CN108645945A - A kind of while 5 kinds of liposoluble vitamins of detection methods and application - Google Patents
A kind of while 5 kinds of liposoluble vitamins of detection methods and application Download PDFInfo
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Abstract
The present invention provides a kind of while 5 kinds of liposoluble vitamins of detection methods and applications, the described method comprises the following steps：(1) pretreated sample is analyzed using liquid chromatography mass combined instrument；(2) according to standard curve, the concentration of 5 kinds of liposoluble vitamins in sample to be tested is calculated；Wherein, the liposoluble vitamin includes retinol, alpha tocopherol, β tocopherols, gama tocopherol and methyltocol, chromatographic column in the liquid chromatography mass combined instrument uses gradient elution mode, the mobile phase of the gradient elution to use ammonium formate acetonitrile mixture.The all technicals such as the rate of recovery, detection limit and the precision of the method for the present invention meet the requirements, using isotopic label as internal standard so that the identification high specificity of target compound, high sensitivity, accuracy are good, and analysis time is short, the method reproducibility of the present invention is good, and error is small.
Description
Technical field
The invention belongs to technical field of analysis and detection, are related to a kind of while 5 kinds of liposoluble vitamins of detection methods and answer
With.
Background technology
Vitamin A and vitamin E are liposoluble vitamins needed by human, are mainly derived from food, vitamin A and Wei Sheng
The shortage of plain E can impact the growth and development of human body.Vitamin A is mainly retinol, synthesis visual purple is participated in, to human body
Growth, breeding, hematopoiesis and the multiple-effect sexual behaviour of the different physiological roles such as immune have effect (Albarhani A A,
Collier F,Greaves R F,et al.Vitamins D and A can be successfully measured by
LC-MS/MS in cord blood diluted plasma.Clinical Biochemistry,2015,48:1105；
Villamor E,Fawzi W W.Effects of vitamin A supplementation on immune responses
and correlation with clinical outcomes.Clinical Microbiology Reviews,2005,18:
446.) A that, is deficient in vitamin may lead to child's blindness, measles and diarrhea, while (Wong A related with the high Pregnancy death of pregnant woman
Y,Chan E W,Chui Cs.The phenomenon of micronutrient deficiency among children
in China:a systematic review of the literature.Public Health Nutrition,2013,
17:2605.).Vitamin E is mainly tocopherol, including tetra- kinds of forms of α, β, γ, δ, wherein alpha-tocopherol content highest, is had
Effect (the Beck M A.Selenium and vitamin E status of anti-aging:impact on viral
pathogenicity.Journal of Nutrition,2007,137:1338-1340.), recent studies indicate that, dimension life
Plain E can prevent the formation of active oxygen when fat oxidation, have and reduce the DNA damage as caused by active oxygen, reduce pathogenesis of cancer
Effect (Zhang Y Y, Lu L, Abliz G, et al.Serum carotenoid, the retinol and of risk
tocopherol concentrations and risk of cervical cancer among Chinese
women.Asian Pacific Journal of Cancer Prevention,2015,16:2981-2986.)。
Currently, the assay method of vitamin A and vitamin E is mainly high performance liquid chromatography (HPLC) and liquid chromatogram-
Second order ms are combined method (LC-MS/MS), for detecting the life of the dimension in formulated infant milk, strengthening model, milk, cheese and food
Cellulose content (Woollard D C, Anja B, Harvey I, et al.Determination of vitamin A and
vitamin E esters in infant formulae and fortified milk powders by HPLC:Use of
internal standardisation.Food Chemistry,2016,197:457；Elsayeds M,Hagrass A B,
Askar A A,et al.Simultaneous determination of retinolα-tocopherol andβ-
carotene contents using HPLC method in some dairy products marketed in Cairo
area.Egyptian Journal of Dairy Science,2012,40:149；Fat soluble vitamin
detection in Food by LC/MS/MS.Stephen Lock,ABsCIEX,Europe.)。
Measure vitamin A in serum, the content of E is rarely reported.The content of vitamin A and vitamin E in serum is low, and
Easily be illuminated by the light, the influence of air and temperature and oxygenolysis occurs, it is therefore desirable to special extraction and measure analysis method.
Albahrani etc. uses the dimension life in the triplex tandem level four bars liquid chromatography tandem mass spectrometry detection human serum of two kinds of models
Plain A, D and E (Albahrani A A, Rotarou V, Roche P J, et al.A simultaneous quantitative
method for vitamins A,D and E in human serum using liquid chromatography-
tandem mass spectrometry.Journal of Steroid Biochemistry&Molecular Biology,
2016,159:41.), however the pretreatment process of this method has used a large amount of organic solvents, including 200 μ L methanol, 1.5mL are just
Hexane and 250 μ L methanol, and the separate run times of detection method are 42-45 minutes, it is time-consuming longer, limit answering for this method
Use range.Sadilek etc. is using the vitamin A and vitamin E in column switching high performance liquid chromatography detection human plasma
(Sadilek P,Satinsky D,Otapka M,et al.Rapid and simple determination of
Vitamin A and Vitamin E in human plasma by column-switching high-performance
liquid chromatography.Current Analytical Chemistry,2009,5:311.), the mode of column switching
Pretreatment process is simplified, but it is higher to instrument requirements, and it is 15 points that this method, which needs a large amount of blood plasma, separate run times,
Clock, time-consuming longer, the specificity and sensitivity of high performance liquid chromatography cannot be satisfied clinical detection requirement.It is very important, this
Two methods only have detected alpha-tocopherol for vitamin E, cannot fully and effectively in appraiser's body vitamin E nutritive water
It is flat.
Therefore it provides a kind of analysis efficiency is high, solvent consumption is few, the inspection of the accurate quantitative analysis of high sensitivity vitamin A, E
Survey method is of great significance in technical field of analysis and detection.
Invention content
In view of the deficiencies of the prior art, it the present invention provides a kind of while 5 kinds of liposoluble vitamins of detection methods and answers
With the method is using the content of Liquid Chromatography-Mass Spectrometry detection vitamin A and vitamin E, analysis efficiency height, solvent
It consumes less, high sensitivity, be suitable for larger scale clinical detection, clinical prevention, diagnosis for liposoluble vitamin relevant disease
Have great importance with treatment, the Nutritional Status for fully assessing human body has the function of not replacing.
For this purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides it is a kind of and meanwhile detect 5 kinds of liposoluble vitamins method, the method includes with
Lower step：
(1) pretreated sample is analyzed using liquid chromatograph-mass spectrometer；
(2) according to standard curve, the concentration of 5 kinds of liposoluble vitamins in sample to be tested is calculated；
Wherein, the liposoluble vitamin includes retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and δ-fertility
Phenol；
Chromatographic column in the liquid chromatograph-mass spectrometer uses gradient elution mode, the flowing of the gradient elution
Mutually use ammonium formate-acetonitrile mixture.
In the present invention, the separation and detection of vitamin A and vitamin E are carried out using Liquid Chromatography-Mass Spectrometry, with liquid
Phase chromatography is compared, sensitivity higher, and interference is less, is conducive to the separation and detection of many kinds of substance, wherein liquid chromatogram uses
Ammonium formate-acetonitrile mixture carries out gradient elution as mobile phase, with the structure of liposoluble vitamin to be separated, polarity, molten
Solution property matching degree is high, is advantageously implemented the separation of retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol.
Preferably, it is 5 μ m, 2.1 μ m 50mm that the chromatographic column, which uses Shimadzu C18 columns, specification,.
Preferably, the temperature of the chromatographic column is 24-26 DEG C, such as can be 24 DEG C, 25 DEG C or 26 DEG C, preferably 25
℃。
Preferably, the time of the gradient elution is 5-7min, such as can be 5min, 6min or 7min, preferably
6min。
Preferably, the volume ratio of ammonium formate and acetonitrile is 1 during the gradient elution:(1-49), such as can be 1:
1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17、1:
18、1:19、1:20、1:21、1:22、1:23、1:24、1:25、1:26、1:27、1:28、1:29、1:30、1:31、1:32、1:
33、1:34、1:35、1:36、1:37、1:38、1:39、1:40、1:41、1:42、1:43、1:44、1:45、1:46、1:47、1:48
Or 1:49.
In the present invention, distribution coefficient of the different component in liquid chromatogram is different, and the delivery time in the chromatography column is not yet
Together, it adjusts the ingredient of mobile phase at any time by gradient elution, the delivery time of different component can be adjusted, realize that each group divides it
Between better separating effect.
In the present invention, the condition of gradient elution is specially：In 0-0.3min, flowing phased constant be 50% ammonium formate and
50% acetonitrile；In 0.3-0.6min, the percent by volume of ammonium formate is continuously reduced by 50% to 5%, the volume basis of acetonitrile
Than being continuously enlarged to 95% by 50%；In 0.6-1.8min, flowing phased constant is 5% ammonium formate and 95% acetonitrile；1.8-
In 2min, the percent by volume of ammonium formate is continuously reduced by 5% to 2%, the percent by volume of acetonitrile by 95% continuously enlarge to
98%；In 2-5min, flowing phased constant is 2% ammonium formate and 98% acetonitrile；In 5-5.2min, the volume basis of ammonium formate
Than being continuously enlarged to 50% by 2%, the percent by volume of acetonitrile is continuously reduced by 98% to 50%；In 5.2-6min, flowing
Phased constant is 50% ammonium formate and 50% acetonitrile.
Preferably, the flow velocity of the mobile phase is 0.4-0.6mL/min, such as can be 0.4mL/min, 0.5mL/min
Or 0.6mL/min, preferably 0.5mL/min.
Preferably, the sample size of the chromatographic column is 6-8 μ L, such as can be 6 μ L, 7 μ L or 8 μ L, preferably 7 μ L.
Preferably, the mass spectrum in step (1) described liquid chromatograph-mass spectrometer uses multiple-reaction monitoring pattern.
Preferably, the mass spectrographic gas curtain air pressure be 32-38psi, such as can be 32psi, 33psi, 34psi,
35psi, 36psi, 37psi or 38psi, preferably 35psi.
Preferably, the mass spectrographic collision air pressure is 5-10psi, such as can be 5psi, 6psi, 7psi, 8psi, 9psi
Or 10psi, preferably 8psi.
Preferably, the mass spectrographic ion source spray voltage be 5200-5800V, such as can be 5200V, 5300V,
5400V, 5500V, 5600V, 5700V or 5800V, preferably 5500V.
Preferably, the mass spectrographic ion source spray pressure be 45-55psi, such as can be 45psi, 46psi,
47psi, 48psi, 49psi, 50psi, 51psi, 52psi, 53psi, 54psi or 55psi, preferably 50psi.
Preferably, the mass spectrographic ion source auxiliary heating air pressure is 45-55psi, for example, can be 45psi, 46psi,
47psi, 48psi, 49psi, 50psi, 51psi, 52psi, 53psi, 54psi or 55psi, preferably 50psi.
Preferably, the mass spectrographic heating temperature is 500-600 DEG C, for example, can be 500 DEG C, 510 DEG C, 520 DEG C, 530
DEG C, 540 DEG C, 550 DEG C, 560 DEG C, 570 DEG C, 580 DEG C, 590 DEG C or 600 DEG C, preferably 550 DEG C.
Preferably, the mass spectrographic entrance potential is 8-12V, such as can be 8V, 9V, 10V, 11V or 12V, preferably
10V。
Preferably, step (1) pretreatment specifically includes following steps：
Sample to be tested is added in internal standard working solution by (1 '), and organic solvent is added after dilution, and isolating protein is removed in centrifugation；
(2 ') extractant, centrifuging and taking supernatant is added；
(3 ') dry up step (2 ') the supernatant nitrogen, are added and redissolve solution, and mixing centrifugation, obtained supernatant is i.e.
For pretreated sample.
In the present invention, sample pretreatment process significantly reduces the usage amount of organic solvent, and process is quick and easy, cost
It is low, small to human body murder by poisoning.
Preferably, step (1 ') the internal standard working solution is the mixed liquor of five deuterated retinols and six deuterated alpha-tocopherols.
Preferably, a concentration of 0.1-0.15 μ g/mL of described five deuterated retinols, such as can be 0.1 μ g/mL, 0.11 μ
G/mL, 0.12 μ g/mL, 0.125 μ g/mL, 0.13 μ g/mL, 0.14 μ g/mL or 0.15 μ g/mL, preferably 0.125 μ g/mL.
Preferably, a concentration of 1-1.5 μ g/mL of described six deuterated alpha-tocopherols, such as can be 1 μ g/mL, 1.1 μ g/
ML, 1.2 μ g/mL, 1.25 μ g/mL, 1.3 μ g/mL, 1.4 μ g/mL or 1.5 μ g/mL, preferably 1.25 μ g/mL.
Preferably, step (the 1 ') organic solvent includes any one in ethyl alcohol, methanol or acetonitrile or at least two
Combination.
Preferably, step (the 2 ') extractant is n-hexane.
Preferably, the formulating method of step (2) described standard curve includes the following steps：
(1 ") standard items of configuration at least various concentration known to 7 parts；
(2 ") it is analyzed using liquid chromatograph-mass spectrometer；
(3 "), with a concentration of abscissa of analyte, draw standard curve using the area of chromatographic peak as ordinate.
Preferably, step (the 1 ") standard items are retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and δ-fertility
The mixed liquor of phenol.
Preferably, the concentration ratio of the retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol is 1:
50:50:10:50。
As optimal technical scheme, the present invention provides method that is a kind of while detecting 5 kinds of liposoluble vitamins, the sides
Method includes the following steps：
(1) standard curve is formulated：The standard items of configuration at least various concentration known to 7 parts, retinol, α-life in standard items
The concentration ratio for educating phenol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol is 1:50:50:10:50, using liquid phase chromatogram-mass spectrometry combination
It is analyzed with instrument, using the area of chromatographic peak as ordinate, with a concentration of abscissa of analyte, draws standard curve；
(2) sample pretreatment：It will include the six deuterated α-fertility of five deuterated retinols of 0.1-0.15 μ g/mL and 1-1.5 μ g/mL
Sample to be tested is added in the internal standard working solution of phenol, and organic solvent is added after dilution, and isolating protein is removed in centrifugation, and extractant, centrifugation is added
Supernatant is taken, the supernatant nitrogen is dried up, is added and redissolves solution, mixing centrifuges, and obtained supernatant is as pretreated
Sample；
(3) liquid chromatography-mass spectrometry：Pretreated sample is divided using liquid chromatograph-mass spectrometer
It analyses, it is 5 μ m, 2.1 μ m 50mm, chromatography that the chromatographic column in the liquid chromatograph-mass spectrometer, which uses Shimadzu C18 columns, specification,
The temperature of column is 24-26 DEG C, and chromatographic column uses gradient elution mode, and the mobile phase of gradient elution is using ammonium formate-acetonitrile mixing
Liquid, time of gradient elution are 5-7min, and the volume ratio of ammonium formate and acetonitrile is 1 during gradient elution:(1-49), mobile phase
Flow velocity be 0.4-0.6mL/min, the sample size of chromatographic column is 6-8 μ L, and the mass spectrum in the liquid chromatograph-mass spectrometer is adopted
With multiple-reaction monitoring pattern, mass spectrographic gas curtain air pressure is 32-38psi, and mass spectrographic collision air pressure is 5-10psi, mass spectrographic ion
Source spray voltage is 5200-5800V, and mass spectrographic ion source spray pressure is 45-55psi, mass spectrographic ion source auxiliary heating gas
Pressure is 45-55psi, and mass spectrographic heating temperature is 500-600 DEG C, and mass spectrographic entrance potential is 8-12V；
(4) concentration calculates：According to standard curve, the concentration of 5 kinds of liposoluble vitamins in sample to be tested is calculated.
In the present invention, liquid chromatogram and mass spectrographic various conditions cooperate, and realize retinol, alpha-tocopherol, β-life
Educate the separation of phenol, Gamma-Tocopherol and Delta-Tocopherol and quantitative detection.
Second aspect, the present invention provides a kind of methods as described in relation to the first aspect in detection serum and/or food
The application of the content of retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol.
Compared with prior art, the present invention has the advantages that：
(1) the method for the present invention can detect vitamin A in blood (retinol) and vitamin E (alpha-tocopherol, β-life simultaneously
Educate phenol, Gamma-Tocopherol and Delta-Tocopherol), using isotopic label as internal standard so that the identification high specificity of target compound,
High sensitivity, accuracy are good, and analysis time is short；
(2) rate of recovery of the method for the present invention is higher than 90%, and precision is higher than 0.85%, and all technical conforms to
It asks, reproducibility is good, and error is small.
Description of the drawings
Fig. 1 be serum sample in retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol, Delta-Tocopherol, five it is deuterated regard Huang
The total ion chromatogram of alcohol and six deuterated alpha-tocopherols.
Specific implementation mode
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, according to technology or condition described in document in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
The configuration and calibration of 1 solution of embodiment
(1) preparation of standard items
Take the alpha-tocopherol of 200 a concentration of 5mg/mL of μ L, the betatocopherol of 20 a concentration of 50mg/mL of μ L, 200 μ L a concentration of
The BHT chromatography methanol of the Gamma-Tocopherol of 1mg/mL, the Delta-Tocopherol of 20 a concentration of 50mg/mL of μ L and 560 μ L a concentration of 0.1%,
It is uniformly mixed, obtains 1mg/mL vitamin E standard items；
Take the retinol of 50 a concentration of 1mg/mL of μ L, the vitamin E standard items of 500 a concentration of 1mg/mL of μ L and 450 μ L dense
Degree is 0.1% BHT chromatography methanol, and vortex mixed is uniform, is obtained containing 50 μ g/mL vitamin As and 500 μ g/mL vitamin Es
Hybrid standard product.
- 80 DEG C be protected from light under the conditions of preserve 3 months, avoid multigelation.
(2) calibration of standard items
The hybrid standard product that 20 μ L contain 50 μ g/mL vitamin As and 500 μ g/mL vitamin Es are drawn, 5mL volumetric flasks are placed in
In, after absolute ethyl alcohol constant volume, standard concentration is demarcated with ultraviolet specrophotometer, respectively in retinol, alpha-tocopherol, β-life
It educates and measures its UV absorption under the UV absorption wavelength of phenol, Gamma-Tocopherol and Delta-Tocopherol, calculated according to formula (1) and (2)
The actual concentrations of standard items, whole process are protected from light operation, wherein C is the actual concentrations of standard items to be calibrated, unit g/
100mL, A are the absorbance values of the standard items measured under UV absorption wavelength, and E is that 1% standard of sample to be tested compares extinction
Coefficient.
C=A/E × extension rate (1)
Extension rate=10000 × 5mL/20 μ L (2)
(3) dilution of standard items
Standard items are subjected to gradient dilution using 0.1%BHT methanol solutions, it is respectively 0.5 μ g/mL, 1 μ g/ to obtain concentration
The standard solution of mL, 2 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL and 50 μ g/mL.- 80 DEG C be protected from light under the conditions of preserve 3
Month, avoid multigelation.
(4) configuration of internal standard working solution
Five deuterated retinols of 2mg accurately are weighed, is placed in 10mL volumetric flasks, is settled to 10mL with absolute ethyl alcohol, obtains 200
Five deuterated retinol solutions of μ g/mL；Six deuterated alpha-tocopherols of 2.5mg accurately are weighed, is placed in 5mL volumetric flasks, uses absolute ethyl alcohol
It is settled to 5mL, obtains 500 μ g/mL, six deuterated alpha-tocopherol solution.Scaling method is identical as the scaling method of standard items.
200 μ g/mL, five deuterated retinol solutions are diluted to 0.25 μ g/mL with serum, dilute 500 μ g/mL, six deuteriums with serum
For alpha-tocopherol solution to 2.5 μ g/mL, the two equal proportion is mixed, obtains containing 0.125 μ g/mL, five deuterated retinols and 1.25 μ
The internal standard working solution of six deuterated alpha-tocopherols of g/mL.- 80 DEG C be protected from light under the conditions of preserve 3 months, avoid multigelation.
2 sample pretreatment of embodiment
It will be added comprising the internal standard working solution of six deuterated alpha-tocopherol of 0.125 μ g/mL, five deuterated retinols and 1.25 μ g/mL
After sample to be tested, pure water or aqueous formic acid dilution, ethyl alcohol, methanol or acetonitrile solution, the removal of vortex mixing high speed centrifugation is added
N-hexane extraction is added in protein, and vortex mixing centrifuging and taking supernatant dries up the supernatant nitrogen at room, is added and redissolves
Solution, the centrifugation of vortex mixing, obtained supernatant is pretreated sample.
3 liquid chromatography-mass spectrometry of embodiment
The pretreated samples of 7 μ L are added in the sample injection bottle of liquid chromatograph-mass spectrometer, chromatographic column uses Shimadzu C18
Column, specification are 5 μ m, 2.1 μ m 50mm, and the temperature of chromatographic column is 25 DEG C, and chromatographic column uses gradient elution mode, gradient elution
Mobile phase to use ammonium formate-acetonitrile mixture, the flow velocity of mobile phase be 0.5mL/min, the time of gradient elution is 6min,
In, the condition of gradient elution is as shown in table 1, specially：In 0-0.3min, flowing phased constant is 50% ammonium formate and 50% second
Nitrile；In 0.3-0.6min, the percent by volume of ammonium formate is continuously reduced by 50% to 5%, the percent by volume of acetonitrile by
50% continuously enlarges to 95%；In 0.6-1.8min, flowing phased constant is 5% ammonium formate and 95% acetonitrile；1.8-2min
Interior, the percent by volume of ammonium formate is continuously reduced by 5% to 2%, and the percent by volume of acetonitrile is continuously enlarged by 95% to 98%；
In 2-5min, flowing phased constant is 2% ammonium formate and 98% acetonitrile；In 5-5.2min, the percent by volume of ammonium formate by
2% continuously enlarges to 50%, and the percent by volume of acetonitrile is continuously reduced by 98% to 50%；In 5.2-6min, mobile phase is permanent
It is set to 50% ammonium formate and 50% acetonitrile.
1 condition of gradient elution of table
Using multiple-reaction monitoring pattern (MRM), mass spectrographic gas curtain air pressure is mass spectrum in liquid chromatograph-mass spectrometer
35psi, mass spectrographic collision air pressure are 8psi, and mass spectrographic ion source spray voltage is 5500V, and mass spectrographic ion source spray pressure is
50psi, mass spectrographic ion source auxiliary heating air pressure is 50psi, and mass spectrographic heating temperature is 550 DEG C, and mass spectrographic entrance potential is
10V, the parameter and appearance time of multiple-reaction monitoring pattern are as shown in table 2.
The parameter and appearance time of 2 multiple-reaction monitoring pattern of table
4 linear relationship of embodiment and quantitative limit
It is respectively 0.5 μ g/mL, 1 μ g/mL, 2 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL and 50 μ g/mL to concentration
Standard solution carries out liquid chromatography-mass spectrometry, using chromatographic peak area as ordinate, with a concentration of horizontal seat of analyte
Mark, draws standard items curve, and linear weight takes 1/x, the results are shown in Table 3.
The equation of linear regression and related coefficient of 3 five kinds of analytes of table
| Object | Regression equation | R |
| Retinol | Y=3.45129x+-0.00506 | 0.999 |
| Alpha-tocopherol | Y=0.17573x+-0.00284 | 0.999 |
| Betatocopherol | Y=0.05136x+0.01238 | 0.999 |
| Gamma-Tocopherol | Y=0.32888x+0.01578 | 0.996 |
| Delta-Tocopherol | Y=0.05501x+0.02208 | 0.993 |
The result shows that retinol, alpha-tocopherol, betatocopherol, the concentration of Gamma-Tocopherol and Delta-Tocopherol are corresponding
Chromatographic peak area is in good linear relationship, and the obtained range of linearity is identical with clinical Reportable range：The linear model of retinol
It encloses and clinical Reportable range is 0.05-5 μ g/mL, the range of linearity of Gamma-Tocopherol and clinical Reportable range are 0.1-10 μ
G/mL, alpha-tocopherol, the range of linearity of betatocopherol and Delta-Tocopherol and clinical Reportable range are 0.5-50 μ g/mL.
4 rate of recovery of embodiment and precision
Vitamin A and vitamin E internal standard working solution are taken respectively, and the sample that 3 kinds of concentration is prepared carries out sample recovery rate
And Precision Experiment.After sample is pre-processed, liquid chromatography-mass spectrometry is carried out, replicate analysis measures 6 batches, dimension
The rate of recovery and precision of raw element A and vitamin E are as shown in table 4.
The recovery of standard addition and precision of 4 internal standard working solution of table
Average recovery rate of the vitamin A in 3 concentration ranges is 93.3-96.2%, precision 1.13-2.41%；
Average recovery rate in 3 concentration ranges of vitamin is 97.6-106.8%, precision 0.89-2.56%.
Vitamin A (retinol) and vitamin E (alpha-tocopherol, betatocopherol, Gamma-Tocopherol and δ-life in serum sample
Educate phenol) total ion chromatogram it is as shown in Figure 1：The retention time and relative abundance of vitamin A and vitamin E and its standard work
It is consistent to make solution.
Comparative example 1
After using the method such as embodiment 2 to be pre-processed in sample, analyzed using high performance liquid chromatography, chromatographic column
Using Shimadzu C18 columns, specification is 5 μ m, 2.1 μ m 50mm, and the temperature of chromatographic column is 25 DEG C, and chromatographic column uses gradient elution side
Formula, the mobile phase of gradient elution use ammonium formate-acetonitrile mixture, and the flow velocity of mobile phase is 0.5mL/min, gradient elution when
Between be 6min, condition of gradient elution is as shown in table 1.
The results show that retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol are isolated not successfully, it is former
Because high performance liquid chromatography carries out sample pretreatment using a large amount of organic solvent, remaining organic solvent influence regards Huang
The separation of alcohol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol.
Comparative example 2
Compared with Example 3, gradient elution uses 0.05% trifluoroacetic acid aqueous solution for mobile phase A, 0.02% trifluoro second
The methanol/ethanol mixed solution of acid is Mobile phase B, and the volume ratio of methanol and ethyl alcohol is 3:1, other conditions are same as Example 3.
The results show that mobile phase A and Mobile phase B are not successfully by retinol, alpha-tocopherol, betatocopherol, γ-life in 6min
Phenol and Delta-Tocopherol separation are educated, after time lengthening to 30min, just realizes retinol, alpha-tocopherol, betatocopherol, γ-fertility
The separation of phenol and Delta-Tocopherol.
Comparative example 3
Compared with Example 3, the condition of gradient elution is as shown in table 5, and other conditions are same as Example 3.
The condition of gradient elution of 5 comparative example 3 of table
| Time (min) | Ammonium formate (%) | Acetonitrile (%) |
| 0 | 15 | 85 |
| 2 | 10 | 90 |
| 4 | 5 | 95 |
| 5.6 | 2 | 98 |
| 8.3 | 2 | 98 |
| 12.5 | 5 | 95 |
| 15 | 8 | 92 |
| 18 | 10 | 90 |
| 23 | 10 | 90 |
| 25 | 15 | 85 |
| 30 | 15 | 85 |
The results show that mobile phase A and Mobile phase B are not successfully by retinol, alpha-tocopherol, betatocopherol, γ-life in 6min
Phenol and Delta-Tocopherol separation are educated, after time lengthening to 30min, just realizes retinol, alpha-tocopherol, betatocopherol, γ-fertility
The separation of phenol and Delta-Tocopherol.
In conclusion all technicals such as the rate of recovery of the method for the present invention, detection limit and precision meet the requirements, it should
Method can detect simultaneously vitamin A in blood (retinol) and vitamin E (alpha-tocopherol, betatocopherol, Gamma-Tocopherol and
Delta-Tocopherol), using isotopic label as internal standard so that the identification high specificity of target compound, high sensitivity, accuracy
Good, analysis time is short, and method reproducibility of the invention is good, and error is small.
Applicant states that the present invention illustrates the method detailed of the present invention, but the present invention not office by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope.
Claims (10)
1. method that is a kind of while detecting 5 kinds of liposoluble vitamins, which is characterized in that the described method comprises the following steps：
(1) pretreated sample is analyzed using liquid chromatograph-mass spectrometer；
(2) according to standard curve, the concentration of 5 kinds of liposoluble vitamins in sample to be tested is calculated；
Wherein, the liposoluble vitamin includes retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol；
Chromatographic column in the liquid chromatograph-mass spectrometer uses gradient elution mode, the mobile phase of the gradient elution to adopt
With ammonium formate-acetonitrile mixture.
2. according to the method described in claim 1, it is characterized in that, it is 5 μ ms that the chromatographic column, which uses Shimadzu C18 columns, specification,
2.1μm×50mm；
Preferably, the temperature of the chromatographic column is 24-26 DEG C, preferably 25 DEG C；
Preferably, the time of the gradient elution is 5-7min, preferably 6min；
Preferably, the volume ratio of ammonium formate and acetonitrile is 1 during the gradient elution:(1-49)；
Preferably, the flow velocity of the mobile phase is 0.4-0.6mL/min, preferably 0.5mL/min；
Preferably, the sample size of the chromatographic column is 6-8 μ L, preferably 7 μ L.
3. method according to claim 1 or 2, which is characterized in that in step (1) described liquid chromatograph-mass spectrometer
Mass spectrum use multiple-reaction monitoring pattern；
Preferably, the mass spectrographic gas curtain air pressure is 32-38psi, preferably 35psi；
Preferably, the mass spectrographic collision air pressure is 5-10psi, preferably 8psi；
Preferably, the mass spectrographic ion source spray voltage is 5200-5800V, preferably 5500V.
4. according to claim 1-3 any one of them methods, which is characterized in that the mass spectrographic ion source spray pressure is
45-55psi, preferably 50psi；
Preferably, the mass spectrographic ion source auxiliary heating air pressure is 45-55psi, preferably 50psi；
Preferably, the mass spectrographic heating temperature is 500-600 DEG C, preferably 550 DEG C；
Preferably, the mass spectrographic entrance potential is 8-12V, preferably 10V.
5. according to claim 1-4 any one of them methods, which is characterized in that step (1) it is described pretreatment specifically include with
Lower step：
Sample to be tested is added in internal standard working solution by (1 '), and organic solvent is added after dilution, and isolating protein is removed in centrifugation；
(2 ') extractant, centrifuging and taking supernatant is added；
(3 ') dry up step (2 ') the supernatant nitrogen, are added and redissolve solution, mixing centrifugation, and obtained supernatant is as pre-
The sample of processing.
6. according to claim 1-5 any one of them methods, which is characterized in that step (1 ') the internal standard working solution is five deuteriums
For the mixed liquor of retinol and six deuterated alpha-tocopherols；
Preferably, a concentration of 0.1-0.15 μ g/mL of described five deuterated retinols, preferably 0.125 μ g/mL；
Preferably, a concentration of 1-1.5 μ g/mL of described six deuterated alpha-tocopherols, preferably 1.25 μ g/mL；
Preferably, step (the 1 ') organic solvent include in ethyl alcohol, methanol or acetonitrile any one or at least two group
It closes；
Preferably, step (the 2 ') extractant is n-hexane.
7. according to claim 1-6 any one of them methods, which is characterized in that the formulation side of step (2) described standard curve
Method includes the following steps：
(1 ") standard items of configuration at least various concentration known to 7 parts；
(2 ") it is analyzed using liquid chromatograph-mass spectrometer；
(3 "), with a concentration of abscissa of analyte, draw standard curve using the area of chromatographic peak as ordinate.
8. according to claim 1-7 any one of them methods, which is characterized in that step (the 1 ") standard items be retinol,
The mixed liquor of alpha-tocopherol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol；
Preferably, the concentration ratio of the retinol, alpha-tocopherol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol is 1:50:50:
10:50。
9. according to claim 1-8 any one of them methods, which is characterized in that the described method comprises the following steps：
(1) standard curve is formulated：The standard items of configuration at least various concentration known to 7 parts, retinol, α-fertility in standard items
Phenol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol concentration ratio be 1:50:50:10:50, using liquid chromatograph mass spectrography
Instrument is analyzed, and using the area of chromatographic peak as ordinate, with a concentration of abscissa of analyte, draws standard curve；
(2) sample pretreatment：Six deuterated alpha-tocopherol of five deuterated retinols of 0.1-0.15 μ g/mL and 1-1.5 μ g/mL will be included
Sample to be tested is added in internal standard working solution, and organic solvent is added after dilution, centrifuges and removes isolating protein, addition extractant, in centrifuging and taking
Clear liquid dries up the supernatant nitrogen, is added and redissolves solution, and mixing centrifugation, obtained supernatant is pretreated sample；
(3) liquid chromatography-mass spectrometry：Pretreated sample is analyzed using liquid chromatograph-mass spectrometer, institute
It is 5 μ m, 2.1 μ m 50mm to state the chromatographic column in liquid chromatograph-mass spectrometer and use Shimadzu C18 columns, specification, chromatographic column
Temperature is 24-26 DEG C, and chromatographic column uses gradient elution mode, the mobile phase of gradient elution to use ammonium formate-acetonitrile mixture, ladder
The time of elution is spent for 5-7min, and the volume ratio of ammonium formate and acetonitrile is 1 during gradient elution:(1-49), the stream of mobile phase
Speed is 0.4-0.6mL/min, and the sample size of chromatographic column is 6-8 μ L, and the mass spectrum in the liquid chromatograph-mass spectrometer is using more
Reaction monitoring pattern, mass spectrographic gas curtain air pressure are 32-38psi, and mass spectrographic collision air pressure is 5-10psi, mass spectrographic ion source spray
Mist voltage is 5200-5800V, and mass spectrographic ion source spray pressure is 45-55psi, and mass spectrographic ion source auxiliary heating air pressure is
45-55psi, mass spectrographic heating temperature are 500-600 DEG C, and mass spectrographic entrance potential is 8-12V；
(4) concentration calculates：According to standard curve, the concentration of 5 kinds of liposoluble vitamins in sample to be tested is calculated.
10. a kind of retinol, α-fertility such as claim 1-9 any one of them method in detection serum and/or food
The application of the content of phenol, betatocopherol, Gamma-Tocopherol and Delta-Tocopherol.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109541087A (en) * | 2018-11-27 | 2019-03-29 | 江苏省中国科学院植物研究所 | A kind of measuring method detecting natural tocopherol level in blackberry, blueberry seed |
| CN110208438A (en) * | 2019-07-12 | 2019-09-06 | 北京和合医学诊断技术股份有限公司 | Method that is a kind of while detecting a variety of liposoluble vitamin contents in blood |
| CN114252530A (en) * | 2021-12-30 | 2022-03-29 | 人福普克药业（武汉）有限公司 | Detection method of tocopherol in vitamin D soft capsules |
| CN115097043A (en) * | 2022-07-01 | 2022-09-23 | 南京海关动植物与食品检测中心 | Method for rapidly and simultaneously determining 7 fat-soluble vitamins in food |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109541087A (en) * | 2018-11-27 | 2019-03-29 | 江苏省中国科学院植物研究所 | A kind of measuring method detecting natural tocopherol level in blackberry, blueberry seed |
| CN110208438A (en) * | 2019-07-12 | 2019-09-06 | 北京和合医学诊断技术股份有限公司 | Method that is a kind of while detecting a variety of liposoluble vitamin contents in blood |
| CN110208438B (en) * | 2019-07-12 | 2023-08-22 | 北京和合医学诊断技术股份有限公司 | Method for simultaneously detecting contents of multiple fat-soluble vitamins in blood |
| CN114252530A (en) * | 2021-12-30 | 2022-03-29 | 人福普克药业（武汉）有限公司 | Detection method of tocopherol in vitamin D soft capsules |
| CN115097043A (en) * | 2022-07-01 | 2022-09-23 | 南京海关动植物与食品检测中心 | Method for rapidly and simultaneously determining 7 fat-soluble vitamins in food |
| CN115097043B (en) * | 2022-07-01 | 2023-09-26 | 南京海关动植物与食品检测中心 | Method for rapidly and simultaneously determining 7 fat-soluble vitamins in food |
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