CN108802213A - Vitamin A detection dried blood spot reagent preparation box and detection method - Google Patents
Vitamin A detection dried blood spot reagent preparation box and detection method Download PDFInfo
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- CN108802213A CN108802213A CN201810360213.3A CN201810360213A CN108802213A CN 108802213 A CN108802213 A CN 108802213A CN 201810360213 A CN201810360213 A CN 201810360213A CN 108802213 A CN108802213 A CN 108802213A
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- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 title claims abstract description 114
- 210000004369 blood Anatomy 0.000 title claims abstract description 108
- 239000008280 blood Substances 0.000 title claims abstract description 106
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 title claims abstract description 23
- 235000019155 vitamin A Nutrition 0.000 title claims abstract description 23
- 239000011719 vitamin A Substances 0.000 title claims abstract description 23
- 229940045997 vitamin a Drugs 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 39
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 20
- 235000006708 antioxidants Nutrition 0.000 claims abstract description 20
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 15
- 235000010354 butylated hydroxytoluene Nutrition 0.000 claims abstract description 13
- 239000005030 aluminium foil Substances 0.000 claims abstract description 6
- 235000019441 ethanol Nutrition 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 19
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 7
- 229940088594 vitamin Drugs 0.000 claims description 7
- 229930003231 vitamin Natural products 0.000 claims description 7
- 235000013343 vitamin Nutrition 0.000 claims description 7
- 239000011782 vitamin Substances 0.000 claims description 7
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 238000010241 blood sampling Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 125000005909 ethyl alcohol group Chemical group 0.000 claims description 5
- 239000012224 working solution Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 4
- 238000007605 air drying Methods 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical class OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims 1
- 239000004322 Butylated hydroxytoluene Substances 0.000 abstract description 10
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 abstract description 10
- 229940095259 butylated hydroxytoluene Drugs 0.000 abstract description 10
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 abstract description 4
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 abstract description 2
- 229930003268 Vitamin C Natural products 0.000 abstract description 2
- 235000019154 vitamin C Nutrition 0.000 abstract description 2
- 239000011718 vitamin C Substances 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 53
- 235000020944 retinol Nutrition 0.000 description 48
- 229960003471 retinol Drugs 0.000 description 45
- 239000011607 retinol Substances 0.000 description 45
- 238000004458 analytical method Methods 0.000 description 13
- 230000008569 process Effects 0.000 description 12
- 239000013062 quality control Sample Substances 0.000 description 11
- 238000007789 sealing Methods 0.000 description 10
- 238000000605 extraction Methods 0.000 description 9
- 238000011835 investigation Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000011159 matrix material Substances 0.000 description 7
- 238000002203 pretreatment Methods 0.000 description 7
- 208000010011 Vitamin A Deficiency Diseases 0.000 description 5
- 239000003223 protective agent Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000005259 measurement Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 150000004347 all-trans-retinol derivatives Chemical class 0.000 description 3
- 235000021196 dietary intervention Nutrition 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 235000003715 nutritional status Nutrition 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- RGCLLPNLLBQHPF-HJWRWDBZSA-N phosphamidon Chemical compound CCN(CC)C(=O)C(\Cl)=C(/C)OP(=O)(OC)OC RGCLLPNLLBQHPF-HJWRWDBZSA-N 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- NCYCYZXNIZJOKI-OVSJKPMPSA-N Retinaldehyde Chemical compound O=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-OVSJKPMPSA-N 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000002723 alicyclic group Chemical group 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- AQHHHDLHHXJYJD-UHFFFAOYSA-N propranolol Chemical compound C1=CC=C2C(OCC(O)CNC(C)C)=CC=CC2=C1 AQHHHDLHHXJYJD-UHFFFAOYSA-N 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000020945 retinal Nutrition 0.000 description 1
- 239000011604 retinal Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- -1 start to decay Chemical compound 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of vitamin A detection dried blood spot reagent preparation box, it includes as antioxidant vitamin C or butylated hydroxytoluene (BHT), the kit may further include alcohol, filter paper, aluminium foil bag.The invention also discloses the non-diagnostic detection methods of vitamin A dried blood spot for using the kit to carry out, wherein when preparing dried blood spot, are dried after 50 microlitres of antioxidants are added dropwise on blood point.
Description
One technical field
The invention belongs to vitamin detection fields, are prepared with dried blood spot specifically, the present invention relates to vitamin A detections
Kit and corresponding method of detection.
Two background technologies
Vitamin A (vitaminA) is also known as retinol (its aldehyde derivatives retinene) or antiophthalmic factor, is a tool
The unsaturated monohydric alcohol for having alicyclic ring is a kind of liposoluble vitamin, plays an important role to children growth and development.World health
As a countries and regions, there are vitamin A deficiency public health problems by 5 years old or less child mortality >=50 ‰ for tissue
Important indicator.About 33.3% preschool child faces vitamin A deficiency in world wide.China is the subclinical dimension life of moderate
Plain A lacks national (children), lacks condition survey as a result, outlying district according to the national vitamin A in children of 1999-2000
Rural area is that vitamin A severe lacks area, and outlying district rural children is the key population of China's vitamin A nutritional intervention.Dimension
Medical treatment of the raw element A Evaluation of Nutrition Status for vitamine A deficiency, nutritional intervention, and guiding area is agriculture, food industries
(the plantation processing for such as promoting homovitamin A content rice crop) developing direction is all significant.
Reliable nutritional status of vitamin A assessment applies high performance liquid chromatography (HPLC) detection serum to regard Huang more at present
Alcohol level, using the μ g/dL of serum retinol≤20 as the standard of vitamin A deficiency.Since retinol stability is poor, serum
It preparing and needs to centrifuge as early as possible after venous blood samples, serum specimen stores and transports the characteristics of being also required to keep low temperature environment,
Nutritional status of vitamin A assessment is significantly limited with intervention to carry out in the rural children of outlying district.
Peripheral whole blood, i.e., be collected on filter paper by dried blood spot sampling technique (DBS), compared with serum, has storage fortune
Defeated convenience requires low, a variety of advantages such as minimally invasive to operating personnel's level professional technology, is highly suitable for a wide range of crowd's dimension
Blood specimen collection when raw element Evaluation of Nutrition Status, be particularly suitable for it is local do not have vitamin and quantify detection device, need to transport
The outlying district of sample.The past DBS combines HPLC and is used in retinol Quantitative Study, the stabilization of retinol in dried blood spot
Property be not resolved always, document report dried blood spot prepare after retinol i.e. start to decay, a few hours to 10 days or so when
In, the retinol content on blood cake reduces by more than 20%, and maintains to stablize thereafter.For effective protection retinol, previously
There is the report using antioxidants such as BHT in vitamin A Quantitative Study, but application method is that BHT is added to liquid blood
In clearly/blood plasma, or by butylated hydroxytoluene (BHT) add to dissolving dried blood spot working solution in, have no anti-oxidant using BHT etc.
Agent pre-processes the report of dried blood spot.In fact, in order to avoid in long-term preservation may with other in scraps of paper ingredient and blood at
The interaction divided influences detection, general in the prior art also not use additional reagent pretreatment dried blood spot (referring to dry blood
The drug evaluations such as point sampling and its application Liu Yong in the dynamic research of medicine study the 5th phase of volume 34).
Three invention contents
The ethanol solution of present invention application antioxidant, especially 0.1g/mL BHT pre-process Blood piece, can be in blood card
Effective protection retinol during process of drying in the shade and storage is allowed to maintain to stablize.Experiments have shown that this processing method can be effective
Keep the stabilization of retinol before testing, detection method that there is good precision and stability, be entirely capable of reaching clinical inspection
The requirement of survey.
On the one hand, the present invention provides a kind of vitamin A detection dried blood spot reagent preparation boxes, including antioxidant.
Further, the antioxidant is 0.02g/mL vitamin Cs or the ethanol solution of 0.1g/mLBHT.
Further, the antioxidant is the ethanol solution of 0.1g/mLBHT.
Further, which further includes alcohol, filter paper, aluminium foil bag.
On the other hand, the application the present invention provides antioxidant in preparing vitamin A detection dried blood spot.
Further, the antioxidant is 0.02g/mL vitamin Cs or the ethanol solution of 0.1g/mLBHT.
Further, the antioxidant is the ethanol solution of 0.1g/mLBHT.
On the other hand, the present invention provides a kind of non-diagnostic detection method of vitamin A dried blood spot using mentioned reagent box,
Including：
(1) blood specimen collection；(2) prepared by dried blood spot；(3) sample detection.
Further, step (2) includes：With alcohol in heel/pad of finger after massage or hot compress heel or finger tip
Disinfection is wiped First Blood away light blood of extruding out, is added dropwise using quantitative capillary tube with disposably blood needle being taken to puncture after air-drying
50 microlitres, to filter paper front, 50 microlitres of antioxidants are added dropwise on each blood point, and the filter paper after blood sampling dries in the air naturally in air
At least two hours dry, the Blood piece after drying is placed in clean aluminium foil bag and vacuumizes preservation.
Further, step (3) includes that a dried blood spot is placed in 2mL centrifuge tubes, and it is molten that the work of 20 μ L internal standards is added
Liquid, 50 μ L deionized waters and the 50 μ L absolute ethyl alcohols containing 0.625mg/mLBHT are added acetonitrile and are protected from light ultrasonic extraction, LC-MS/MS
Analysis.
Four description of the drawings
Fig. 1：Blank Blood piece retinol test map.
Fig. 2：Retinol test map in dried blood spot.
Fig. 3：Retinol standard working curve (0.04~3.00 μ g/mL).
Fig. 4：Whole blood dried blood spot room temperature is protected from light shelf stability figure.
Five specific implementation modes
With reference to embodiment, the present invention is described in further detail, and but the scope of the present invention is not limited thereto.
It is specifically sampled in following embodiment, detection method is as follows：
Blood specimen collection method：
1. massage or hot compress heel/finger tip 3-5min increase blood flow, sterilized in heel/pad of finger with alcohol,
After air-drying, with disposably blood needle being taken to puncture.
2. First Blood is wiped away.
3. dripped in circle on filter paper using quantitative capillary tube upper 50 μ L blood (have 3 circles of taking a blood sample on filter paper, it is desirable that
50 μ L to two blood sampling circles are at least added dropwise respectively), then 50 microlitres of antioxidants are added dropwise on each blood point.
4. if venous blood sampling samples, cannot be avoided from just taking a blood sample in the pipeline of intravenous administration or TPN using containing adding
Add the disposable syringe of agent.
5. filter paper naturally dry at least two hours in air after blood sampling, by the slightly curved Qu Yifang Hemostatic Oral Liquids of filter paper card
It contacts and loses with other surfaces when being permeated on filter paper, the Blood piece after drying is placed in clean aluminium foil bag and vacuumizes preservation.
Sample detection methods：
1. blank filter paper piece and dried blood spot (1 blood cake) are placed in 2mL centrifuge tubes (whole process under dim light into
Row)；
2. the preparation of standard series sample and quality-control sample：50 μ L retinol standard solution working solutions and matter are pipetted respectively
Solution is controlled, is added in the centrifuge tube of the piece containing blank filter paper, 50 μ L deionized waters are added；
3. other that 20 μ L internal standard working solutions are added other than double blank samples, dried blood spot sample mends 50 μ L deionizations
Water and 50 μ L absolute ethyl alcohols (containing 0.625mg/mLBHT), 50 μ L deionized waters and 70 μ L absolute ethyl alcohols are added in double blank samples
(containing 0.625mg/mLBHT)；
4. 180 μ L acetonitriles are added in all samples, be vortexed concussion 30s, and liquid level is made to impregnate dried blood spot；
5. being protected from light ultrasonic extraction 15min, vortex oscillation 30s.
5min is centrifuged under 6.13000rpm, takes 10 μ L to carry out LC-MS/MS analyses, condition is as follows：
Vitamin A detecting instrument analysis condition
The 1 vitamin A detection method range of linearity of embodiment is investigated：
The range of linearity of vitamin A is 0.04~3.00 μ g/mL, a concentration of 500ng/mL of Isotopic Internal Standard in dried blood spot.
The preparation of standard curve sample is that 3.00,2.40,1.50,1.00,0.40 is separately added into blank filter paper piece,
0.20,0.08 and 0.04 μ g/mL series concentrations standard working solution, 50 μ L, then be separately added into 20 μ L 500ng/mL internal standard it is molten
Liquid carries out sample treatment after vortex mixing.Treated, and sample carries out LC-MS/MS points according to the sequence of concentration from low to high
The average value of analysis, linearly dependent coefficient is 0.99932, and the average value of slope is 2.452.The linearly related system of all analyses batch
Number both greater than 0.9900.Specific data are shown in Table 1 and attached drawing 3
1 retinol standard curve parameter of table (0.04~3.00 μ g/mL)
2 matrix effect of embodiment is investigated：
Retinol matrix effect：Retinol peak area deducts in dried blood spot background in mark-on sample after investigation dried blood spot extraction
The peak area of retinol with retinol peak area ratio in the standard solution of concentration, three concentration samples parallel determination values
Relative standard deviation is respectively less than 15%, and the matrix effect factor is between 89.89% to 105.54%, average value 98.01%；
Internal standard matrix effect：Retinol internal standard peak area is regarded with the standard solution of same concentration in mark-on sample after investigation dried blood spot extraction
Flavol internal standard peak area ratio, the relative standard deviations of three concentration samples parallel determination values are respectively less than 15%, matrix effect because
For son between 87.45% to 90.62%, average value 89.42%, data refer to table 2-1,2-2.
Table 2-1 retinol matrix effects investigate data
ME (%)=(background mark-on peak area-background peak area)/standard liquid peak area * 100%
Table 2-2 retinol internal standard matrix effects investigate data
3 rate of recovery of embodiment is investigated
Rate of recovery investigation be by three concentration mark-ons extract retinol peak area in samples deduct dried blood spot background with not
The ratio that retinol peak area in sample deducts dried blood spot background is extracted, three concentration are respectively to be added into 1mL new bloods
0.2,0.4,0.6 μ g retinol standard items, the relative standard deviation of sample parallel determination value are respectively less than 15%, and the rate of recovery exists
Between 86.48% to 98.13%, average value 90.45%, the relative standard deviation of dried blood spot background parallel sample measured value
It is 4.51%, the relative standard deviation of mark-on parallel sample measured value adds between 1.12% to 2.16% after extraction before extracting
The relative standard deviation of parallel sample measured value is marked between 0.60% to 1.50%, is satisfied by corresponding requirements, determination data is shown in
Table 3-1.
The table 3-1 retinol rate of recovery investigates data
A：Mark-on sample before extraction；B：Mark-on sample after extraction；C：Dried blood spot background.
The investigation of 4 quality-control sample of embodiment and standard curve series of samples preci-sion and accuracy
According to the requirement that method is confirmed, the quality-control sample of each concentration and the opposite mark of standard curve parallel sample measured value
Quasi- deviation is no more than 15%, and average value relative error is between ± 15%, the relative standard of minimum quantitative limit parallel determination value
Deviation is no more than 20%, and relative error is within ± 20%.
The withinrun precision of quality-control sample and accuracy are investigated：
The retinol quality-control sample of a concentration of 0.12,0.90 and 2.25 μ g/mL, 6 parallel samples of each concentration, same
It is measured in one batch, substitutes into retinue standard curve and seek calculation measured concentration, the experimental results showed that, parallel quality-control sample measures
The relative standard deviation of value is between 3.69% to 5.26%, and the relative error of average value is between 0.63% to 5.69%,
Meet corresponding requirements.Data result for details see attached table 4-1.
The betweenrun precision and accuracy of quality-control sample and standard curve sample are investigated：
To the standard curve sample of a concentration of 0.04,0.08,0.20,0.40,1.00,1.50,2.40 and 3.00 μ g/mL
It is respectively the quality-control sample of 0.12,0.90 and 2.25 μ g/mL with three concentration, is investigated in 5 independent analyses batch,
Measurement result shows the relative standard deviation of parallel quality-control sample measured value between 3.38% to 7.08%, the phase of average value
To error between -0.50% to 0.81%, it is satisfied by corresponding requirements.Determination data for details see attached table 4-2.Standard curve is parallel
Between 1.57% to 4.60%, the relative error of average value arrives the relative standard deviation of sample measurements -3.50%
Between 1.90%, data result is listed in subordinate list 4-3.
The preci-sion and accuracy of minimum quantitative limit (LLOQ) is investigated：
Six samples parallel to standard curve minimum point are investigated in 3 batches.The experimental results showed that parallel in criticizing
The relative standard of sample measurements is 4.60% partially, the relative error 5.42% of average value；The phase of parallel sample measured value between batch
It is 6.30% to standard deviation, the relative error of average value is 1.11%, is satisfied by LLOQ and requires accordingly.Measurement result exists
In subordinate list 4-4,4-5.
Data are investigated in table 4-1 retinol quality-control sample withinrun precisions and accuracy
Allowable error：Relative standard deviation≤15%, relative error is between ± 15%.
" * " indicates that by Dixon statistical decisions be exceptional value, is added without statistics and calculates.
Data are investigated in table 4-2 retinol quality-control sample betweenrun precisions and accuracy
Allowable error：Relative standard deviation≤15%, relative error is between ± 15%.
" * " indicates that by Dixon statistical decisions be exceptional value, is added without statistics and calculates.
Data are investigated in table 4-3 retinol standard curve betweenrun precisions and accuracy
Allowable error：Relative standard deviation≤15%, relative error is between ± 15%；
LLOQ：Relative standard deviation≤20%, relative error is between ± 20%.
Data are investigated in the minimum quantitative limit withinrun precision of table 4-4 retinols and accuracy
Allowable error：Relative standard deviation≤20%, relative error is between ± 20%.
Data are investigated in the minimum quantitative limit betweenrun precision of table 4-5 retinols and accuracy
Allowable error：Relative standard deviation≤20%, relative error is between ± 20%.
Sample stability is investigated after embodiment 6 is extracted
Automatic sample injector, which is placed, after extraction repeats the investigation of sample introduction reproducibility：
Sample carries out LC-MS/MS analyses immediately after pre-treatment, after sample introduction all samples be positioned over automatically into
In sample device, new standard curve is used after about 25 hours, sample introduction is analyzed again to above-mentioned sample.Parallel sample repeats sample introduction and measures
The relative standard deviation of value is 4.79%, and the relative error of average value is -3.33%, the results showed that the sample after extraction is certainly
It is stable that at least 25 hours are placed in dynamic injector.Data result refers to table 5.
The investigation of -20 DEG C of refrigerator shelf-stabilities of sample after extraction：
Sample is allocated as two parts after pre-treatment, and a part carries out LC-MS/MS analyses immediately, and another part is -20
After placing about 25 hours in DEG C refrigerator, one new standard curve of retinue, which is measured, asks calculation.Refrigerator places sample parallel determination
The relative standard deviation of value is 1.88%, and the relative error of average value is -5.89%, the results showed that the sample after extraction is -20
DEG C refrigerator place at least 25 hours be stable.Data result refers to table 5.
Sample repeats sample introduction reproducibility and refrigerator shelf-stability data after table 5 extracts
Allowable error：Relative standard deviation≤15%, relative error is between ± 15%.
A：Sample introduction Retinol concentration immediately after dried blood spot processing；
B：It is positioned over after sample introduction in autosampler after about 25 hours and repeats sample introduction Retinol concentration；
C：Sample introduction Retinol concentration after -20 DEG C of refrigerators are placed about 25 hours after processing.
" -- " indicates not generate data.
7 dried blood spot of embodiment prepare (different protective agent investigations) dry in the shade to vacuum plastic sealing (2 hours) be protected from light and be placed at room temperature for
Process stability is investigated
Blood point is prepared, 50 μ L ethanol solutions containing protective agent are added dropwise in dried blood spot, and (protective agent type and concentration are shown in Table
6) the 2h blood point and with 4 degree of batch placement whole bloods of drying in the shade, is taken, LC-MS/MS analyses are carried out after sample pre-treatments, the results showed that
The vitamin C of 0.02g/mL and the BHT of 0.1g/mL can realize that preferable stablizing effect, data result refer to table 7.
6 protective agent type of table and concentration setting
8 dried blood spot vacuum plastic sealing of embodiment, which is protected from light, is placed at room temperature for stability
Prepare blood point, dry in the shade (2 hours), immediately vacuum plastic sealing in aluminium plastic packaging bag, after preparation 0h, 2h, 1d,
2d, 5d, 9d, 15d, 30d take out, and after sample pre-treatments, carry out LC-MS/MS analyses, investigate dried blood spot vacuum plastic sealing and keep away
Light room temperature shelf-stability, the results showed that dried blood spot is protected from light that be placed at room temperature for 30d be stable in vacuum plastic sealing.Data result is detailed
It is shown in Table 8.
8 dried blood spot long-time stability data of table
" -- " indicates not generate data.
9 dried blood spot of embodiment preparation dry in the shade to vacuum plastic sealing (2 hours) be protected from light be placed at room temperature for process stability investigation
Blood point is prepared, 0min, 15min, 30min, 60min, 90min, 120min blood point are taken, after sample pre-treatments,
Carry out LC-MS/MS analyses, the results showed that dried blood spot preparation dry in the shade to vacuum plastic sealing (2 hours) be protected from light the process of being placed at room temperature for
In, Retinol concentration is gradually decrease to the 75.86% of initial concentration in sample.Data result refers to table 9
9 dried blood spot of table preparation dry in the shade to vacuum plastic sealing (2h) be protected from light room temperature stability investigate data
A%：Dried blood spot placement process Retinol concentration and 0min Retinol concentration ratios；
" -- " indicates not generate data.
10 dried blood spot of embodiment prepares (be added absolute ethyl alcohol (contain 0..1g/mLBHT)) dry in the shade to vacuum plastic sealing that (2 is small
When) be protected from light be placed at room temperature for process stability investigation
Prepare blood point, 50 μ L absolute ethyl alcohols (contain 0.1g/mLBHT) solution be added dropwise in dried blood spot, take 0min, 15min,
30min, 60min, 90min, 120min blood point carry out LC-MS/MS analyses, the results showed that dried blood spot after sample pre-treatments
Prepare (be added absolute ethyl alcohol (contain 0.1g/mLBHT)) dry in the shade to vacuum plastic sealing (2 hours) be protected from light the process of being placed at room temperature for, sample
It is stable.Data result refers to table 10.
10 dried blood spot of table adds protective agent stability data
A%：Dried blood spot (absolute ethyl alcohol (containing 0.1g/mLBHT) is added) placement process Retinol concentration and 0min retinols
Concentration ratio；
" -- " indicates not generate data.
11 homologous whole blood of embodiment is investigated with retinol content balance in blood point
It is synchronous with the whole blood for preparing this dried blood spot after sample pre-treatments to prepare blood point, carries out LC-MS/MS analyses, examines
Examine retinol content difference.The result shows that homologous whole blood is no apparent poor with retinol content in the fresh blood point handled immediately
It is different.Data result refers to table 11.
11 homologous whole blood of table investigates data with retinol content balance in blood point
X%：Retinol and retinol ratio in homologous whole blood in blood point；A compares Retinol concentration, and B and C compare peak area
Than.
In summary：Present invention application BHT pre-processes blood collecting card, and retinol is tieed up in can effectively protect Peripheral whole blood dried blood spot
It is fixed to keep steady.Minimally invasive, the advantage of micro blood collection and room temperature solid-state transport can be realized to outlying area countryside children vitamin
The assessment of A nutrition conditions, the vitamin A supplement that crowd is lacked for China's vitamin A emphasis is intervened and corresponding agricultural industry rule
It draws and foundation is provided.In world wide, vitamin A deficiency crowd concentrates on the poverty-stricken area in Africa and Southeast Asia, and the present invention is same
Reference can be provided for the nutritional intervention of vitamin A deficiency crowd in world wide and crop varieties selection.
Claims (10)
1. a kind of vitamin A detection dried blood spot reagent preparation box, including antioxidant.
2. the kit of claim 1, wherein the antioxidant is 0.02g/mL vitamin Cs (VC) or 0.1g/mL butyl hydroxyls
The ethanol solution of base toluene (BHT).
3. the kit of claim 2, wherein the antioxidant is the ethanol solution of 0.1g/mL butylated hydroxytoluenes.
4. the kit of any one of claim 1-3 further includes alcohol, filter paper, aluminium foil bag.
5. application of the antioxidant in preparing vitamin A detection dried blood spot.
6. the application of claim 5, wherein the antioxidant is molten for the absolute ethyl alcohol of 0.02g/mL V C or 0.1g/mLBHT
Liquid.
7. the application of claim 6, wherein the antioxidant is the ethanol solution of 0.1g/mL BHT.
8. a kind of non-diagnostic detection method of vitamin A dried blood spot using any one of claim 1-4 kits, including：
(1) blood specimen collection；(2) prepared by dried blood spot；(3) sample detection.
9. the method for claim 8, wherein step (2) include：Massage or hot compress heel or finger tip after with alcohol heel/
Pad of finger sterilizes, and with disposably blood needle being taken to puncture after air-drying, wipes First Blood away light blood of extruding out, uses quantitative capillary
50 μ L are added dropwise to filter paper front in pipe, are added dropwise 50 microlitres of antioxidants on each blood point, filter paper after blood sampling in air from
It so dries at least two hours, the Blood piece after drying is placed in clean aluminium foil bag and vacuumizes preservation.
10. the method for claim 9, wherein step (3) include, a dried blood spot is placed in 2mL centrifuge tubes, is added in 20 μ L
Working solution, 50 μ L deionized waters and the 50 μ L absolute ethyl alcohols containing 0.625mg/mLBHT are marked, acetonitrile is added and is protected from light ultrasonic extraction,
LC-MS/MS is analyzed.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115343379A (en) * | 2022-04-20 | 2022-11-15 | 杭州谱聚医疗科技有限公司 | Preparation method of blank blood matrix and standard substance and detection method of vitamin |
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