CN113391008B - Fat-soluble vitamin sample pretreatment method based on automatic pipetting workstation - Google Patents

Abstract

The invention provides a pretreatment method for a fat-soluble vitamin detection sample based on an automatic pipetting workstation, which is mainly used for detecting fat-soluble vitamins in the sample by high performance liquid chromatography tandem mass spectrometry, and by completely new application of the automatic pipetting workstation, proper operation modes and program parameters are selected, so that the high throughput, automation and low cost of liquid-liquid extraction of the fat-soluble vitamin sample are realized, the problem that the liquid-liquid extraction needs a large amount of manual operation is solved, and the method can perfectly replace an SLE plate extraction method with high cost and suitable for high throughput detection; and the mixture of the isooctane and the isopropanol is used as an extracting agent, the isopropanol is used as a dissolving solution, and the methanol and the water are used as a complex solvent, meanwhile, the proportion relation is further optimized, the recovery rate of the 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 the K1 in the sample meets the actual requirement of a clinical reference interval.

Description

Fat-soluble vitamin sample pretreatment method based on automatic pipetting workstation

Technical Field

The invention belongs to the field of vitamin detection, and particularly relates to a sample pretreatment method for fat-soluble vitamin detection, in particular to a pretreatment method for a serum sample requiring fat-soluble vitamin detection through an automatic pipetting workstation.

Background

Fat-soluble vitamins have important effects on human health, especially on growth and development of children, and vitamin A deficiency can lead to xerophthalmia and nyctalopia, and researches show that obvious correlation exists between vitamin A level and cancer risk; the primary 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 relevant for immune system function and the onset of a variety of diseases, such as the occurrence of tuberculosis, heart disease and endocrine disease. Vitamin E is also called tocopherol, mainly plays an antioxidant function and can remove 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, and there are two natural forms (vitamin K1 and vitamin K2) in the body, and clinical assays mainly detect vitamin K1.

The existing 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 small-polarity organic extraction solvent is added into a blood sample, and fat-soluble vitamins are obtained through extraction in a vortex oscillation uniformly mixing mode, so that the method has the remarkable defects of low detection flux, low efficiency, large amount of manual operation in the pretreatment process, difficulty in converting the method into a 96-hole plate high-flux method, difficulty in realizing high flux and automation, and limitation of clinical application and popularization. CN106504947a provides a method for measuring fat-soluble vitamins by using semi-automatic sample processing liquid chromatography, and adopts a traditional vortex oscillation mixing mode to perform liquid-liquid extraction by using a semi-automatic liquid-liquid extractor, so that the number of channels for simultaneous extraction by the semi-automatic liquid-liquid extractor is very limited, seamless butt joint with a 96-well plate cannot be realized, a lot of complicated manual operation processes are brought to experimental operation, the operation time is increased, and a lot of manpower is required, so that the method is not suitable for large-scale high-throughput fat-soluble vitamin extraction.

(2) The solid-liquid extraction method, namely the solid-phase support liquid-liquid extraction method, is based on special diatomite filler particles to realize liquid-liquid distribution and enrich and extract fat-soluble vitamins in blood samples, has the advantages of easy realization of 96-well plate high-throughput detection, commercial products of consumable materials, high detection throughput, less manual operation, high consumable material cost and the like. CN110763788A discloses a method for realizing high-throughput detection of fat-soluble vitamins in plasma by adopting a solid-phase supported liquid-liquid extraction method (SLE method), and the sample pretreatment method is suitable for high-throughput detection of a 96-well plate, and also can be matched with an automatic pipetting workstation to realize automatic operation, but the sample extraction process is mainly finished by means of a SLE plate, and the automatic pipetting workstation can only replace manual pipetting function, cannot replace the extraction function of the SLE plate, and also has the problem of high cost of the SLE plate.

Therefore, the fat-soluble vitamin sample pretreatment method capable of truly realizing high-flux and automatic operation is urgently needed, so that the method can overcome the defects and the shortcomings of the existing method, and has the advantages of short sample treatment time, high treatment efficiency, high flux, low consumable 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 the 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, and in the existing sample pretreatment method, the extraction agent selected can meet the requirement of simultaneously detecting various fat-soluble vitamins in a sample, but the extraction recovery rate of the vitamin K1 is low, so that the detection sensitivity of the vitamin K1 is limited, and whether K1 lacks clinically cannot be accurately judged.

Therefore, an improved pretreatment method of the fat-soluble vitamin sample is urgently needed for quantitative analysis of fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry, and on one hand, high throughput and automatic operation can be truly realized, and high efficiency and convenience are realized; on the other hand, the consumable 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 clinical reference intervals 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 preprocessing a sample requiring fat-soluble vitamin detection through an automatic pipetting workstation, and the method can perfectly replace an expensive SLE plate extraction method suitable for high-throughput detection by completely new application of the automatic pipetting workstation and selecting proper operation modes and program parameters, thereby realizing high throughput, automation and low cost of liquid-liquid extraction of the fat-soluble vitamin sample, which are difficult to realize in the past, solving the problem that the liquid-liquid extraction needs a large amount of manual operation; meanwhile, by selecting a more proper extractant, a diluent and a redissolution, the recovery rate of vitamin K1 with the lowest content in human serum is obviously improved, so that 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 simultaneously meets the actual requirement of a clinical reference interval, and the method can be used for clinically and accurately judging whether the vitamin K1 with lower normal content in a human body is deficient or not.

The existing high-flux pretreatment method of the fat-soluble vitamin detection sample is to perform solid-liquid extraction through an SLE plate, so that 96-well plate high-flux detection can be realized; the liquid-liquid extraction is difficult to be converted into a 96-pore plate high-flux method, high flux and automation are difficult to realize, although vortex mixed liquid-liquid extraction is carried out by a semi-automatic liquid-liquid extraction instrument, the number of channels is very limited, the operation is complex, the device still cannot be in butt joint with the 96-pore plate, because the structure of the 96-pore plate is not suitable for vortex mixing of a large volume of solvent, the single pore of the 96-pore plate is generally of a slender columnar structure, the inner diameter is small, the inner volume is small, and when the large volume of two immiscible solvents are added, the purpose of effective mixed extraction cannot be achieved through a traditional vortex oscillation mode, so the device is not suitable for vortex mixing of the two immiscible solvents, and is difficult to be applied to fat-soluble vitamin detection.

An automated pipetting station is generally used for high throughput pipetting instead of manual pipetting, wherein a "mixing mode" is provided for mixing two mutually soluble solvents together, such as dilution, pipetting, liquid adding, mixing, etc., so that the pipetting station is conventionally used as an auxiliary operation device and is widely used in sample pretreatment methods for protein precipitation, liquid-liquid extraction, solid-phase extraction, etc. The research group surprisingly discovers that the liquid-liquid extraction of the fat-soluble vitamin detection sample can be successfully completed by the automatic pipetting workstation, especially by the 'mixing mode' in the automatic pipetting workstation, and the liquid-liquid extraction between two immiscible solvents, and the high flux and automation of the liquid-liquid extraction of the fat-soluble vitamin detection sample can be truly realized by the serial arrangement of different operation modes and operation parameters, so that the pretreatment process is more convenient and efficient, the treatment effect is good, the recovery rate of the fat-soluble vitamin is high, and the SLE plate with high cost can be completely replaced.

The principle of liquid-liquid extraction by utilizing the 'mixing mode' of an automatic pipetting workstation is shown in fig. 1, a biological sample and an extraction solvent are distributed at the lower part of a hole due to density difference and incompatibility, the extraction solvent is distributed at the upper part of the hole, the mixing mode is used for sucking a sample layer into the pipetting gun head by setting the liquid suction depth of the pipetting gun head, then the liquid discharge depth is regulated, the biological sample is transferred to the upper part of a single hole in the above mode, the extraction solvent is transferred to the lower part, then the two phases are distributed to an initial state again according to the density difference, the two phases are efficiently mixed through the circulation process, and the fat-soluble vitamins in the biological sample are extracted into the extraction solvent, so that the technical effect which cannot be realized in the 96-channel treatment process of the traditional vortex mixing mode is realized.

In one aspect, the invention provides a pretreatment method of a fat-soluble vitamin high-throughput detection sample 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 extractant adopted is a mixture of isooctane and isopropanol.

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

The sample pretreatment method comprises the processes of diluting, extracting and drying and re-dissolving the sample.

The mixing mode disclosed by the invention is equivalent to the rapid cyclic liquid suction and discharge operation of an automatic liquid-transferring workstation. Through the mixing mode of the automatic pipetting 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 high flux effect, and the defects of the existing liquid-liquid extraction method (which cannot realize high flux detection) and the solid-liquid extraction method (which are high in cost) are overcome.

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

The automatic pipetting workstation is suitable for simultaneous pipetting of a large number of samples, is usually 96 channels, 384 channels and other types, has high pipetting efficiency, can greatly improve experimental efficiency, and can liberate experimental staff immersed in high-throughput repeated pipetting work from experiments. In order to correspond to a 96-well plate in the experimental process, the invention preferably selects an automatic pipetting workstation with 96 channels, but does not indicate that the automatic pipetting workstations with other channels are not applicable.

In the liquid-liquid extraction, the extractant is a mixture of isooctane and isopropanol, and a great deal of researches prove that when the extractant is a mixture of isooctane and isopropanol, the recovery rate of vitamin K1 in a serum sample is obviously improved.

Furthermore, in the processes of diluting, extracting and drying and re-dissolving the sample, an automatic pipetting workstation can adopt a mixing mode; in the extraction process of the sample, the program setting parameters of the mixing mode of the automatic pipetting workstation comprise: the mixing times are 50-100 times, and the mixing volume is 0.3-0.9ml.

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

In the existing sample extraction process, the extraction agent is generally adopted for carrying out multiple extractions, and the steps are complicated. In the extraction process, liquid suction and liquid discharge are rapidly carried out through a mixing mode of an automatic pipetting workstation, the mixing times of each liquid suction and liquid discharge are 50-100 times according to one mixing time, the mixing volume of a sample 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 "mixing mode" program setting parameters of the automatic pipetting workstation further include: 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 volume ratio of the isooctane to the isopropanol in the extractant is 9:1.

Further, in the dilution process of the sample, the "mixing mode" program setting parameters of the automatic pipetting workstation include: the mixing times are 10-30 times, and the mixing volume is 100-200ul.

When the automatic pipetting workstation is used for diluting serum samples, the mixing times are 10-30 times, and when the mixing volume of the samples in each channel is 100-200ul, the dilution effect is optimal, and the sample recovery rate is higher.

Further, in the dilution process of the sample, the "mixing mode" program setting parameters of the automatic pipetting workstation further include: 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 dilution process of the sample, the dilution solution is a solution of 0.1-1 mg/mL BHT (2, 6-di-tert-butyl-4-methylphenol), and the dissolution solvent is one or more selected from methanol, ethanol, acetonitrile and isopropanol.

Further, the dissolution solvent is isopropyl alcohol.

Further, in the process of drying and redissolving the sample, the redissolving 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 pipetting is needed, a 'pipetting/evacuating mode' of an automatic pipetting 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%.

In the pretreatment process of serum samples, including dilution, extraction and re-dissolution processes, a "pipetting/draining mode" is used.

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

Further, the method comprises the steps of:

1) Dilution: taking 50-200ul of samples 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 uniform mixing mode of an automatic pipetting workstation;

2) Extraction: adding 0.5-1.5mL of extractant, and performing liquid-liquid extraction by adopting a 'mixing mode' of an automatic pipetting workstation;

3) Drying and re-dissolving: and taking 0.5-1 mL of the upper extraction solvent after centrifugation, drying by nitrogen, adding the redissolution, and re-dissolving by adopting a 'mixing mode' of an automatic pipetting workstation.

Further, a quality control material may be added to the sample.

Further, the operation process of adding 10-100ul internal standard in the step 1) is as follows: after a sample is added into a 96-well plate, the sample is placed in a tray 1 of a pipetting workstation, a 96-well sample adding groove for placing an internal standard working solution is placed in a tray 2 of the pipetting workstation, the pipetting workstation adopts a liquid sucking/evacuating mode program, a proper amount of internal standard working solution is sucked from the sample adding groove in the tray 2 and added into the sample 96-well plate of the tray 1, and program parameters are set to 80% -90% of liquid sucking speed and liquid discharging speed, 90% of liquid sucking depth and 50% of liquid discharging depth.

Further, the operation process of taking the upper extraction solvent after centrifugation in the step 3) is as follows: sample 96 well plate is placed on tray 1 of 96 channel pipetting workstation, another empty 96 well plate is placed on tray 2 of pipetting workstation, pipetting program selects "pipetting/evacuating mode", 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%.

Further, the operation process of adding the redissolution in the step 3) and re-dissolving by adopting a 'mixing mode' of an automatic pipetting workstation is as follows: placing a 96-well plate containing a sample in a tray 1, placing a sample adding groove containing a redissolved solvent in a tray 2, and transferring and adding the redissolved solution into the sample plate by a liquid-sucking/exhausting mode in a liquid-transferring workstation; then running a mixing mode program to mix the residues uniformly.

Further, the operation of adding the internal standard working solution and the diluted solution in the step 1) can be completed by adopting any mode of a manual multi-channel liquid dispenser, a manual continuous liquid dispenser or a 96-channel liquid dispenser workstation; or the uniform mixing operation in the step 1) can be completed by adopting any mode of a 96-hole vortex oscillator or a 96-channel pipetting workstation; or the supernatant liquid removal in the step 3) can be finished by selecting any mode of a manual multi-channel pipette or a 96-channel pipetting workstation; or the re-dissolution process in the step (3) can be completed by selecting any mode of a manual multi-channel pipette 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 percent: 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

Further, the mass spectrum adopted by the liquid chromatography tandem mass spectrometry method is a triple quadrupole mass spectrometer, and the mass spectrum detection is carried out by adopting an atmospheric pressure chemical ionization source, a positive ion mode (APCI+) and a multi-reaction monitoring (MRM) mode.

In another aspect, the invention provides the use of a mixture of isooctane and isopropanol for preparing an extractant for liquid-liquid extraction of a sample for high throughput detection of 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 yet another aspect, the present invention provides a test kit for determining human serum fat-soluble vitamins, the kit comprising a standard working fluid, a quality control sample, an internal standard working fluid, a diluent, an extractant, a reconstitution solution and a liquid phase elution solution; the extractant 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 diluting solution and a dissolving solution, wherein the diluting solution is 0.1-1 mg/mL of BHT (2, 6-di-tert-butyl-4-methylphenol) solution; the solvent is selected from one or more of methanol, ethanol, acetonitrile and isopropanol.

Further, the redissolution 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 concentration, low, medium and high; the internal standard working solution is as follows: the isotope internal standard solution with specific concentration comprises VA1-d6, VD3-d6, VD2-d6, VK1-d4 and VE-d6.

Further, the standard working solution concentration is as follows: vitamin A1, 200-10000 ng/mL; 25-hydroxy vitamin D3, 40-2000 ng/mL; 25-hydroxy vitamin D2, 20-1000 ng/mL; vitamin E, 5000-250000 ng/mL; vitamin K1, 1-50 ng/mL.

Further, the quality control sample is a serum matrix sample containing three different levels of concentration.

Further, the liquid chromatography eluting solution comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.05-0.2% formic acid water solution by volume, and the mobile phase B is 0.05-0.2% formic acid methanol solution by volume.

The invention has the following beneficial effects:

(1) Through the automatic pipetting workstation, the high throughput, automation and low cost of the liquid-liquid extraction of the fat-soluble vitamin sample, which have been difficult in the past, are realized, the difficult 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 throughput detection can be perfectly replaced.

(2) The optimal extractant is selected, the dilution and re-dissolution processes are further optimized, 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 requirements of a clinical reference interval can be met, and the method can be used for accurately judging whether the vitamin K1 with lower normal content in a human body is deficient or not.

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

Drawings

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

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

FIG. 3 is a chromatogram of the detection of vitamin K1 in a human serum sample of 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, it being noted that the examples described below are intended to facilitate an understanding of the invention and are not intended to limit the invention in any way.

Example 1 sample preparation, pretreatment and detection

The following formulation is based on the use of solid standards, such as standard stock solutions commercially available, with the formulation method being adjusted to the actual standard stock solution concentration used.

1. Sample preparation

1. Preparation of stock solution

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

First-order stock solutionIs prepared from the following raw materials: precisely weighing Vitamin A (VA), 25 hydroxy vitamin D3 (VD 3), 25 hydroxy vitamin D2 (VD 2) and Vitamin E (VE), and adding a proper volume of BHT methanol solution to obtain a primary stock solution, wherein the details are shown in Table 1;

TABLE 1 preparation of Primary stock

Compounds of formula (I)	Weight mg	BHT methanol solution volume (mL)	Primary stock concentration (mg/mL)
				Vitamin A1	2	1	2
Vitamin D3	1	1	1
				Vitamin D2	1	1	1
Vitamin E	5	1	5
				Vitamin K	2	1	2

Secondary stock solutionIs prepared from the following raw materials: a suitable amount of the primary stock solution was taken, and then a BHT methanol solution was added to obtain secondary stock solutions of the test substances, respectively, as shown in table 2.

Table 2 preparation of secondary stock solutions

Compounds of formula (I)	First order stock solution volume (μL)	Add BHT methanol solution volume (μL)	Concentration of secondary stock solution (μg/mL)
				Vitamin A1	100	900	200
Vitamin D3	50	950	50
				Vitamin D2	25	975	50
Vitamin K1	20	980	40

Three-stage stock solutionIs prepared from the following raw materials: taking a proper volume of the secondary stock solution, and then adding a BHT methanol solution to obtain a tertiary stock solution of the detection substance, wherein the tertiary stock solution of the vitamin K1 is prepared as shown in Table 3.

TABLE 3 preparation of three-stage stock solutions

Note that: the stock solutions prepared above are stored in a refrigerator at-80 ℃ and protected from light.

2. Preparation of standard working solution

Mixed MIX stock solutionIs prepared from the following raw materials: an appropriate volume of each standard stock solution was mixed and added, and then BHT methanol solution was added for dilution, to obtain a mixed MIX stock solution of 5 vitamins, as shown in table 4.

TABLE 4 preparation of Mixed MIX stock solutions

Standard working solution Is configured with: appropriate volumes of the mixed MIX stock solution were taken and serially diluted to obtain a series of standard working solutions as shown in table 5.

TABLE 5 preparation of standard working solutions

Note that: the standard working solutions prepared above are stored in a refrigerator at-80 ℃ and protected from light.

3. Preparation of internal standard working solution

Internal standard primary stock solutionIs prepared from the following raw materials: precisely weighing a proper amount of internal standard substance, adding a proper amount of 0.1mg/mL BHT methanol solution for dissolution to obtain an internal standard primary stock solution, and detailing in Table 6.

TABLE 6 preparation of Primary stock

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

Internal standard secondary stock solutionIs prepared from the following raw materials: an appropriate volume of the internal standard primary stock solution was taken, and an appropriate volume of 0.1mg/mL of BHT methanol solution was added to obtain an internal standard secondary stock solution, and the details are shown in Table 7.

Preparation method of secondary stock solution of internal standard in table 7

Compounds of formula (I)	First order stock solution volume (μL)	Add BHT solution volume (μL)	Concentration of secondary stock solution (μg/mL)
				Vitamins A1-d6	10	990	100
Vitamin D3-D6	50	950	50
				Vitamin D2-D6	50	950	50
Vitamins K1-d4	10	990	100

Internal standard three-level stock solutionIs prepared from the following raw materials: an appropriate volume of internal standard secondary stock solution is taken, and an appropriate volume of 0.1mg/mL BHT methanol solution is added to obtain an internal standard tertiary stock solution, and the details are shown in Table 8.

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

Compounds of formula (I)	Volume of secondary stock solution (μL)	BHT solution volume (μL)	Three-stage stock solution concentration (μg/mL)
				Vitamins K1-d4	100	900	10

Internal standard working fluidIs configured with: and mixing a proper amount of each internal standard stock solution, and adding a proper amount of BHT methanol for dilution to obtain an internal standard working solution.

Table 9 internal standard working solution configuration method

2. Sample pretreatment

(1) Adding a sample: taking 100 mu L of serum sample from a sample blood collection tube and adding the serum sample into a single hole of a 96-well 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 semiautomatic pipetting workstation, simultaneously placing a 96-well sample adding groove for placing an internal standard working solution into a tray position 2 of the automatic pipetting workstation, and adopting a liquid suction/evacuation mode to suck 50 mu L of the internal standard working solution from the tray position 2 sample adding groove for transferring and adding the internal standard working solution into the 96-well plate of a sample in the tray position 1, wherein program parameters are set as liquid suction speed and liquid discharge speed of 80% -90%, liquid suction depth of 90% and liquid discharge depth of 50%; the method comprises the steps of carrying out a first treatment on the surface of the

(3) Adding a diluting solvent: in the same step (2), 200 mu L of 0.1mg/mL of BHT methanol solution is sucked by adopting a 'liquid sucking/exhausting mode' of a liquid transferring work station and transferred and added into a sample 96-well plate;

(4) Mixing process A: and (3) running a mixing mode program of the 96-channel automatic pipetting workstation, repeatedly sucking and emptying the sample solution in the tray 1 to uniformly mix the sample and the added reagent, wherein the program parameters of the mixing process A are as follows: mixing the mixture to a volume of 150 mu L; mixing for 10 times; liquid suction speed and liquid discharge speed, 90%; liquid absorption depth, 90%; depth of liquid discharge, 50%;

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

(6) And (3) uniformly mixing process B: and (3) running a mixing mode program of the 96-channel automatic pipetting workstation, repeatedly sucking and evacuating the sample solution in the tray 1 to uniformly mix the sample and the extraction solution, wherein the program parameters of the mixing process B are as follows: mixing the mixture into 0.6mL; mixing for 100 times; liquid suction speed and liquid discharge speed, 90%; liquid absorption depth, 90%; depth of liquid discharge, 50%;

(7) Taking supernatant: centrifuging the sample 96-well plate at 4000rpm for 10min, placing the sample 96-well plate at a tray position 1 of a pipetting workstation, placing another empty 96-well plate at a tray position 2 of the pipetting workstation, and transferring 0.9mL of upper extraction solvent from the sample 96-well plate to another 96-well plate by adopting a 'liquid sucking/emptying mode' in the 96-channel pipetting workstation; 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%;

(8) Introducing nitrogen into the removed supernatant to blow dry;

(9) And (3) a re-dissolving process: placing a sample 96-well plate in a tray position 1, placing a sample adding groove of 90% methanol of a redissolution solution in a tray position 2, adopting a liquid suction/evacuation mode by a liquid transfer workstation, and transferring and adding 100 mu L of redissolution into the sample plate; then, running a 'mixing mode' program of the pipetting workstation to completely re-dissolve, and setting program parameters: the mixing volume was 60. Mu.L and the number of mixing was 30.

3. Sample detection

And (3) carrying out 20 mu L of analysis on the re-dissolved sample by using a high performance liquid chromatography-tandem mass spectrometry system. When the liquid chromatography tandem mass spectrometry analysis is carried out, the liquid chromatography adopts gradient elution, and the reverse phase chromatography establishes the separation conditions of the to-be-detected object as follows: the chromatographic column was Phenomenex Kinetex C (2.6, 100A,50X2.1 mm), the flow rate was 0.7mL/min, and the column temperature was 40 ℃; wherein the mobile phase A is formic acid aqueous solution with the volume ratio of 0.1%, the mobile phase B is 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%. Gradient procedure is shown in table 10 below; the retention time of vitamin A and its isotope internal standard is 2.75min, the retention time of 25 hydroxy vitamin D3 and its isotope internal standard is 2.68min, the retention time of 25 hydroxy vitamin D2 and its isotope internal standard is 2.72min, the retention time of vitamin E and its isotope internal standard is 3.67min, and the retention time of vitamin K1 and its isotope internal standard is 4.10min.

Table 10: gradient elution procedure

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

The fat-soluble vitamins separated on the liquid chromatograph enter a mass spectrum for detection, a triple quadrupole mass spectrometer is adopted for detection and quantification in mass spectrum analysis, the model of the apparatus is SCIEX 4500MD, a positive ion mode (APCI+) of an atmospheric pressure chemical ionization source and a multi-reaction monitoring MRM mode are adopted for mass spectrum detection, 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: mass spectrum detection method parameters

Compounds of formula (I)	Q1	Q3	TIME	DP focus voltage	EP entry 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 conditions for mass spectral parameters

By adopting a triple quadrupole mass spectrometer, the ion pairs in the MRM mode and the corresponding retention time can be monitored through multiple reactions to detect different kinds of lipid-soluble vitamins, the matrix effect of a sample is eliminated by utilizing an internal standard of an isotope, so that the content of various lipid-soluble vitamins is accurately quantified, and a standard graph is drawn.

As shown in fig. 2-1 to 2-5, after the standard yeast sample is separated by liquid chromatography, different fat-soluble vitamins show peaks at different elution times, and the content of the different fat-soluble vitamins is quantified by detecting in an MRM mode, wherein the left graph is a chromatogram of a to-be-detected substance of the different fat-soluble vitamins in the standard yeast sample, and the right graph is a chromatogram of a corresponding isotope internal standard. Fig. 3 is a chromatogram of a human serum sample for detecting vitamin K1, wherein the left image is a chromatogram of vitamin K1 in the sample, and the right image is a chromatogram of an isotopic internal standard thereof.

After the sample is separated by liquid chromatography, different fat-soluble vitamins show peaks at different elution times and are detected by a mass spectrum MRM mode, so that the content of the fat-soluble vitamins is detected. And preparing a sample to be detected according to a certain standard sample concentration for detection to obtain a standard curve.

4. Data processing and analysis

1. Drawing a standard curve

The standard curve is obtained by linear regression with the concentrations of the five fat-soluble vitamins as the abscissa and the peak area ratios of the internal standards of the five fat-soluble vitamins as the ordinate, and the standard curves are shown in figures 4-1, 4-2, 4-3, 4-4 and 4-5, and the standard curves and the correlation coefficients are shown in table 13, so that the linear relationship is good in the concentration range shown in table 13.

Table 13 standard curve regression equation and detection range

2. Calculating the daily precision and the daytime precision

(1) Precision of

The ratio of the area of the to-be-detected object to the area of the internal standard peak in the quality control sample is substituted into the established standard curve of the fat-soluble vitamins, the concentrations of the five fat-soluble vitamins in the quality control sample are calculated, the accuracy and precision results of the quality control samples with three concentrations of at least 6 analysis batches in the day and the precision results of the quality control samples with three concentrations of at least 5 analysis batches in the day are calculated, the acceptance criteria are the daily precision and the daytime precision (CV) and the deviation Bias are less than or equal to 15%, and the detection results are shown in tables 14-23.

Table 14 within day precision of 14 VA

Table 15 VA batch to batch precision

Table 16 within-day precision of VD3

Table 17 daytime precision of VD3

Table 18 within-day precision of VD2

Table 19 daytime precision of VD2

Table 20 within day precision of 20 VE

Daytime precision data of Table 21 VE

Table 22 within day precision of 22 VK

Table 23 VK daytime precision

By combining the data, we examine the daily precision and the daytime precision of the three quality control levels of the 5 fat-soluble vitamin analytes VA, VD3, VD2, VE and VK1, and the results show that the daily precision and the daytime precision of the method are less than 15 percent, which indicates that the method meets the clinical detection requirement.

3. Calculating the concentration of various fat-soluble vitamins in the sample to be measured

And detecting by mass spectrum 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 sample preparation according to the above procedure, different sample pretreatment methods were used, detection was performed using the above-described lc tandem mass spectrometry conditions, and the following examples were arranged for the different sample pretreatment methods.

Example 2 Effect of different extractants on detection recovery

After sample preparation was completed according to the procedure of example 1, 100 μl of a standard with a vitamin K1 concentration of 0.1ng/ml was taken for sample pretreatment according to the sample pretreatment procedure of example 1, and the samples were divided into four groups according to the extraction agents collected therein, the volume ratio of the extraction agents to the serum samples was 25:10, and after pretreatment was detected using the liquid chromatography tandem mass spectrometry conditions of example 1, the effect of the use of the different extraction agents on the recovery rate of vitamin K1 in the plasma samples was examined, and the results are shown in table 24.

TABLE 24 influence of different extractants on the recovery of vitamin K1 detection

As can be seen from table 24, there is a significant difference in the extraction results of vitamin K1 from the different extractants, resulting in a large difference in the sensitivity of the final detection results.

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

In addition, the effect of different ratio relationships between isooctane and isopropanol on the extraction result of vitamin K1 was also compared, and it was found that when isooctane: isopropyl alcohol was 8:2 or 7:3, the recovery rate of vitamin K1 is reduced back to 65.8% and 55.4%, and the ratio relationship between isooctane and isopropanol has very important influence on the extraction result of vitamin K1, and the ratio relationship between isooctane and isopropanol must 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 influence of the mixing volume of an automatic pipetting station on the detection recovery during extraction

In this example, the method for preparing and preprocessing a sample as provided in example 1 was adopted, wherein in the preprocessing process, relevant parameters of a mixing mode during extraction were set to be that mixing volumes are respectively 0.3mL,0.6mL and 0.9mL, and after preprocessing is completed, detection was performed by using the conditions of liquid chromatography tandem mass spectrometry in example 1, and effects of different mixing volumes on recovery rate of vitamin K1 in a plasma sample in the extraction process were examined, and the results are shown in table 25.

TABLE 25 influence of different mixing volumes on the recovery rate of vitamin K1 detection during extraction

As can be seen from table 25, in the extraction process, the automatic pipetting workstation adopts different mixing volumes, and the influence on the recovery rate of vitamin K1 is small, and the volatilization of the organic solvent in the actual mixing process is considered, so that the mixing volume B is set to 0.3mL.

Example 4 influence of the number of mixing times of the automatic pipetting station on the detection recovery during extraction

In this example, the method for preparing and preprocessing a sample as provided in example 1 was adopted, wherein in the preprocessing process, the relevant parameters of the "mixing mode" during extraction were set to be that the mixing times were respectively 50 times, 100 times and 200 times, and the liquid chromatography-tandem mass spectrometry condition in example 1 was adopted to detect after the preprocessing was completed, and the influence of different mixing times on the recovery rate of vitamin K1 in the serum sample in the extraction process was examined, and the results are shown in table 26.

TABLE 26 influence of different mixing times on the recovery rate of vitamin K1 detection during extraction

/>

As can be seen from Table 26, the number of mixing times during the extraction process has a slight effect on the detection recovery rate of vitamin K1, and the recovery rate of 100 times is slightly higher, so that the number of mixing times during the extraction process is preferably 100 times.

EXAMPLE 5 Effect of different dissolution solvents on recovery

In this example, the sample preparation and pretreatment method as provided in example 1 was used, wherein in the pretreatment dilution process, the diluent was 0.1-1 mg/mL BHT (2, 6-di-tert-butyl-4-methylphenol) solution, and the dissolution solvent was one of methanol, ethanol, acetonitrile and isopropanol. After the pretreatment was completed, the effect of using different extractants on the recovery rate of vitamin K1 in the plasma sample was examined by using the liquid chromatography-tandem mass spectrometry conditions in example 1, and the results are shown in Table 27.

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

As can be seen from Table 27, the use of different dissolution solvents during dilution also has an effect on the detection recovery of vitamin K1, wherein acetonitrile is used, the recovery is reduced, no significant difference is observed between methanol and ethanol, and isopropanol is used, so that isopropanol is the preferred dissolution solvent during dilution.

Example 6 influence of the number of mixing times of the automatic pipetting station on the detection recovery during dilution

In this example, the method for preparing and pre-treating a sample as provided in example 1 was adopted, wherein in the pre-treatment process, the relevant parameters of the "mixing mode" during dilution were set to be that the mixing times were respectively 10 times, 20 times and 30 times, and the liquid chromatography-tandem mass spectrometry condition in example 1 was adopted to detect after the pre-treatment was completed, and the influence of different mixing times on the recovery rate of vitamin K1 in the plasma sample during dilution was examined, and the results are shown in table 28.

TABLE 28 influence of different mixing times on the recovery rate of vitamin K1 detection at dilution

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

EXAMPLE 7 Effect of different reconstitution solutions and dosage ratios on recovery

After sample preparation was completed by the procedure of example 1, 100. Mu.L of a standard having a vitamin K1 concentration of 0.1ng/ml was sampled according to the sample pretreatment procedure of example 1 and divided into 5 groups according to the reconstituted solutions collected therein, and after pretreatment was completed, the effect on the recovery rate of vitamin K1 in serum samples was examined by using different diluents under the conditions of liquid chromatography tandem mass spectrometry of example 1, and the results are shown in Table 29.

TABLE 29 influence of different reconstituted solutions on the recovery rate of vitamin K1 detection

As can be seen from table 29, the recovery rate of vitamin K1 detected by using different redissolved solutions has a certain effect, and acetonitrile used in the prior art: water: formic acid (80:20:0.1) was used as a reconstituted solution, which was not ideal for vitamin K1 recovery when methanol was used: when water (9:1) is used as a redissolution, the recovery rate of vitamin K1 can be improved; meanwhile, the proportion relation between methanol and water has a certain influence on the recovery rate of vitamin K1, and most preferably methanol: water (9:1) was used as the reconstitution solution.

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

This example used SLE plate solid-liquid extraction (available from angel bonna Ai Jieer technologies, model clearert SLE 96 well plate, 300 mg) and the automated pipetting workstation based method provided in example 1 for sample pretreatment, respectively.

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

Table 30 results of the solid liquid extraction method and the automatic pipetting workstation method (quality control samples)

The results in Table 30 show that the method of the automatic pipetting workstation provided by the invention has no obvious deviation from the results of the existing solid-liquid extraction SLE extraction method, the result error is within 80-120%, and the detection results are basically consistent.

Example 9 comparison with sample pretreatment methods for detection of fat-soluble vitamins

1. Comparison of sample pretreatment time

The existing detection method of fat-soluble vitamins comprises 4 basic steps of sample addition, solvent extraction, nitrogen blowing and redissolution. Taking 96 samples to be processed as an example, the effects of 3 methods are analyzed, and the results are shown in Table 31; 1) The sample addition process was performed for substantially consistent time for the 3 methods, with manual sample addition completed within about 20 minutes. 2) In the solvent extraction process, the conventional liquid-liquid extraction method adopts a single centrifuge tube method, and needs detection personnel to perform operations such as solvent addition, transfer, extraction and the like on samples one by one, thus being time-consuming and labor-consuming and needing to be completed in about 50 minutes; solid-liquid extraction (SLE) can use a multi-channel pipette and a 96-well plate, has high operation efficiency, does not need to be transferred, but generally needs to add the extraction solvent for 4 times or 5 times, so the total time is about 25 minutes; according to the automatic pipetting workstation method, extraction solvents are added only once, the adding, transferring and extracting processes of the solvents are automatically completed by a pipetting workstation according to the set program, manual operation is little, and the extraction solvents can be completed only for 15 minutes. In summary, the automatic pipetting workstation based method has a great advantage over the existing methods in terms of the time consumption of the pre-treatment.

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

2. Cost comparison

Further comparing the detection costs of 3 methods, 1) instrument cost: the conventional liquid-liquid extraction and solid-liquid extraction methods have relatively low cost for a pretreatment instrument, generally only need to purchase low-cost conventional equipment such as a vortex oscillator, a high-speed centrifuge, a nitrogen blower, a nitrogen positive pressure instrument and the like, but the automatic pipetting workstation method needs to purchase pipetting workstation equipment with higher price; 2) The labor cost is as follows: the conventional liquid-liquid extraction method needs a large amount of manual processing operation, and solid-liquid extraction can be carried out by adopting a plurality of pipettes and 96-well plates for batch addition, or can be finished by using automatic sample adding equipment compatible with the 96-well plates, and for the automatic pipetting workstation method, the operation of the step can be finished by only operating an interface touch button, so that the solid-liquid extraction and the automatic pipetting workstation method need little labor cost; 3) Extraction solvent, standard substance and internal standard substance: the cost of the three methods is low; 4) Cost of consumable: the solid-liquid extraction method relies on the use of a commercialized SLE extraction plate, and the SLE extraction plate belongs to disposable consumables, so that the consumable cost of the method is high, and the consumable is not needed in the conventional liquid-liquid extraction and automatic pipetting workstation methods.

In combination with the above cost analysis, see table 32, the detection costs for conventional liquid-liquid extraction and solid-liquid extraction are lower when the sample size detected is smaller, while the equipment costs for the automated pipetting workstation method are higher and are not applicable. However, for clinical laboratory tests requiring daily large sample volumes to be tested for long periods of time, or for laboratories already equipped with programmable automatic pipetting workstation instruments, the total cost of testing for the automatic pipetting workstation method is significantly lower than the cost of other testing methods available today. In addition, the pipetting workstation can also be used for other detection projects in a laboratory, and equipment cost can be partially shared.

Table 32 cost comparison of three detection methods

	Conventional liquid extraction (LLE)	Solid-liquid extraction (SLE)	Automatic pipetting workstation method
				Equipment cost of pretreatment	Low cost	Low cost	High cost
Manual work load	High workload	The workload is small	The workload is small
				Standard substance, internal standard substance and extraction solvent	Low cost	Low cost	Low cost
Commercial SLE extraction 96-well plate	Does not need	High cost	Does not need
				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 the peripheral blood of children

The collected EDTA anticoagulated pediatric peripheral blood sample was placed in a centrifuge and centrifuged at 3000rpm for 10min to aspirate 50. Mu.L of the upper plasma, and then the content of fat-soluble vitamins in peripheral blood was measured by the sample preparation and pretreatment method as provided in example 1. The results showed that vitamins A, D2, D3, E, K ₁ The detection precision of (2) is less than 15 percent, and vitamin K as low as 0.04ng/ml can be accurately detected ₁ The content of vitamin K is greatly improved ₁ The pretreatment recovery rate of the plasma is greatly improved, and the actual requirement of the clinical reference interval on the detection capability of vitamin K1 in the plasma is completely met.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.

Claims (4)

1. A pretreatment method of a fat-soluble vitamin high-throughput detection sample based on an automatic pipetting workstation is characterized in that the fat-soluble vitamin is vitamin K1, the vitamin K1 is extracted from the sample by adopting a 'mixing mode' of the automatic pipetting workstation, and an extracting agent adopted by the pretreatment method is a mixture of isooctane and isopropanol; in the processes of diluting, extracting and drying and re-dissolving the sample, an automatic pipetting workstation adopts a mixing mode; in the extraction process of the sample, the program setting parameters of the mixing mode of the automatic pipetting workstation are as follows: the mixing times are 100 times, and the mixing volume is 0.3ml; the volume ratio of the isooctane to the isopropanol in the extractant is 9:1;

In the dilution process of the sample, the program setting parameters of the mixing mode of the automatic pipetting workstation are as follows: the mixing times are 10 times, and the mixing volume is 100-200ul; in the dilution process of a sample, the dilution solution is 0.1-1 mg/mL of BHT (2, 6-di-tert-butyl-4-methylphenol) solution, and the dissolution solvent is isopropanol;

in the drying and re-dissolving process of the sample, the re-dissolving solution is a mixture of methanol and water, wherein the volume ratio of the methanol to the water is 9:1.

2. The method of claim 1, wherein the "mix mode" programming parameters of the automated pipetting station during extraction of the sample 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%.

3. The method of any one of claims 1-2, wherein the sample is human serum, plasma, or peripheral whole blood.

4. A method according to claim 3, characterized in that the method comprises the steps of:

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

b) Extraction: adding 0.5-1.5mL of extractant, and performing liquid-liquid extraction by adopting a 'mixing mode' of an automatic pipetting workstation;

c) Drying and re-dissolving: and (3) taking 0.5-1 mL of the upper extraction solvent after centrifugation, drying by nitrogen, adding a redissolution, and re-dissolving by adopting a 'mixing mode' of an automatic pipetting workstation.