CN108802221A - The Sparklet testing method of vitamin A and vitamin E in peripheral blood - Google Patents

The Sparklet testing method of vitamin A and vitamin E in peripheral blood Download PDF

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Publication number
CN108802221A
CN108802221A CN201810603414.1A CN201810603414A CN108802221A CN 108802221 A CN108802221 A CN 108802221A CN 201810603414 A CN201810603414 A CN 201810603414A CN 108802221 A CN108802221 A CN 108802221A
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vitamin
solution
sample
peripheral blood
testing method
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高飞
海云
陈亮
栾羽
张志栋
黄新良
徐可丰
李冰
罗示齐
王万强
黄曙
刘礼欢
周响响
应海媚
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Hangzhou Du An Medical Laboratory Laboratory Co Ltd
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Hangzhou Du An Medical Laboratory Laboratory Co Ltd
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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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid 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
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

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  • Health & Medical Sciences (AREA)
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  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The present invention announces the Sparklet testing method of vitamin A and vitamin E in a peripheral blood, which is characterized in that the pre-treatment including sample to be tested：The addition of tip blood sample is taken to contain inner mark solution, albumen precipitation is completed in vortex concussion, and tertiary butyl methyl ether is added and is vortexed and shakes extraction, and centrifuged supernatant, nitrogen dries up the supernatant at room temperature, and redissolution liquid is added, and is centrifuged after the concussion that is vortexed and obtains sample to be tested；And ultra performance liquid chromatography tandem mass spectrum detection：The rapidly and efficiently detection of vitamin A and vitamin E in micro tip blood sample can be completed in 2 minutes by obtaining the peak area of determinand and corresponding internal standard peak area, Sparklet testing method provided by the invention in the sample to be tested by the detection of ultra performance liquid chromatography tandem mass spectrum.

Description

The Sparklet testing method of vitamin A and vitamin E in peripheral blood
Technical field
The present invention relates to vitamin detection fields, and in particular to vitamin A and vitamin E is micro in a kind of peripheral blood Detection method.
Background technology
It is to maintain normal physiological function and must be obtained from food a kind of micro organic that vitamin, which is humans and animals, Substance plays an important role in human growth, metabolism, growth course, although it and be not involved in composition human body cell, Energy is not provided for human body, but is a kind of organic compound necessary to maintaining health.
Vitamin A (Vitamin A) is also known as retinol, is a kind of liposoluble vitamin, and function is to maintain animal just Normal visual performance safeguards the health of Epithelial cell and promotes the synthesis of immunoglobulin, promotes growth and reproduction.Generally may be used To be absorbed from animal's liver, dairy produce or eggs, the vegetable and fruit rich in carrotene can also be taken in, by closing in vivo At the vitamin A come
Vitamin E (Vitamin E) is also known as tocopherol and a kind of liposoluble vitamin, and function is to promote sex hormone Secretion makes man's sperm motility and quantity increase；So that woman's female hormone concentration is increased, improve fecundity, prevention of miscarriage is gone back It can be used for preventing male sterility, burn, frostbite, capillary hemorrhage, climacteric syndrome, beauty etc..Recently it also sends out Existing vitamin E makes peripheral vasodilation, improves blood circulation, pre- Anti-myopic eye occurrence and development.
Vitamin A and vitamin E deficiency and excess in human body can all lead to different physiological maladies, so to internal two The detection and monitoring of kind vitamine concentration are just particularly important.Currently, in vitamin detection field, vitamin in serum The detection method of A and E mainly has liquid chromatography tandem mass spectrometry LC-MS/MS methods, immune detection, HPLC-UV methods etc., In, liquid chromatography tandem mass spectrometry has the separation method of chromatography to have mass spectrographic specificity identification method again, in small-molecule substance There is great advantage in measurement, have the characteristics that high specificity, accuracy is high, analysis time is short, blood is widely used in by industry The measurement of vitamin A and E in clear.
CN108107133A discloses a kind of " method that Liquid Chromatography-Tandem Mass Spectrometry measures vitamin A and E in serum ", packet Include following steps：1) vitamin A and vitamin E are extracted by sample preparation liquid, the sample sampling after extraction is entered into chromatography matter Spectra system is entered by the initial gross separation of chromatographic column in mass spectrum, qualitative detection vitamin A and vitamin E.2) isotope mark is used Retinol-the d6 and alpha-tocopherol-d6 of note are used as internal standard, prepare standard curve.3) signal strength of detection sample to be tested substitutes into Standard curve obtains the quantitative values of vitamin A and vitamin E.
However in this technical solution：
1, it is the serum for needing venous blood collection to obtain 1mL or so to extract vitamin A and the method for vitamin E, but for For children, especially newborn's venous blood collection with for has certain difficulty, so that the practical application of this method exists Limitation.
Specifically, it when by the way of venous blood collection, needs through disposable blood taking needle and vacuum blood collection tube One milliliter or more of venous blood is extracted, acquisition position multidigit is quiet in the superficial vein of human body table, hand back vein, internal malleolus vein or stock Arteries and veins, children's Chang Cai vena jugularis externa blood (obvious blood vessel).When actually detected, it is also desirable to add separation into venous blood Glue obtains supernatant, is then detected with supernatant, and 1 milliliter of venous blood at most obtains 0.5 milliliter of supernatant, therefore in reality Border is also required to acquire enough blood volumes when taking a blood sample to ensure the progress of detection.
2, although the technical solution compares more previous technology can accomplish spy in Liquid Chromatography-Tandem Mass Spectrometry detecting step Anisotropic raising, but its detection time has no optimization improvement, it is still low so as to cause detection efficiency.
3, needed in the pre-treatment step of the technical solution be added internal standard compound, methanol solution, solution of zinc sulfate, n-hexane, Redissolution liquid, pre-treatment step trouble so that detection efficiency is low are added after nitrogen drying.
To sum up, vitamin detection field still needs a kind of inspection of the vitamin A and vitamin E of efficient high throughput at present Survey method.
Invention content
It is described micro the purpose of the present invention is to provide the Sparklet testing method of vitamin A and vitamin E in a peripheral blood Detection method obtains the blood sample of only a few in such a way that tip acquires Trace Blood, you can efficiently detects vitamin with high throughput The content of A and vitamin E, improve the detection efficiency of vitamin, and the mode of tip blood sampling is especially suitable for children, pregnant woman with And the blood sampling of the crowds such as old man, reduce the sense of discomfort of infant's venous blood collection.
It is described micro the purpose of the present invention is to provide the Sparklet testing method of vitamin A and vitamin E in a peripheral blood The peripheral blood of detection method acquisition testing person, amount for taking blood is less than 0.5 milliliter, and peripheral blood need not be detached, Improve detection efficiency.
The Sparklet testing method is compared traditional Liquid Chromatography-Tandem Mass Spectrometry mode and is improved, before detecting sample Processing step is simpler easily and effectively, tandem mass spectrum method it is efficient, point of vitamin A and E can be completed in 2 minutes Analysis detection, meets the needs of clinical detection.
It is described micro the purpose of the present invention is to provide the Sparklet testing method of vitamin A and vitamin E in a peripheral blood The tandem mass spectrum testing conditions for adding optimal design-aside in detection method using the Sample pretreatment step after improvement, realize high pass Amount, the quantitative detection of high-precision vitamin A and vitamin E and qualitative detection.
In order to realize any of the above goal of the invention, the present invention provides the micro of vitamin A and vitamin E in a peripheral blood Detection method includes the following steps：
S1:The pre-treatment of sample to be tested：
The addition of tip blood sample is taken to contain inner mark solution, albumen precipitation is completed in the concussion that is vortexed, and tertiary butyl first is added
Ether, which is vortexed, shakes extraction, and centrifuged supernatant, and nitrogen dries up the supernatant at room temperature, and is added multiple
Solution obtains sample to be tested after the concussion that is vortexed；And
S2:Ultra performance liquid chromatography tandem mass spectrum detects：
The peak area and correspondence of determinand in the sample to be tested are obtained by the detection of ultra performance liquid chromatography tandem mass spectrum Internal standard peak area.
In some embodiments, including：
S3:The ratio of the peak area of determinand and internal standard peak area in the sample to be tested is substituted into obtain to regression equation The content of sample to be tested, wherein the regression equation reaction normal product peak area and internal standard peak area ratio and concentration of standard solution Between relationship.
In some embodiments, in the step S2, mobile phase A：0.1% aqueous formic acid；Mobile phase B：2mM first Sour ammonium and 0.1% formic acid methanol solution, coutroi velocity 0.5mL/min.
In some embodiments, gradient is set as：
Time (min) Mobility A (%) Mobility B (%)
0.0 12 88
0.1 12 88
0.5 2 98
1.5 2 98
1.51 12 88
2.0 12 88
In some embodiments, ion source：Electric spray ion source, positive ion mode；Spray capillary voltage： 1.0- 2.5KV；Desolvention gas velocity：400-600℃；Desolvention gas velocity：800-1000L/Hr；Ion source temperature：130-150℃； Desolventizing gas pressure：8bar；Collide atmospheric pressure：0.7bar.
In some embodiments, the inner mark solution is the d configured containing useful methanol4The inner mark solution of retinol and/or The d of the configuration containing useful methanol6The inner mark solution of-α-retinols.
In some embodiments, described to redissolve the methanol solution that liquid is 75-85%, the inner mark solution of the d4- retinols A concentration of 500-1000ng/mL, a concentration of 8-12 μ g/mL of the inner mark solution of the d6- α-retinol, the tertiary butyl Methyl ether single extraction.
In some embodiments, the preparation of the regression equation includes the following steps：
A1:The standard items storing solution that vitamin A and/or vitamin E are prepared with methanol, is then added artificial serum, prepares The vitamin A standard solution and/or vitamin E standard solution, Cord blood of various concentration are spare；
A2:The standard solution carries out the operation of step S1 and step S2 as the sample in step S1；
A3：Various criterion product and corresponding internal standard peak area are obtained, it is described with a concentration of X axis of the standard solution The ratio of titer peak area and corresponding internal standard peak area is Y-axis, obtains standard curve, is carried out to the standard curve linear Regression analysis obtains the regression equation by " 1/X " weight factor.
In some embodiments, the concentration control of the vitamin A standard solution is in 20-2000ng/ml, the dimension life The concentration of plain E standard solutions is controlled in 0.2-20 μ g/mL.
In some embodiments, the blood sample is derived from peripheral blood, volume 30-60ul, the ultra high efficiency liquid phase color It composes tandem mass spectrum and completes detection in 2 minutes.
Description of the drawings
Fig. 1 be an embodiment according to the present invention peripheral blood in the Sparklet testing method of vitamin A and vitamin E examining Survey the MRM chromatograms of the vitamin A shown in the process.
Fig. 2 be an embodiment according to the present invention peripheral blood in the Sparklet testing method of vitamin A and vitamin E examining Survey the MRM chromatograms of the vitamin E shown in the process.
Fig. 3 be an embodiment according to the present invention peripheral blood in the Sparklet testing method of vitamin A and vitamin E examining Survey the quantitative criterion working curve of the vitamin A shown in the process.
Fig. 4 be an embodiment according to the present invention peripheral blood in the Sparklet testing method of vitamin A and vitamin E examining Survey the quantitative criterion working curve of the vitamin E shown in the process.
Fig. 5 be an embodiment according to the present invention peripheral blood in the Sparklet testing method of vitamin A and vitamin E examining Survey the chromatogram of the vitamin A shown in the process.
Fig. 6 be an embodiment according to the present invention peripheral blood in the Sparklet testing method of vitamin A and vitamin E examining Survey the chromatogram of the vitamin E shown in the process.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, the every other embodiment that those of ordinary skill in the art are obtained belong to what the present invention protected Range.
It is understood that term " one " is interpreted as " at least one " or " one or more ", i.e., in one embodiment, The quantity of one element can be one, and in a further embodiment, the quantity of the element can be multiple, and term " one " is no It can be interpreted as the limitation to quantity.
Experimental method used in the following example is conventional method unless otherwise specified.
Material, reagent used in the following example etc. are commercially available such as figure specified otherwise.
Present invention seek to address that problem of the existing technology, when improving detection vitamin A in the prior art and vitamin E Need using defect existing for venous blood collection, provide it is a kind of can be in such a way that tip acquires Trace Blood come with high throughput efficiently The method for detecting vitamin A and vitamin E, so that the method for the detection vitamin A and vitamin E can be fitted well Blood sampling for children, especially infant.In addition, the preceding place of the mode of detection vitamin A provided by the invention and vitamin E It is simple and convenient to manage step, tandem mass spectrum testing conditions optimal design-aside, it is more preferable to compare traditional detection efficiency, can be complete in 2 minutes At the qualitative and quantitative detection of vitamin A and vitamin E, detection efficiency is greatly improved.
The present invention provides the Sparklet testing method of vitamin A and vitamin E in a peripheral blood, includes the following steps：
Step S1:The pre-treatment of sample to be tested：
The addition of blood sampling product contains inner mark solution, and albumen precipitation is completed in vortex concussion, and tertiary butyl methyl ether is added and is vortexed and shakes Simultaneously centrifuged supernatant is extracted, nitrogen dries up the supernatant at room temperature, and obtains sample to be tested after redissolution liquid vortex concussion is added.
What is particularly worth mentioning is that in an embodiment of the present invention, the blood sample is peripheral blood, it is only necessary to very micro Blood sample, peripheral blood includes Heel blood, Ear lobe blood and finger tip blood.
In clinical application, the third finger of selection blood sampling object is first sterilized with cotton ball soaked in alcohol, then with disposable blood sampling Pin puncture allows blood to flow out naturally, after gently wiping the first drop away with sterilized cotton ball, by blood collection in 0.5 milliliter of centrifuge tube (EDTAK2 anticoagulant tubes), about 0.2-0.3 milliliters of the blood volume collected at this time, then pushes down site of puncture with dry cotton ball, advises blood sampling pair As pressing 2-3 minutes.
Compare traditional venous blood collection, the mode of tip blood sampling only need against tester it is nameless blood was collected i.e. Can, and need to obtain the nearly serum of 1ml or so when traditional venous blood collection.For children, venous blood is acquired Be have it is certain difficult and also more painful for tester.Since Sparklet testing method provided by the invention only needs Trace Blood, about 50ul are wanted, therefore the Sparklet testing method can be completed by way of tip blood sampling.That is, the step In S1, the blood sample is can be controlled in no more than 50ul, and the volume of the blood sample is selected as 30-60ul.
Specifically, in the step S1, the inner mark solution is selected as containing d4The inner mark solution of retinol and Contain d6The inner mark solution of alpha-tocopherol, it is described to contain d4The inner mark solution of retinol and/or described contain d6Alpha-tocopherol The preparation process of inner mark solution be：D is configured with methanol4A concentration of 500-1000ng/mL of retinol configures d6Retinol Concentration 8-12 μ g/mL, be sealed in low temperature environment after constant volume, wherein the concentration of the internal standard compound is suitable for, this is specific Embodiment, but without limitation.Internal standard of the present invention has with vitamin A (retinol) and vitamin E (alpha-tocopherol) There is similar physicochemical property, the signal in inaccurate factor and the chromatographic mass spectrometry in sample process can be offset and inhibit or increase By force.
In addition, when configuring the inner mark solution, using the methanol of the BHT containing 1g/L, concentration is controlled in 0.5- 1.5g/L。
From the foregoing, it will be observed that the inner mark solution can only include d4Retinol can also only include d6Alpha-tocopherol, or simultaneously Including d4Retinol and d6Alpha-tocopherol, concrete condition is depending on specific experiment.I.e. if in an embodiment of the present invention, only needed When detecting vitamin A, just it not be arranged including d6Alpha-tocopherol inner mark solution.
In one embodiment of this invention, the vortex concussion time in the step S1 controls at 3-5 minutes, specifically For, preferably 4 minutes, when obtaining the supernatant and addition it is described redissolve liquid after centrifugal condition be：4 DEG C of items It is centrifuged 5 minutes under part 12000rpm, the condition of the nitrogen drying is to complete the drying of nitrogen at room temperature, the redissolution liquid selection For the methanol solution of 75-85%, it is preferable that be 80% methanol, it is notable that the selection of these conditions is to be suitable for The optimal selection of the present embodiment, but limited not as special.
The Sample pretreatment step mentioned in the prior art needs that solution of zinc sulfate is added and methanol solution carries out protein Precipitation, in the step S1, the process that inner mark solution and albumen precipitation is added is carried out at the same time, and in addition the prior art is adopted With n-hexane extraction, and the present invention by tertiary butyl methyl ether extracted in the way of, and the people for being familiar with this technology should be understood that Flux and analysis time when the selection of extractant detects follow-up vitamin A and vitamin E influence very great, this hair The bright pre-treatment step in sample to be tested is improved the prior art, so that subsequent high throughput liquid chromatographic detection mistake Journey performance is good.Also, since the sample volume of the blood sample is few, correspondingly, the use examination in the step S1 Dosage is also few, so that the process becomes more simple.
It is stressed again that since the embodiment of the present invention uses micro peripheral blood, the volume of peripheral blood that can be not more than 50ul, Therefore correspondingly, the amount of reagent in the step S1 is reduced, as long as the tertiary butyl methyl ether also carries out single extraction, It is added after redissolving liquid and can be applied to sample introduction without centrifugation.
S2:Ultra performance liquid chromatography tandem mass spectrum detects：
Ultra performance liquid chromatography tandem mass spectrum completes the detection sample to be tested in 2 minutes, obtains the sample to be tested The peak area of middle determinand and corresponding internal standard peak area：
The sample to be tested is entered by the initial gross separation of chromatographic column in mass spectrum, in chromatographic mass spectrometry system further according to matter Spectrum generates different mass-to-charge ratioes and detects in the sample to be tested whether have vitamin A and vitamin E and corresponding internal standard peak.
The present invention is using Waters Acquity UPLC BEH C18 chromatographic columns, the technical parameter of the chromatographic column For 2.1mm x 50mm, 1.7 μm.
Instrumental Analysis liquid contains：
Mobile phase A：0.1% aqueous formic acid；
Mobile phase B：2mM ammonium formates and 0.1% formic acid methanol solution.
Coutroi velocity is：0.5mL/min, column temperature are 38-45 DEG C, sample introduction 5-15 μ L.
In a preferred embodiment, the column temperature is 40 degrees Celsius, 10 μ L of sample introduction.
Liquid chromatogram mobile phase A and Mobile phase B are by setting as shown in Table 1：
Table 1：Table is arranged in the gradient of mobile phase A and Mobile phase B
Time (min) Mobility A (%) Mobility B (%)
0.0 12 88
0.1 12 88
0.5 2 98
1.5 2 98
1.51 12 88
2.0 12 88
By table, it can be seen that, the disengaging time of chromatography described in the embodiment of the present invention only needs control within 2 minutes, Compared with the prior art for 4-5 minutes, its detection efficiency is greatly increased.
Mass spectrum alternative condition：
Ion source：Electric spray ion source (ESI), positive ion mode；
Spray capillary voltage (Capliary)：1.0-2.5KV；
Desolvention gas velocity (Desolvation Temp)：400-600 DEG C, preferably 500 DEG C；
Desolvention gas velocity (Desolvation)：800-1000L/Hr；
Ion source temperature (Source Temp)：130-150℃；
Desolventizing gas pressure (decompression table)：8bar；
Collide atmospheric pressure (decompression table)：0.7bar；
In a preferred embodiment, mass spectrum alternative condition is as follows：
Ion source：Electric spray ion source (ESI), positive ion mode；
Spray capillary voltage (Capliary)：1.1KV；
Desolvention gas velocity (Desolvation Temp)：400-600 DEG C, preferably 500 DEG C；
Desolvention gas velocity (Desolvation)：1000L/Hr；
Ion source temperature (Source Temp)：150℃；
Desolventizing gas pressure (decompression table)：8bar；
Collide atmospheric pressure (decompression table)：0.7bar.
Scan pattern：Multiple-reaction monitoring, wherein MRM：Multiple Reaction Monitor, what is referred to is exactly more reactions Detect ion scan.Specific condition is as shown in table 2：
Table 2：Condition is arranged in vitamin A and vitamin E and corresponding interior target MRM detection ions and collision energy
Simple introduction explanation is carried out to table：
The case where multiple-reaction monitoring ion scan MRM of target quota ion pair, is as follows：
d4The mass-to-charge ratio that retinol quantifies the parent ion of parent ion is 237.2, and the mass-to-charge ratio of corresponding daughter ion is 94.2；
d6The mass-to-charge ratio that alpha-tocopherol quantifies the parent ion of parent ion is 437.4, and the mass-to-charge ratio of corresponding daughter ion is 57.1；
The case where multiple-reaction monitoring ion scan MRM of target quota ion, is as follows：
The mass-to-charge ratio of the quantitative parent ion of retinol-is 269.2, and the mass-to-charge ratio of corresponding quota ion is 93.1；
The mass-to-charge ratio of the quantitative parent ion of alpha-tocopherol-is 431.4, and the mass-to-charge ratio of corresponding quota ion is 165.1；
The mass-to-charge ratio of the qualitative parent ion of retinol-is 269.2, and the mass-to-charge ratio of corresponding qualitative ion is 92.5；
The mass-to-charge ratio of the qualitative parent ion of alpha-tocopherol-is 431.4, and the mass-to-charge ratio of corresponding qualitative ion is 139.6.
It is understood that the ultra performance liquid chromatography tandem mass spectrum is provided solely for a specific performance, such as Fig. 1 And Fig. 2, under this condition, the MRM chromatograms of the vitamin A and the vitamin E are demonstrated, but without limitation.
It is noted that in an embodiment of the present invention, the chromatographic mass spectrometry system is in detection vitamin A and Wei Sheng When plain E between two needles detection sample without waiting, that is, shift to an earlier date pipette samples in injector, when to further save detection Between.
Step S3:Quantitative determination：
The ratio of the peak area of determinand and internal standard peak area in the sample to be tested is substituted into obtain to be measured to regression equation The content of sample, wherein between the regression equation reaction normal product peak area and internal standard peak area ratio and concentration of standard solution Relationship.
In the step S3, once obtain the area of determinand and internal standard peak area in regression equation and sample to be tested Ratio, you can the content for obtaining the sample to be tested is quickly compared, wherein the sample to be tested includes vitamin A or vitamin E。
As shown in Figure 5 and Figure 6, the chromatogram of vitamin A and vitamin E is demonstrated in peripheral blood, to obtain dimension life The area at internal standard peak in plain A and the vitamin E, and be updated in the regression equation and obtain the vitamin A and/or institute State the content of vitamin E.
However in the step S3, the regression equation can be pre-existing, can also be that weight is freshly prepd, The preparation process of the regression equation is as follows：
A1:The standard items storing solution that vitamin A and/or vitamin E are prepared with methanol, is then added artificial serum, prepares The standard solution of various concentration, subzero 10-30 DEG C saves backup.
The concentration of the wherein described vitamin A standard solution is controlled in 20-2000ng/ml, the vitamin E standard items The concentration control of solution is in 0.2-20 μ g/mL, statistics indicate that the concentration when the standard solution controls within the scope of this When the performance of its linear relationship it is good, wherein the methanol is the methanol configuration containing BHT (1g/L), a concentration of 0.5-1.5g/L.
A2:The standard solution carries out the operation of step S1 and step S2 as the sample in step S1, at this point, The standard solution and the sole difference of tip blood sample are only in that the concentration of the standard solution is known, and last The concentration of vitamin A and vitamin E is unknown in tip blood sample.
A3：The corresponding internal standard peak area of standard items peak area for obtaining various concentration, with the concentration of the standard solution For X-axis, the ratio of the standard items peak area and corresponding internal standard peak area is Y-axis, obtains standard curve, to standard song Line carries out linear regression analysis, and the equation of linear regression is obtained by " 1/X " weight factor.
As shown in Figure 3 and Figure 4, the quantitative criterion working curve of the vitamin A and the vitamin E is demonstrated, and is such as schemed It is seen that vitamin A standard solution linear relationship within the scope of 20-2000ng/ml is good, the vitamin E mark Quasi- product solution interior linear relationship in the range of 0.2-20 μ g/mL is good.
In addition, if a. cannot be detected and must tip blood sample be placed in 2-8 DEG C and be kept in dark place immediately；
B. the QC solution prepared, after packing, -80 DEG C of long-term preservations.
C. standard reserving solution and inner mark solution use the methanol containing BHT (1g/L) to configure, and it is spare to be stored in -20 DEG C of refrigerators.
D. standard solution and sample preparation are operated in darkroom.
It is illustrated by taking specific embodiment as an example below, the experimentation of embodiment is as follows：
Embodiment one：
Experiment reagent：Sample preparation liquid, standard items store liquid, Instrumental Analysis liquid.
Sample preparation liquid includes：
Inner mark solution：The d configured with the methanol containing BHT1g/L4The inner mark solution of retinol, concentration 850ng/ mL；The d configured with the methanol containing BHT1g/L6The inner mark solution of alpha-tocopherol, 10 μ g/mL of concentration；
Tertiary butyl methyl ether：Volume 500mL；
Redissolve liquid：80% methanol；
Standard items store liquid：
The vitamin A standard items storing solution that methanol to contain BHT (1g/L) is prepared, a concentration of 15,100 after dilution, 150,200,500,1000,1200,1500,2000ng/ml；
The vitamin E standard items storing solution that methanol to contain BHT (1g/L) is prepared, a concentration of 0.2,0.5 after dilution, 0.85,1,1.5,5,10,12.5,16,20 μ g/mL；
Instrumental Analysis liquid：
Mobile phase A:0.1% aqueous formic acid；
Mobile phase B：2mM ammonium formates and 0.1% formic acid methanol solution.
Laboratory apparatus：Waters Acquity UPLC BEH C18 chromatographic columns, centrifuge tube, centrifugation apparatus, 96 orifice plates
Experimental procedure：
1. precision measures standard solution, quality-control sample, peripheral blood 30-50 μ L are placed in clean centrifuge tube；It is added 100 μ L's Inner mark solution, the concussion mixing that is vortexed 4min；Then 500 μ L tertiary butyl methyl ethers are added and are vortexed and shake 4min, 4 DEG C of condition 12000rpm Centrifuge 5min；300 μ L of Aspirate supernatant are set in clean 96 orifice plates；Nitrogen dries up at room temperature；The redissolution of 100 μ L, 80% methanol is added, Be vortexed concussion 4min, you can sample introduction；
It is arranged shown in the condition Tables 1 and 2 as above of chromatographic mass spectrometry instrument, obtains corresponding typical spectrogram, obtain corresponding The area of the peak area and corresponding internal standard peak of vitamin A and E, substitutes into regression equation and vitamin A and/or dimension life is calculated The content of plain E.
Laboratory report：In the embodiment, vitamin A and E contents are respectively 535ng/mL and 10.2 μ g/ in peripheral blood mL。
Embodiment two, with embodiment one difference lies in：
Mass Spectrometry Conditions：Electric spray ion source (ESI), positive ion mode；
Spray capillary voltage (Capliary)：1.0KV；
Desolvention gas velocity (Desolvation Temp)：650℃；
Desolvention gas velocity (Desolvation)：800L/Hr；
Ion source temperature (Source Temp)：125℃；
In this embodiment, actually detected result does not have significant change.
Embodiment three, with embodiment one difference lies in：
Sampling volume improves, and actually detected result is also without significant change.
The present invention is not limited to above-mentioned preferred forms, anyone can show that other are various under the inspiration of the present invention The product of form, however, make any variation in its shape or structure, it is every that there is skill identical or similar to the present application Art scheme, is within the scope of the present invention.

Claims (10)

1. the Sparklet testing method of vitamin A and vitamin E in peripheral blood, which is characterized in that include the following steps：
S1:The pre-treatment of sample to be tested：
The addition of tip blood sample is taken to contain inner mark solution, albumen precipitation is completed in vortex concussion, and tertiary butyl methyl ether is added and is vortexed and shakes Extraction, and centrifuged supernatant, nitrogen dries up the supernatant at room temperature, and redissolution liquid is added and is vortexed after concussion, obtains waiting for test sample This；And
S2:Ultra performance liquid chromatography tandem mass spectrum detects：
The peak area of determinand and corresponding internal standard in the sample to be tested are obtained by the detection of ultra performance liquid chromatography tandem mass spectrum Peak area.
2. the Sparklet testing method of vitamin A and vitamin E in peripheral blood according to claim 1, which is characterized in that packet It includes：
S3:The ratio of the peak area of determinand and internal standard peak area in the sample to be tested is substituted into obtain to be measured to regression equation The content of sample, wherein between the regression equation reaction normal product peak area and internal standard peak area ratio and concentration of standard solution Relationship.
3. the Sparklet testing method of vitamin A and vitamin E in peripheral blood according to claim 2, which is characterized in that institute It states in step S2, mobile phase A：0.1% aqueous formic acid；Mobile phase B：2mM ammonium formates and 0.1% formic acid methanol solution, control Flow velocity processed is 0.5mL/min.
4. the Sparklet testing method of vitamin A and vitamin E in peripheral blood according to claim 3, which is characterized in that ladder Degree is set as：
Time (min) Mobility A (%) Mobility B (%) 0.0 12 88 0.1 12 88 0.5 2 98 1.5 2 98 1.51 12 88 2.0 12 88
5. the Sparklet testing method of vitamin A and vitamin E in peripheral blood according to claim 4, which is characterized in that from Component：Electric spray ion source, positive ion mode；Spray capillary voltage：1.0-2.5KV；Desolvention gas velocity：400-600℃； Desolvention gas velocity：800-1000L/Hr；Ion source temperature：130-150℃；Desolventizing gas pressure：8bar；Collide atmospheric pressure： 0.7bar。
6. according to the Sparklet testing method of vitamin A and vitamin E in any peripheral blood of claim 1 to 5, feature It is, the inner mark solution is the d configured containing useful methanol4The inner mark solution of retinol and/or the d of the configuration containing useful methanol6- The inner mark solution of α-retinol.
7. the Sparklet testing method of vitamin A and vitamin E in peripheral blood according to claim 6, which is characterized in that institute State the methanol solution for redissolving that liquid is 75%-85%, the d4A concentration of 500-1000ng/mL of the inner mark solution of retinol, institute State d6A concentration of 8-12 μ g/mL of the inner mark solution of-α-retinols, the tertiary butyl methyl ether single extraction.
8. according to the Sparklet testing method of vitamin A and vitamin E in any peripheral blood of claim 2 to 5, feature It is, the preparation of the regression equation includes the following steps：
A1:Then artificial serum is added in the standard items storing solution that vitamin A and/or vitamin E are prepared with methanol, prepare different The vitamin A standard solution and/or vitamin E standard solution, Cord blood of concentration are spare；
A2:The standard solution carries out the operation of step S1 and step S2 as the sample in step S1；
A3：The corresponding internal standard peak area of various criterion product solution is obtained, with a concentration of X-axis of the standard solution, the mark The ratio of quasi- product peak area and corresponding internal standard peak area is Y-axis, obtains standard curve, is linearly returned to the standard curve Return analysis, the regression equation is obtained by " 1/X " weight factor.
9. the Sparklet testing method of vitamin A and vitamin E in peripheral blood according to claim 8, which is characterized in that institute The concentration control of vitamin A standard solution is stated in 20-2000ng/ml, the concentration control of the vitamin E standard solution exists In 0.2-20 μ g/mL.
10. special according to the Sparklet testing method of vitamin A and vitamin E in any peripheral blood of claim 1 to 5 Sign is that the volume of the peripheral blood is 30-60ul, and the ultra performance liquid chromatography tandem mass spectrum completes detection in 2 minutes.
CN201810603414.1A 2018-06-12 2018-06-12 The Sparklet testing method of vitamin A and vitamin E in peripheral blood Pending CN108802221A (en)

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CN114778727A (en) * 2022-04-19 2022-07-22 天津国科医工科技发展有限公司 Method for detecting fat-soluble vitamins in cryopreserved breast milk
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Application publication date: 20181113