CN110146628A - A kind of high performance liquid chromatography string mass-spectrometric technique detects the kit of folic acid in blood cake - Google Patents
A kind of high performance liquid chromatography string mass-spectrometric technique detects the kit of folic acid in blood cake Download PDFInfo
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- CN110146628A CN110146628A CN201910526620.1A CN201910526620A CN110146628A CN 110146628 A CN110146628 A CN 110146628A CN 201910526620 A CN201910526620 A CN 201910526620A CN 110146628 A CN110146628 A CN 110146628A
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- 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
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Abstract
The invention discloses the kits of folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake, including standard items, quality-control product, mixing internal standard solution, eluent A, eluent B, extracting solution, solution additive, joint antioxidant and redissolution liquid are extracted, the folic acid is respectively folic acid and 5-methyltetrahydrofolate.It being detected using kit of the present invention, high sensitivity, high specificity, accurate and pre-treating method are easy, and it is time-consuming short, time cost is greatly saved, precision and the rate of recovery substantially meet requirement.
Description
Technical field
The present invention relates to the quantitative detection of folic acid, the method that liquid chromatogram string mass spectrometry detects folic acid in human body blood cake,
Dry blood cake folic acid detects finished product kit.
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.Accordingly, it is desirable to provide a kind of using blood cake as the kit of the simple operations of object.
Summary of the invention
The problem to be solved by the invention is to provide folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake
Kit.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
A kind of high performance liquid chromatography string mass-spectrometric technique detects the kit of folic acid in blood cake,
The folic acid is respectively folic acid and 5-methyltetrahydrofolate；
The kit includes following reagent:
(1) standard items: the blood cake containing folic acid and 5-methyltetrahydrofolate；
(2) quality-control product: the blood cake containing folic acid and 5-methyltetrahydrofolate；
(3) internal standard solution: folic acid-is mixed13The aqueous solution of C5 and 5-methyltetrahydrofolate-d3；
(4) eluent
The aqueous solution of A:0.1%vt elution solution additive；
The acetonitrile solution of B:0.1%vt elution solution additive；
(5) extracting solution: the aqueous solution of 70%vt methanol；
(6) solution additive: ammonium hydroxide is extracted；
(7) combine antioxidant: the mixture of dithiothreitol (DTT), ascorbic acid and citric acid；
(8) liquid is redissolved: the aqueous solution containing joint antioxidant.
Wherein, the kit further include: 96 hole microwell plates, operational manual and separation simultaneously concentrate these reagents of packaging
Any one or a few combination in the packing box of box or pipe.
Wherein, standard items are the blood cake containing folic acid and 5-methyltetrahydrofolate, and specific concentration is shown in Table 1,
The corresponding concentration of 1 standard items of table (ng/mL)
| Folic acid | C1 | C2 | C3 |
| Folic acid | 50 | 5 | 0.5 |
| 5-methyltetrahydrofolate | 50 | 5 | 0.5 |
。
Wherein, quality-control product is the blood cake containing folic acid and 5-methyltetrahydrofolate, is divided to high and low two concentration, is respectively
QCH,QCL；Wherein the corresponding concentration of folic acid quality-control product is shown in Table 2 in QCH, QCL,
The corresponding concentration of 2 quality-control product of table (ng/mL)
| Folic acid | QCH | QCL |
| Folic acid | 30 | 6 |
| 5-methyltetrahydrofolate | 30 | 6 |
。
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 elution solution additive is formic acid；
Wherein, it is 1:1:1 mixed that the joint antioxidant, which is dithiothreitol (DTT), ascorbic acid and citric acid with mass ratio,
The mixture of conjunction.
Wherein, the redissolution liquid is that 1%vt combines aqueous antioxidant solution.
The preparation method of the kit of folic acid in above-mentioned high performance liquid chromatography string mass-spectrometric technique detection blood cake
(1) standard items:
A. it is cleaned rabbit erythrocyte 3 times using physiological saline, it is spare；
B. configuration 50mg/mL combines the human serum albumin solution of 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；
C. rabbit erythrocyte is sufficiently mixed according to the volume ratio of 55:45 and matrix is made in human serum albumin solution；
D. 100 μ g/mL joint aqueous antioxidant solution is prepared；
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；
E. 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；
(2) quality-control product: taking the folic acid that concentration is C1 to store up standard items and each 3mL of 5-methyltetrahydrofolate standard items respectively, point
5mL and 25mL, which is not settled to, with matrix obtains the folic acid and 5-methyltetrahydrofolate of 30ng/mL and 6ng/mL；Take the matter of each concentration
66 μ L drop of control product on speciality filter paper, dry collect -20 DEG C be kept in dark place；
(3) mix internal standard solution: preparation contains the folic acid-of 10 μ g/mL13The aqueous solution of C5 and 5-methyltetrahydrofolate-d3.
(4) eluent: the aqueous solution of 70%vt formic acid；
A: the aqueous solution of preparation formic acid containing 0.1%vt；
B: the acetonitrile solution of preparation formic acid containing 0.1%vt；
(5) extracting solution: the aqueous solution of 70%vt methanol is prepared；
(6) solution additive: ammonium hydroxide is extracted；
(7) combine antioxidant: preparing the mixing of dithiothreitol (DTT), ascorbic acid and citric acid that mass ratio is 1:1:1
Object；
(8) redissolve liquid: preparation 1%vt combines aqueous antioxidant solution.
The use of the kit of folic acid includes the following steps: in above-mentioned high performance liquid chromatography string mass-spectrometric technique detection blood cake
(1) solution additive will preparation liquid: be extracted by the volume ratio of 1:10:10:1000: joint antioxidant: mixing
Internal standard solution: extracting solution mixes well；
(2) sample pre-treatments: taking 2~8mm of diameter sample in 2mL EP pipe respectively with punch, and 100~500 μ L are added
Working solution, 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；
(3) liquid relief: solution in step (2) each orifice plate is transferred in 96 microwell plate sample introduction plate holes；
(4) liquid phase string Mass Spectrometry Conditions are set: high performance liquid chromatography-tandem mass instrument is respectively set according to actual instrumentation model
Running parameter and condition；
(5) 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；
(6) interpretation of result: drawing standard curve and fit standard Regression Equations, calculates folic acid and 5- methyl in sample
The concentration of tetrahydrofolic acid.
The application of mentioned reagent box folic acid in using superelevation liquid chromatogram string mass-spectrometric technique detection blood cake.
Wherein, the liquid phase string Mass Spectrometry Conditions of step (4) are specific as follows:
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 eluent A；Mobile phase B is to wash
De- liquid B；Column temperature is 40 DEG C；Sample volume is 5 μ L；By the way of gradient mode elution, it is shown in Table 3；
3 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 4；
4 multiple-reaction monitoring ion pair of table and corresponding voltage parameter
The utility model has the advantages that the method for this kit folic acid suitable for high performance liquid chromatography string mass-spectrometric technique detection blood cake, it should
Method can be with:
1, this kit can extract folic acid therein and 5-methyltetrahydrofolate from blood cake.
2, the sample pre-treatments for high performance liquid chromatography string mass spectrography, quantitatively to be divided by Liquid Chromatography-Tandem Mass Spectrometry instrument
Analysis is prepared to obtain concentration levels, so that mass spectrometry clinical is detected.
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.
Instrument and material source used in following embodiment are as follows:
(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 (PLSCONFM), and purity 99%, 5-methyltetrahydrofolate are purchased from USP, pure
Degree is 99%, and internal standard folic acid -13C5 and 5-methyltetrahydrofolate-d3 are purchased from TRC, purity > 99%.
Embodiment 1
Assay kit each component is shown in Table 5.
The preparation of 5 assay kit component of table
Mentioned reagent box the preparation method is as follows:
(1) standard items:
A. it is cleaned rabbit erythrocyte 3 times using physiological saline, it is spare；
B. configuration 50mg/mL combines the human serum albumin solution of 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；
C. rabbit erythrocyte is sufficiently mixed according to the volume ratio of 55:45 and matrix is made in human serum albumin solution；
D. 100 μ g/mL joint aqueous antioxidant solution is prepared；
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；
E. 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；
(2) quality-control product: taking the folic acid that concentration is C1 to store up standard items and each 3mL of 5-methyltetrahydrofolate standard items respectively, point
5mL and 25mL, which is not settled to, with matrix obtains the folic acid and 5-methyltetrahydrofolate of 30ng/mL and 6ng/mL；Take the matter of each concentration
66 μ L drop of control product on speciality filter paper, dry collect -20 DEG C be kept in dark place.
(3) internal standard solution is mixed: the aqueous solution of folic acid -13C5 and 5-methyltetrahydrofolate-d3 of the preparation containing 10g/mL.
(4) eluent
A: the aqueous solution of preparation formic acid containing 0.1%vt；
B: the acetonitrile solution of preparation formic acid containing 0.1%vt；
(5) extracting solution: the aqueous solution of 70%vt methanol is prepared；
(6) solution additive: ammonium hydroxide is extracted；
(7) combine antioxidant: preparing the mixing of dithiothreitol (DTT), ascorbic acid and citric acid that mass ratio is 1:1:1
Object；
(8) redissolve liquid: preparation 1%vt combines aqueous antioxidant solution.
Embodiment 2
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, pre-treatment
Standard items processing[2][3]: it takes 2~8mm of diameter sample in 2mL EP pipe respectively with punch, 100~500 μ is added
L working solution, 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.
Liquid relief: by hole each in each orifice plate the above-mentioned sample handled well and standard items and control panel solution be transferred to 96
In microwell plate sample introduction plate hole；
3, take the standard items, quality-control product and test sample solution handled well inject high performance liquid chromatography-tandem mass instrument into
Row detection, and record chromatogram and detect the peak area and its internal standard peak area of sample folic acid and 5-methyltetrahydrofolate, specifically
Chromatographic condition is[1]:
(1) liquid phase chromatogram condition
Chromatographic column is Phenomenex2.6μm F5 100A°；Mobile phase A is eluent A；Mobile phase B is to wash
De- liquid B；Column temperature is 40 DEG C；Sample volume is 5 μ L；By the way of gradient mode elution, it is shown in Table 6；
6 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 7；
7 multiple-reaction monitoring ion pair of table and corresponding voltage parameter
Embodiment 3:
1, 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 each dense of 2 standard items
The ratio for spending sample and the peak area of its internal standard compound is ordinate (y), draws standard curve, and calculate above-mentioned folic acid and 5- methyl
The content of tetrahydrofolic acid.The linear fit equation of folic acid and 5-methyltetrahydrofolate in respective range, linear good, phase relation
Number meets quantitative requirement, is shown in Table 8 within 0.999.
8 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 9~table 10.
The calculating of the Specification Curve of Increasing and quality-control product and test sample concentration of 9 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 10 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.
2, 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.
3, 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 it connects
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.
4, 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 (10)
1. the kit of folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake, which is characterized in that
The folic acid is respectively folic acid and 5-methyltetrahydrofolate；
The kit includes following reagent:
(1) standard items: the blood cake containing folic acid and 5-methyltetrahydrofolate；
(2) quality-control product: the blood cake containing folic acid and 5-methyltetrahydrofolate；
(3) internal standard solution: folic acid-is mixed13The aqueous solution of C5 and 5-methyltetrahydrofolate-d3；
(4) eluent
The aqueous solution of A:0.1%vt elution solution additive；
The acetonitrile solution of B:0.1%vt elution solution additive；
(5) extracting solution: the aqueous solution of 70%vt methanol；
(6) solution additive: ammonium hydroxide is extracted；
(7) combine antioxidant: the mixture of dithiothreitol (DTT), ascorbic acid and citric acid；
(8) liquid is redissolved: the aqueous solution containing joint antioxidant.
2. the kit of folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake according to claim 1,
It is characterized in that, the kit further include: 96 orifice plates, operational manual and separation simultaneously concentrate and pack these kits or pipe
Any one or a few combination in packing box.
3. the kit of folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake according to claim 1,
It being characterized in that, standard items are the blood cake containing folic acid and 5-methyltetrahydrofolate, and specific concentration is shown in Table 1,
The corresponding concentration of 1 standard items of table (ng/mL)
。
4. the kit of folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake according to claim 1,
Be characterized in that, quality-control product be the blood cake containing folic acid and 5-methyltetrahydrofolate, be 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 2 in QCH, QCL,
The corresponding concentration of 2 quality-control product of table (ng/mL)
。
5. the kit of folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake according to claim 1,
It is characterized in that, the folic acid-containing 10 μ g/mL is designated as in the mixing135-methyltetrahydrofolate-the d3's of C5 and 10 μ g/mL
Aqueous solution.
6. the kit of folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake according to claim 1,
It is characterized in that, the elution solution additive is formic acid；The joint antioxidant is dithiothreitol (DTT), ascorbic acid and lemon
Lemon acid is with the mixture that mass ratio is that 1:1:1 is mixed.
7. the kit of folic acid in a kind of high performance liquid chromatography string mass-spectrometric technique detection blood cake according to claim 1,
It is characterized in that, the redissolution liquid is that 1%vt combines aqueous antioxidant solution.
8. the preparation method of kit described in claim 1~7 any one, which is characterized in that
(1) standard items:
A. rabbit erythrocyte is cleaned using physiological saline, it is spare；
B. configuration 50mg/mL combines the human serum albumin solution of 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；
C. rabbit erythrocyte is sufficiently mixed according to the volume ratio of 55:45 and matrix is made in human serum albumin solution；
D. 100 μ g/mL joint aqueous antioxidant solution is prepared；
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；
E. 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；
(2) it quality-control product: takes the folic acid that concentration is C1 to store up standard items and each 3mL of 5-methyltetrahydrofolate standard items respectively, uses respectively
Matrix is settled to 5mL and 25mL obtains the folic acid and 5-methyltetrahydrofolate of 30ng/mL and 6ng/mL；Take the quality-control product of each concentration
66 μ L drops on speciality filter paper, dry collect -20 DEG C be kept in dark place；
(3) mix internal standard solution: preparation contains the folic acid-of 10 μ g/mL13The aqueous solution of C5 and 5-methyltetrahydrofolate-d3.
(4) eluent: the aqueous solution of 70%vt formic acid；
A: the aqueous solution of preparation formic acid containing 0.1%vt；
B: the acetonitrile solution of preparation formic acid containing 0.1%vt；
(5) extracting solution: the aqueous solution of 70%vt methanol is prepared；
(6) solution additive: ammonium hydroxide is extracted；
(7) combine antioxidant: preparing the mixture that mass ratio is the dithiothreitol (DTT) of 1:1:1, ascorbic acid and citric acid；
(8) redissolve liquid: preparation 1%vt combines aqueous antioxidant solution.
9. kit described in claim 1~7 any one, which is characterized in that the use of the kit includes the following steps:
(1) solution additive will preparation liquid: be extracted by the volume ratio of 1:10:10:1000: joint antioxidant: mixing internal standard
Liquid: extracting solution mixes well；
(2) sample pre-treatments: taking 2~8mm of diameter sample in 2mL EP pipe respectively with punch, and 100~500 μ L work is added
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；
(3) liquid relief: solution in step (2) each orifice plate is transferred in 96 microwell plate sample introduction plate holes；
(4) liquid phase string Mass Spectrometry Conditions are set: the work of high performance liquid chromatography-tandem mass instrument being respectively set according to actual instrumentation model
Make parameter and condition；
(5) sample detection: the standard items, quality-control product and test sample solution handled well is taken to inject high performance liquid chromatography-series connection matter
Spectrometer is detected, and is recorded chromatogram and detected peak area and its internal standard peak face of sample folic acid and 5-methyltetrahydrofolate
Product；
(6) interpretation of result: drawing standard curve and fit standard Regression Equations, calculates folic acid and 5- methyl tetrahydro in sample
The concentration of folic acid.
10. kit described in claim 1~7 any one is in using superelevation liquid chromatogram string mass-spectrometric technique detection blood cake
The application of folic acid.
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| CN110514625A (en) * | 2019-09-12 | 2019-11-29 | 宁波奥丞生物科技有限公司 | A kind of measuring method of human serum folic acid |
| CN112083108A (en) * | 2020-09-23 | 2020-12-15 | 辽宁润生康泰医学检验实验室有限公司 | Accurate detection method and kit for folic acid in blood |
| CN113009033A (en) * | 2021-03-02 | 2021-06-22 | 广东南芯医疗科技有限公司 | Liquid phase tandem mass spectrum detection kit and detection method for testing folic acid metabolic derivatives of human body |
| CN114646713A (en) * | 2022-04-29 | 2022-06-21 | 北京豪思生物科技股份有限公司 | Sample pretreatment method, detection method and kit for folic acid and metabolites thereof |
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| CN115326957A (en) * | 2022-08-09 | 2022-11-11 | 苏州和合医学检验有限公司 | Method for measuring blood concentration of folic acid and 5-methyl tetrahydrofolic acid in serum |
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| CN112083108A (en) * | 2020-09-23 | 2020-12-15 | 辽宁润生康泰医学检验实验室有限公司 | Accurate detection method and kit for folic acid in blood |
| CN112083108B (en) * | 2020-09-23 | 2021-04-27 | 辽宁润生康泰医学检验实验室有限公司 | Accurate detection method and kit for folic acid in blood |
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| CN114646713A (en) * | 2022-04-29 | 2022-06-21 | 北京豪思生物科技股份有限公司 | Sample pretreatment method, detection method and kit for folic acid and metabolites thereof |
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| CN114994218B (en) * | 2022-05-07 | 2024-05-03 | 凯莱谱科技股份有限公司 | Detection kit for detecting 4 fat-soluble vitamins in dry blood spots by liquid chromatography-tandem mass spectrometry and detection method thereof |
| CN114935620A (en) * | 2022-05-20 | 2022-08-23 | 上海市精神卫生中心（上海市心理咨询培训中心） | Kit for simultaneously and quantitatively detecting 78 neuropsychiatric drugs |
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| CN115326957A (en) * | 2022-08-09 | 2022-11-11 | 苏州和合医学检验有限公司 | Method for measuring blood concentration of folic acid and 5-methyl tetrahydrofolic acid in serum |
| CN115436540A (en) * | 2022-09-26 | 2022-12-06 | 汤臣倍健股份有限公司 | Method and kit for simultaneously determining contents of folic acid and homocysteine in blood |
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