CN110208436A - A kind of method of farnoquinone content in detection blood - Google Patents
A kind of method of farnoquinone content in detection blood Download PDFInfo
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Abstract
The present invention provides a kind of methods of farnoquinone content in detection blood, this method comprises: detecting at least three standard solution using efficient liquid phase tandem mass spectrometer, the chromatogram of each standard solution is obtained, the standard solution is containing farnoquinone and internal standard compound known to concentration；With the concentration proportion of farnoquinone in standard solution and internal standard compound for a two-dimensional coordinate, in the chromatogram of the standard solution, the two peak area ratio is another two-dimensional coordinate, fitting obtains calibration curve equation；It handles blood to be detected and obtains blood sample, and internal standard compound known to concentration is added, through sample pre-treatments to obtain sample to be tested；Sample to be tested is detected under the conditions of same detection obtains chromatogram；The peak area ratio of farnoquinone and internal standard compound in the chromatogram is updated in calibration curve equation, and according to the concentration of added internal standard compound, calculates the content of farnoquinone in blood to be detected.The present invention can accurately detect the farnoquinone content in blood.
Description
Technical field
The present invention relates to technical field of chemistry, in particular to the method for farnoquinone content in a kind of detection blood.
Background technique
Farnoquinone is liposoluble vitamin, and the normal coagulation of body is participated in as glutamic acid-gamma-carboxylation enzyme coenzyme
Process.Clinically, farnoquinone can be widely used for treatment newborn, baby and the pregnant woman bleeding as caused by vitamin K deficiency
Disease treats and prevents osteoporosis.Farnoquinone generates bone protein, then generates sclerotin jointly with calcium, increases bone density, prevents
Only fracture.Meanwhile it farnoquinone appropriate being added in diet can be effectively reduced coronary calcification.In addition, grinding in recent years
Study carefully discovery, farnoquinone has growth inhibition effect to Several Kinds of Malignancy cell, and inducible hepatocellular carcinoma, oophoroma etc. are a variety of
The Apoptosis of solid tumor and Leukemic Bone Marrow hyperplasia exception syndrome, has an apparent antitumor action, and with it is a variety of anti-swollen
Tumor medicine has synergistic effect.
Currently, using HPLC (High Performance Liquid Chromatography, high performance liquid chromatography
Method), the blood after farnoquinone related drugs is taken for the preparation of effective component, patient to farnoquinone bulk pharmaceutical chemicals, farnoquinone
The types sample such as liquid sample carries out the detection of farnoquinone content.
But this kind of detection is lower to sensitivity requirement, generally can not detect the farnoquinone content in blood, or detection standard
True rate is lower.
Summary of the invention
The present invention provides a kind of method of farnoquinone content in detection blood, the dimension that can accurately detect in blood is raw
Plain K2 content.
In order to achieve the above object, the present invention is achieved through the following technical solutions:
The present invention provides a kind of methods of farnoquinone content in detection blood, comprising:
Using efficient liquid phase tandem mass spectrometer, under certain testing conditions, N number of standard solution is detected respectively, is obtained each
The chromatogram of standard solution, wherein containing internal standard known to farnoquinone known to concentration and concentration in any standard solution
Object, the concentration of farnoquinone is different in various criterion solution, and N >=3；
With in standard solution, the ratio of the concentration of the concentration and internal standard compound of farnoquinone is a two-dimensional coordinate, and with this
In the chromatogram of standard solution, the ratio of the peak area of the peak area and internal standard compound of farnoquinone is another two-dimensional coordinate, fitting
Obtain calibration curve equation；
It handles blood to be detected and obtains blood sample；
By a certain amount of internal standard working solution containing internal standard compound known to concentration, it is added in the blood sample, goes forward side by side
Row sample pre-treatments are to obtain sample to be tested；
Using efficient liquid phase tandem mass spectrometer, under the conditions of same detection, the sample to be tested is detected, is obtained described to be measured
The chromatogram of sample；
By in the chromatogram of the sample to be tested, the ratio of the peak area of the peak area and internal standard compound of farnoquinone is substituted into
Into the calibration curve equation, and according to the concentration of added internal standard compound, vitamin in the blood to be detected is calculated
The content of K2；
Wherein, the calibration curve equation are as follows: y=k × x+b, wherein y characterizes peak area ratio, and x characterizes concentration proportion,
K and b characterize coefficient.
Preferably, the testing conditions include: liquid-phase condition and tandem mass spectrum condition；
Wherein, the liquid-phase condition include: the length of chromatographic column be 50mm, internal diameter 3.0mm, packing material size are 2.7 μm,
Mobile phase is containing the pure water that volume ratio is 0.05%-0.25% formic acid and containing volume ratio be 0.05%-0.25% formic acid methanol,
Analysis time is 7.5min, and column temperature is 27 DEG C -33 DEG C, and sample volume is 10 μ L-30 μ L, flow velocity 0.55mL/min-0.65mL/
min；
Wherein, the tandem mass spectrum condition includes: using atmosphere pressure chemical ion source APCI, cation scan pattern, choosing
Reaction monitoring mode is selected, dry temperature degree is 300 DEG C, and dry gas stream speed is 6L/min, and atomization gas pressure is 20psi, capillary
Voltage is 4500V.
Preferably, the liquid-phase condition further include: 120 EC-C18 chromatographic column of Poroshell.
Preferably, it is 30 DEG C that the liquid-phase condition, which includes: column temperature,.
Preferably, it is to be containing the pure water that volume ratio is 0.2% formic acid and containing volume ratio that the liquid-phase condition, which includes: mobile phase,
The methanol of 0.2% formic acid, flow velocity 0.6mL/min；
Elution process are as follows: the percentage amount of the methanol containing formic acid and the pure water containing formic acid ratio, in washing for 4.1min-6.0min
It is 99-100:1-0 in de- time range, is 90-98:10-2 within the scope of other elution times.
Preferably, the internal standard working solution by a certain amount of containing internal standard compound known to concentration, is added to the blood
In sample, and sample pre-treatments are carried out to obtain sample to be tested, comprising:
A certain amount of internal standard working solution containing internal standard compound known to concentration is pipetted with liquid-transfering gun, is placed in centrifuge tube, then
Blood sample described in 50 μ L-300 μ L is added, a certain amount of protein precipitation reagent is added, in the revolving speed of 1500rpm-2500rpm
Lower vortex mixed 2min-5min；
A certain amount of extraction agent is added after vortex mixed in centrifuge tube, the whirlpool under the revolving speed of 1500rpm-2500rpm
Rotation mixing 3min-10min, then the high speed centrifugation 4min-6min under the revolving speed of 10000rpm-15000rpm；
Supernatant after pipetting a certain amount of centrifugation is transferred to and is dried with nitrogen on device to blow supernatant into centrifuge tube
It is dry；
It pipettes in the centrifuge tube that a certain amount of redissolution liquid is dried up to supernatant, the whirlpool under the revolving speed of 1500rpm-2500rpm
Rotation mixing 1min-3min, then the high speed centrifugation 4min-6min under the revolving speed of 10000rpm-15000rpm, obtain sample to be tested.
Preferably, the amount of pipetting of the internal standard working solution is 10 μ L.
Preferably, the protein precipitation reagent is ethyl alcohol, and the dosage of ethyl alcohol is 100 μ L-500 μ L.
Preferably, the extraction agent is n-hexane, and the dosage of n-hexane is 800 μ L-1200 μ L.
Preferably, the liquid that redissolves is the acetonitrile for being 0.05%-0.25% formic acid containing volume ratio, or, being containing volume ratio
The methanol of 0.05%-0.25% formic acid, the dosage for redissolving liquid are 50 μ L-200 μ L.
Preferably, processing blood to be detected obtains blood sample, comprising: take a certain amount of blood to be detected, through from
The heart takes supernatant using as blood sample.
Preferably, the dosage of the blood to be detected is no less than 5mL；
Centrifugal speed is 3500rpm；
Centrifugation duration is 10min；
The preservation condition of the blood sample is to be placed at -20 DEG C to save until spare before analysis.
Preferably, it is described detect N number of standard solution respectively before, further comprise:
Internal standard working solution described in 10 μ L standard working solutions, 10 μ L and 80 μ L acetonitriles are pipetted into centrifuge tube using pipettor,
Wherein, farnoquinone known to concentration is contained in standard working solution；
The centrifuge tube is vortexed after mixing 1min-2min under the revolving speed of 1500rpm-2500rpm, pipettes supernatant to obtain
To standard solution.
Preferably, it is described obtain standard solution before, further comprise:
The standard items for accurately weighing the farnoquinone of 10mg, are placed in 10mL volumetric flask, are dissolved and determined with methanol
Hold, to obtain standard reserving solution, and saved at -80 DEG C, validity period is 6 months；
A certain amount of standard reserving solution is taken to be placed in the volumetric flask of certain volume, it is water-soluble with the methanol of 85%-95%
Liquid is diluted and constant volume, to obtain the standard working solution, and in the standard working solution farnoquinone concentration between
It between 0.5ng/mL-100ng/mL, and saves, is valid for three months at -80 DEG C；
The internal standard compound of 2mg is taken, which is the Isotopic Internal Standard object of farnoquinone, is placed in 10mL volumetric flask, uses first
Alcohol dissolve and constant volume, to obtain internal standard stock solution, and saves at -80 DEG C, and validity period is 1 year；
It takes a certain amount of internal standard stock solution to be placed in the volumetric flask of certain volume, is carried out with 90% methanol aqueous solution
Simultaneously constant volume is diluted, to obtain the internal standard working solution, and is saved at -80 DEG C, validity period is 1 year.
Preferably, internal standard compound is the Isotopic Internal Standard object of farnoquinone.
Preferably, in N number of standard solution the concentration of farnoquinone include: 0.5ng/mL, 1ng/mL, 2ng/mL,
At least three in 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL.
The present invention provides a kind of methods of farnoquinone content in detection blood, this method comprises: utilizing efficient liquid phase
Tandem mass spectrometer detects at least three standard solution, obtains the chromatogram of each standard solution, and the standard solution is containing known to concentration
Farnoquinone and internal standard compound；With the concentration proportion of farnoquinone in standard solution and internal standard compound for a two-dimensional coordinate, with the mark
In the chromatogram of quasi- solution, the two peak area ratio is another two-dimensional coordinate, and fitting obtains calibration curve equation；It handles to be detected
Blood obtains blood sample, and internal standard compound known to concentration is added, through sample pre-treatments to obtain sample to be tested；In same detection
Under the conditions of detection sample to be tested obtain chromatogram；The peak area ratio of farnoquinone and internal standard compound in the chromatogram is updated to mark
In directrix curve equation, and according to the concentration of added internal standard compound, the content of farnoquinone in blood to be detected is calculated.The present invention
The farnoquinone content in blood can accurately be detected.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the flow chart of the method for farnoquinone content in a kind of detection blood of one embodiment of the invention offer；
Fig. 2 is the chemical structural formula for the farnoquinone (MK4) that one embodiment of the invention provides；
Fig. 3 is the chromatogram of farnoquinone (MK4) in a standard solution of one embodiment of the invention offer；
Fig. 4 is the chromatogram of internal standard compound in a standard solution of one embodiment of the invention offer；
Fig. 5 is the chromatogram of farnoquinone (MK4) in a sample to be tested of one embodiment of the invention offer；
Fig. 6 is the chromatogram of internal standard compound in a sample to be tested of one embodiment of the invention offer.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments, based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
Embodiment one
As shown in Figure 1, may include the embodiment of the invention provides a kind of method of farnoquinone content in detection blood
Following steps:
Step 101: N number of standard solution is detected respectively under certain testing conditions using efficient liquid phase tandem mass spectrometer,
Obtain the chromatogram of each standard solution, wherein contained farnoquinone known to concentration and concentration in any standard solution
The internal standard compound known, the concentration of farnoquinone is different in various criterion solution, and N >=3.
Step 102: in standard solution, the ratio of the concentration of the concentration and internal standard compound of farnoquinone is a two-dimensional coordinate,
And in the chromatogram of the standard solution, the ratio of the peak area of the peak area and internal standard compound of farnoquinone is that another two dimension is sat
Mark, fitting obtain calibration curve equation, wherein the calibration curve equation are as follows: y=k × x+b, wherein y characterizes peak area ratio
Value, x characterize concentration proportion, and k and b characterize coefficient.
Step 103: handling blood to be detected and obtain blood sample.
Step 104: by a certain amount of internal standard working solution containing internal standard compound known to concentration, being added to the blood sample
In, and sample pre-treatments are carried out to obtain sample to be tested.
Step 105: detecting the sample to be tested under the conditions of same detection using efficient liquid phase tandem mass spectrometer, obtain
The chromatogram of the sample to be tested.
Step 106: by the chromatogram of the sample to be tested, the ratio of the peak area of the peak area and internal standard compound of farnoquinone
Value, is updated in the calibration curve equation, and according to the concentration of added internal standard compound, calculate in the blood to be detected
The content of farnoquinone.
Preferably, internal standard compound is the Isotopic Internal Standard object of farnoquinone.
In detail, step 101 defines the detection of standard solution, and step 102 defines the acquisition of calibration curve equation, step
Rapid 103 define the processing of blood to be detected, and step 104 defines the acquisition of sample to be tested, and step 105 defines sample to be tested
Detection, step 106 defines the calculating of farnoquinone content in blood to be detected, to complete to contain farnoquinone in blood
The detection of amount.
In detail, coefficient is characterized since y=k × x+b can be deformed into x=y/k-b/k, 1/k and-b/k, therefore step 102
In, it both can be ordinate and using concentration proportion as abscissa using peak area ratio, it can also be using peak area ratio as abscissa
And using concentration proportion as ordinate.
In detail, since the pretreatment process of sample can be related to sample blending, therefore farnoquinone in blood to be detected
The content of farnoquinone is consistent in content, with blood sample and sample to be tested.
In detail, farnoquinone here can be farnoquinone (MK4), farnoquinone (MK7), farnoquinone (MK9).
Referring to FIG. 2, Fig. 2 shows the chemical structural formulas of farnoquinone (MK4).
In the embodiment of the present invention, standard solution is detected using efficient liquid phase tandem mass spectrometer, is obtained with fitting
Represent the calibration curve equation of farnoquinone, then take blood sample to be detected, the blood sample to be detected after pre-treatment,
Sample to be measured is detected using identical efficient liquid phase tandem mass spectrometer, obtains blood y value to be measured, by blood sample to be measured
The y of product is substituted into above-mentioned standard curvilinear equation, and farnoquinone (MK4) content and internal standard in blood sample to be measured is obtained by calculation
The ratio x of object concentration, since internal standard compound concentration is known, therefore by calculating the vitamin in available blood sample to be measured
The concentration of K2.
As it can be seen that in this detection blood provided in an embodiment of the present invention farnoquinone content method, be by internal standard method with
High performance liquid chromatography Mass Spectrometry combines, and greatly reduces disturbing factor, and high specificity, high sensitivity, testing result
It is more accurate, while analysis time shortens.
In detail, correspond to above-mentioned steps 101, before examination criteria solution, it is necessary first to configure each standard solution.
Further, before configuring each standard solution, configuration standard stock solution and each standard working solution, internal standard stock solution are also needed
With internal standard working solution.
In detail, for the configuration of standard solution, at least may exist following embodiments two.
Embodiment two
The embodiment of the present invention is basically the same as the first embodiment, and something in common repeats no more, the difference is that: in the step
Before rapid 101, further comprise:
Step A1: using pipettor pipette internal standard working solution described in 10 μ L standard working solutions, 10 μ L and 80 μ L acetonitriles to from
In heart pipe, wherein contain farnoquinone known to concentration in standard working solution；
Step A2: the centrifuge tube is vortexed after mixing 1min-2min under the revolving speed of 1500rpm-2500rpm, is pipetted
Clear liquid is to obtain standard solution.For example, the value of revolving speed can be 1500,1700,1900,2100,2300 or 2500；Whirlpool
The value for revolving the time can be 1,1.2,1.4,1.6,1.8 or 2.
In detail, for the configuration of stock solution and working solution, at least may exist following embodiments three.
Embodiment three
The embodiment of the present invention and embodiment two are essentially identical, and something in common repeats no more, the difference is that: in the step
Before rapid A1, further comprise:
Step B1: the standard items of the farnoquinone of 10mg are accurately weighed, is placed in 10mL volumetric flask, is carried out with methanol molten
Simultaneously constant volume is solved, to obtain standard reserving solution, and is saved at -80 DEG C, validity period is 6 months；
Step B2: taking a certain amount of standard reserving solution to be placed in the volumetric flask of certain volume, with the first of 85%-95%
Alcohol solution is diluted and constant volume, to obtain the standard working solution, and in the standard working solution farnoquinone concentration
It saves, is valid for three months between 0.5ng/mL-100ng/mL, and at -80 DEG C；
Step B3: taking the internal standard compound of 2mg, which is the Isotopic Internal Standard object of farnoquinone, is placed in 10mL capacity
Bottle dissolve simultaneously constant volume with methanol, to obtain internal standard stock solution, and saves at -80 DEG C, and validity period is 1 year；
Step B4: a certain amount of internal standard stock solution is taken to be placed in the volumetric flask of certain volume, with 90% methanol-water
Solution is diluted and constant volume, to obtain the internal standard working solution, and saves at -80 DEG C, and validity period is 1 year.
When due to one calibration curve equation of fitting, three coordinate points is at least needed just to can guarantee that most basic fitting is accurate
Degree, therefore a series of standard working solution of different farnoquinone concentration should be obtained, and then it is dense that a series of different farnoquinones can be obtained
The standard solution of degree.Based on this, at least may exist following example IVs.
Example IV
The embodiment of the present invention is basically the same as the first embodiment, and something in common repeats no more, the difference is that: it is described N number of
In standard solution the concentration of farnoquinone include: 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL,
At least three in 50ng/mL, 100ng/mL.
In detail, the existing detection to farnoquinone absorbs or joins with multi-wavelength after high performance liquid chromatography separation can be used
The detection method that conjunction absorbs or fluorescent absorption combines, but easily there is poor specificity in this method, sample treatment complexity leads to sample
The variation of vitamin content, analysis time are long during present treatment, method poor specificity, the problem of entire detection process time length.
And farnoquinone detection method provided in an embodiment of the present invention, it is to be examined using efficient liquid phase tandem mass spectrometry
It surveys, can solve the above problems.It is thus desirable to certain testing conditions be controlled, to reach good detection effect.It, can based on this
There are following embodiments five.
Embodiment five
The embodiment of the present invention is basically the same as the first embodiment, and something in common repeats no more, the difference is that: the detection
Condition includes: liquid-phase condition and tandem mass spectrum condition；
Wherein, the liquid-phase condition include: the length of chromatographic column be 50mm, internal diameter 3.0mm, packing material size are 2.7 μm,
Mobile phase is containing the pure water that volume ratio is 0.05%-0.25% formic acid and containing volume ratio be 0.05%-0.25% formic acid methanol,
Analysis time is 7.5min, and column temperature is 27 DEG C -33 DEG C, and sample volume is 10 μ L-30 μ L, flow velocity 0.55mL/min-0.65mL/
min；
Wherein, the tandem mass spectrum condition includes: using atmosphere pressure chemical ion source APCI (Advanced
Configuration and Power Interface, advanced configuration and power interface), cation scan pattern, selection reaction
Monitoring pattern, dry temperature degree are 300 DEG C, and dry gas stream speed is 6L/min, and atomization gas pressure is 20psi, and capillary voltage is
4500V。
For example, for this value range of 0.05%-0.25%, specific value can for 0.05,0.1,0.15,
0.2 or 0.25.In addition, the value of column temperature can be 27,28,29,30,31,32 or 33；The value of sample volume can for 10,15,
20,25 or 30；The value of flow velocity can be 0.55,0.57,0.59,0.61,0.63 or 0.65.
On the optimized integration of above-described embodiment five, at least there may be following embodiments six to embodiment eight or its
The combination of middle one or more embodiment.
Embodiment six
The embodiment of the present invention and embodiment five are essentially identical, and something in common repeats no more, the difference is that: the liquid phase
Condition further include: 120 EC-C18 chromatographic column of Poroshell.
Embodiment seven
The embodiment of the present invention and embodiment five are essentially identical, and something in common repeats no more, the difference is that: the liquid phase
Condition includes: that column temperature is 30 DEG C.
Embodiment eight
The embodiment of the present invention and embodiment five are essentially identical, and something in common repeats no more, the difference is that: the liquid phase
Condition includes: the methanol of mobile phase to be containing the pure water that volume ratio is 0.2% formic acid and containing volume ratio be 0.2% formic acid, and flow velocity is
0.6mL/min；
Elution process are as follows: the percentage amount of the methanol containing formic acid and the pure water containing formic acid ratio, in washing for 4.1min-6.0min
It is 99-100:1-0 in de- time range, is 90-98:10-2 within the scope of other elution times.
As it can be seen that this type of elution is gradient elution, and elution process can refer to following table 1.
Table 1
| Time/min | Methanol (containing 0.2% formic acid)/% | Water (containing 0.2% formic acid)/% |
| 0 | 90-98 | 10-2 |
| 4.0 | 90-98 | 10-2 |
| 4.1 | 99-100 | 1-0 |
| 6 | 99-100 | 1-0 |
| 6.1 | 90-98 | 10-2 |
| 7.5 | 90-98 | 10-2 |
For example, for this value range of 90-98, specific value can be 90,92,94,96 or 98；For 99-
100 this value range, specific value can be 99,99.2,99.4,99.6,99.8 or 100.
In detail, corresponding to above-mentioned steps 103, for how to handle blood to be detected, at least may exist following implementations
Example nine.
Embodiment nine
The embodiment of the present invention is basically the same as the first embodiment, and something in common repeats no more, the difference is that: the step
103 include: to take a certain amount of blood to be detected, is centrifuged, and takes supernatant using as blood sample.
Under normal conditions, this supernatant can be serum or blood plasma.
On the optimized integration of above-described embodiment nine, at least may exist following embodiments ten.
Embodiment ten
The embodiment of the present invention and embodiment nine are essentially identical, and something in common repeats no more, the difference is that: it is described to be checked
The dosage for surveying blood is no less than 5mL (for example can be 6mL, 8mL or 10mL)；
Centrifugal speed is 3500rpm；
Centrifugation duration is 10min；
The preservation condition of the blood sample is to be placed at -20 DEG C to save until spare before analysis.
In detail, corresponding to above-mentioned steps 104, for how to obtain sample to be tested, at least may exist following embodiments
11.
Embodiment 11
The embodiment of the present invention is basically the same as the first embodiment, and something in common repeats no more, the difference is that: the step
104, comprising:
A certain amount of internal standard working solution containing internal standard compound known to concentration is pipetted with liquid-transfering gun, is placed in centrifuge tube, then
50 μ L-300 μ L (such as 50 μ L, 100 μ L, 150 μ L, 200 μ L, 250 μ L or 300 μ L) described blood sample is added, adds one
Quantitative protein precipitation reagent, under the revolving speed of 1500rpm-2500rpm vortex mixed 2min-5min (such as 2min, 3min,
4min or 5min)；
A certain amount of extraction agent is added after vortex mixed in centrifuge tube, the whirlpool under the revolving speed of 1500rpm-2500rpm
Rotation mixing 3min-10min (such as 3min, 5min, 7min or 10min), then the high speed under the revolving speed of 10000rpm-15000rpm
It is centrifuged 4min-6min (such as 4min, 5min or 6min)；
Supernatant after pipetting a certain amount of centrifugation is transferred to and is dried with nitrogen on device to blow supernatant into centrifuge tube
It is dry；
It pipettes in the centrifuge tube that a certain amount of redissolution liquid is dried up to supernatant, the whirlpool under the revolving speed of 1500rpm-2500rpm
Rotation mixing 1min-3min (such as 1min, 2min or 3min), then the high speed centrifugation under the revolving speed of 10000rpm-15000rpm
4min-6min (such as 4min, 5min or 6min), obtains sample to be tested.
For example, for this value range of 1500rpm-2500rpm, specific value can for 1500,1700,
2000,2300 or 2500；For this value range of 10000rpm-15000rpm, specific value can for 10000,11000,
12000,13000,14000 or 15000.
In the embodiment of the present invention, by simplifying pre-treatment step, shorten sample processing time.
On the optimized integration of above-described embodiment 11, at least there may be following embodiments 12 to embodiment ten
Five, or in which the combination of one or more embodiments.
Embodiment 12
The embodiment of the present invention is identical as embodiment hendecyl sheet, and something in common repeats no more, the difference is that: in described
The amount of pipetting for marking working solution is 10 μ L.
Embodiment 13
The embodiment of the present invention is identical as embodiment hendecyl sheet, and something in common repeats no more, the difference is that: it is described heavy
Shallow lake protein reagent is ethyl alcohol, and the dosage of ethyl alcohol is 100 μ L-500 μ L.For example, this dosage value can for 100,200,300,
400 or 500.
Embodiment 14
The embodiment of the present invention is identical as embodiment hendecyl sheet, and something in common repeats no more, the difference is that: the extraction
Taking reagent is n-hexane, and the dosage of n-hexane is 800 μ L-1200 μ L.For example, this dosage value can for 800,900,
1000,1100 or 1200.
Embodiment 15
The embodiment of the present invention is identical as embodiment hendecyl sheet, and something in common repeats no more, the difference is that: it is described multiple
Solution is the acetonitrile for being 0.05%-0.25% formic acid containing volume ratio, or, be the methanol of 0.05%-0.25% formic acid containing volume ratio,
The dosage for redissolving liquid is 50 μ L-200 μ L.
For example, for this value range of 0.05%-0.25%, specific value can for 0.05,0.1,0.15,
0.2 or 0.25.In addition, the dosage value for redissolving liquid can be 50,100,150 or 200.
One to embodiment 15 based on the above embodiments, at least may exist following embodiments 16.
Embodiment 16
The embodiment of the invention provides a kind of methods of farnoquinone content in detection blood, may comprise steps of.
(1) calibration of standard solution
First with pipettor respectively by 10 μ L, eight kinds of various concentration standard working solutions, 10 μ L internal standard working solutions and 80 μ L acetonitriles
It is placed in centrifuge tube, to obtain eight standard solution.Eight standard solution are vortexed in the case where revolving speed is 1500rpm respectively and are mixed
After 1min, supernatant 80uL is pipetted respectively, supernatant is detected using efficient liquid phase tandem mass spectrometer, obtains eight standards
The chromatogram of farnoquinone (MK4) and internal standard compound of solution.Farnoquinone is respectively obtained from the chromatogram of each standard solution
(MK4) chromatographic peak area and internal standard compound chromatographic peak area, respectively with farnoquinone (MK4) chromatographic peak of above-mentioned eight various concentrations
Ordinate y of the ratio of area and internal standard compound chromatographic peak area as calibration curve equation, in above-mentioned eight kinds of standard working solutions
Farnoquinone (MK4) concentration and the internal standard compound concentration in internal standard working solution abscissa x of the ratio as calibration curve equation,
Resulting eight groups of data will be detected above and carry out linear regression, and it is y=a × x+b that fitting, which obtains calibration curve equation, and is obtained
Coefficient a, b.
Above-mentioned standard working solution is the solution containing farnoquinone (MK4), and above-mentioned internal standard working solution is to contain farnoquinone
(MK4) solution of Isotopic Internal Standard.
(a) preparation of standard reserving solution A:
Accurately weigh farnoquinone (MK4) standard items 10mg and be placed in 10mL volumetric flask, dissolved with methanol, and constant volume in
10mL obtains standard reserving solution A, and farnoquinone (MK4) concentration is 1000 μ g/mL, and is saved under the conditions of -80 DEG C, effectively
Phase is 6 months.
(b) preparation of standard working solution A
Appropriate step (a) Plays stock solution A is taken, is diluted, is obtained containing 0.5ng/ with 90% methanol aqueous solution
The standard of the farnoquinone (MK4) of mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL
Working solution A, and saved under the conditions of -80 DEG C, it is valid for three months.
(c) preparation of internal standard stock solution
Take farnoquinone (MK4)-d7 standard items 2mg to be placed in 10mL volumetric flask, dissolved with methanol, and constant volume in
10mL obtains standard reserving solution B, and saves under the conditions of -80 DEG C, and validity period is 1 year.
(d) preparation of internal standard working solution
Internal standard stock solution B in appropriate step (c) is taken, is diluted, is obtained containing 10ng/mL with 90% methanol aqueous solution
Farnoquinone (MK4)-d7 internal standard working solution C, and saved under the conditions of -80 DEG C, validity period is 1 year.
(2) 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, supernatant is taken to obtain serum, blood
It is spare to before analyzing that it is placed in the lower preservation of -20 DEG C of freezings clearly.
(3) sample to be tested is handled
(e) then 300 μ L step is added in 2mL centrifuge tube in the internal standard working solution for pipetting 10 μ L steps (d) with liquid-transfering gun
Suddenly serum described in (two) adds 400 μ L dehydrated alcohols, under the revolving speed of 1500rpm after vortex mixed 3min, is added 1100
μ L n-hexane, under the revolving speed of 1500rpm after vortex mixed 5min, then the high speed centrifugation 5min under the revolving speed of 12000rpm, it moves
It takes the centrifuged supernatant of 1000 μ L into clean 1.5mL centrifuge tube, the 1.5mL centrifuge tube for filling supernatant is transferred to nitrogen
On air-blowing equipment for drying, supernatant is dried up, pipettes the 1.5mL that 100 μ L are dried up containing the acetonitrile that volume ratio is 0.2% formic acid to supernatant
In centrifuge tube, under the revolving speed of 1500rpm after vortex mixed 1min, then the high speed centrifugation 5min under the revolving speed of 12000rpm, it moves
Taking 80 μ L is sample to be tested.
(4) detection of sample to be tested
Sample to be tested in above-mentioned steps (e) is detected using efficient liquid phase tandem mass spectrometer, at the same obtain it is above-mentioned to
The chromatogram of farnoquinone (MK4) and internal standard compound of sample.Available farnoquinone (MK4) chromatographic peak from the chromatogram
Area and internal standard compound chromatographic peak area substitute into the ratio y of farnoquinone (MK4) chromatographic peak area and internal standard compound chromatographic peak area
In calibration curve equation y=a × x+b of above-mentioned steps (one), be obtained by calculation farnoquinone in sample to be tested (MK4) with
The relative concentration x of internal standard compound.Concentration of the internal standard compound in internal standard working solution be it is known, the sample to be tested is thus calculated
The concentration of farnoquinone (MK4).Certainly, the concentration of the farnoquinone (MK4) of sample to be tested, the vitamin of blood as to be detected
The concentration of K2 (MK4).
Liquid-phase condition are as follows: chromatographic column: the 120 EC-C18 chromatographic column (50*3.0mm of Poroshell of agilent company
2.7um)；Gradient elution: mobile phase be the pure water of 0.2% formic acid containing volume ratio, and be the first of 0.2% formic acid containing volume ratio
Alcohol；Analysis time: 7.5min；Column temperature: 30 DEG C；Sample volume: 20ul；Flow velocity: 0.6mL/min.
Tandem mass spectrum condition are as follows: use atmosphere pressure chemical ion source APCI, cation scan pattern, Selective reaction monitoring mould
Formula, dry 300 DEG C of temperature degree, dry gas stream speed 6L/min, atomization gas pressure 20psi, capillary voltage 4500V.
In addition, tandem mass spectrum condition further includes content shown in following table 2.
Table 2
| Substance title | Farnoquinone (MK4) | Farnoquinone (MK4) 4-d7 |
| Parent ion | 445.3 | 452.3 |
| Daughter ion | 187 | 194 |
| Retention time (min) | 3.5 | 3.4 |
| Dwell | 200 | 200 |
| Fragmentor | 150 | 150 |
| CE | 22 | 22 |
| CAV | 5 | 5 |
Technical method demonstration is as follows in the embodiment of the present invention:
One, the linear relationship and quantitative limit of the embodiment of the present invention
By farnoquinone (MK4) standard working solution of the 10 each concentration of μ l, it is separately added into 10 μ l internal standard working solutions and 80 μ l
Acetonitrile mixes sample introduction, is measured from low to high by determination condition of embodiment of the present invention concentration, with quantitative ion chromatography peak face
The mapping of product-concentration, obtains standard curve.The result shows that the range of linearity and quantitative limit of farnoquinone (MK4) are as follows:
(1) detection limit (LOD): 0.017ng/mL.
(2) quantitative limit (LOQ): 0.05ng/mL.
(3) range of linearity: farnoquinone (MK4) is linear good in 0.05ng/mL to 10ng/mL range, related coefficient
R2 ﹥ 0.9900.
Two, the rate of recovery and precision of the embodiment of the present invention
It takes farnoquinone (MK4) standard working solution to be configured to high, medium and low 3 kinds of concentration respectively and carries out sample recovery rate and essence
Density experiment is measured by present invention method, replicate analysis measure 3 batches, the rate of recovery of farnoquinone (MK4) and
Precision is respectively such as following Table 3.Average recovery rate of the farnoquinone (MK4) within the scope of 3 basic, normal, high pitch-based spheres be
95.5%~99.1%, relative standard deviation is 1.5%~3.1%.
Table 3
| Scalar quantity | 0.3ng/mL | 1.0ng/mL | 5.0ng/mL |
| Average recovery rate | 99.1% | 95.3% | 95.5% |
| Precision RSD | 3.0% | 1.5% | 3.1% |
In summary verification test is it is found that every technology such as the rate of recovery, detection limit and precision of the embodiment of the present invention refers to
Mark meets the requirements, and can detect the content of farnoquinone (MK4) in blood, and reproducibility is good, and sample recovery rate is good, thus
The accuracy of testing result is improved, systematic error is eliminated.
In addition, in embodiments of the present invention, the chromatogram of farnoquinone (MK4) can be as shown in Figure 3 in standard solution；Mark
The chromatogram of farnoquinone (MK4) Isotopic Internal Standard can be as shown in Figure 4 in quasi- solution；Farnoquinone (MK4) in serum sample
Chromatogram can be as shown in Figure 5；The chromatogram of farnoquinone (MK4) Isotopic Internal Standard can be as shown in Figure 6 in serum sample.
By Fig. 3-6 it is found that the retention time of farnoquinone (MK4) is consistent with its standard working solution in serum sample.This method is with same
The plain marker in position is internal standard compound, so that the identification of target compound is more accurate, analysis time is short, interference is small, and interior scalar quantity is fitted
Suitable high specificity, accuracy and high sensitivity.
In conclusion the embodiment of the present invention have it is at least following the utility model has the advantages that
1, in the embodiment of the present invention, internal standard method is combined with high performance liquid chromatography Mass Spectrometry, keeps disturbing factor big
It is big to reduce, and high specificity, high sensitivity, testing result are more accurate, while analysis time shortens.
2, in the embodiment of the present invention, using isotopic label as internal standard compound, so that the identification of target compound is more accurate,
Analysis time is short, interference is small, and interior scalar quantity appropriate specificity is strong, accuracy and high sensitivity.
It should be noted that, in this document, such as first and second etc relational terms are used merely to an entity
Or operation is distinguished with another entity or operation, is existed without necessarily requiring or implying between these entities or operation
Any actual relationship or order.Moreover, the terms "include", "comprise" or its any other variant be intended to it is non-
It is exclusive to include, so that the process, method, article or equipment for including a series of elements not only includes those elements,
It but also including other elements that are not explicitly listed, or further include solid by this process, method, article or equipment
Some elements.In the absence of more restrictions, the element limited by sentence " including a 〃 〃 ", it is not excluded that
There is also other identical factors in the process, method, article or apparatus that includes the element.
Finally, it should be noted that the foregoing is merely presently preferred embodiments of the present invention, it is merely to illustrate skill of the invention
Art scheme, is not intended to limit the scope of the present invention.Any modification for being made all within the spirits and principles of the present invention,
Equivalent replacement, improvement etc., are included within the scope of protection of the present invention.
Claims (10)
1. a kind of method of farnoquinone content in detection blood characterized by comprising
Using efficient liquid phase tandem mass spectrometer, under certain testing conditions, N number of standard solution is detected respectively, obtains each standard
The chromatogram of solution, wherein containing internal standard compound known to farnoquinone known to concentration and concentration in any standard solution, no
It is different with the concentration of farnoquinone in standard solution, and N >=3；
With in standard solution, the ratio of the concentration of the concentration and internal standard compound of farnoquinone is a two-dimensional coordinate, and with the standard
In the chromatogram of solution, the ratio of the peak area of the peak area and internal standard compound of farnoquinone is another two-dimensional coordinate, and fitting obtains
Calibration curve equation；
It handles blood to be detected and obtains blood sample；
By a certain amount of internal standard working solution containing internal standard compound known to concentration, it is added in the blood sample, and carry out sample
Product pre-treatment is to obtain sample to be tested；
Using efficient liquid phase tandem mass spectrometer, under the conditions of same detection, the sample to be tested is detected, obtains the sample to be tested
Chromatogram；
By in the chromatogram of the sample to be tested, the ratio of the peak area of the peak area and internal standard compound of farnoquinone is updated to institute
It states in calibration curve equation, and according to the concentration of added internal standard compound, calculates farnoquinone in the blood to be detected
Content；
Wherein, the calibration curve equation are as follows: y=k × x+b, wherein y characterizes peak area ratio, and x characterizes concentration proportion, k and b
Characterize coefficient.
2. the method according to claim 1, wherein
The testing conditions include: liquid-phase condition and tandem mass spectrum condition；
Wherein, the liquid-phase condition include: the length of chromatographic column be 50mm, internal diameter 3.0mm, packing material size are 2.7 μm, flowing
Xiang Weihan volume ratio be the pure water of 0.05%-0.25% formic acid and containing volume ratio be 0.05%-0.25% formic acid methanol, analysis
Time is 7.5min, and column temperature is 27 DEG C -33 DEG C, and sample volume is 10 μ L-30 μ L, flow velocity 0.55mL/min-0.65mL/min；
Wherein, the tandem mass spectrum condition includes: using atmosphere pressure chemical ion source APCI, cation scan pattern, and selection is anti-
Monitoring pattern is answered, dry temperature degree is 300 DEG C, and dry gas stream speed is 6L/min, and atomization gas pressure is 20psi, capillary voltage
For 4500V.
3. according to the method described in claim 2, it is characterized in that,
The liquid-phase condition further include: 120 EC-C18 chromatographic column of Poroshell；
And/or
The liquid-phase condition includes: that column temperature is 30 DEG C；
And/or
It is containing the pure water that volume ratio is 0.2% formic acid and containing volume ratio is 0.2% formic acid that the liquid-phase condition, which includes: mobile phase,
Methanol, flow velocity 0.6mL/min；
Elution process are as follows: the percentage amount of the methanol containing formic acid and the pure water containing formic acid ratio, in the elution of 4.1min-6.0min
Between be 99-100:1-0 in range, be 90-98:10-2 within the scope of other elution times.
4. the method according to claim 1, wherein
The internal standard working solution by a certain amount of containing internal standard compound known to concentration, is added in the blood sample, goes forward side by side
Row sample pre-treatments are to obtain sample to be tested, comprising:
A certain amount of internal standard working solution containing internal standard compound known to concentration is pipetted with liquid-transfering gun, is placed in centrifuge tube, adds
Blood sample described in 50 μ L-300 μ L, adds a certain amount of protein precipitation reagent, the whirlpool under the revolving speed of 1500rpm-2500rpm
Rotation mixing 2min-5min；
A certain amount of extraction agent is added after vortex mixed in centrifuge tube, is vortexed under the revolving speed of 1500rpm-2500rpm mixed
Close 3min-10min, then the high speed centrifugation 4min-6min under the revolving speed of 10000rpm-15000rpm；
Supernatant after pipetting a certain amount of centrifugation is transferred to and is dried with nitrogen on device to dry up supernatant into centrifuge tube；
It pipettes in the centrifuge tube that a certain amount of redissolution liquid is dried up to supernatant, is vortexed under the revolving speed of 1500rpm-2500rpm mixed
1min-3min, then the high speed centrifugation 4min-6min under the revolving speed of 10000rpm-15000rpm are closed, sample to be tested is obtained.
5. according to the method described in claim 4, it is characterized in that,
The amount of pipetting of the internal standard working solution is 10 μ L；
And/or
The protein precipitation reagent is ethyl alcohol, and the dosage of ethyl alcohol is 100 μ L-500 μ L；
And/or
The extraction agent is n-hexane, and the dosage of n-hexane is 800 μ L-1200 μ L；
And/or
The redissolution liquid, which is containing volume ratio, is the acetonitrile of 0.05%-0.25% formic acid, or, containing volume ratio is 0.05%-0.25%
The methanol of formic acid, the dosage for redissolving liquid are 50 μ L-200 μ L.
6. the method according to claim 1, wherein
The processing blood to be detected obtains blood sample, comprising: takes a certain amount of blood to be detected, is centrifuged, takes supernatant
Using as blood sample.
7. according to the method described in claim 6, it is characterized in that,
The dosage of the blood to be detected is no less than 5mL；
Centrifugal speed is 3500rpm；
Centrifugation duration is 10min；
The preservation condition of the blood sample is to be placed at -20 DEG C to save until spare before analysis.
8. the method according to claim 1, wherein
It is described detect N number of standard solution respectively before, further comprise:
Internal standard working solution described in 10 μ L standard working solutions, 10 μ L and 80 μ L acetonitriles are pipetted into centrifuge tube using pipettor, wherein
Contain farnoquinone known to concentration in standard working solution；
The centrifuge tube is vortexed after mixing 1min-2min under the revolving speed of 1500rpm-2500rpm, pipettes supernatant to be marked
Quasi- solution.
9. according to the method described in claim 8, it is characterized in that,
It is described obtain standard solution before, further comprise:
The standard items for accurately weighing the farnoquinone of 10mg, are placed in 10mL volumetric flask, dissolve simultaneously constant volume with methanol, with
Standard reserving solution is obtained, and is saved at -80 DEG C, validity period is 6 months；
Take a certain amount of standard reserving solution to be placed in the volumetric flask of certain volume, with the methanol aqueous solution of 85%-95% into
Row dilution and constant volume, to obtain the standard working solution, and in the standard working solution farnoquinone concentration between 0.5ng/
It between mL-100ng/mL, and saves, is valid for three months at -80 DEG C；
Take the internal standard compound of 2mg, the internal standard compound be farnoquinone Isotopic Internal Standard object, be placed in 10mL volumetric flask, with methanol into
Row dissolution and constant volume, to obtain internal standard stock solution, and save at -80 DEG C, and validity period is 1 year；
It takes a certain amount of internal standard stock solution to be placed in the volumetric flask of certain volume, is diluted with 90% methanol aqueous solution
And constant volume, it to obtain the internal standard working solution, and is saved at -80 DEG C, validity period is 1 year.
10. according to claim 1 to any method in 9, which is characterized in that
Internal standard compound is the Isotopic Internal Standard object of farnoquinone；
And/or
The concentration of farnoquinone includes: 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/ in N number of standard solution
At least three in mL, 20ng/mL, 50ng/mL, 100ng/mL.
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