CN110887924A - Method for simultaneously detecting contents of retinol and α -tocopherol in blood - Google Patents
Method for simultaneously detecting contents of retinol and α -tocopherol in blood Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/62—Detectors specially adapted therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- 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
- G01N2030/8813—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 biological materials
- G01N2030/8822—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 biological materials involving blood
Abstract
The invention provides a method for simultaneously detecting contents of retinol and α -tocopherol in blood, which comprises the steps of respectively detecting at least three standard solutions by utilizing a high performance liquid chromatograph based on specific liquid phase conditions to obtain chromatograms of the standard solutions, wherein each standard solution contains standard products and internal standard substances of the retinol and α -tocopherol with known concentrations, fitting the standard curve equations of the retinol and α -tocopherol according to the chromatograms of the standard solutions, adding mixed internal standard working solution into a blood sample obtained by processing at least 2mL of blood to be detected, preprocessing the sample to be detected to obtain a sample to be detected, detecting the sample to be detected to obtain the chromatogram thereof, and calculating the contents of the retinol and α -tocopherol in the blood sample according to the chromatograms and each standard curve equation.
Description
Technical Field
The invention relates to the technical field of clinical chemistry, in particular to a method for simultaneously detecting the contents of retinol and α -tocopherol in blood.
Background
Retinol, also known as vitamin a (vitamine a), or anti-xerophthalmia factor, is an alicyclic unsaturated monohydric alcohol, belongs to fat-soluble vitamins, and is a constituent of rhodopsin which is sensitive to weak light in visual cells. Vitamin A can maintain normal visual function and normal growth and development of bones of human bodies, maintain the health of epithelial tissue cells, promote the synthesis of immunoglobulin, promote growth and reproduction and the like. When a human body lacks vitamin A, symptoms such as dry skin, desquamation, alopecia and the like can occur, night blindness can occur when the human body is seriously deficient, and normal growth and development of bones are influenced by destroying the balance between osteoblasts and osteoclasts, or enabling bone to be excessively proliferated, or enabling formed bone not to be absorbed. If the pregnant women lack the vitamin A, the development of the fetus can be directly influenced, and even dead fetus can occur.
α -tocopherol is the most widely distributed, abundant and active vitamin E form in nature, and is also a fat-soluble vitamin, which can increase the activity and quantity of sperms of men by promoting sex hormone secretion, so that the concentration of female estrogen of women is increased, the fertility is improved, and abortion is prevented, symptoms such as testicular atrophy, epithelial cell degeneration, abnormal pregnancy and the like can be caused when vitamin E is deficient.
For example, a method for simultaneously detecting the contents of vitamin A and vitamin E in blood, which is disclosed in publication No. CN106442754A, has retention times of vitamin A, an internal standard of vitamin A, vitamin E and an internal standard of vitamin E of 0.57min, 0.90min, 2.25min and 2.90min respectively, and an analysis time of 4.2 min.
Disclosure of Invention
The invention provides a method for simultaneously detecting the contents of retinol and α -tocopherol in blood, which can more quickly and simultaneously detect the contents of retinol and α -tocopherol in blood.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides a method for simultaneously detecting the content of retinol and α -tocopherol in blood, which comprises the following steps:
respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a standard product and an internal standard substance of retinol with known concentrations, a standard product and an internal standard substance of α -tocopherol, and the concentrations of the same standard product in different standard solutions are different;
fitting to obtain a standard curve equation of retinol and a standard curve equation of α -tocopherol according to the chromatogram of each standard solution;
adding a certain amount of mixed internal standard working solution into a certain amount of blood sample, and performing sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by processing at least 2mL of blood to be detected, and the mixed internal standard working solution contains an internal standard substance of retinol and an internal standard substance of α -tocopherol, wherein the concentration of the internal standard substance is known;
detecting a certain amount of samples to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the samples to be detected;
calculating the contents of retinol and α -tocopherol in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation obtained by fitting;
wherein the liquid phase condition in the detection conditions comprises: the Poroshell120SB-C18 chromatographic column has a length of 50mm, an inner diameter of 3.0mm, a filler particle size of 2.7 μm, a mobile phase of methanol and water, an analysis time of 2.9-3.5min, a column temperature of 25-35 ℃, a sample injection amount of 0.1-10 μ L and a flow rate of 0.5-1 mL/min.
Preferably, the detector switching mode of the fluorescence detector in the high performance liquid chromatograph comprises the following steps:
preferably, the liquid phase conditions include: the elution mode is gradient elution;
the elution process comprises the following steps:
| time (min) | Methanol (%) | Water (%) |
| 0 | 92 | 8 |
| 0.2 | 92 | 8 |
| 0.3 | 99 | 1 |
| 1.9 | 99 | 1 |
| 2 | 92 | 8 |
| 3 | 92 | 8 |
。
Preferably, the liquid phase conditions include: the column temperature was 35 ℃ and the amount of sample was 5. mu.L at a flow rate of 0.8 mL/min.
Preferably, the internal standard for retinol is retinol acetate, α -internal standard for tocopherol, α -tocopheryl acetate.
The invention provides a method for simultaneously detecting contents of retinol and α -tocopherol in blood, which comprises the steps of respectively detecting at least three standard solutions by utilizing a high performance liquid chromatograph based on specific liquid phase conditions to obtain chromatograms of the standard solutions, wherein each standard solution contains standard products and internal standard substances of the retinol and α -tocopherol with known concentrations, fitting the standard curve equations of the retinol and α -tocopherol according to the chromatograms of the standard solutions, adding mixed internal standard working solution into a blood sample obtained by processing at least 2mL of blood to be detected, preprocessing the sample to be detected to obtain a sample to be detected, detecting the sample to be detected to obtain the chromatogram thereof, and calculating the contents of the retinol and α -tocopherol in the blood sample according to the chromatograms and each standard curve equation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flow chart of a method for simultaneously detecting retinol and α -tocopherol in blood according to an embodiment of the present invention;
FIG. 2 is a chemical formula of retinol provided in accordance with one embodiment of the present invention;
FIG. 3 is a chemical structural formula of α -tocopherol provided by an embodiment of the present invention;
FIG. 4 is a chromatogram of a standard solution of retinol and α -tocopherol standard;
FIG. 5 is a chromatogram of an internal standard for retinol and an internal standard for α -tocopherol in a standard solution provided in accordance with an embodiment of the present invention;
FIG. 6 is a chromatogram of retinol and α -tocopherol in a sample provided by an embodiment of the present invention;
FIG. 7 is a chromatogram of an internal standard for retinol and an internal standard for α -tocopherol in a sample provided in accordance with an embodiment of the present invention;
FIG. 8 is a graph of the linear relationship of retinol provided in accordance with one embodiment of the present invention;
FIG. 9 is a graph of α -tocopherol linearity provided by an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.
As shown in fig. 1, an embodiment of the present invention provides a method for simultaneously detecting the contents of retinol and α -tocopherol in blood, which may include the following steps:
and 101, respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a standard product and an internal standard substance of retinol with known concentrations, and a standard product and an internal standard substance of α -tocopherol, and the concentrations of the same standard product in different standard solutions are different.
In an embodiment of the present invention, the liquid phase condition in the detection condition includes: the Poroshell120SB-C18 chromatographic column has a length of 50mm, an inner diameter of 3.0mm, a filler particle size of 2.7 μm, a mobile phase of methanol and water, an analysis time of 2.9-3.5min, a column temperature of 25-35 ℃, a sample injection amount of 0.1-10 μ L and a flow rate of 0.5-1 mL/min.
For example, the value of the analysis time may be 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5, preferably 3.0 min; the column temperature can take the value of 25, 27, 29, 30, 31, 33 or 35; the value of the sample amount can be 0.1, 0.5, 1, 3, 5 or 10; the flow rate may take on the value 0.5, 0.6, 0.7, 0.8, 0.9 or 1.
And 102, fitting to obtain a standard curve equation of the retinol and a standard curve equation of α -tocopherol according to the chromatogram of each standard solution.
103, adding a certain amount of mixed internal standard working solution into a certain amount of blood sample, and performing sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by processing at least 2mL of blood to be detected, and the mixed internal standard working solution contains an internal standard substance of retinol with known concentration and an internal standard substance of α -tocopherol.
In detail, the blood to be tested is usually venous blood taken from a human body.
Step 104: and detecting a certain amount of samples to be detected by using a high performance liquid chromatograph under the same detection condition to obtain the chromatogram of the samples to be detected.
And 105, calculating the contents of retinol and α -tocopherol in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation obtained by fitting.
Researches show that the time for separating impurities by using a C8 column and other types of C18 columns is longer, and a Poroshell120SB-C18 chromatographic column which is a core-shell chromatographic column has high separation efficiency and low back pressure and is beneficial to shortening the analysis time. In addition, a Poroshell120SB-C18 column having a size of 3.0 mm. times.50 mm 2.7 μm is preferable in combination with the flow rate, separation effect, analysis time and the like.
In the embodiment of the invention, based on the liquid phase condition, the analysis time can be shortened on the premise of ensuring the separation effect, and the method is particularly suitable for the condition of more blood samples and is beneficial to the rapid and accurate detection of a large number of blood samples.
Preferably, the column temperature is 35 ℃, the sample size is 5 μ L, and the flow rate is 0.8 mL/min.
In the embodiment of the invention, the high performance liquid chromatography is selected to detect the contents of retinol and α -tocopherol in blood, compared with the high performance liquid mass spectrometry, the detection instrument used by the high performance liquid chromatography is a high performance liquid chromatograph, the cost investment of the instrument is greatly reduced, the popularization rate of the instrument is higher, and the detection method is easier to standardize.
In the embodiment of the invention, a high performance liquid chromatography method is used for content detection, the blood consumption can be as low as 2mL, and the method is particularly suitable for people with small blood sampling difficulty, such as most adults. Considering that the number of adults is large, and the analysis time used by the embodiment of the invention is shorter, the application prospect of the embodiment of the invention has certain advantages.
Referring to fig. 2 and 3, fig. 2 shows the chemical structural formula of retinol, and fig. 3 shows the chemical structural formula of α -tocopherol.
Usually, at least three coordinate points are required for establishing the standard curve equation so as to ensure the accuracy of the established equation, so at least three standard solutions are required to be prepared in advance, so that the standard curve equation of retinol and the standard curve equation of α -tocopherol can be fitted according to the chromatogram obtained by detecting each standard solution.
In detail, taking retinol as an example, the standard curve equation of retinol obtained by fitting may be generally that y is k × x + b, wherein, the two variables x and y may be the peak area ratio of the standard product of retinol and the corresponding internal standard substance in the chromatogram of each standard solution, and the concentration ratio of the standard product of retinol and the corresponding internal standard substance in each standard solution.
Preferably, the internal standard for retinol is retinol acetate, α -internal standard for tocopherol, α -tocopheryl acetate.
Generally, after at least 2mL of blood to be detected is taken, the blood to be detected is processed, for example, centrifuged at 3500rpm for 10min, and the supernatant is taken to obtain serum or plasma, thus obtaining the blood sample. Serum or plasma samples were stored frozen at-20 ℃ until needed for analysis.
After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded. In one embodiment of the present invention, the implementation process of sample preprocessing may include:
(1) transferring 10 mu L of mixed internal standard working solution into a 1.5mL centrifuge tube by using a liquid transfer gun, then adding 50-200 mu L of blood sample, adding a certain amount of diluent, and carrying out vortex mixing for 0.5-1.5min at the rotating speed of 1500-2000 rpm;
(2) adding a certain amount of protein precipitation reagent, and carrying out vortex mixing for 0.5-2min at the rotating speed of 1500-;
(3) adding a certain amount of extractant, carrying out vortex mixing for 3-10min at the rotating speed of 1500-2000rpm, and then carrying out high-speed centrifugation for 8-12min at the rotating speed of 10000-15000 rpm;
(4) transferring a certain amount of centrifuged supernatant into a clean 1.5mL centrifuge tube, transferring the centrifuge tube containing the supernatant into a nitrogen blow-drying device, and blow-drying the supernatant;
(5) transferring a certain amount of redissolution to a 1.5mL centrifuge tube for blowing the supernatant, carrying out vortex mixing for 0.5-1.5min at the rotating speed of 1500 plus 2000rpm, then carrying out high-speed centrifugation for 4-6min at the rotating speed of 10000 plus 15000rpm, and transferring the supernatant, namely the sample to be detected. For example, 80. mu.L of the supernatant can be removed as a sample to be tested.
Preferably, in the sample pretreatment, the diluent is pure water, and the amount of the diluent is 50-200 μ L; the precipitated protein reagent is absolute ethyl alcohol, and the amount of the protein precipitant is 100-; the extraction reagent is n-hexane, and the amount of the extraction reagent is 300-800 mu L; the compound solution is methanol, and the amount of the compound solution is 50-200 μ L.
Considering the strong specificity and high sensitivity of the fluorescence detector in the hplc-coupled detector, in one embodiment of the present invention, the detector switching method of the fluorescence detector in the hplc is shown in table 1 below.
TABLE 1
In the embodiment of the invention, the excitation wavelength corresponding to retinol is 325nm, and the emission wavelength is 470 nm; the excitation wavelength corresponding to the alpha-tocopherol is 295nm, and the emission wavelength is 330 nm. In the embodiment of the invention, the maximum absorption wavelength is selected, and the sensitivity is highest at the wavelength.
In detail, retinol and α -tocopherol can be eluted generally with equal elution with 85% or less of the organic phase, with good separation from interfering substances, but with greatly extended retention time of the target, resulting in extended analysis time.
Based on this, considering the separation degree of the target and the interferent and the shortest possible analysis time, in one embodiment of the present invention, preferably, the liquid phase conditions include: the elution mode is gradient elution; the elution process is shown in Table 2 below.
Based on the elution mode, the separation degree of the target object and the interfering object can be ensured, the analysis time is shortened, and meanwhile, the procedures of washing the chromatographic column and balancing the chromatographic column are added, so that the interference of strongly-retained substances is reduced, the consistency of the sample injection states of the chromatographic columns of a plurality of samples in front and back is ensured, the sample detection accuracy is higher, and the reproducibility is better.
TABLE 2
| Time (min) | Methanol (%) | Water (%) |
| 0 | 92 | 8 |
| 0.2 | 92 | 8 |
| 0.3 | 99 | 1 |
| 1.9 | 99 | 1 |
| 2 | 92 | 8 |
| 3 | 92 | 8 |
In addition, in the embodiment of the invention, the conversion speed of the elution gradient is slow, so that the influence on the detection stability and the detection accuracy caused by the excessively fast conversion speed when the flow speed is high can be avoided.
In one embodiment of the present invention, before the separately detecting at least three standard solutions, further comprising:
preparing at least three standard working solutions, wherein the standard working solutions contain a known concentration of a retinol standard and a α -tocopherol standard, the concentration of the retinol standard is 0.0625-4.00mg/L, and the concentration of the α -tocopherol standard is 0.625-40.00 mg/L;
using a pipettor to transfer 90 mu L of standard working solution and 10 mu L of the mixed internal standard working solution into a centrifuge tube;
uniformly mixing the centrifugal tube at the rotating speed of 1500-2000rpm for 0.5-2min in a vortex manner, and then transferring supernatant to obtain a standard solution;
wherein, in the at least three standard working solutions, the concentration of the retinol standard substance is at least three of 0.0625mg/L, 0.125mg/L, 0.25mg/L, 0.50mg/L, 1.00mg/L, 2.00mg/L and 4.00mg/L, and the concentration of the α -tocopherol standard substance is at least three of 0.625mg/L, 1.25mg/L, 2.50mg/L, 5.00mg/L, 10.00mg/L, 20.00mg/L and 40.00 mg/L.
In detail, a linear range can be set by combining the population to be tested, the amount of blood to be tested, and the approximate content range of retinol and α -tocopherol in the human body, so as to ensure that most of the test results of the clinical samples fall within a reportable range.
Preferably, the number of standard working fluids is 7.
In summary, the method for simultaneously detecting the contents of retinol and α -tocopherol in blood provided by the embodiment of the invention combines the internal standard method and the high performance liquid chromatography, so that the interference factors are greatly reduced, the specificity is strong, the sensitivity is high, the detection result is accurate, and the analysis time is shortened.
The present invention will be described in detail below by way of examples, but the present invention is not limited to the following examples.
Example 1
The embodiment of the invention is used for obtaining the standard curve equation.
1.1 preparation of Standard stock solutions
Standard stock solution a: retinol standard solution (107.1 + -5.4 mg/L), was used immediately after decapsulation.
And (3) accurately weighing α -tocopherol standard substance 25mg, placing the weighed standard substance in a 5mL volumetric flask, dissolving the weighed standard substance in absolute ethyl alcohol, and fixing the volume to 5mL to obtain standard stock solution B, storing the standard stock solution B at the temperature of minus 80 ℃, wherein the effective period is 6 months, and correcting the concentration of the standard stock solution B by using an ultraviolet spectrophotometer before use.
1.2 preparation of stock solutions for internal standards
Internal standard stock C: taking 10mg of retinol acetate standard substance, placing in a 10mL volumetric flask, dissolving with anhydrous ethanol, and diluting to 10mL to obtain retinol acetate mother liquor (1000mg/L), storing at-80 deg.C, keeping out of the sun, and having an effective period of 6 months.
And (3) taking α -tocopheryl acetate standard substance 1500mg, placing the standard substance in a 100mL volumetric flask, dissolving the standard substance in absolute ethyl alcohol, and fixing the volume to 100mL to obtain α -tocopheryl acetate mother liquor (15000mg/L), storing the mother liquor at 80 ℃, keeping out of the sun, and prolonging the effective period for 6 months.
1.3 Instrument for detection
Shimadzu LC-20A.
1.4 Detector conversion mode
As in table 1 above.
1.5 liquid phase conditions
1.5.1 chromatography columns
Poroshell120SB-C18 column from Agilent having a length of 50mm, an internal diameter of 3.0mm and a packing particle size of 2.7 μm.
1.5.2 Mobile phase
Methanol and pure water.
1.5.3 elution mode
Gradient elution was used and the elution was as shown in table 2 above.
1.5.4 others
The analysis time is 3min, the column temperature is 35 ℃, the sample injection amount is 5 mu L, and the flow rate is 0.8 mL/min.
1.6 preparation of Standard working solution
And (3) taking a proper amount of the standard stock solution A and the standard stock solution B, diluting and mixing the standard stock solution A and the standard stock solution B by using absolute ethyl alcohol to obtain seven standard work solutions containing 0.0625-4.00mg/L of retinol and 0.625-40.00mg/L of α -tocopherol, and storing the seven standard work solutions at the temperature of-80 ℃ for 3 months.
In seven standard working solutions, the concentrations of the retinol standard substances are respectively 0.0625mg/L, 0.125mg/L, 0.25mg/L, 0.50mg/L, 1.00mg/L, 2.00mg/L and 4.00mg/L, and the concentration of the α -tocopherol standard substance is respectively 0.625mg/L, 1.25mg/L, 2.50mg/L, 5.00mg/L, 10.00mg/L, 20.00mg/L and 40.00 mg/L.
1.7 preparation of Mixed internal standard working solution
And (3) taking a proper amount of internal standard stock solution C and internal standard stock solution D, diluting with absolute ethyl alcohol to obtain mixed internal standard work solution containing retinol acetate 7.5mg/L and α -tocopheryl acetate 750mg/L, storing at the temperature of minus 80 ℃, keeping out of the sun, and prolonging the effective period for 3 months.
1.8 preparation of Standard solution
And for each standard working solution, transferring 90 mu L of the standard working solution and 10 mu L of the mixed internal standard working solution by using a pipette, respectively placing the standard working solution and the mixed internal standard working solution into a centrifuge tube, uniformly mixing the standard working solution and the mixed internal standard working solution in a vortex manner at the rotating speed of 2000rpm for 1min, and taking the supernatant as the standard solution to be detected.
Thus, seven standard solutions can be obtained for seven standard working solutions.
1.9 detecting the standard solution to generate a standard curve equation
After obtaining each standard solution, the high performance liquid chromatograph can be used for respectively detecting the seven standard solutions, and the chromatogram of each standard solution is correspondingly obtained.
Referring to fig. 4 and 5, fig. 4 shows chromatograms of a retinol standard and an α -tocopherol standard in a standard solution, and fig. 5 shows chromatograms of an internal standard of retinol (i.e., the retinol acetate standard described above) and an internal standard of α -tocopherol (i.e., the α -tocopherol acetate standard described above) in a standard solution.
From the chromatogram of the standard solution, the chromatographic peak areas of the retinol standard, the α -tocopherol standard and each internal standard substance can be obtained, and then the standard curve equation of retinol and the standard curve equation of α -tocopherol can be obtained by combining the known concentrations of the standard substance and the internal standard substance in each standard solution.
Referring to fig. 8 and 9, fig. 8 shows the linear relationship of retinol and fig. 9 shows the linear relationship of α -tocopherol.
Corresponding to fig. 8, the standard curve equation for retinol was found to be Y4.42921 × X + (-0.00123629), correlation coefficient R2＝0.9997534。
Corresponding to fig. 9, the standard curve equation of α -tocopherol is obtained as Y2355.28 × X +0.0282314, correlation coefficient R2＝0.9992942。
It can be seen that retinol is in the linear range of 0.05625-3.6mg/L, the correlation coefficient R2Greater than 0.9900, indicating good linear relation, α -tocopherol is in the linear range of 0.5625-36mg/L, and the correlation coefficient R2And the linear relation is good when the content of the retinol and the α -tocopherol in the blood is calculated based on the two standard curve equations, the accuracy is high, and the error is small.
And (3) after obtaining the standard curve equation, pretreating the blood sample to obtain a sample to be detected, detecting the sample to be detected under the same detection condition, and combining the obtained standard curve equation to obtain the contents of retinol and α -tocopherol in the blood sample.
Example 2
The embodiment of the invention is used for detecting the content of the retinol and the α -tocopherol in the human venous blood.
2.1 obtaining blood samples
The blood sample is obtained by treating at least 2mL of blood to be tested. After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded.
2.2 blood sample pretreatment
Transferring 10 mu L of mixed internal standard working solution into a 1.5mL centrifuge tube by using a pipette gun, then adding 100 mu L of blood sample, adding 100 mu L of pure water, carrying out vortex mixing at the rotating speed of 2000rpm for 1min, then adding 200 mu L of absolute ethyl alcohol, carrying out vortex mixing at the rotating speed of 2000rpm for 1min, then adding 400 mu L of n-hexane, carrying out vortex mixing at the rotating speed of 2000rpm for 5min, then carrying out high-speed centrifugation at the rotating speed of 12000rpm for 10min, transferring 350 mu L of centrifuged supernatant into a clean 1.5mL centrifuge tube, transferring 1.5mL centrifuge tube containing the supernatant into a nitrogen blow-drying device, carrying out blow-drying on the supernatant, transferring 100 mu L of methanol into the blow-dried 1.5mL centrifuge tube, carrying out vortex mixing at the rotating speed of 2000rpm for 1min, carrying out high-speed centrifugation at the rotating speed of 12000rpm for 5min, and transferring 80 mu L of supernatant, thus obtaining the sample to be detected.
2.3 detection of samples to be tested
Under the detection conditions of example 1, the same high performance liquid chromatograph is used to detect the sample to be detected, and the chromatogram of the sample to be detected is obtained.
Referring to fig. 6 and 7, fig. 6 shows chromatograms of retinol and α -tocopherol in the test sample, and fig. 7 shows chromatograms of an internal standard of retinol (i.e., the above retinol acetate standard) and an internal standard of α -tocopherol (i.e., the above α -tocopherol acetate standard) in the test sample.
Referring to fig. 4 to 7, the retention time of retinol in the sample to be detected is consistent with that of the retinol standard in the standard solution, the retention time of α -tocopherol in the sample to be detected is consistent with that of α -tocopherol standard in the standard solution, and retinol acetate and α -tocopherol acetate are used as internal standard substances, respectively, so that the identification of the target compound is more accurate, the analysis time is short, the interference is small, the internal standard is appropriate in quantification, the specificity is strong, and the accuracy and the sensitivity are high.
2.4 calculation of retinol and α -tocopherol content in the samples to be tested
And (3) correspondingly substituting the chromatographic peak areas of the retinol, α -tocopherol and each internal standard substance in the chromatogram of the sample to be detected and the known concentration of each internal standard substance in the sample to be detected into the 2 standard curve equations to calculate the contents of the retinol and α -tocopherol in the sample to be detected.
Example 3
The embodiment of the invention is used for determining the quantitative limit and the detection limit.
Serum/plasma samples from different sources are prepared and diluted with physiological saline to different degrees, so as to prepare serum/plasma sample dilutions with different concentrations, and the serum/plasma sample dilutions are measured according to the blood sample pretreatment method and measurement conditions in example 2, and the detection limit and the quantification limit of retinol and α -tocopherol are shown as follows:
retinol
(1) Limit of detection (LOD): 0.02 mg/L.
(2) Limit of quantitation (LOQ): 0.04 mg/L.
α -tocopherol
(1) Limit of detection (LOD): 0.075 mg/L.
(2) Limit of quantitation (LOQ): 0.4 mg/L.
As can be seen from the present example, the detection limit of retinol can be as low as 0.02mg/L, the quantification limit can be as low as 0.04mg/L, the detection limit of α -tocopherol can be as low as 0.075mg/L, the quantification limit can be as low as 0.4mg/L, the sensitivity is very high, the accurate quantification can be performed on biological samples with very low content of retinol or α -tocopherol, and the high accuracy and the wide applicability of the detection method are ensured.
Example 4
The embodiment of the invention is used for measuring the recovery rate and the precision.
The standard working solution containing retinol and α -tocopherol was prepared into high, medium and low concentrations, and sample recovery and precision experiments were performed according to the method in example 2, and 3 batches were analyzed and determined, wherein the recovery and precision of retinol and α -tocopherol are shown in table 3.
TABLE 3
It can be seen that α -tocopherol and retinol have an average recovery rate of 93.90% -101.63% in the range of 3 addition levels of low, medium and high, have good reproducibility and good sample recovery rate, have a precision of 0.37% -1.17%, have high accuracy of detection results, and can eliminate system errors.
Comparative example 1
A method for simultaneously detecting the contents of vitamin A and vitamin E in blood is disclosed as CN 106442754A.
In the comparison document 1, the retention time of the vitamin a, the internal standard of the vitamin a, the retention time of the vitamin E, and the internal standard of the vitamin E are 0.57min, 0.90min, 2.25min, and 2.90min, respectively, and the analysis time is 4.2 min.
In the embodiment of the present invention, referring to fig. 4 to fig. 7, the time of the retinol peak is 0.86min, the time of the retinol peak is 1.27min, the time of the α -tocopherol peak is 2.08min, the time of the α -tocopherol peak is 2.5min, and the analysis time is 3 min.
Therefore, the analysis time of the embodiment of the invention is obviously shortened.
Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (5)
1. A method for simultaneously detecting the contents of retinol and α -tocopherol in blood, comprising:
respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a standard product and an internal standard substance of retinol with known concentrations, a standard product and an internal standard substance of α -tocopherol, and the concentrations of the same standard product in different standard solutions are different;
fitting to obtain a standard curve equation of retinol and a standard curve equation of α -tocopherol according to the chromatogram of each standard solution;
adding a certain amount of mixed internal standard working solution into a certain amount of blood sample, and performing sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by processing at least 2mL of blood to be detected, and the mixed internal standard working solution contains an internal standard substance of retinol and an internal standard substance of α -tocopherol, wherein the concentration of the internal standard substance is known;
detecting a certain amount of samples to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the samples to be detected;
calculating the contents of retinol and α -tocopherol in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation obtained by fitting;
wherein the liquid phase condition in the detection conditions comprises: the Poroshell120SB-C18 chromatographic column has a length of 50mm, an inner diameter of 3.0mm, a filler particle size of 2.7 μm, a mobile phase of methanol and water, an analysis time of 2.9-3.5min, a column temperature of 25-35 ℃, a sample injection amount of 0.1-10 μ L and a flow rate of 0.5-1 mL/min.
2. The method of claim 1,
the detector switching mode of the fluorescence detector in the high performance liquid chromatograph comprises the following steps:
3. the method of claim 1,
the liquid phase conditions include: the elution mode is gradient elution;
the elution process comprises the following steps:
。
4. The method of claim 1,
the liquid phase conditions include: the column temperature was 35 ℃ and the amount of sample was 5. mu.L at a flow rate of 0.8 mL/min.
5. The method according to any one of claims 1 to 4,
the internal standard of retinol is retinol acetate, α -internal standard of tocopherol, α -tocopherol acetate.
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| CN111426775A (en) * | 2020-05-29 | 2020-07-17 | 中国烟草总公司郑州烟草研究院 | Method for simultaneously measuring vitamin E and vitamin E acetate in electronic cigarette liquid |
| CN113640395A (en) * | 2020-04-27 | 2021-11-12 | 深圳波顿香料有限公司 | Synthetic phase chromatographic analysis method of tocopherol acetate in electronic cigarette oil |
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