CN111929394A

CN111929394A - Warfarin detection method

Info

Publication number: CN111929394A
Application number: CN202010869106.0A
Authority: CN
Inventors: 贾永娟; 刘春冉; 翟瑞雪; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Hefei Hehe Medical Technology Co ltd
Priority date: 2020-08-25
Filing date: 2020-08-25
Publication date: 2020-11-13
Anticipated expiration: 2040-08-25
Also published as: CN111929394B

Abstract

The invention provides a warfarin detection method, which comprises the following steps: preparing a standard solution having at least three concentrations of warfarin and an internal standard; respectively detecting each standard solution by using a liquid chromatography-mass spectrometer to obtain a first detection result of each standard solution; fitting a standard curve equation of warfarin according to the first detection results, the warfarin in the standard solution and the concentration of the internal standard substance; centrifuging a sample to be processed, and taking a centrifuged first supernatant; adding a precipitated protein reagent and an internal standard substance into the first supernatant, carrying out vortex mixing and high-speed centrifugation, and taking the centrifuged second supernatant as a sample to be detected; detecting the sample to be detected by using a liquid chromatography-mass spectrometer under the detection condition to obtain a second detection result of the sample to be detected; and obtaining the concentration of warfarin in the sample to be detected based on the standard curve equation and the second detection result. The scheme can shorten the sample detection time.

Description

Warfarin detection method

Technical Field

The invention relates to the technical field of biological detection, in particular to a warfarin detection method.

Background

Warfarin is one of coumarin anticoagulants, and has effect in resisting vitamin K. Can inhibit the synthesis of blood coagulation factors II, VII, IX and X in liver, which are involved in vitamin K.

At present, the method generally adopted for detecting the warfarin content in a sample is high performance liquid chromatography. The high performance liquid chromatography method for detecting warfarin generally needs to carry out more complex pretreatment on a sample to be detected, and consumes more time, so that the sample detection time is longer.

Disclosure of Invention

The invention provides a warfarin detection method, which can shorten the sample detection time.

In order to solve the above problem, an embodiment of the present invention provides a warfarin detection method, including:

preparing standard solutions with at least three concentrations, wherein the standard solutions are solutions with warfarin and internal standard substances, and the internal standard substances in the standard solutions with at least three concentrations are the same in amount;

respectively detecting each standard solution by using a liquid chromatography-mass spectrometer under a preset detection condition to obtain a first detection result corresponding to each standard solution;

fitting a standard curve equation of warfarin according to the first detection result, the concentration of warfarin in the standard solution and the concentration of an internal standard substance;

centrifuging a sample to be processed, and taking a centrifuged first supernatant;

adding a precipitated protein reagent and an internal standard working solution containing an internal standard substance into the first supernatant, mixing in a vortex manner, centrifuging at a high speed, and taking the centrifuged second supernatant as a sample to be detected;

detecting the sample to be detected by using a liquid chromatography-mass spectrometer under the detection condition to obtain a second detection result of the sample to be detected;

and obtaining the concentration of warfarin in the sample to be detected based on the standard curve equation and the second detection result.

The first supernatant is serum or plasma.

Specifically, in order to improve compliance of the person to be sampled, the amount of the sample to be treated is 2 to 10. mu.L.

The concentration of the internal standard substance in the sample to be detected is the same as that in the standard solution.

Preferably, the internal standard is warfarin-D5. The isotope of the target is used as the internal standard substance, so that the internal standard substance and the target substance can be prevented from reacting to influence the detection of the target substance when the target substance is detected.

Preferably, the first and second electrodes are formed of a metal,

the liquid phase condition among the detection conditions includes:

the elution mobile phase comprises an organic phase and an aqueous phase, wherein the organic phase comprises a methanol solution and/or an acetonitrile solution;

the column temperature is 18-50 ℃;

the flow rate is 0.2-0.6 mL/min.

Preferably, the sample to be tested is introduced in an amount of 0.1-10. mu.L. When the sample volume is 0.1 muL, the signal-to-noise ratio is 16.1, the quantitative requirement can be met, and when the sample volume exceeds 10 muL, the test signal is saturated.

Specifically, the columns under liquid phase conditions include Waters Atlantis dC18 column (inner diameter 2.1X column length 50mm, particle size of packing 5 μm), Aglient extended-C18 (inner diameter 2.1X column length 50mm, particle size of packing 5 μm), Aglient XDB-C8 (inner diameter 2.1X column length 50mm, particle size of packing 5 μm), SHIMADZU-SP-C18 (inner diameter 2.1X column length 50mm, particle size of packing 2.6 μm), and Aglient SB-Aq (inner diameter 2.1X column length 50mm, particle size of packing 5 μm).

The elution mobile phase may be composed of a methanol solution and an aqueous phase, may be composed of an acetonitrile solution and an aqueous phase, and may be composed of an aqueous phase mixed with a methanol solution and an acetonitrile solution at an arbitrary ratio.

In terms of the column temperature, 18 ℃ to 50 ℃ means any value in the range of 18 ℃ to 50 ℃, for example, 18 ℃, 20 ℃, 23 ℃, 25 ℃, 30 ℃, 33 ℃, 35 ℃, 40 ℃, 43 ℃, 45 ℃ and 50 ℃.

With respect to the flow rate, 0.2-0.6mL/min refers to any value in the range of 0.2mL/min to 0.6mL/min, such as 0.2mL/min, 0.25mL/min, 0.3mL/min, 0.35mL/min, 0.4mL/min, 0.45mL/min, 0.5mL/min, 0.55mL/min, and 0.6 mL/min.

Preferably, the ratio of organic phase to aqueous phase in the elution mobile phase is:

0-0.5min：20-30％:80-70％；

0.51-1.5min：90-95％:10-5％；

1.51-2.5min：20-30％:80-70％。

specifically, the sum of the proportions of the organic phase and the aqueous phase in the elution mobile phase was 1, and therefore,

20-30% and 80-70% of the elution mobile phase in 0-0.5min are 20%: any ratio in the range of 80% to 30% to 70%. For example, the ratio of organic phase to aqueous phase is 20%: 80% and 25%: 75% or 30%: 70 percent.

90-95% and 10-5% of elution mobile phase at the ratio of 0.51-1.5min refers to that the elution mobile phase has the following weight percentage: 10% to 95%: any ratio within the range of 5%, for example, a ratio of organic phase to aqueous phase of 90%: 10%, 93%: 7% or 95%: 5 percent.

20-30% and 80-70% of the elution mobile phase in the proportion of 1.51-2.5min refer to 20%: any ratio in the range of 80% to 30% to 70%. For example, the ratio of organic phase to aqueous phase is 20%: 80% and 25%: 75% or 30%: 70 percent.

Preferably, the organic phase in the elution mobile phase contains 0-1mmol/L of buffer salt and 0-0.5% of formic acid;

the aqueous phase in the elution mobile phase contains 0-10mmol/L of buffer salt and 0-0.5% of formic acid.

Wherein the buffer salt in the elution mobile phase comprises ammonium formate or ammonium acetate.

Specifically, when the amount of formic acid added to the elution mobile phase is greater than 1%, the pH of the mobile phase is less than 2.5, and detection of the elution mobile phase at this pH causes irreversible damage to the column, and therefore, the sum of the formic acid contents in the organic phase and the aqueous phase of the elution mobile phase is not greater than 1%. When the addition amount of the buffer salt in the organic phase of the elution mobile phase is more than 1mmol/L or the addition amount of the buffer salt in the aqueous phase of the elution mobile phase is more than 10mmol/L, the buffer salt in the organic phase and the buffer salt in the aqueous phase are in a saturated state, and the buffer salt is precipitated, and if the detection is carried out by using the organic phase, the precipitated buffer salt blocks a chromatographic column and influences the sample test.

0-0.5% for formic acid in the elution mobile phase means any proportion in the range of 0-0.5%, such as 0, 0.01%, 0.03%, 0.05%, 0.07%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%.

0 to 1mmol/L for the buffer salt in the organic phase of the elution mobile phase means any value in the range of 0mmol/L to 1mmol/L, such as 0mmol/L, 0.05mmol/L, 0.1mmol/L, 0.3mmol/L, 0.5mmol/L, 0.7mmol/L, 0.9mmol/L and 1 mmol/L.

0 to 10mmol/L for the buffer salt in the aqueous phase in the elution mobile phase means any value in the range of 0mmol/L to 10mmol/L, such as 0mmol/L, 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L and 10 mmol/L.

Preferably, the mass spectrometric conditions in the detection conditions comprise:

a mass spectrum detector in the liquid chromatography-mass spectrometer adopts an ESI (+) detection mode;

the ion source parameters in the liquid chromatography-mass spectrometer are as follows: heating air flow rate (L/min) is 8-12, atomizing air flow rate (L/min) is 30-35, heating air temperature (DEG C) is 300-.

With respect to the heating gas flow rate, 8-12L/min refers to any value in the range of 8L/min to 12L/min, such as 8L/min, 9L/min, 10L/min, 11L/min, and 12L/min.

With respect to the atomizing gas flow rate, 30-35L/min refers to any value in the range of 30L/min to 35L/min, such as 30L/min, 31L/min, 32L/min, 33L/min, 34L/min, and 35L/min.

For the heating gas temperature, 300-350 ℃ refers to any value in the range of 300 ℃ to 350 ℃, such as 300 ℃, 310 ℃, 320 ℃, 330 ℃, 340 ℃ and 350 ℃.

For capillary voltage. 3000-4000V means any value in the range of 3000V to 4000V, such as 3000V, 3100V, 3200V, 3300V, 3400V, 3500V, 3600V, 3700V, 3800V, 3900V, and 4000V.

Preferably, the two variables of the standard curve equation are respectively: the ratio of the chromatographic peak area of warfarin to the chromatographic peak area of the internal standard substance in the first detection result, and the ratio of the concentration of warfarin in the standard solution to the concentration of the internal standard substance.

It is understood that, when the ratio of the chromatographic peak area of warfarin in the standard solution to the chromatographic peak area of the internal standard substance in the standard solution is taken as the ordinate y of the standard curve equation, the ratio of the concentration of warfarin in the standard solution to the concentration of the internal standard substance in the standard solution is taken as the abscissa x of the standard curve equation.

And if the ratio of the chromatographic peak area of the warfarin in the standard solution to the chromatographic peak area of the internal standard substance in the standard solution is taken as the abscissa x of the standard curve equation, the ratio of the concentration of the warfarin in the standard solution to the concentration of the internal standard substance in the standard solution is taken as the ordinate y of the standard curve equation.

Preferably, the precipitated protein reagent comprises: at least one of a methanol solution, an acetonitrile solution, trichloroacetic acid, sulfosalicylic acid, and perchloric acid.

The protein precipitation reagent can be a methanol solution, an acetonitrile solution, trichloroacetic acid, sulfosalicylic acid or perchloric acid, and can also be a solution formed by mixing any two protein precipitation reagents according to any proportion.

The acetonitrile solution and the methanol solution are used as protein precipitation reagents, so that warfarin and internal standards thereof are relatively stable, and degradation is prevented.

Preferably, the volume ratio of the precipitated protein reagent to the first supernatant is from 5:1 to 500: 1. So that under the condition that the protein in the first supernatant is completely precipitated, the first supernatant is prevented from being excessively diluted when the ratio of the amount of the precipitated protein reagent to the first supernatant is more than 500:1, so that the target substance in the diluted first supernatant is lower than the detection limit of a liquid chromatography-mass spectrometer, and the signal-to-noise ratio at the point L1 is 25.4, so that the target substance cannot be detected.

By volume ratio of precipitated protein reagent to first supernatant, 5:1-500:1 is meant any ratio in the range of 5:1 to 500:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 50:1, 70:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, and 500: 1.

Preferably, the adding a precipitated protein reagent and an internal standard working solution containing an internal standard substance into the first supernatant, vortex mixing, and high-speed centrifugation, and taking the centrifuged second supernatant as a sample to be detected comprises:

adding an internal standard working solution containing an internal standard substance into the first supernatant, and mixing for 30s-1min by vortex oscillation at the rotating speed of 1500-2500 rpm;

adding the precipitated protein reagent into the mixed first supernatant in sequence, mixing for 2-4min by vortex oscillation at the rotating speed of 1200-2000rpm, centrifuging at the rotating speed of 10000-15000rpm for 5-10min at high speed, and taking the centrifuged second supernatant as a sample to be detected.

Specifically, after the internal standard working solution is added into the first supernatant, the mixed solution can be subjected to vortex mixing to ensure that the first supernatant and the internal standard substance are uniformly mixed. And sequentially adding the precipitated protein reagent, carrying out vortex mixing to precipitate the protein in the first supernatant in the vortex process through the precipitated protein reagent, and then carrying out high-speed centrifugation to obtain a second supernatant which is the sample to be detected after the protein is precipitated.

Specifically, a standard solution can be prepared by the following steps:

(1) preparation of standard working solution

Accurately weighing 4.38mg of warfarin standard substance, placing the warfarin standard substance in a 2mL volumetric flask, dissolving the warfarin standard substance in a diluent, and determining the volume of the warfarin standard substance in 2mL to obtain standard stock solution, wherein the concentration of warfarin is 2190 mu g/mL; diluting with water as diluent to obtain warfarin standard working solution with concentration of 15.00 μ g/mL, 7.50 μ g/mL, 3.75 μ g/mL, 1.88 μ g/mL, 0.94 μ g/mL, 0.47 μ g/mL, 0.23 μ g/mL, respectively, and storing at-80 deg.C for 3 months.

(2) Preparation of internal standard working solution

0.5mg of internal standard was dissolved in 1mL of a diluent containing water to give a stock solution of standard internal standard, a solution at a concentration of 500. mu.g/mL, and stored at-20 ℃. Adding 4990. mu.L of diluent into 10. mu.L of stock solution B for dilution to obtain 1. mu.g/mL of internal standard working solution, and storing at-80 ℃.

(3) Calibration of standard solutions

And (3) transferring at least three standard solutions with different concentrations in the step (1), the internal standard working solution and the diluent in the step (2) by using a liquid transfer device, respectively placing the standard solutions in a centrifuge tube, respectively uniformly mixing the standard solutions for 30s-1min in a vortex mode at the rotation speed of 1000-2000rpm, and mixing to prepare at least three standard solutions.

The standard working solution, the internal standard working solution and the diluent in the standard solution comprise a methanol aqueous solution with the water content of 0-30%, namely, the ratio of the methanol solution to the aqueous solution in the methanol aqueous solution is 100:0-70: 30. The highest water content in the diluent is 30%, so that the problem that the solution is solidified due to over-low temperature when the standard working solution, the internal standard working solution and the standard solution are stored due to over-high water content can be avoided.

The invention provides a warfarin detection method, which comprises the steps of detecting standard solutions containing warfarin with different concentrations through a liquid chromatography-mass spectrometer, obtaining a first detection result corresponding to the standard solution with each concentration, fitting the standard solutions with various concentrations based on the concentration of the warfarin, the concentration of an internal standard substance and a plurality of detection results to obtain a standard curve equation of the warfarin, carrying out high-speed centrifugation on a sample to be processed to obtain centrifuged serum or plasma, and sequentially adding a precipitated protein reagent and an internal standard substance for protein precipitation to obtain a sample to be detected, wherein the sample to be detected can be detected. And detecting the sample to be detected by using the same detection method as the standard solution to obtain a second detection result of the sample to be detected, and obtaining the warfarin content of the sample to be detected based on the standard curve equation and the second detection result. The sample to be tested can be obtained by centrifuging and precipitating the protein of the sample to be treated, the pretreatment of the sample is relatively simple, the consumed time is less, and the sample detection time can be shortened.

Drawings

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

FIG. 1 is a flow chart of a warfarin detection method according to an embodiment of the present invention;

FIG. 2 is a chromatogram of warfarin in a sample to be tested according to an embodiment of the present invention;

FIG. 3 is a chromatogram of warfarin in a standard solution provided by an embodiment of the invention;

FIG. 4 is a chromatogram of a sample to be tested at a flow rate of 0.2mL/min and a column temperature of 18 ℃ according to an embodiment of the present invention;

FIG. 5 is a chromatogram of a sample to be tested at a flow rate of 0.3mL/min and a column temperature of 25 ℃ according to an embodiment of the present invention;

FIG. 6 is a chromatogram of a sample to be tested at a flow rate of 0.4mL/min and a column temperature of 30 ℃ according to an embodiment of the present invention;

FIG. 7 is a chromatogram of a sample to be tested at a flow rate of 0.4mL/min and a column temperature of 45 ℃ according to an embodiment of the present invention;

FIG. 8 is a chromatogram of a sample to be tested at a flow rate of 0.6mL/min and a column temperature of 50 ℃ according to an embodiment of the present invention;

FIG. 9 is a chromatogram at a flow rate of 0.1mL/min and a column temperature of 15 ℃ provided by an embodiment of the present invention;

FIG. 10 is a chromatogram of acetonitrile as the protein precipitation reagent provided by an embodiment of the invention;

FIG. 11 is a chromatogram of 5% trichloroacetic acid as the protein-precipitating reagent provided by an embodiment of the invention;

FIG. 12 is a chromatogram of 6% sulfosalicylic acid as a precipitated protein reagent provided by an embodiment of the present invention;

FIG. 13 is a chromatogram of a sample volume of 0.1 μ L of a sample to be tested according to an embodiment of the present invention;

FIG. 14 shows a ratio of precipitated protein reagent to first supernatant of 500 according to an embodiment of the present invention: 1 chromatogram of;

FIG. 15 is a chromatogram of an Aglientextend-C18 column according to an embodiment of the present invention;

FIG. 16 is a chromatogram from an AglientXDB-C8 column according to one embodiment of the present invention;

FIG. 17 is a chromatogram from a column of SHIMADZU-SP-C18 according to an embodiment of the present invention;

FIG. 18 is a chromatogram of an agent-SB-Aq chromatographic column provided in accordance with an embodiment of the present invention;

FIG. 19 is a chromatogram of a mobile phase provided by an embodiment of the invention;

FIG. 20 is a chromatogram of another mobile phase provided by an embodiment of the invention;

FIG. 21 is a chromatogram of yet another mobile phase provided by an embodiment of the invention;

fig. 22 is a chromatogram of yet another mobile phase provided by an embodiment of the invention.

Detailed Description

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

At present, a derivatization method is generally adopted for detecting warfarin in a sample to be detected, a derivatization reagent is utilized to react with a target functional group in a warfarin structure to generate a derivative, and the warfarin content in the sample to be detected is judged based on the mass ratio of the derivative to warfarin. However, the stability of the derivatization reagent is generally poor, so that the storage difficulty of the derivatization reagent is increased, and the difficulty of detecting warfarin in a sample to be detected is increased. And the reaction of the derivatization reagent and warfarin in the sample to be detected needs to be carried out under the conditions of certain temperature, pH value and the like, so that the detection difficulty of warfarin in the sample to be detected is further increased, the time needed by derivatization reaction is longer, and the overall detection time of warfarin in the sample to be detected is prolonged.

Based on the above problem, an embodiment of the present invention provides a warfarin detection method, as shown in fig. 1, including:

step 101: preparing standard solutions with at least three concentrations, wherein the standard solutions are solutions with warfarin and internal standard substances, and the internal standard substances in the standard solutions with at least three concentrations are the same in amount;

step 102: respectively detecting each standard solution by using a liquid chromatography-mass spectrometer under a preset detection condition to obtain a first detection result corresponding to each standard solution;

step 103: fitting a standard curve equation of warfarin according to the first detection result, the concentration of warfarin in the standard solution and the concentration of an internal standard substance;

step 104: centrifuging a sample to be processed, and taking a centrifuged first supernatant;

step 105: adding a precipitated protein reagent and an internal standard working solution containing an internal standard substance into the first supernatant, mixing in a vortex manner, centrifuging at a high speed, and taking the centrifuged second supernatant as a sample to be detected;

step 106: detecting the sample to be detected by using a liquid chromatography-mass spectrometer under the detection condition to obtain a second detection result of the sample to be detected;

step 107: and obtaining the concentration of warfarin in the sample to be detected based on the standard curve equation and the second detection result.

In the embodiment of the invention, the liquid chromatography-mass spectrometer is used for detecting the standard solutions containing warfarin with different concentrations, so that a first detection result corresponding to the standard solution with each concentration can be obtained, then a standard curve equation of warfarin is obtained by fitting based on the concentration of warfarin, the concentration of an internal standard substance and a plurality of detection results in the standard solutions with various concentrations, then the sample to be processed is subjected to high-speed centrifugation, centrifuged serum or plasma can be obtained, and a precipitated protein reagent and the internal standard substance are sequentially added for protein precipitation, so that the sample to be detected can be obtained. And detecting the sample to be detected by using the same detection method as the standard solution to obtain a second detection result of the sample to be detected, and obtaining the warfarin content of the sample to be detected based on the standard curve equation and the second detection result. The sample to be tested can be obtained by centrifuging and precipitating the protein of the sample to be treated, the pretreatment of the sample is relatively simple, the consumed time is less, and the sample detection time can be shortened.

The warfarin detection method is described in detail below with reference to several examples.

Example 1: preparation of Standard solutions of series of concentrations

(a) Preparation of standard stock solution

Accurately weighing 4.38mg of warfarin standard substance, placing the warfarin standard substance in a 2mL volumetric flask, dissolving the warfarin standard substance in a methanol aqueous solution with the water content of 30%, and determining the warfarin standard substance in 2mL to obtain a standard stock solution A, wherein the warfarin concentration is 2190 mug/mL; diluting with methanol water solution with water content of 30% to obtain warfarin standard working solution with concentration of 15.00 μ g/mL, 7.50 μ g/mL, 3.75 μ g/mL, 1.88 μ g/mL, 0.94 μ g/mL, 0.47 μ g/mL, and 0.23 μ g/mL, respectively, and storing at-80 deg.C for 3 months.

(b) Preparation of internal standard working solution

0.5mg of internal standard was dissolved in 1mL of aqueous methanol solution containing 30% water to give stock solution B as a standard internal standard, which was stored at-20 ℃ in a solution of 500. mu.g/mL. Adding 4990 μ L of aqueous methanol solution with water content of 30% into 10 μ L of stock solution B, diluting to obtain 1 μ g/mL internal standard working solution, and storing at-80 deg.C.

(c) Calibration of standard solutions

And (3) transferring 10 mu L of the seven standard working solutions with different concentrations in the step (a), 10 mu L of the internal standard working solution in the step (b) and 80 mu L of methanol aqueous solution with the water content of 30% by using a liquid transfer machine, respectively placing the seven standard working solutions, the 10 mu L of the internal standard working solution and the 80 mu L of methanol aqueous solution in the step (b) into 1.5mL centrifuge tubes, respectively uniformly mixing the seven standard working solutions and the 80 mu L of methanol aqueous solution in a vortex manner at the rotating speed of 2000rpm for 30s, and mixing to prepare seven standard solutions containing the warfarin standard substances.

Example 2: fitting standard curve equation

And (3) respectively detecting the seven standard solutions with different concentrations in the example 1 by using a liquid chromatography-mass spectrometer to obtain chromatograms of the seven standard solutions with different concentrations of warfarin.

Respectively obtaining the chromatographic peak area of warfarin and the chromatographic peak area of an internal standard substance in seven standard solutions from the chromatogram of the standard solution of the warfarin, taking the ratio of the chromatographic peak area of the warfarin obtained from the chromatogram of the standard solution of each concentration to the chromatographic peak area of the internal standard substance as the ordinate y of a standard curve equation, taking the ratio of the concentration of the warfarin and the concentration of the internal standard substance in the standard solution as the abscissa x of the standard curve equation, linearly returning seven kinds of data with different concentrations obtained by detection, and fitting to obtain the standard curve equation y which is a x + b, wherein a and b are weight coefficients, a is the slope of the standard curve equation, and b is the intercept of the standard curve equation.

The detection conditions include:

a chromatographic column: waters Atlantis dC18 column (2.1X 50mm, 5 μm).

The instrument comprises the following steps: agilent 1290-6430 high performance liquid chromatography-mass spectrometry combination instrument.

Elution conditions: an aqueous phase and a methanol solution;

column temperature: 30 ℃, sample introduction: 2 μ L, flow rate: 0.4 mL/min.

The mass spectrometer detector is in ESI (+) detection mode, and the ion source parameters are: heating air flow rate (L/min) is 10, atomizing air flow rate (L/min) is 35, heating air temperature (DEG C) is 350, and capillary voltage (V) is 4000.

Wherein, the ratio of eluting mobile phase is shown in the following table 1:

TABLE 1

Time/min	Methanol/% of	Water/%)
			0	30	70
0.5	30	70
			0.51	90	10
1.5	90	10
			1.51	30	70
2.5	30	70

The ion pair parameters of the mass spectrometer are shown in the following table 2:

TABLE 2

Name of substance	Parent ion	Daughter ions	Dwell	Fragmentor	CE	CAV
							Warfarin (quantitative)	309	251	150	120	14	7
Warfarin (nature)	309	121	150	120	42	7
							Warfarin-d 5	314	256	150	120	14	7

Wherein Dewll is the scanning time, fragment is the fragmentation voltage, CE is the collision voltage, CAV 6: is a linear acceleration voltage.

Example 3: treatment of samples to be tested

3.1 taking at least 50-80 μ L of the sample to be processed, centrifuging at 3500rpm for 10min, taking the supernatant to obtain serum or plasma, and storing the serum or plasma at-20 deg.C until the serum or plasma is ready for analysis.

3.2 using a pipette to pipette 10 μ L of the internal standard working solution of step (b) of example 1 into 1.5mL of a centrifuge tube, then adding 10 μ L of the supernatant of step 3.1, mixing by vortex at 2000rpm for 30s, sequentially adding 80 μ L of methanol into the centrifuge tube, mixing by vortex at 2000rpm for 3min, centrifuging at 12000rpm for 10min at high speed, and pipetting 100 μ L of the supernatant into a clean inner cannula, wherein the pipetted supernatant is the sample to be measured.

Example 4: detection of a sample to be tested

And (3) transferring 80 mu L of the sample to be detected in the step (3.2), and detecting the sample to be detected by using a liquid chromatography-mass spectrometer under the detection conditions in the embodiment (2) to obtain a chromatogram of the sample to be detected.

The chromatogram of the sample to be detected can be used for obtaining the chromatographic peak areas of the warfarin and the internal standard substance in the sample to be detected, the ratio of the chromatographic peak area of the warfarin and the chromatographic peak area of the internal standard substance in the sample to be detected is used as a longitudinal coordinate y1 and is substituted into the standard curve equation of the embodiment 2, which is y, a, x + b, because the weight coefficients a and b are known, the ratio of the concentration of the warfarin in the sample to be detected to the concentration of the internal standard substance can be obtained, and because the concentration of the internal standard substance in the sample to be detected is known, the concentration of the warfarin in the sample to be detected can be calculated.

In conclusion, the warfarin content in the sample to be detected is detected by the detection method, and the sample to be detected can be obtained only by taking 50-80 mu L of the sample to be processed, so that the compliance of a user is better.

Example 5: linear relationship and quantitative limits of warfarin detection methods

Transferring 10 mu L of the seven warfarin standard working solutions with the concentrations in the step (a), adding 10 mu L of the internal standard working solution in the step (b) and 80 mu L of methanol into each transferred warfarin standard working solution with each concentration, mixing uniformly, and detecting by using a liquid chromatography-mass spectrometer according to the detection conditions in the embodiment 2, wherein the detection is performed according to the sequence from low to high in the embodiment, so that the influence of the mixed solution with high concentration on the mixed solution with low concentration during detection is avoided. And then, drawing by using the peak area-concentration of the quantitative chromatogram to obtain a standard curve, wherein the result shows that the linear range and the quantitative limit of warfarin are as follows:

(1) limit of detection (LOD): 0.077ng/mL

(2) Limit of quantitation (LOQ): 0.23ng/mL

(3) Linear range: warfarin is in the range of 0.023 mu g/mL to 1.5 mu g/mL, the linearity is good, and the correlation coefficient R²﹥0.999。

Example 6: recovery rate and precision of warfarin detection method

Transferring the warfarin standard working solution in the step (a) to prepare high, medium and low concentrations for sample adding recovery rate experiments and precision experiments, measuring according to the detection conditions in the example (2), and repeatedly analyzing and measuring for 3 batches, wherein the recovery rate and precision are shown in the following table 3:

TABLE 3

Adding quantity of scalar	0.47μg/mL	1.88μg/mL	7.5μg/mL
				Average recovery rate	99.91％	103.61％	105.06％
Precision RSD	2.51％	1.92％	0.73％

By combining the verification tests, the detection limit, the recovery rate, the precision and other technical indexes of the embodiment meet the requirements, the method for detecting the concentration of warfarin in blood has good reproducibility and high sample recovery rate, and the accuracy of the detection result is improved.

FIG. 2 is a chromatogram of warfarin in a sample to be tested, and FIG. 3 is a chromatogram of warfarin in a standard solution. The retention times of warfarin and internal standard were consistent. In fig. 2 and 3, the chromatograms located above are internal standards and below are warfarin chromatograms;

the unit length of the abscissa in FIG. 2 is 0.1, and the unit length of the ordinate in the chromatogram located above in FIG. 2 is 1X 10³The unit length of the ordinate of the chromatogram lying below is 1X 10³；

The unit length of the abscissa in FIG. 3 is 0.1, and the unit length of the ordinate of the chromatogram located above in FIG. 3 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.5X 10³。

As can be seen from FIGS. 2 and 3, the method of the present embodiment has accurate target recognition, short analysis time, less interference, and high specificity.

Example 7: description of column temperature and flow rate of mobile phase

The tests corresponding to fig. 4 to 9 are parallel tests corresponding to the tests in example 3 and example 4, respectively, and are different from the detection of the sample to be measured in example 3 and example 4 in the flow rate of the mobile phase and the column temperature. Among the chromatograms in fig. 4 to 9, the chromatogram of the average internal standard warfarin-D5 located at the upper part is the chromatogram of warfarin located at the lower part.

FIG. 4 is a chromatogram at a flow rate of 0.2mL/min and a column temperature of 18 ℃, the unit length on the abscissa of FIG. 4 being 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 4 being 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.1X 10³；

FIG. 5 is a chromatogram at a flow rate of 0.3mL/min and a column temperature of 25 ℃, the unit length on the abscissa of FIG. 5 being 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 5 being 0.25X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10³；

FIG. 6 is a chromatogram at a flow rate of 0.4mL/min and a column temperature of 30 ℃, the unit length on the abscissa of FIG. 6 being 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 6 being 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.1X 10⁴；

FIG. 7 is a chromatogram at a flow rate of 0.4mL/min and a column temperature of 45 ℃, the unit length on the abscissa of FIG. 7 is 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 7 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 1X 10³；

FIG. 8 is a chromatogram at a flow rate of 0.6mL/min and a column temperature of 50 ℃, the unit length on the abscissa of FIG. 8 being 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 8 being 0.1X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.5X 10³。

FIG. 9 is a chromatogram at a flow rate of 0.1mL/min and a column temperature of 15 ℃, the unit length on the abscissa of FIG. 9 being 0.2, and the unit length on the ordinate of the chromatogram located above in FIG. 9 being 0.5X 10³The unit length of the ordinate of the chromatogram lying below is 0.1X 10⁴。

As can be seen from fig. 4 to 9, when the column temperature is 15 ℃ and the flow rate is less than 0.1mL/min, the retention time of warfarin and the internal standard substance is greater than 4.2min, which may cause the detection time of the whole sample to be detected to be too long, and affect the timeliness of the sample detection. And because the flow velocity of the mobile phase is too low, the ionization efficiency of the sample to be detected in the high performance liquid chromatography-mass spectrometer is poor, so that the detection signal is poor, and the detection of the sample to be detected is not facilitated.

When the flow rate is more than 0.6mL/min and the column temperature is more than 50 ℃, the column pressure of the chromatographic column exceeds the pressure which can be borne by the chromatographic column, when the detection quantity of the samples to be detected is large, irreversible damage can be caused to the chromatographic column, and the detection accuracy of the samples to be detected is also reduced. Moreover, the column temperature at this time exceeds the temperature that the chromatographic column can bear, which may cause irreversible damage to the packing in the chromatographic column and affect the detection effect of the sample.

Example 8: description of the Agents for precipitating proteins

The assays corresponding to FIGS. 10 to 12 are parallel assays corresponding to the assays in examples 3 and 4, respectively, and differ from the detection of the test sample in examples 3 and 4 by the precipitated protein reagent. Among the chromatograms in fig. 10 to 12, the chromatogram of the average internal standard warfarin-D5 located above is the chromatogram of warfarin located below.

FIG. 10 is a chromatogram showing the case where the protein-precipitating reagent is acetonitrile, and the unit length on the abscissa of FIG. 10 is 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 10 is 0.25X 10³The unit length of the ordinate of the chromatogram lying below is 0.5X 10³；

FIG. 11 is a chromatogram of 5% trichloroacetic acid as a protein-precipitating reagent, in which the unit length on the abscissa of FIG. 11 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 11 is 0.5X 10³The unit length of the ordinate of the chromatogram lying below is 1X 10³；

FIG. 12 is a 6% sulfosalicylic acid chromatogram of a precipitated protein reagent, with a unit length of 0.1 on the abscissa of FIG. 12, in FIG. 12The unit length of the ordinate of the chromatogram lying above is 0.5X 10³The unit length of the ordinate of the chromatogram lying below is 0.1X 10⁴。

Example 9: description of sample size

The test corresponding to fig. 13 is a parallel test corresponding to the tests in examples 3 and 4, and differs from the test of the sample to be tested in examples 3 and 4 in the amount of sample to be taken. In the chromatogram in fig. 13, the chromatogram of the average internal standard warfarin-D5 located above is the chromatogram of warfarin located below.

FIG. 13 is a chromatogram showing that the amount of sample to be measured is 0.1. mu.L, and the unit length on the abscissa and the unit length on the ordinate of FIG. 13 are 0.1 and 0.2X 10, respectively¹。

Specifically, the signal-to-noise ratio is 16.1 when the sample volume of the sample to be detected is 0.1 μ L, which can meet the quantitative requirement, and the signal is saturated by more than 10ul, therefore, the sample volume of the scheme is 0.1-10 μ L.

Example 10: instructions for the amount of protein-precipitating agent

The assay corresponding to FIG. 14 is a parallel assay corresponding to the assays in examples 3 and 4, and differs from the assays of the samples to be tested in examples 3 and 4 in the amount of precipitated protein reagent used. In the chromatogram in fig. 14, the chromatogram of the average internal standard warfarin-D5 located above is the chromatogram of warfarin located below.

FIG. 14 is a ratio of precipitated protein reagent to first supernatant of 500:1, the unit length of the abscissa in FIG. 14 is 0.1, and the unit length of the ordinate is 0.2X 10¹。

Specifically, when the ratio of the precipitated protein reagent to the first supernatant is less than 5 times, the first supernatant is used in a smaller amount without being removed. When the ratio of the precipitated protein reagent to the first supernatant was 500 times, the signal-to-noise ratio at L1 point was 25.4, and when the ratio of the precipitated protein reagent to the first supernatant was more than 500, the first supernatant was excessively diluted with the precipitated protein reagent, which resulted in failure to ensure quantitative detection.

Example 11: description of the column

The tests corresponding to fig. 15 to 18 are parallel tests corresponding to the tests in examples 3 and 4, respectively, and are different from the tests for a sample to be tested in examples 3 and 4 in that the test is performed using a different column. Among the chromatograms in fig. 15 to 18, the chromatogram of the average internal standard warfarin-D5 located above is the chromatogram of warfarin located below.

FIG. 15 is a chromatogram of a chromatographic column of Aglientextend-C18, with the unit length on the abscissa of FIG. 15 being 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 15 being 0.5X 10³The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

FIG. 16 is a chromatogram obtained by subjecting a chromatographic column of AglientXDB-C8 to chromatography, in which the abscissa of FIG. 16 has a unit length of 0.1 and the ordinate of the chromatogram located above in FIG. 16 has a unit length of 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.25X 10³；

FIG. 17 is a chromatogram obtained by subjecting a chromatographic column to SHIMADZU-SP-C18, wherein the unit length on the abscissa of FIG. 17 is 0.1 and the unit length on the ordinate of the chromatogram located above in FIG. 17 is 1X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

FIG. 18 is a chromatogram obtained by subjecting a column to active-SB-Aq chromatography, wherein the unit length on the abscissa of FIG. 18 is 0.1, and the unit length on the ordinate of the chromatogram located above in FIG. 18 is 0.2X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.5X 10³。

Example 12: description of the Mobile phase

The tests corresponding to fig. 19 to 22 are parallel tests corresponding to the tests in examples 3 and 4, respectively, and differ from the tests of the samples to be tested in examples 3 and 4 in the mobile phase. Among the chromatograms in fig. 19 to 22, the chromatogram of the average internal standard warfarin-D5 located above is the chromatogram of warfarin located below.

FIG. 19 shows the mobile phase is H₂O-0.1Chromatogram of% FA with MeOH-0.1% FA, unit length on abscissa of FIG. 19 is 0.1, unit length on ordinate of chromatogram located above in FIG. 19 is 0.1X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

FIG. 20 shows the mobile phase is H₂O-10mM NH₄Chromatogram of AC and MeOH, unit length of abscissa of FIG. 20 was 0.1, and unit length of ordinate of chromatogram located above in FIG. 20 was 0.1X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

Fig. 21 shows the mobile phases:

H₂O-0.1％FA+1mM NH₄FA and MeOH-0.1% FA +1mM NH₄In the chromatogram of FA, the unit length of the abscissa in FIG. 21 is 0.1, and the unit length of the ordinate of the chromatogram located above in FIG. 21 is 0.1X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³；

FIG. 22 shows the mobile phase is H₂O-0.2％FA+4mM NH₄FA and MeOH-0.2% FA, the unit length of the abscissa in FIG. 22 is 0.1, and the unit length of the ordinate of the chromatogram located above in FIG. 22 is 0.1X 10⁴The unit length of the ordinate of the chromatogram lying below is 0.2X 10³。

As can be seen from fig. 19 to 22, whether to add buffer salt and formic acid to the organic phase and the aqueous phase of the elution mobile phase has a small influence on the detection of the sample to be detected, and has a small influence on the peak shapes of the chromatographic peaks of the target substance and the internal standard substance, but the addition of buffer salt and formic acid to the elution mobile phase can promote the ionization of the target substance and the internal standard substance, which is beneficial to the detection of the sample to be detected.

It should be noted that the abscissa of fig. 2 to 22 is the acquisition time, the ordinate is the ion signal intensity, and the missing graph in the chromatogram does not affect the technical content of the present solution.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

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

Claims

1. The warfarin detection method is characterized by comprising the following steps:

2. The warfarin detection method of claim 1,

the liquid phase condition among the detection conditions includes:

the column temperature is 18-50 ℃;

the flow rate is 0.2-0.6 mL/min.

3. The warfarin detection method of claim 2,

the ratio of the organic phase to the aqueous phase in the elution mobile phase is:

0-0.5min：20-30％:80-70％；

0.51-1.5min：90-95％:10-5％；

1.51-2.5min：20-30％:80-70％；

and/or the presence of a gas in the gas,

the organic phase in the elution mobile phase contains 0-1mmol/L of buffer salt and 0-0.5% of formic acid;

4. The warfarin detection method of claim 1,

mass spectrometry conditions in the detection conditions comprising:

5. The warfarin detection method of claim 1,

the two variables of the standard curve equation are respectively: the ratio of the chromatographic peak area of warfarin to the chromatographic peak area of the internal standard substance in the first detection result, and the ratio of the concentration of warfarin in the standard solution to the concentration of the internal standard substance.

6. The warfarin detection method of claim 1,

the precipitated protein reagent comprises: at least one of a methanol solution, an acetonitrile solution, trichloroacetic acid, sulfosalicylic acid, and perchloric acid.

7. The warfarin detection method of claim 1,

the volume ratio of the precipitated protein reagent to the first supernatant is 5:1-500: 1.

8. The warfarin detection method of claim 1,

the internal standard substance is warfarin-D5.

9. The warfarin detection method of any one of claims 1 to 8,

adding a precipitated protein reagent and an internal standard working solution containing an internal standard substance into the first supernatant, mixing in a vortex manner, centrifuging at a high speed, and taking the centrifuged second supernatant as a sample to be detected, wherein the method comprises the following steps: