CN109959740A - Detect the liquid matter analysis method of vitamin K1 content in blood - Google Patents
Detect the liquid matter analysis method of vitamin K1 content in blood Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
Abstract
The liquid matter analysis method that the present invention detects vitamin K1 content in blood is to be demarcated using the triple quadrupole tandem mass spectrometers of high performance liquid chromatography to standard solution, it is y=a*x+b that fitting, which obtains calibration curve equation, take blood sample to be measured, the blood to be detected after treatment, equally sample to be measured is detected using the triple quadrupole tandem mass spectrometers of high performance liquid chromatography, obtain blood y value to be measured, blood y to be measured is substituted into calibration curve equation, the relative concentration x of vitamin K1 in blood sample to be measured is obtained by calculation, vitamin K1 isotopes concentration is known in internal standard compound working solution, thus the concentration of the vitamin K1 in the sample in blood to be detected is calculated, sample introduction is analyzed after liquid-liquid extraction pre-treatment for serum sample of the present invention, detection process is easy quickly, detection sensitivity is high , more conducively the vitamin K1 content of patient's body is measured in clinical treatment.
Description
Technical field
The present invention relates to the clinical content concn monitoring technical field of liposoluble vitamin more particularly to a kind of detection blood
The method of middle vitamin K1 content.
Background technique
Vitamin K1, molecular formula C31H46O2, particular chemical are illustrated in fig. 1 shown below.Vitamin K1 is synthesis in liver
The necessary material of factor can cause blood coagulation disorders when lacking.When vitamin K 1 injection prothrombin deficiency in blood
When, it is slow that the solidification of blood just will appear, and at this moment supplementing suitable vitamin K1 can promote liver to synthesize factor, plays only
The effect of blood.Vitamin K1 is clinically applied to the too low disease of fibrin ferment, vitamin K1 deficiency disease, new life as pharmaceutical preparation
Bleeding caused by the prevention and treatment of youngster's nature haemorrhage and obstructive jaundice, leak, chronic diarrhea etc., Coumarins, sodium salicylate etc.
Caused Hypoprothrombinemia.Vitamin also have the function of analgesia, alleviate bronchial spasm, to visceral smooth muscle colic pain,
Colic pain caused by cholepathia spastica, enterospasm has obvious effects on, and vitamin K1 can be also used for the additive of multidimensional food.
The measuring method of currently used vitamin K1 is numerous, respectively there is an advantage and disadvantage, including high performance liquid chromatography, immune
Method, liquid chromatography tandem mass spectrometry etc..When liquid chromatography detects, pretreatment process is complicated, and sensitivity is low.Immunization inspection
When survey, pretreatment process is simpler, and analysis time is short but sensitivity is low, specificity is poor.Liquid chromatography tandem mass spectrometry is surveyed
Determining vitamin content has many advantages, such as that quantitatively accurately favorable reproducibility, high sensitivity, specificity are strong, but there are still pre-treatments at present
The problems such as complexity, analysis time is longer.
Summary of the invention
In view of the above technical problems, it is an object of the present invention to provide a kind of liquid matter analyses of vitamin K1 content in detection blood
Method, making the measurement of vitamin K1 content, quick and precisely pre-treatment is easy to operate, high sensitivity, and accurate quantification related substances are simultaneously
And it can be generally applicable.
The technical solution adopted by the present invention is that:
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: it the following steps are included:
(1), the calibration of standard solution
(a), the preparation of standard working solution:
It is accurate that vitamin K1 standard items 15.46mg is claimed to be placed in 10mL volumetric flask, it is dissolved and is settled to the pure methanol of 10mL
Scale obtains standard reserving solution A, and standard reserving solution A is diluted with the methanol solution that water content is 5%-15%, is existed respectively
The standard working solution of each concentration is made in concentration range containing 0.5-5000ng/mL vitamin K1, and under the conditions of -80 DEG C
It saves；
(b), the preparation of standard internal standard solution:
Vitamin K1 Isotopic Internal Standard standard items 1.00mg accurately is weighed in 1mL volumetric flask, simultaneously with the pure methanol dissolution of 1mL
It is settled to scale, obtains internal standard stock solution B, internal standard stock solution B is diluted with the methanol solution that water content is 5%-15%,
It obtains the concentration containing vitamin K1 isotope and is the standard internal standard solution of 10ng/mL, and saved under the conditions of -80 DEG C；
(c), it is demarcated with standard solution, obtains calibration curve equation
The 10 μ L standard working solutions for pipetting at least three kinds various concentrations respectively with liquid-transfering gun are dense by above-mentioned at least three kinds of differences
The standard working solution of degree is mixed with the standard internal standard solution of 10 μ L and 80 μ L substitution matrix respectively, and above-mentioned substitution matrix is to contain 4%
Bovine serum albumin(BSA), be placed in 2.0mL centrifuge tube and at least three kinds of standard solution be made, above-mentioned standard solution is respectively in revolving speed
After the mixing 30s-1min that is vortexed under 1000-2000rpm, the methanol of 200-400 μ L is added in Xiang Shangshu centrifuge tube, and in revolving speed
3-5min is mixed to be vortexed under 1000-2000rpm, then the n-hexane of 400-800 μ L is added into above-mentioned centrifuge tube, and in revolving speed
5-10min is mixed to be vortexed under 1000-2000rpm, then 5-10min is centrifuged in the case where revolving speed is 12000-14000rpm, obtains
Supernatant pipettes the above-mentioned supernatant of 400-550 μ L respectively and is placed in another centrifuge tube, blows 10min- with nitrogen at normal temperature
20min is separately added into 100 μ L into the centrifuge tube of drying and redissolves liquid, the redissolution liquid after above-mentioned supernatant is all dried
It is the formic acid containing 0.05%-2% and the methanol solution containing 5%-15% water, is vortexed in the case where revolving speed is 1000-2000rpm mixed
After even 30s-1min, then 5-10min is centrifuged in the case where revolving speed is 12000-14000rpm and obtains supernatant, take above-mentioned supernatant respectively
100 μ L detect above-mentioned supernatant with the triple quadrupole tandem mass spectrometers of high performance liquid chromatography, obtain respectively it is above-mentioned at least
Vitamin K1 chromatogram and corresponding vitamin K1 isotope chromatogram in three kinds of standard solution, with above-mentioned at least three standard
Ordinate y of the vitamin K1 peak area of solution with corresponding vitamin K1 isotopic peak area ratio as canonical plotting,
To contain the ratio between vitamin K1 isotopes concentration in the concentration containing vitamin K1 in above-mentioned standard working solution and standard internal standard solution
As the abscissa x of canonical plotting, gained at least three groups of data will be detected above and carry out linear regression, fitting obtains standard song
Line equation is y=a*x+b, and obtains weight coefficient a and b；
(2), the centrifugation of blood is detected
Blood to be detected at least 5mL is taken, 10min is centrifuged in the case where centrifugal speed is 3500rpm, obtains supernatant i.e. serum,
It is spare to before analyzing that above-mentioned serum is placed in the lower preservation of -80 DEG C of freezings；
(3), sample to be tested is handled
(d), step is then added in the centrifuge tube of 1.5mL with 10 μ L of the standard internal standard solution of liquid-transfering gun removing step (c)
(2) serum of 100uL in above-mentioned centrifuge tube, revolving speed be 1000-2000rpm under be vortexed mix 30s-1min, then to
200-400 μ L methanol is added in above-mentioned centrifuge tube, and after the mixing 3-5min that is vortexed under revolving speed is 1000-2000rpm, then upwards
Addition 400-800 μ L n-hexane in centrifuge tube is stated, after the mixing 5-10min that is vortexed under revolving speed is 1000-2000rpm, then is being turned
Speed is to be centrifuged 5-10min under 12000-14000rpm, obtains supernatant, pipettes the above-mentioned supernatant of 400-550 μ L respectively and be placed in separately
In one centrifuge tube, after blowing 10min-20min until above-mentioned supernatant is all dried with nitrogen at normal temperature, to drying from
100 μ L are added in heart pipe and redissolve liquid, which is that the formic acid containing 0.05%-2% and the methanol containing 5%-15% water are molten
Liquid is centrifuged 5- after the mixing 30s-1min that is vortexed under revolving speed is 1000-2000rpm, then in the case where revolving speed is 12000-14000rpm
10min, obtained liquid are sample to be tested；
(4), the detection of sample to be tested
100 μ L of removing step (d) sample to be tested, using the triple quadrupole tandem mass spectrometers of high performance liquid chromatography to it is above-mentioned to
Sample is detected, and obtains the vitamin K1 chromatogram and vitamin K1 isotope chromatogram of above-mentioned sample to be tested, will be above-mentioned
Vitamin K1 peak area and vitamin K1 isotopic peak area ratio y in chromatogram substitute into the standard curve of above-mentioned steps (c)
In equation y=a*x+b, vitamin K1 concentration and the same position of vitamin K1 in standard internal standard solution in sample to be tested is obtained by calculation
The ratio between plain concentration x, the concentration of vitamin K1 isotope is known in standard internal standard solution, is obtained by calculation in blood to be detected
Vitamin K1 concentration.
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: used in step (c)
The standard working solution of nine kinds of various concentrations, the standard working solutions of nine kinds of various concentrations be respectively contain 0.5,1,2,5,20,100,
500, the vitamin K1 solution of 2000 and 5000ng/mL concentration.
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: in step (c), institute
State substitution matrix be by 4g bovine serum albumin(BSA) in 100mL physiological saline, it is manufactured by ultrasonic dissolution.
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: in step (a) and (b)
Described in methanol solution be made of the water and methanol of 10:90.
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: in step (c) and step
(d) the redissolution liquid that the redissolution liquid described in is made of formic acid, water and methanol that ratio is 0.1:10:89.9.
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: in step (c) and step
(d) in, analysis chromatographic column used in the triple quadrupole tandem mass spectrometers of high performance liquid chromatography is Poroshell 120-
SB-C18, used pot strainer are 1290 Infinity In-line Filter, the triple level Four of high performance liquid chromatography
The column temperature of bar tandem mass spectrometer setting is 40 DEG C, and sample volume is 20 μ L.
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: the analysis chromatographic column
Mobile phase contain the water of 4:96 ratio: methanol, analysis column flow rate is 0.6mL/min, and analysis chromatographic column is washed using gradient
Off-square formula.
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: the high-efficient liquid phase color
The ion source for composing triple quadrupole tandem mass spectrometers is the source APCI, and acquisition mode is cation acquisition, and quota ion pair is
451.20-186.60。
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, in which: the concentration refers to body
The concentration of product ratio, the ratio refer to the ratio of volume ratio.
The invention has the advantages that:
The liquid matter analysis method of vitamin K1 content in detection blood of the present invention, serum is through precipitating and liquid-liquid extraction
Sample introduction is analyzed afterwards, reduces the interference of endogenous impurity, improves the accuracy of quantitative result, shortens analysis time, makes to examine
Survey process is easy to be quickly more conducively in clinical treatment monitored the vitamin K1 content of patient's body, is vitamin K1
Reasonable injection provide experiment basis.
Detailed description of the invention
Fig. 1 is vitamin K1 chemical structural formula；
Fig. 2 is vitamin K1 chromatogram in embodiment Plays solution；
Fig. 3 is vitamin K1 chromatogram in mark-on serum sample in embodiment.
In figure 2 and figure 3, mark 1 is the chromatographic peak of vitamin K1；Mark 2 is target chromatographic peak in vitamin K1.
Below in conjunction with specific embodiments and the drawings, the invention will be further described.
Specific embodiment
The liquid matter analysis method of vitamin K1 content in a kind of detection blood of the invention, it the following steps are included:
(1), the calibration of standard solution
(a), the preparation of standard working solution:
It is accurate that vitamin K1 standard items 15.46mg is claimed to be placed in 10mL volumetric flask, scale is dissolved and is settled to pure methanol,
Standard reserving solution A is obtained, the concentration of vitamin K1 is 1500 μ g/mL, the methanol for being 10% by standard reserving solution A water content
Solution is diluted, and makes the standard work of nine kinds of concentration in the range of containing 0.5-5000ng/mL vitamin K1 respectively
Liquid, the standard working solution of nine kinds of various concentrations are respectively to contain 0.5,1,2,5,20,100,500,2000 and 5000ng/mL concentration
Vitamin K1 solution, and saved under the conditions of -80 DEG C；
(b), the preparation of standard internal standard solution:
Vitamin K1 Isotopic Internal Standard standard items 1.00mg accurately is weighed in 1mL volumetric flask, is dissolved and is determined with pure methanol
Hold to scale, obtain internal standard stock solution B, vitamin K1 Isotopic Internal Standard concentration is 1000 μ g/mL, and internal standard stock solution B is used
The methanol solution that water content is 10% is diluted, and obtains the standard internal standard solution for being 10ng/mL containing vitamin K1 internal standard concentration,
And it is saved under the conditions of -80 DEG C；
(c), it is demarcated with standard solution, obtains calibration curve equation:
With liquid-transfering gun by 10 μ L of standard working solution of nine kinds of various concentrations, 10 μ L of standard internal standard solution and 80 μ L, 4% cow's serum
Albumin, which is respectively placed in 2.0mL centrifuge tube, is mixed and made into nine kinds of standard solution, and above-mentioned standard solution is in revolving speed respectively
It is vortexed under 1500rpm and mixes 30s, 400 μ L methanol are added in Xiang Shangshu centrifuge tube, then be vortexed and mix in the case where revolving speed is 1500rpm
3min, then 600 μ L n-hexanes are added into above-mentioned centrifuge tube, it is then vortexed in the case where revolving speed is 1500rpm and mixes 5min, in revolving speed
To be centrifuged 5min under 12000rpm, 550 μ L of supernatant is taken respectively, and then nitrogen blows 15min to doing at normal temperature, and it is multiple that 100 μ L are added
Solution, the redissolution liquid are the formic acid containing 0.1% and methanol solution containing 10% water, and in the case where revolving speed is 1000-2000rpm
It is vortexed and mixes 30s-1min, centrifugation 5-10min obtains supernatant in the case where revolving speed is 12000-14000rpm, takes above-mentioned supernatant respectively
100 μ L detect above-mentioned supernatant with the triple quadrupole tandem mass spectrometers of high performance liquid chromatography, obtain above-mentioned nine kinds respectively
Vitamin K1 chromatogram and corresponding vitamin K1 isotope chromatogram in standard solution, with the dimension of above-mentioned nine standard solution
The ordinate y of raw element K1 peak area and corresponding vitamin K1 isotopic peak area ratio as canonical plotting, with above-mentioned mark
The conduct of the ratio between the concentration containing vitamin K1 isotope in concentration and standard internal standard solution in quasi- working solution containing vitamin K1
The abscissa x of canonical plotting will detect nine groups of data of gained above and carry out linear regression, and fitting obtains calibration curve equation and is
Y=a*x+b, and obtain weight coefficient a and b；
(2), the centrifugation of blood is detected
Blood to be detected at least 5mL is taken, 10min is centrifuged in the case where centrifugal speed is 3500rpm, obtains supernatant i.e. serum,
It is spare to before analyzing that above-mentioned serum is placed in the lower preservation of -80 DEG C of freezings；
(3), sample to be tested is handled
(d), step is then added in the centrifuge tube of 1.5mL with 10 μ L of the standard internal standard solution of liquid-transfering gun removing step (b)
(2) serum of 100uL is vortexed in the case where revolving speed is 1500rpm in above-mentioned centrifuge tube and mixes 30s, add in Xiang Shangshu centrifuge tube
Enter 400 μ L methanol, is vortexed in the case where revolving speed is 1500rpm and mixes 3min, then 600 μ L n-hexanes are added into above-mentioned centrifuge tube,
Revolving speed is to be vortexed to mix 5min under 1500rpm, then be centrifuged 5-10min in the case where revolving speed is 12000rpm, takes 550 μ L of supernatant then
Nitrogen blows 15min to dry, and 100 μ L are added and redissolve liquid, which is molten containing 0.1% formic acid and methanol containing 10% water
Liquid, and be vortexed in the case where revolving speed is 1000-2000rpm and mix 30s-1min, 5- is centrifuged in the case where revolving speed is 12000-14000rpm
10min obtains supernatant, and obtained liquid is sample to be tested；
(4), the detection of sample to be tested
100 μ L of removing step (c) sample to be tested, using the triple quadrupole tandem mass spectrometers of high performance liquid chromatography to it is above-mentioned to
Sample is detected, and obtains the vitamin K1 chromatogram and vitamin K1 isotope chromatogram of above-mentioned sample to be tested, will be above-mentioned
Vitamin K1 peak area and vitamin K1 isotopic peak area ratio y in chromatogram substitute into the standard curve of above-mentioned steps (one)
In equation y=a*x+b, vitamin K1 concentration and the same position of vitamin K1 in standard internal standard solution in sample to be tested is obtained by calculation
The ratio between plain concentration x, vitamin K1 isotopes concentration is known in standard internal standard solution, is obtained by calculation in blood to be detected
Vitamin K1 concentration.
Sample treatment includes: that pot strainer used in the triple quadrupole tandem mass spectrometers of high performance liquid chromatography is 1290
Infinity In-line Filter, used analysis chromatographic column are Poroshell 120-SB-C18, and the column temperature of setting is
40 DEG C, sample volume 20uL, analysis column Mobile phase contains the water containing 4:96 ratio: methanol, and analysis column flow rate is
0.6mL/min, analysis chromatographic column use gradient elution mode；The ion source of the triple quadrupole tandem mass spectrometers of high performance liquid chromatography
For the source APCI, acquisition mode is cation acquisition, quota ion pair 451.20-186.60；The concentration refers to volume ratio
Concentration, the ratio refer to the ratio of volume ratio.
Technical method demonstration is as follows in the present embodiment:
One, the linear relationship and quantitative limit of this method
By the hybrid standard working solution of the vitamin K1 of each concentration (0.5-5000ng/mL) of 10 μ L of above-mentioned preparation,
The mixing of 80 μ L, 4% bovine serum albumin(BSA) is added, the sample introduction after albumen precipitation and liquid-liquid extraction, vitamin K1 concentration is in 0.05ng/
In mL to 500ng/mL range, by the present embodiment determination condition, it is measured from low to high by concentration, with quantitative chromatographic peak face
The mapping of product-concentration, obtains standard curve, the results showed that the range of linearity and quantitative limit of vitamin K1 are as follows:
(1) detection limit (LOD): 0.0167ng/mL.
(2) quantitative limit (LOQ): 0.05ng/mL.
(3) range of linearity:
Vitamin K1 is linear good in 0.05ng/mL to 500ng/mL range, coefficient R2﹥ 0.9900.
Two, the rate of recovery and precision of this method
Taking vitamin K1 standard working solution to be configured to, high, medium and low 3 kinds of concentration carries out sample recovery rate experiment and precision is real
It tests, is measured by the present embodiment method, replicate analysis measures 3 batches, and the rate of recovery and precision are respectively such as table 1.Its it is low,
Average recovery rate within the scope of 3 middle and high pitch-based spheres is 93.77%~109.04%, relative standard deviation 0.36%
~3.21%, table 1 is the recovery of standard addition and precision of vitamin K1.
Table 1
In summary verification test, the detection limit of the present embodiment, all technicals such as the rate of recovery and precision meet
It is required that vitamin K1 content in method detection blood, reproducibility is good, and sample recovery rate is high, improves the accurate of testing result
Degree.
Vitamin K1 chromatogram is shown in Fig. 3 in serum sample, and vitamin K1 chromatogram is shown in Fig. 2, vitamin K1 in standard solution
Retention time be 2.58min, the identification by the present embodiment method target compound known to Fig. 2 and Fig. 3 is accurate, and analysis time
Short, small, the high specificity of interference.
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.
Claims (9)
1. it is a kind of detection blood in vitamin K1 content liquid matter analysis method, it is characterised in that: it the following steps are included:
(1), the calibration of standard solution
(a), the preparation of standard working solution:
It is accurate that vitamin K1 standard items 15.46mg is claimed to be placed in 10mL volumetric flask, scale is dissolved and is settled to the pure methanol of 10mL,
Standard reserving solution A is obtained, standard reserving solution A is diluted with the methanol solution that water content is 5%-15%, is being contained respectively
The standard working solution of each concentration is made in the concentration range of 0.5-5000ng/mL vitamin K1, and is protected under the conditions of -80 DEG C
It deposits；
(b), the preparation of standard internal standard solution:
Vitamin K1 Isotopic Internal Standard standard items 1.00mg accurately is weighed in 1mL volumetric flask, with the pure methanol dissolution of 1mL and constant volume
To scale, internal standard stock solution B is obtained, internal standard stock solution B is diluted with the methanol solution that water content is 5%-15%, is obtained
Concentration containing vitamin K1 isotope is the standard internal standard solution of 10ng/mL, and saves under the conditions of -80 DEG C；
(c), it is demarcated with standard solution, obtains calibration curve equation
The 10 μ L standard working solutions for pipetting at least three kinds various concentrations respectively with liquid-transfering gun, by above-mentioned at least three kinds of various concentrations
Standard working solution is mixed with the standard internal standard solution of 10 μ L and 80 μ L substitution matrix respectively, and above-mentioned substitution matrix is the ox containing 4%
Seralbumin is placed in 2.0mL centrifuge tube and at least three kinds of standard solution, above-mentioned standard solution is made is in revolving speed respectively
It is vortexed after mixing 30s-1min under 1000-2000rpm, the methanol of 200-400 μ L is added in Xiang Shangshu centrifuge tube, and be in revolving speed
It is vortexed under 1000-2000rpm and mixes 3-5min, then the n-hexane of 400-800 μ L is added into above-mentioned centrifuge tube, and be in revolving speed
It is vortexed under 1000-2000rpm and mixes 5-10min, be then centrifuged 5-10min in the case where revolving speed is 12000-14000rpm, obtain
Clear liquid pipettes the above-mentioned supernatant of 400-550 μ L respectively and is placed in another centrifuge tube, blows 10min- with nitrogen at normal temperature
20min is separately added into 100 μ L into the centrifuge tube of drying and redissolves liquid, the redissolution liquid after above-mentioned supernatant is all dried
It is the formic acid containing 0.05%-2% and the methanol solution containing 5%-15% water, is vortexed in the case where revolving speed is 1000-2000rpm mixed
After even 30s-1min, then 5-10min is centrifuged in the case where revolving speed is 12000-14000rpm and obtains supernatant, take above-mentioned supernatant respectively
100 μ L detect above-mentioned supernatant with the triple quadrupole tandem mass spectrometers of high performance liquid chromatography, obtain respectively it is above-mentioned at least
Vitamin K1 chromatogram and corresponding vitamin K1 isotope chromatogram in three kinds of standard solution, with above-mentioned at least three standard
Ordinate y of the vitamin K1 peak area of solution with corresponding vitamin K1 isotopic peak area ratio as canonical plotting,
To contain the ratio between vitamin K1 isotopes concentration in the concentration containing vitamin K1 in above-mentioned standard working solution and standard internal standard solution
As the abscissa x of canonical plotting, gained at least three groups of data will be detected above and carry out linear regression, fitting obtains standard song
Line equation is y=a*x+b, and obtains weight coefficient a and b；
(2), the centrifugation of blood is detected
Blood to be detected at least 5mL is taken, 10min is centrifuged in the case where centrifugal speed is 3500rpm, obtains supernatant i.e. serum, it will be upper
State serum be placed in -80 DEG C of freezings it is lower save it is spare to before analyzing；
(3), sample to be tested is handled
(d), step (2) is then added in the centrifuge tube of 1.5mL with 10 μ L of the standard internal standard solution of liquid-transfering gun removing step (c)
100uL serum in above-mentioned centrifuge tube, revolving speed be 1000-2000rpm under be vortexed mix 30s-1min, state then up
In centrifuge tube be added 200-400 μ L methanol, and revolving speed be 1000-2000rpm under be vortexed mix 3-5min after, then to it is above-mentioned from
400-800 μ L n-hexane is added in heart pipe, is after the mixing 5-10min that is vortexed under revolving speed is 1000-2000rpm, then in revolving speed
It is centrifuged 5-10min under 12000-14000rpm, obtains supernatant, the above-mentioned supernatant of 400-550 μ L is pipetted respectively and is placed in another
In centrifuge tube, 10min-20min is blown after above-mentioned supernatant is all dried with nitrogen at normal temperature, to the centrifuge tube of drying
100 μ L of middle addition redissolve liquid, which is the formic acid containing 0.05%-2% and the methanol solution containing 5%-15% water,
Revolving speed is to be vortexed after mixing 30s-1min under 1000-2000rpm, then be centrifuged 5-10min in the case where revolving speed is 12000-14000rpm,
Obtained liquid is sample to be tested；
(4), the detection of sample to be tested
100 μ L of removing step (d) sample to be tested, using the triple quadrupole tandem mass spectrometers of high performance liquid chromatography to above-mentioned to test sample
Product are detected, and the vitamin K1 chromatogram and vitamin K1 isotope chromatogram of above-mentioned sample to be tested are obtained, by above-mentioned chromatography
Vitamin K1 peak area and vitamin K1 isotopic peak area ratio y in figure substitute into the calibration curve equation y of above-mentioned steps (c)
In=a*x+b, it is dense that vitamin K1 concentration and vitamin K1 isotope in standard internal standard solution in sample to be tested is obtained by calculation
The ratio between degree x, the concentration of vitamin K1 isotope is known in standard internal standard solution, and the dimension in blood to be detected is obtained by calculation
Raw element K1 concentration.
2. detecting the liquid matter analysis method of vitamin K1 content in blood as described in claim 1, it is characterised in that: in step
(c) using the standard working solution of nine kinds of various concentrations in, the standard working solutions of nine kinds of various concentrations be respectively contain 0.5,1,2,
5, the vitamin K1 solution of 20,100,500,2000 and 5000ng/mL concentration.
3. detecting the liquid matter analysis method of vitamin K1 content in blood as claimed in claim 2, it is characterised in that: in step
(c) in, the substitution matrix be by 4g bovine serum albumin(BSA) in 100mL physiological saline, it is manufactured by ultrasonic dissolution.
4. detecting the liquid matter analysis method of vitamin K1 content in blood as claimed in claim 3, it is characterised in that: in step
(a) it is made of with methanol solution described in (b) the water and methanol of 10:90.
5. detecting the liquid matter analysis method of vitamin K1 content in blood as claimed in claim 4, it is characterised in that: in step
(c) the redissolution liquid being made of with redissolution liquid described in step (d) formic acid, water and methanol that ratio is 0.1:10:89.9.
6. the liquid matter analysis method of detection blood vitamin K1 content as claimed in claim 5, it is characterised in that: in step
(c) and in step (d), analysis chromatographic column used in the triple quadrupole tandem mass spectrometers of high performance liquid chromatography is
Poroshell 120-SB-C18, used pot strainer are 1290 Infinity In-line Filter, efficient liquid
The column temperature of the triple quadrupole tandem mass spectrometer settings of phase chromatography is 40 DEG C, and sample volume is 20 μ L.
7. detecting the liquid matter analysis method of vitamin K1 content in blood as claimed in claim 6, it is characterised in that: described point
The mobile phase of analysis chromatographic column contains the water of 4:96 ratio: methanol, and analysis column flow rate is 0.6mL/min, and analysis chromatographic column is adopted
Use gradient elution mode.
8. detecting the liquid matter analysis method of vitamin K1 content in blood as claimed in claim 7, it is characterised in that: the height
The ion source of the triple quadrupole tandem mass spectrometers of effect liquid phase chromatogram is the source APCI, and acquisition mode is cation acquisition, quota ion
To for 451.20-186.60.
9. detecting the liquid matter analysis method of vitamin K1 content in blood as claimed in claim 8, it is characterised in that: described dense
Degree refers to that the concentration of volume ratio, the ratio refer to the ratio of volume ratio.
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