, and transferring and adding 100 mu L of the redissolution into the sample plate by a pipetting/emptying workstation in a liquid suction/emptying mode; then, operating a 'blending mode' program of the pipetting workstation to completely redissolve, and setting program parameters: the volume of the mixture was 60. mu.L, and the number of mixing times was 30.

Third, sample detection

And (4) injecting 20 mu L of sample into a high performance liquid chromatography-tandem mass spectrometry system for analysis after redissolution. When the liquid chromatogram tandem mass spectrometry is carried out, the liquid chromatogram adopts gradient elution, and the reverse phase chromatography establishes the separation conditions of the substances to be detected as follows: the chromatographic column is Phenomenex Kinetex C18(2.6,100A,50x2.1mm), the flow rate is 0.7mL/min, and the column temperature is 40 ℃; wherein the mobile phase A is a formic acid aqueous solution with the volume ratio of 0.1%, the mobile phase B is a methanol solution with the volume ratio of 0.1%, and the volume ratio of the mobile phase A to the mobile phase B is 60-0%: 40-100%. The gradient program is shown in table 10 below; the retention time of vitamin A and the isotope internal standard thereof is 2.75min, the retention time of 25 hydroxy vitamin D3 and the isotope internal standard thereof is 2.68min, the retention time of 25 hydroxy vitamin D2 and the isotope internal standard thereof is 2.72min, the retention time of vitamin E and the isotope internal standard thereof is 3.67min, and the retention time of vitamin K1 and the isotope internal standard thereof is 4.10 min.

Table 10: gradient elution procedure

Fat-soluble vitamins separated from the liquid chromatogram enter a mass spectrum for detection, the mass spectrum analysis adopts a triple quadrupole mass spectrometer for detection and quantification, the instrument model is SCIEX 4500MD, the mass spectrum detection is carried out by adopting an atmospheric pressure chemical ionization source positive ion mode (APCI +) and a multi-reaction monitoring MRM mode, the corresponding mass spectrum detection method is set as shown in the following table 11, and the mass spectrum conditions are shown in the table 12.

Table 11: parameters of mass spectrometric detection method

Compound (I)	Q1	Q3	TIME	DP focusing voltage	EP inlet voltage	CE collision energy	CXP collision cell exit voltage
								VA	269.2	119.1	30	72	10	43	10
VA-d6	275.3	122.1	30	60	10	25	11
								VD3	383.4	365.3	30	131	10	21	20
VD3-d6	389.4	371.4	30	96	10	16	11
								VD2	395.4	209	30	116	10	35	12
VD2-d6	416.3	398.2	30	89	10	12	12
								VE	431.4	111	30	116	10	45	10
VE-d6	437.1	171	30	100	10	39	13
								VK	451.4	187.1	30	121	10	35	10
VK1-d4	455.4	191.1	30	100	10	31	13

TABLE 12 Mass Spectrometry parameter conditions

The triple quadrupole mass spectrometer can be used for detecting different fat-soluble vitamins by monitoring ion pairs in an MRM mode through multiple reactions and corresponding retention time, and the matrix effect of a sample is eliminated by utilizing an internal standard of an isotope, so that the content of the fat-soluble vitamins is accurately quantified, and a standard curve graph is drawn.

As shown in fig. 2-1 to 2-5, the left chart is a chromatogram of an analyte of different fat-soluble vitamins in the labeled sample, and the right chart is a chromatogram of a corresponding isotope internal standard, wherein the labeled sample is separated by liquid chromatography, different fat-soluble vitamins peak at different elution times, and the content of the different fat-soluble vitamins is quantified by detection in an MRM mode. FIG. 3 is a chromatogram of a human serum sample for detecting vitamin K1, wherein the left graph is a chromatogram of vitamin K1 in the sample, and the right graph is a chromatogram of an isotope internal standard thereof.

After the sample is separated by liquid chromatography, different fat-soluble vitamins peak at different elution times and are detected by mass spectrum MRM mode, thereby detecting the content of the vitamins. And (4) configuring a sample to be detected according to a certain standard sample concentration for detection to prepare a standard curve.

Fourth, data processing and analysis

1. Drawing a standard curve

The standard curve graph is obtained by performing linear regression with the concentrations of the five fat-soluble vitamin standard substances as abscissa and peak areas of respective internal standards of the five fat-soluble vitamins as ordinate, as shown in fig. 4-1, 4-2, 4-3, 4-4, and 4-5, and the standard curves and correlation coefficients are shown in table 13, and it can be seen that the linear relationship is good in the concentration range shown in table 13.

TABLE 13 regression equation of standard curve and detection range

2. Calculating the intra-day precision and the inter-day precision

(1) Precision degree

Substituting peak area ratios of the substance to be detected in the quality control sample and the internal standard thereof into the established fat-soluble vitamin standard curve, calculating the concentrations of the five fat-soluble vitamins in the quality control sample, then calculating the accuracy and precision results of the quality control samples with three concentrations of at least 6 analysis batches in a day, and the precision results of the quality control samples with three concentrations of at least 5 analysis batches in the day, wherein the acceptance standards are daily precision, daily precision (CV) and deviation Bias which are less than or equal to 15%, and the detection results are shown in tables 14-23.

TABLE 14 in-day precision of VA

TABLE 15 inter-batch precision of VA

TABLE 16 in-day precision of VD3

TABLE 17 daytime precision of VD3

TABLE 18 daily precision of VD2

TABLE 19 daytime precision of VD2

TABLE 20 in-day precision of VE

TABLE 21 daytime precision data for VE

TABLE 22 daily precision of VK

TABLE 23 daytime precision of VK

By combining the data, the intra-day precision and the inter-day precision of the 5 fat-soluble vitamin analytes VA, VD3, VD2, VE and VK1 at three quality control levels of low, medium and high are investigated, and the result shows that the intra-day precision and the inter-day precision of the method are both less than 15%, which indicates that the method meets the clinical detection requirements.

3. Calculating the concentration of multiple fat-soluble vitamins in the sample to be detected

And detecting by mass spectrometry to obtain the ratio of the fat-soluble vitamin to the internal standard, substituting the ratio into a quantitative correction equation, and calculating to obtain the content of the fat-soluble vitamin in the sample.

After the samples are prepared according to the steps, different sample pretreatment methods are adopted, the conditions of the liquid chromatography tandem mass spectrometry are adopted for detection, and the following embodiments of the different sample pretreatment methods are arranged.

EXAMPLE 2 Effect of different extractants on assay recovery

After the sample preparation was completed according to the procedure of example 1, 100. mu.L of a standard substance having a vitamin K1 concentration of 0.1ng/ml was subjected to sample pretreatment according to the sample pretreatment procedure of example 1, and divided into four groups according to the different extraction agents used therein, the volume ratio of the extraction agent to the serum sample was 25: 10, and after the pretreatment was completed, the influence of the different extraction agents on the recovery rate of vitamin K1 in the plasma sample was examined, and the results are shown in Table 24.

TABLE 24 Effect of different extractants on vitamin K1 assay recovery

As can be seen from Table 24, the extraction results of vitamin K1 with different extractants are significantly different, resulting in a great difference in the sensitivity of the final detection result.

In the prior art, normal hexane is usually adopted as an extracting agent of vitamin K1, but experimental results show that when normal hexane is adopted as the extracting agent, the recovery rate of vitamin K1 can only reach 55.8%; when isooctane is used as an extracting agent, the recovery rate of vitamin K1 can reach 66.3 percent, which is slightly higher than that of n-hexane; when isopropanol is used as an extracting agent, the extraction result of vitamin K1 is not ideal, and the recovery rate is only 50.1%; and when a mixed solvent of isooctane and isopropanol is used as an extracting agent, the ratio of isooctane: isopropanol is 9: at 1, the recovery rate of the vitamin K1 is obviously increased to 90.9 percent, which is obviously higher than that of other extracting agents.

In addition, the influence of different proportion relations between isooctane and isopropanol on the extraction result of vitamin K1 is simultaneously compared, and the results are found when the ratio of isooctane: isopropanol is 8: 2 or 7: at the time of 3, the recovery rate of the vitamin K1 is reduced back to 65.8 percent and 55.4 percent, and the proportion relation between the isooctane and the isopropanol has very important influence on the extraction result of the vitamin K1, and the proportion relation between the isooctane and the isopropanol has to be strictly controlled to be 9: 1, the recovery rate of vitamin K1 can be obviously improved, thereby obviously improving the detection sensitivity.

Example 3 Effect of blending volume at automated pipetting station on assay recovery during extraction

This example employs the sample preparation and pretreatment methods as provided in example 1, wherein the parameters related to the "blending mode" during the extraction are set as 0.3mL, 0.6mL and 0.9mL for the blending volume, respectively, and the liquid chromatography tandem mass spectrometry conditions in example 1 are used for the detection after the pretreatment is completed, and the influence of different blending volumes on the recovery rate of vitamin K1 in the plasma sample during the extraction is examined, and the results are shown in table 25.

TABLE 25 Effect of different mix volumes on vitamin K1 recovery in the extraction

As can be seen from Table 25, in the extraction process, the automatic pipetting station adopted different blending volumes, which had little effect on the recovery rate of vitamin K1, taking into account the volatilization of the organic solvent in the actual blending process, so the blending volume B was set to 0.3 mL.

Example 4 Effect of blending frequency of automatic pipetting station on recovery detection in extraction Process

The sample preparation and pretreatment method provided in example 1 was used in this example, wherein the parameters related to the "blending mode" during the extraction were set as 50 times, 100 times and 200 times respectively for the number of blending times during the pretreatment, and the conditions of lc tandem mass spectrometry in example 1 were used for the detection after the pretreatment was completed, and the influence of different blending times on the recovery rate of vitamin K1 in the serum sample during the extraction process was examined, and the results are shown in table 26.

TABLE 26 Effect of different mixing times during extraction on vitamin K1 recovery in detection

As can be seen from table 26, the number of mixing times during the extraction process slightly affects the recovery rate of vitamin K1 detection, and the recovery rate of 100 times is slightly higher, so the number of mixing times during the extraction process is preferably 100 times.

Example 5 Effect of different dissolving solvents on recovery

In this embodiment, the sample preparation and pretreatment methods as in embodiment 1 are adopted, wherein in the dilution process of the pretreatment, the diluent is 0.1 to 1mg/mL BHT (2, 6-di-tert-butyl-4-methylphenol) solution, and the dissolving solvent is one of methanol, ethanol, acetonitrile, and isopropanol. After the pretreatment, the results of the tests using the conditions of LC-MS/MS in example 1 were shown in Table 27, and the effect of different extractants on the recovery of vitamin K1 in the plasma samples was examined.

TABLE 27 Effect of different dissolution solvents on recovery of vitamin K1 assay at dilution

As can be seen from table 27, the recovery rate of vitamin K1 was also affected by using different dissolution solvents during the dilution process, wherein the recovery rate was decreased with acetonitrile, no significant difference was observed between methanol and ethanol, and slightly increased with isopropanol, so the dissolution solvent during the dilution process was preferably isopropanol.

Example 6 Effect of number of mixes at automated pipetting station on assay recovery in dilution

This example employed the sample preparation and pretreatment methods as provided in example 1, wherein the parameters related to the "blending mode" during dilution during pretreatment were set as 10 times, 20 times and 30 times respectively, and the liquid chromatography tandem mass spectrometry conditions in example 1 were used for detection after pretreatment was completed, and the effects of different blending times during dilution on the recovery rate of vitamin K1 in the plasma sample were examined, and the results are shown in table 28.

TABLE 28 Effect of different number of blendings on vitamin K1 recovery in dilution

As can be seen from table 28, the number of mixing times during the dilution process had no significant effect on the recovery rate of vitamin K1, and therefore the selection was set to 10 times to reduce the time for pretreatment of this step.

Example 7 Effect of different reconstitution solutions and dose ratios on recovery

After the sample preparation was completed according to the procedure of example 1, 100. mu.L of a standard substance having a vitamin K1 concentration of 0.1ng/ml was subjected to sample pretreatment according to the sample pretreatment procedure of example 1, and divided into 5 groups according to the difference in reconstitution solution employed therein, and after the pretreatment was completed, the influence of the liquid chromatography tandem mass spectrometry conditions of example 1 on the recovery rate of vitamin K1 in a serum sample was examined, and the results are shown in Table 29.

TABLE 29 Effect of different reconstitution solutions on vitamin K1 assay recovery

As can be seen from table 29, there is a certain effect on the recovery rate of vitamin K1 detected using different reconstitution solutions, acetonitrile used in the prior art: water: formic acid (80: 20: 0.1) as a reconstituted solution, in fact, did not give an ideal recovery of vitamin K1, when methanol: when water (9: 1) is used as a redissolution, the recovery rate of vitamin K1 can be improved; meanwhile, the ratio relationship between methanol and water also has certain influence on the recovery rate of vitamin K1, and the most preferable ratio is methanol: water (9: 1) as a reconstitution solution.

Example 8 comparison of test results with SLE plate solid-liquid extraction method

In this example, sample pretreatment was performed by using SLE plate solid-liquid extraction (purchased from tianjin banner technologies ltd, model clearert SLE 96 well plate, 300mg) and the method based on automatic pipetting workstation provided in example 1.

Both processes use isopropanol as the dissolution solvent, the ratio of isooctane to isopropanol being 9: 1 is an extractant, methanol: and (3) taking water (9: 1) as a redissolution, and detecting the content of fat-soluble vitamins in the serum quality control sample by liquid chromatography tandem mass spectrometry after the pretreatment is finished. Wherein, when the solid-liquid extraction of the SLE plate is carried out for each time, the volume ratio of the extracting agent to the plasma sample is 25: 10, and the extraction is carried out for 5 times totally; based on the method of an automatic pipetting workstation, the blending frequency in the dilution process is 10 times, the blending volume is 100 mu L, the blending frequency in the extraction process is 100 times, and the blending volume is 0.3 ml. The samples used were quality control samples, and the test results are shown in table 30.

TABLE 30 comparison of results of solid-liquid extraction method and automatic pipetting station method (quality control sample)

The results in table 30 show that the method for automatically pipetting the work station provided by the invention has no obvious deviation with the existing solid-liquid extraction method for SLE, the error of the results is within 80-120%, and the detection results are basically consistent.

Example 9 comparison with the conventional sample pretreatment method for fat-soluble vitamin detection

1. Comparison of sample pretreatment times

The existing detection method of fat-soluble vitamins comprises 4 basic steps of sample adding, solvent extraction, nitrogen blowing and redissolution. Taking 96 samples as an example, the effect of 3 methods is analyzed, and the result is shown in table 31; 1) the sample adding process is basically consistent in the time of 3 methods, and the manual sample adding cost is completed within about 20 min. 2) The solvent extraction process adopts a single centrifugal tube method in the conventional liquid-liquid extraction method, and detection personnel are required to perform operations of solvent addition, transfer, extraction and the like on samples one by one, so that the method is time-consuming and labor-consuming and needs about 50min to complete; the solid-liquid extraction method (SLE) can use a multi-channel pipettor and a 96-pore plate, has high operation efficiency, does not need transfer, but generally needs to add an extraction solvent for 4 times or 5 times, so the total time is about 25 min; according to the automatic liquid transfer workstation method, the extraction solvent only needs to be added once, the adding, transferring and extracting processes of the solvent are automatically completed by the liquid transfer workstation according to the set program, manual operation is few, and the process can be completed only by 15 min. In summary, the method based on an automated pipetting station has a great advantage in terms of the time consumption of the pre-treatment compared to the existing methods.

TABLE 31 comparison of vitamin detection methods-sample pretreatment time (96 samples)

2. Cost comparison

Further comparing the detection costs of the 3 methods, 1) the instrument equipment cost: the conventional liquid-liquid extraction and solid-liquid extraction methods use pretreatment instruments with relatively low cost, generally only need to purchase conventional devices with relatively low price such as a vortex oscillator, a high-speed centrifuge, a nitrogen blowing instrument, a nitrogen positive pressure instrument and the like, but the automatic liquid transfer workstation method needs to purchase liquid transfer workstation devices with higher price; 2) labor cost: the conventional liquid-liquid extraction method needs a large amount of manual processing operation, while the solid-liquid extraction can adopt a multi-channel pipettor and a 96-pore plate for batch addition, or can also use automatic sample adding equipment compatible with the 96-pore plate for completing the operation, and for the automatic liquid-transferring workstation method, the operation of the step can be completed only by operating an interface touch button, so that the solid-liquid extraction and the automatic liquid-transferring workstation method need little labor cost; 3) extraction solvent, standard, internal standard substance: the cost of the three methods is lower; 4) cost of consumables: the solid-liquid extraction method relies on the use of a commercial SLE extraction plate, which is a disposable consumable, so the method has high consumable cost, and the conventional liquid-liquid extraction and automatic pipetting workstation methods do not need the consumable.

From the above cost analysis, see table 32, when the amount of the sample to be detected is small, the detection cost by the conventional liquid-liquid extraction and solid-liquid extraction is low, and the equipment cost of the automatic pipetting workstation method is high, which is not suitable. However, for clinical testing laboratories that require daily large sample size testing for long runs, or for laboratories that have been equipped with programmable automated pipetting workstation instruments, the total cost of testing for automated pipetting workstation methods is significantly lower than the cost of other existing testing methods. In addition, the pipetting workstation can also be used for other detection items in a laboratory, and the equipment cost can be partially shared.

TABLE 32 cost comparison of the three test methods

	Conventional liquid-liquid extraction (LLE)	Solid-liquid extraction (SLE)	Automatic pipetting workstation method
				Equipment cost of pretreatment	Low cost	Low cost	High cost
Workload of manpower	The workload is large	The workload is low	The workload is low
				Standard substance, internal standard substance, extraction solvent	Low cost	Low cost	Low cost
Commercial SLE extraction 96-well plate	Does not need to use	High cost	Does not need to use
				Other detection reagents	Low cost	Low cost	Low cost

Example 10 application of the method to the detection of the content of fat-soluble vitamins in peripheral blood of children

The collected children peripheral blood sample anticoagulated by EDTA is placed in a centrifuge to be centrifuged at 3000rpm for 10min, 50 μ L of upper plasma is absorbed, and then the content of fat-soluble vitamins in the peripheral blood is detected by adopting the sample preparation and pretreatment methods provided in example 1. The results show that vitamins A, D2, D3, E, K₁The detection precision of the method is less than 15 percent, and the vitamin K can be accurately detected to be as low as 0.04ng/ml₁The content of vitamin K is greatly improved₁The recovery rate of the pretreatment is greatly improved, thereby greatly improving the high performance liquid chromatography stringThe sensitivity of the mass spectrometry detection result completely meets the actual requirement of a clinical reference interval on the detection capability of vitamin K1 in blood plasma.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A pretreatment method for high-throughput detection of a sample by using fat-soluble vitamins based on an automatic pipetting workstation is characterized in that liquid-liquid extraction is carried out on the sample by using a 'mixing mode' of the automatic pipetting workstation, wherein an adopted extracting agent is a mixture of isooctane and isopropanol.

2. The method of claim 1, wherein the automated pipetting station is capable of using a "puddle mode" during the dilution, extraction, blow-drying and reconstitution of the sample; in the extraction process of the sample, the program setting parameters of the 'blending mode' of the automatic pipetting workstation comprise: mixing for 50-100 times, and mixing volume of 0.3-0.9 ml; the volume ratio of isooctane to isopropanol in the extractant is 9: 1.

3. The method of claim 2, wherein the "puddle mode" programming parameters of the automated pipetting station during the extraction of the sample further comprises: the liquid suction speed and the liquid discharge speed are 80-90%, the liquid suction depth is 90% and the liquid discharge depth is 50%.

4. The method of any one of claims 1-3, wherein the "homogenize mode" programming parameters of the automated pipetting station during dilution of the sample comprise: the mixing times are 10-30 times, and the mixing volume is 100-.

5. The method of claim 4, wherein during the dilution of the sample, the diluted solution is 0.1-1 mg/mL BHT (2, 6-di-tert-butyl-4-methylphenol) solution, and the dissolving solvent is selected from one or more of methanol, ethanol, acetonitrile and isopropanol.

6. The method of any one of claims 1 to 5, wherein during blow drying reconstitution of the sample, the reconstitution solution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 9: 1.

7. The method of any one of claims 1-6, wherein the sample is human serum, plasma, or peripheral whole blood.

8. The method of claim 7, wherein the method comprises the steps of:

a) diluting: taking 50-200ul of sample to a 96-well plate, adding 10-100ul of internal standard and 100-500 ul of diluted solution, and uniformly mixing by adopting a 'uniform mixing mode' of an automatic pipetting workstation;

b) and (3) extraction: adding 0.5-1.5mL of extracting agent, and performing liquid-liquid extraction by adopting a 'mixing mode' of an automatic liquid transfer workstation;

c) drying and redissolving: and (3) centrifuging, taking 0.5-1 mL of upper-layer extraction solvent, drying by using nitrogen, adding a redissolution, and redissolving by using a uniform mixing mode of an automatic liquid transfer workstation.

9. Use of a mixture of isooctane and isopropanol for preparing an extractant for liquid-liquid extraction of a sample for high-throughput detection of a fat-soluble vitamin by high performance liquid chromatography-tandem mass spectrometry, wherein the volume ratio of isooctane to isopropanol is 9: 1, and the fat-soluble vitamin is vitamin K1.

10. A detection kit for measuring human serum fat-soluble vitamins is characterized by comprising a standard working solution, a quality control sample, an internal standard working solution, a diluent, an extracting agent, a redissolution solution and a liquid-phase elution solution; wherein the extracting agent is a mixture of isooctane and isopropanol, and the volume ratio of the isooctane to the isopropanol is 9: 1; the diluent comprises a diluted solution and a dissolved solution, wherein the diluted solution is a 0.1-1 mg/mL BHT (2, 6-di-tert-butyl-4-methylphenol) solution, and the dissolved solvent is selected from one or more of methanol, ethanol, acetonitrile and isopropanol; the redissolution solution is a mixture of methanol and water, wherein the volume ratio of the methanol to the water is 9: 1.