CN110146626A - A kind of method of folic acid in high performance liquid chromatography string mass Spectrometry for Determination blood cake - Google Patents
A kind of method of folic acid in high performance liquid chromatography string mass Spectrometry for Determination blood cake Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The present invention provides a kind of methods of folic acid in high performance liquid chromatography string mass Spectrometry for Determination blood cake.First folic acid and 5-methyltetrahydrofolate are separated with high performance liquid chromatography, mass spectrum Isotopic Internal Standard sizing technique is recycled, using the concentration of standard items as X-axis, the peak area of standard items and internal standard compound is that ratio is Y-axis, calibration curve is established, the content of above-mentioned folic acid and 5-methyltetrahydrofolate is calculated.The method of the present invention high sensitivity, high specificity, accurate and pre-treating method are easy, time-consuming short, and time cost is greatly saved.
Description
Technical field
The invention belongs to technical field of analysis and detection, specifically, the present invention relates to a kind of high performance liquid chromatography string mass spectrums
The method that technology used in conjunction measures folic acid in blood cake.
Background technique
Folic acid is also Vitamin B9, is a kind of water soluble vitamin, and one of the complex of vitamin B, is equivalent to butterfly in fact
Acyl glutamic acid.Epidemiology and experimental study show that folic acid deficiency and dysbolism seriously affect human health, are various diseases
The inducement coexisted, as megaloblastic anemia, leukopenia, cardiovascular disease, congenital heart disease and nerve channel are abnormal
Shape etc..In addition, folic acid is even more important to pregnant woman, it is common recognition that pregnant woman, which requires supplementation with folic acid,.Such as lack leaf in pregnancy head 3 months
Acid can lead to fetal neural tube developmental defect, Yi Yinqi Fetal neurotubules malformation.
Folic acid is existed in the form of three kinds in vivo, and 5-methyltetrahydrofolate content is high, and activity is maximum, therefore measures leaf
Sour and 5-methyltetrahydrofolate can prompt clinical meaning and the intervention of testing result by the network analysis to testing result
It adjusts and suggests, further diagnosed to doctor and nutritionist, offer reference is provided.But it since blood spot sample matrix is complicated, needs
It is extracted and is detected.Therefore, high, easy to operate and can be same there is an urgent need in the art to develop new detection sensitivity
When quantitative detection folic acid and 5-methyltetrahydrofolate technology.
Summary of the invention
The problem to be solved by the invention is to provide a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake middle periods
The method of acid.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
A kind of method of folic acid in high performance liquid chromatography string mass Spectrometry for Determination blood cake,
The folic acid is respectively folic acid and 5-methyltetrahydrofolate；
Included the following steps: first using the method for folic acid in high performance liquid chromatography string mass Spectrometry for Determination blood cake with height
Effect liquid phase chromatogram separates folic acid and 5-methyltetrahydrofolate, mass spectrum Isotopic Internal Standard sizing technique is recycled, with the dense of standard items
Spending is X-axis, and it is Y-axis that the peak area of standard items and internal standard compound, which is ratio, establishes calibration curve, calculates above-mentioned folic acid and 5- methyl four
The content of hydrogen folic acid, specific chromatographic condition are as follows:
(1) chromatographic condition
Chromatographic column is Phenomenex2.6μm F5 100A°；Mobile phase A is the water-soluble of 0.1vt% formic acid
Liquid；Mobile phase B is the acetonitrile solution of 0.1vt% formic acid；Column temperature is 40 DEG C；Sample volume is 5 μ L；It is eluted, is seen using gradient mode
Table 1；
1 eluent gradient elution parameters of table
| Time/min | Flow velocity/μ L/min | A/% | B/% |
| 0.01 | 500 | 90 | 10 |
| 4.00 | 500 | 50 | 50 |
| 4.05 | 500 | 10 | 90 |
| 4.85 | 500 | 10 | 90 |
| 4.90 | 500 | 90 | 10 |
| 7.00 | 500 | 90 | 10 |
(2) Mass Spectrometry Conditions
Under electron spray ion positive ion detection mode, using MRM scanning analysis, source parameters: atomization gas flow is
3L/min；Dry gas stream amount is 2L/min；Heating throughput is 15L/min；DL tube temperature degree is 100-400 DEG C；Interface temperature is
100-400℃；Heating deblocking temperature is 100-400 DEG C, CID gas: 100-300kPa；Precursor and product ion channel are as shown in table 2；
2 multiple-reaction monitoring ion pair of table and corresponding voltage parameter
Wherein, the blood cake is prepared as follows to obtain: (1) with punch take 2~8mm of diameter sample in
In 2mLEP pipe, 100~500 μ L working solutions are added, 500~2000rpm, which is vortexed, mixes 10~60min；(2) take supernatant 50~
In 96 hole deep-well plates, 10~100L/min of room temperature is dried with nitrogen 450 μ L；(3) 50~400 μ L of addition redissolution liquid, 500~
2000rpm shakes 5~60min.
Wherein, the working solution is prepared as follows to obtain: by the volume ratio of 1:10:10:1000 by extracting solution
Additive: joint antioxidant: mixing internal standard solution: extracting solution mixes well.
Wherein, the extraction solution additive is ammonium hydroxide, and extracting solution is 70vt% methanol aqueous solution.
Wherein, the joint antioxidant is the mixture of dithiothreitol (DTT), ascorbic acid and citric acid, wherein two
The mass ratio of sulphur threitol, ascorbic acid and citric acid is 1:1:1.
Wherein, the folic acid-containing 10 μ g/mL is designated as in the mixing13The 5-methyltetrahydrofolate-of C5 and 10 μ g/mL
The aqueous solution of d3.
Wherein, the redissolution liquid is that 1%vt combines aqueous antioxidant solution.
Wherein, the standard items are the blood cake containing folic acid and 5-methyltetrahydrofolate, and specific concentration is shown in Table 3,
The corresponding concentration of 3 standard items of table (ng/mL)
| Folic acid | C1 | C2 | C3 |
| Folic acid | 50 | 5 | 0.5 |
| 5-methyltetrahydrofolate | 50 | 5 | 0.5 |
。
Wherein, the standard items are prepared in accordance with the following steps:
(1) it is cleaned rabbit erythrocyte 3 times using physiological saline, it is spare；
(2) human serum albumin solution of configuration 50mg/mL joint antioxidant containing 100 μ g/mL:
2g human serum albumins powder is weighed, the physiological saline 40mL of 100 μ g/mL joint antioxidant is added, after mixing
Being configured to concentration is 50mg/mL human serum albumin solution；
(3) rabbit erythrocyte is sufficiently mixed according to the volume ratio of 55:45 and matrix is made in human serum albumin solution；
(4) 100 μ g/mL of preparation combine aqueous antioxidant solution；
The configuration of stock solution: weighing the folic acid of 50mg and the 5-methyltetrahydrofolate of 50mg respectively, is separately added into 100 μ g/
ML combines aqueous antioxidant solution 1L, is configured to folic acid stock solution and 50 μ g/mL5- methyl tetrahydrofolates that concentration is 50 μ g/mL
Stock solution；
Concentration is the preparation of C1 standard items: taking folic acid stock solution and each 10 μ L of 5-methyltetrahydrofolate stock solution respectively, uses
Diluted matrix is settled to the standard items that 1000 μ L are 500ng/mL to get concentration；Taking concentration is 500ng/mL folic acid and 500ng/
Each 300 μ L of mL 5-methyltetrahydrofolate is settled to the standard items that 3000 μ L are C1 to get concentration with diluted matrix；
Concentration is the preparation of C2 standard items: folic acid that concentration is C1 and 5-methyltetrahydrofolate each 300 μ L are taken, it is dilute with matrix
It releases and is settled to the standard items that 3000 μ L are C2 to get concentration；
Concentration is the preparation of C3 standard items: folic acid that concentration is C2 and 5-methyltetrahydrofolate each 300 μ L are taken, it is dilute with matrix
It releases and is settled to the standard items that 3000 μ L are C3 to get concentration；
(5) take the 66 μ L drop of standard items of each concentration on speciality filter paper, dry collect -20 DEG C be kept in dark place.
The utility model has the advantages that
(1) the method for the present invention high sensitivity, high specificity, accurate；(2) pre-treating method of the present invention is easy, time-consuming short, greatly
Time cost has been saved greatly；(3) subject's sample is simple for production, and blood sampling volume is few, and convenience greatly improves；(4) dried blood spot sample
Storage it is more easy compared with traditional liquid matrix sample, many compounds unstable in liquid matrix are opposite in dry blood cake
Stablize, sample can save amount transport expense without freezing, especially in the clinical research for generating a large amount of clinical samples
Later period, bring economic benefit are more significant；(5) compared with fluid blood, the potential of dry blood cake pathogenic is substantially reduced.
Detailed description of the invention
Fig. 1 is the ion stream chromatogram of the mark product and Isotopic Internal Standard of folic acid and 5-methyltetrahydrofolate.
Fig. 2 is the canonical plotting of 5-methyltetrahydrofolate.
Fig. 3 is the canonical plotting of folic acid.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.
Embodiment 1
1, sample acquires
(1) have an impact because of food to measurement result, subject needs empty stomach sample drawn, and blood cake collector cleans both hands
And wear sterile gloves；
(2) blood sampling drop avoids repeating to bleed, at least on folic acid speciality filter paper, making blood naturally osmotic to the filter paper back side
Acquire 3 blood cakes；
(3) by the hanging horizontal of blood cake, naturally dry to dark brown avoids sunlight and ultraviolet light from irradiating, baking, volatility
Learn the pollution such as substance；
(4) all blood cakes should be treated according to Hematogenic infectious disease sample, be answered special infectious disease specimen, such as AIDS
It packs as expression and individually；
(5) qualified dry blood cake is answered are as follows: at least three blood cake, and each blood cake diameter is greater than 8 millimeters；Drop of blood naturally osmotic, filter
Paper front and back sides blood cake is consistent；Blood cake is pollution-free；Blood cake is without oozing of blood ring；
(6) qualified dry blood cake sample should be immediately placed in hermetic bag after drying, and be sealed in -20 DEG C of refrigerators.
Remarks: it must use folic acid speciality filter paper that dry blood cake is made after blood sampling.
2, material
Methodological study experiment sample from Shanghai can Li Meita biological medicine Science and Technology Ltd. sample.
(1) instrument: mass spectrum (NovaClin LCMS-4500+)；Liquid chromatogram (Shimadzu LC20)；Vortex concussion instrument (Hangzhou
MTV-100 contains in Austria)；Nitrogen evaporator (Agela NV96-G-S)；Glass apparatus etc..
(2) reagent consumptive material: methanol, acetonitrile are purchased from Merck company；Dithiothreitol (DTT), ascorbic acid, citric acid buying are certainly
In Sigma company；Ammonium hydroxide is purchased from Chinese medicines group Hu Shi company；Chromatographic column are as follows: Phenomenex2.6μm F5
100A°50*3.0mm。
(3) standard items: folic acid is purchased from USP, and purity 99%, 5-methyltetrahydrofolate are purchased from USP, and purity is
99%, internal standard folic acid-13C5 and 5-methyltetrahydrofolate-d3 is purchased from TRC, purity > 99%.
(4) quality-control product: the blood cake containing folic acid and 5-methyltetrahydrofolate is divided to high and low two concentration, be respectively QCH,
QCL；Wherein the corresponding concentration of folic acid quality-control product is shown in Table 4 in QCH, QCL,
The corresponding concentration of 4 quality-control product of table (ng/mL)
| Type | QCH | QCL |
| Folic acid | 30 | 6 |
| 5-methyltetrahydrofolate | 30 | 6 |
3, method[1]
(1) chromatographic condition: chromatographic column Phenomenex2.6μm F5 100A°；Mobile phase A is 0.1vt%
Elute the aqueous solution of solution additive；Mobile phase B is the acetonitrile solution that Mobile phase B is 0.1vt% elution solution additive；Column temperature is 40
℃；；Sample volume is 5 μ L；It is eluted using gradient mode, is shown in Table 1,
Wherein, the elution solution additive is formic acid.
(2) Mass Spectrometry Conditions: NovaClin LCMS-4500+, under electron spray ion positive ion detection mode, using MRM
Scanning analysis, source parameters: atomization gas flow is 3L/min；Dry gas stream amount is 2L/min；Heating throughput is 15L/
min；DL tube temperature degree is 100-400 DEG C；Interface temperature is 100-400 DEG C；Heating deblocking temperature is 100-400 DEG C, CID gas: 100-
300kPa；Precursor and product ion channel are as shown in table 2.
(3) standard items configure
A. the configuration of folic acid and 5-methyltetrahydrofolate:
A1 is cleaned rabbit erythrocyte 3 times using physiological saline, spare；
A2 configures the human serum albumin solution of 50mg/mL joint antioxidant containing 100 μ g/mL:
2g human serum albumins powder is weighed, the physiological saline 40mL of 100 μ g/mL joint antioxidant is added, after mixing
Being configured to concentration is 50mg/mL human serum albumin solution；
A3 is sufficiently mixed rabbit erythrocyte according to the volume ratio of 55:45 and matrix is made in human serum albumin solution；
A4 prepares 100 μ g/mL and combines aqueous antioxidant solution；
The configuration of stock solution: weighing the folic acid of 50mg and the 5-methyltetrahydrofolate of 50mg respectively, is separately added into 100 μ g/
ML combines aqueous antioxidant solution 1L, is configured to folic acid stock solution and 50 μ g/mL5- methyl tetrahydrofolates that concentration is 50 μ g/mL
Stock solution；
Concentration is the preparation of C1 standard items: taking folic acid stock solution and each 10 μ L of 5-methyltetrahydrofolate stock solution respectively, uses
Diluted matrix is settled to the standard items that 1000 μ L are 500ng/mL to get concentration；Taking concentration is 500ng/mL folic acid and 500ng/
Each 300 μ L of mL 5-methyltetrahydrofolate is settled to the standard items that 3000 μ L are C1 to get concentration with diluted matrix；
Concentration is the preparation of C2 standard items: folic acid that concentration is C1 and 5-methyltetrahydrofolate each 300 μ L are taken, it is dilute with matrix
It releases and is settled to the standard items that 3000 μ L are C2 to get concentration；
Concentration is the preparation of C3 standard items: folic acid that concentration is C2 and 5-methyltetrahydrofolate each 300 μ L are taken, it is dilute with matrix
It releases and is settled to the standard items that 3000 μ L are C3 to get concentration；
A5 take the 66 μ L drop of standard items of each concentration on speciality filter paper, dry collect -20 DEG C be kept in dark place.
B. target configuration in mixing: preparation contains the folic acid-of 10 μ g/mL13The aqueous solution of C5 and 5-methyltetrahydrofolate-d3.
(4) configuration of quality-control product: take concentration each for the folic acid storage standard items and 5-methyltetrahydrofolate standard items of C1 respectively
3mL is settled to 5mL and 25mL with matrix respectively and obtains the folic acid and 5-methyltetrahydrofolate of 30ng/mL and 6ng/mL；It takes each dense
The 66 μ L drop of quality-control product of degree on speciality filter paper, dry collect -20 DEG C be kept in dark place；
(5) working solution configures: will extract solution additive by the volume ratio of 1:10:10:1000: joint antioxidant: mixing
Internal standard solution: extracting solution mixes well；Wherein extracting solution additive is ammonium hydroxide, and extracting solution is 70vt% methanol aqueous solution, and joint is anti-
Oxidant is the mixture of dithiothreitol (DTT), ascorbic acid and citric acid, wherein dithiothreitol (DTT), ascorbic acid and citric acid
Mass ratio be 1:1:1.
(6) sample treatment
Standard items processing[2][3]: it takes 2~8mm of diameter sample in 2mL EP pipe with punch, 100~500 μ L works is added
Make liquid, 500~2000rpm, which is vortexed, mixes 10~60min；Take 50~450 μ L of supernatant in 96 hole deep-well plates, room temperature
10~100L/min is dried with nitrogen；50~400 μ L are added and redissolve liquid, 500~2000rpm shakes 5~60min；
Detect sample process: consistent with the processing method of standard items, details are not described herein again；
Quality-control product processing: consistent with the processing method of standard items, details are not described herein again.
(7) liquid relief: each hole solution in step (6) each orifice plate is transferred in 96 microwell plate sample introduction plate holes；
(8) sample detection: the standard items, quality-control product and test sample solution handled well is taken to inject high performance liquid chromatography-string
Connection mass spectrograph is detected, and is recorded chromatogram and detected the peak area and its internal standard peak of sample folic acid and 5-methyltetrahydrofolate
Area.
Each component is shown in Table 5 in assay kit.
The preparation of 5 kit forms of table
4, method validation
(1) the ion stream chromatogram of the mark product and Isotopic Internal Standard of folic acid and 5-methyltetrahydrofolate
Fig. 1 is respectively the chromatogram of 5-methyltetrahydrofolate standard items, 5-methyltetrahydrofolate-d3 isotope chromatogram,
The chromatogram of folic acid standard items, folic acid-13C5 Isotopic Internal Standard chromatogram, these four substances can satisfy the detection of LC-MS/MS
Quantitative requirement.
(2) standard curve
Use Isotopic Internal Standard sizing technique using 3 concentration of 2 standard items as abscissa (x), with 2 each concentration of standard items
The ratio of the peak area of sample and its internal standard compound is ordinate (y), draws standard curve, and calculate above-mentioned folic acid and 5- methyl four
The content of hydrogen folic acid.The linear fit equation of folic acid and 5-methyltetrahydrofolate in respective range, linear good, related coefficient
Within 0.999, meet quantitative requirement, is shown in Table 6.
6 folic acid of table and 5-methyltetrahydrofolate equation of linear regression and linearly dependent coefficient
Detect the calculating of sample results: the ratio of the practical peak area and internal standard peak area that will test sample substitutes into above-mentioned mark
Directrix curve equation calculates the concentration of untested compound in detection sample, is shown in Table 7~table 8.
The calculating of the Specification Curve of Increasing and quality-control product and test sample concentration of 7 5-methyltetrahydrofolate of table
Calculation specifications: the peak area and internal standard peak area of record standard product calculate peak area ratio.According to peak area ratio
(y) and mark concentration (x) in data point linear regression straight line y=a+bx (see Fig. 2).
The calculation method of b (slope) and a (intercept) in formula are as follows:
Quality-control product and sample content calculating process: the peak area and internal standard peak area of record quality-control product and sample to be tested, meter
Peak area ratio is calculated, standard curve regression equation is substituted into, is calculated according to formula x=(y-a)/b.
The calculating of 8 folic acid Specification Curve of Increasing of table and quality-control product and test sample concentration
Calculation specifications: the peak area and internal standard peak area of reference substance are recorded, peak area ratio is calculated.According to peak area ratio
(y) and mark concentration (x) in data point linear regression straight line y=a+bx (see Fig. 3).
The calculation method of b (slope) and a (intercept) in formula are as follows:
Quality-control product and sample content calculating process: the peak area and internal standard peak area of record quality-control product and sample to be tested, meter
Peak area ratio is calculated, standard curve regression equation is substituted into, is calculated according to formula x=(y-a)/b.
Detect the calculating of sample results: the ratio of the practical peak area and internal standard peak area that will test sample substitutes into above-mentioned mark
Directrix curve equation calculates the concentration of untested compound in detection sample.
(3) calculating of the rate of recovery: the ratio of the practical peak area of compound of quality-control product detection and internal standard peak area is substituted into
Above-mentioned standard curvilinear equation calculates the concentration of compound in quality-control product.The calculation of the quality-control product rate of recovery are as follows: the rate of recovery (%)
=measurement concentration/mark concentration × 100%.It is required that the rate of recovery % of quality-control product detection should be in 100 ± 20% ranges.
(4) accuracy of standard items and quality-control product content:
Standard items: the inaccuracy of each reference substance content, relative deviation % (RE%) are indicated with relative deviation % (RE%)
± 20% should be not more than.
Quality-control product: the inaccuracy of each reference substance content, relative deviation % (RE%) are indicated with relative deviation % (RE%)
± 20% should be not more than.
Accuracy: the inaccuracy of testing result is indicated with relative deviation % (RE%), relative deviation % (RE%) should not
Greater than ± 20%.
(5) precision:
Withinrun precision: the coefficient of variation (CV%)≤20%.
Betweenrun precision: the coefficient of variation (CV%)≤20%.
(6) minimum detection limit: folic acid 0.5ng/mL；5-methyltetrahydrofolate is 0.5ng/mL.With the rate of recovery (%) table
Show the accuracy of measurement result, it is desirable that the rate of recovery (%) is in 80~120% ranges.
(7) stability: kit is protected from light at 2~8 DEG C, saves under the condition of storage that seals to effective end of term, reagent performance
Appearance, the accuracy of reference substance and quality-control product content, the range of linearity, accuracy, withinrun precision, minimum detection limit etc. should be met
The requirement of item clause.
6, the appearance of product and loading amount index
(1) appearance: packing box is without breakage, clear writing；Reagent packaging external appearance is clean and tidy, and mark is clear, and reagent each component is answered
For colorless clear liquid, without floccule and precipitating, the intact no breakage of filter paper.
(2) loading amount: loading amount should be not less than sign value.
7, application notice
(1) different lot numbers, same producer's different cultivars reagent please don't be used with.
(2) kit is saved to -25 DEG C to -18 DEG C, and validity period 12 months, please in being used in validity period.
(3) stringent by specification operation, the volume etc. that reaction temperature, time and each component pipette must be strictly controlled, change
Test result may be will affect by becoming test procedure.
(4) any sample all should be used as treating with infectious sample.
(5) there may be certain stimulation or toxicity for some reagents, please don't directly contact skin, eyes.Once connecing
Touching is rinsed with a large amount of clear water, please don't be swallowed.
(6) this product is only used for in-vitro diagnosis use.
8, bibliography
[1]van Haandel L,Stobaugh J F.Folate determination in human health:
UPLC–MS/MS is the emerging methodology of choice[J].Bioanalysis,2013,5(24):
3023~3031.
[2] Gao Shihong, Wang Yujue, Hu Bei, wait using HPLC-MS/MS joint technology quantitative determination human plasma in folic acid and
Methodological study [J] mass spectrum journal of 5-methyltetrahydrofolate concentration, 2004,25 (3): 145~149.
[3]Kirsch S H,Knapp J P,Geisel J,et al.Simultaneous quantification of
S-adenosyl methionine and S-adenosyl homocysteine in human plasma by stable-
isotope dilution ultra performance liquid chromatography tandem mass
spectrometry[J].Journal of Chromatography B,2009,877(30):3865-3870.
Claims (9)
1. a kind of method of folic acid in high performance liquid chromatography string mass Spectrometry for Determination blood cake, which is characterized in that
The folic acid is respectively folic acid and 5-methyltetrahydrofolate；
Method using folic acid in high performance liquid chromatography string mass Spectrometry for Determination blood cake includes the following steps: first with efficient liquid
Phase chromatography separates folic acid and 5-methyltetrahydrofolate, recycles mass spectrum Isotopic Internal Standard sizing technique, the concentration with standard items is X
The peak area of axis, standard items and internal standard compound is that ratio is Y-axis, establishes calibration curve, calculates above-mentioned folic acid and 5- methyl tetrahydro leaf
The content of acid, specific chromatographic condition are as follows:
(1) chromatographic condition
Chromatographic column is Phenomenex2.6μm F5 100A°；Mobile phase A is the aqueous solution of 0.1vt% formic acid；Flowing
Phase B is the acetonitrile solution of 0.1vt% formic acid；Column temperature is 40 DEG C；Sample volume is 5 μ L；It is eluted using gradient mode, is shown in Table 1；
1 eluent gradient elution parameters of table
(2) Mass Spectrometry Conditions
Under electron spray ion positive ion detection mode, using MRM scanning analysis, source parameters: atomization gas flow is 3L/
min；Dry gas stream amount is 2L/min；Heating throughput is 15L/min；DL tube temperature degree is 100-400 DEG C；Interface temperature is 100-
400℃；Heating deblocking temperature is 100-400 DEG C, CID gas: 100-300kPa；Precursor and product ion channel are as shown in table 2；
2 multiple-reaction monitoring ion pair of table and corresponding voltage parameter
2. the method for folic acid in a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake according to claim 1,
It is characterized in that, the blood cake is prepared as follows to obtain: (1) taking 2~8mm of diameter sample in 2mL EP with punch
100~500 μ L working solutions are added in Guan Zhong, and 500~2000rpm, which is vortexed, mixes 10~60min；(2) 50~450 μ L of supernatant is taken
In 96 hole deep-well plates, 10~100L/min of room temperature is dried with nitrogen；(3) 50~400 μ L are added and redissolve liquid, 500~2000rpm vibration
Shake 5~60min.
3. the method for folic acid in a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake according to claim 2,
It is characterized in that, the working solution is prepared as follows to obtain: adding extracting solution by the volume ratio of 1:10:10:1000
Add agent: joint antioxidant: mixing internal standard solution: extracting solution mixes well.
4. the method for folic acid in a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake according to claim 3,
It is characterized in that, the extraction solution additive is ammonium hydroxide, extracting solution is 70vt% methanol aqueous solution.
5. the method for folic acid in a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake according to claim 3,
It is characterized in that, the joint antioxidant is the mixture of dithiothreitol (DTT), ascorbic acid and citric acid, wherein two sulphur
The mass ratio of threitol, ascorbic acid and citric acid is 1:1:1.
6. the method for folic acid in a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake according to claim 3,
It is characterized in that, being designated as the folic acid-containing 10 μ g/mL in the mixing135-methyltetrahydrofolate-the d3 of C5 and 10 μ g/mL
Aqueous solution.
7. the method for folic acid in a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake according to claim 2,
It is characterized in that, the redissolution liquid is that 1%vt combines aqueous antioxidant solution.
8. the method for folic acid in a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake according to claim 1,
It is characterized in that, the standard items are the blood cake containing folic acid and 5-methyltetrahydrofolate, specific concentration is shown in Table 3,
The corresponding concentration of 3 standard items of table (ng/mL)
。
9. the method for folic acid in a kind of high performance liquid chromatography string mass Spectrometry for Determination blood cake according to claim 1,
It is characterized in that, the standard items are prepared in accordance with the following steps:
(1) rabbit erythrocyte is cleaned using physiological saline, it is spare；
(2) human serum albumin solution of configuration 50mg/mL joint antioxidant containing 100 μ g/mL:
2g human serum albumins powder is weighed, the physiological saline 40mL of 100 μ g/mL joint antioxidant is added, is prepared after mixing
It is 50mg/mL human serum albumin solution at concentration；
(3) rabbit erythrocyte is sufficiently mixed according to the volume ratio of 55:45 and matrix is made in human serum albumin solution；
(4) 100 μ g/mL of preparation combine aqueous antioxidant solution；
The configuration of stock solution: weighing the folic acid of 50mg and the 5-methyltetrahydrofolate of 50mg respectively, is separately added into 100 μ g/mL connection
Aqueous antioxidant solution 1L is closed, folic acid stock solution and 50 μ g/mL5- methyl tetrahydrofolate deposits that concentration is 50 μ g/mL are configured to
Liquid；
Concentration is the preparation of C1 standard items: taking folic acid stock solution and each 10 μ L of 5-methyltetrahydrofolate stock solution respectively, uses matrix
Dilution is settled to the standard items that 1000 μ L are 500ng/mL to get concentration；Taking concentration is 500ng/mL folic acid and 500ng/mL5-
Each 300 μ L of methyl tetrahydrofolate is settled to the standard items that 3000 μ L are C1 to get concentration with diluted matrix；
Concentration is the preparation of C2 standard items: the folic acid and each 300 μ L of 5-methyltetrahydrofolate that concentration is C1 are taken, it is fixed with diluted matrix
Hold the standard items for being C2 to get concentration to 3000 μ L；
Concentration is the preparation of C3 standard items: the folic acid and each 300 μ L of 5-methyltetrahydrofolate that concentration is C2 are taken, it is fixed with diluted matrix
Hold the standard items for being C3 to get concentration to 3000 μ L；
(5) take the 66 μ L drop of standard items of each concentration on speciality filter paper, dry collect -20 DEG C be kept in dark place.
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| CN114858932A (en) * | 2022-03-15 | 2022-08-05 | 昆明和合医学检验所有限公司 | Method for determining nervonic acid content in dry blood filter paper sheet by high performance liquid chromatography tandem mass spectrometry |
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