CN113075305A - Method for quantitatively detecting content of lipid-soluble vitamins in peripheral blood sample - Google Patents

Method for quantitatively detecting content of lipid-soluble vitamins in peripheral blood sample Download PDF

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CN113075305A
CN113075305A CN202110226116.7A CN202110226116A CN113075305A CN 113075305 A CN113075305 A CN 113075305A CN 202110226116 A CN202110226116 A CN 202110226116A CN 113075305 A CN113075305 A CN 113075305A
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vitamin
internal standard
peripheral blood
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刘鹏云
刘妍妍
马金飞
刘华芬
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Hangzhou Calibra Diagnostics Co ltd
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    • G01N30/02—Column chromatography
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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
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Abstract

The invention relates to a method for quantitatively detecting the content of antiviral drugs in a plasma sample, which comprises the following steps: (1) collecting micro peripheral blood; (2) mixing a peripheral blood whole blood sample with a protein precipitation solution containing an internal standard, centrifuging after vortexing, and diluting a supernatant to obtain a solution to be detected; (3) and detecting and analyzing the liquid to be detected by adopting a high performance liquid chromatography-tandem mass spectrometry system. The invention provides a method for simultaneously detecting vitamin A, vitamin D2, vitamin D3 and vitamin E in a peripheral blood sample, which has the advantages of simple pretreatment, low detection cost and high detection efficiency.

Description

Method for quantitatively detecting content of lipid-soluble vitamins in peripheral blood sample
Technical Field
The invention relates to the technical field of detection, and mainly relates to a method for quantitatively detecting the content of fat-soluble vitamins in a peripheral blood sample.
Background
The fat-soluble vitamin is a kind of vitamin which is easily dissolved in an organic solvent and is not dissolved in water, and mainly comprises vitamin A, vitamin D, vitamin E and the like. The vitamin can be dissolved in fat, is not easy to be excreted, can be stored in vivo, and plays an important and indispensable role in human metabolism and function. The vitamin A, D, E participates in the synthesis, decomposition and transformation of protein, enzyme, hormone and vitamin in the form of enzyme and prosthetic group activator, respectively, and influences physiological activities such as growth and development, metabolism, immunoregulation, etc. Therefore, it is important to maintain the body's fat-soluble vitamin levels in the appropriate range, and A, D, E deficiency can cause a variety of diseases, while excessive intake can lead to toxicity.
At present, methods for clinically detecting a plurality of fat-soluble vitamins (including vitamin a, vitamin D3, vitamin D2, vitamin E and the like) mainly comprise an immunization method, an electrochemical method, a chromatography method, a liquid chromatography tandem mass spectrometry method and the like, wherein a mainstream fat-soluble vitamin detection sample type is serum, and the amount of blood samples required for detection is relatively large, but because the infants, particularly newborn groups, have the condition of inconvenient blood collection, sufficient serum is difficult to separate for detection. However, fat-soluble vitamin deficiency is a common disease in children, so the fat-soluble vitamin detection item of trace peripheral blood whole blood has a very important meaning, but documents and patents aiming at the fat-soluble vitamin detection of peripheral blood whole blood are few.
CN110488008A and CN 110488004A respectively disclose a kit for detecting 25-hydroxyvitamin D3 in peripheral blood and 25-hydroxyvitamin D in peripheral blood, a preparation method and an application thereof, wherein the two kits respectively comprise a 25-hydroxyvitamin D3 immunochromatographic detection card and a releasing agent, and a 25-hydroxyvitamin D immunochromatographic detection card and a releasing agent, the immunochromatographic detection card comprises a test strip, the test strip comprises a detection line and a quality control line, the detection line is coated with a 25-hydroxyvitamin D3 or a 25-hydroxyvitamin D complete antigen, and the quality control line is coated with a goat anti-rabbit polyclonal antibody. The disadvantage of both methods is that only 25-hydroxyvitamin D3 or 25-hydroxyvitamin D in peripheral blood can be detected, and other types of fat-soluble vitamins cannot be detected.
CN108802221A discloses a trace detection method of vitamin A and vitamin E in peripheral blood, which comprises the following detection steps: 1. adding the peripheral blood sample into a methanol solution containing an internal standard and BHT to complete protein precipitation; 2. adding tert-butyl methyl ether for liquid-liquid extraction, blowing the extracted supernatant with nitrogen at room temperature, adding 80% methanol for redissolution, performing vortex oscillation, and centrifuging to obtain a sample to be detected; 3. detecting by ultra performance liquid chromatography tandem mass spectrometry: and (3) obtaining the peak area of the object to be detected in the sample to be detected and the peak area of the corresponding internal standard through ultra performance liquid chromatography tandem mass spectrometry detection, and completing the detection of vitamin A and vitamin E in the trace peripheral blood sample. CN 110208435a discloses a method for simultaneously detecting the content of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human peripheral blood, which has similar steps to the sample pretreatment and detection steps disclosed in CN108802221A, but uses different types of reagents: 1. adding an ethanol solution containing an internal standard into a peripheral blood sample to perform protein precipitation; 2. adding n-hexane for liquid-liquid extraction, blowing the extracted supernatant with nitrogen at room temperature, adding a methanol-water composite solution containing formic acid, and centrifuging after vortex oscillation to obtain a sample to be detected; 3. and (3) detecting the vitamin D2 and the vitamin D3 in the trace peripheral blood sample by high performance liquid chromatography-tandem mass spectrometry. The two detection methods can only detect the lipid-soluble vitamin D2 and the vitamin D3 in the same batch, and the pretreatment steps are very complicated, time-consuming and labor-consuming, and difficult to realize high-throughput automatic operation.
At present, the type of a common sample for fat-soluble vitamin detection by using liquid chromatography tandem mass spectrometry is serum, the sample size generally needs at least 0.5mL of serum, and the amount of venous blood to be collected is more than 1 mL. For infants, particularly newborns and other people with venipuncture, venous blood of more than 1mL is difficult to collect, so that peripheral blood collection is particularly important. Compared with a serum sample, when fat-soluble vitamin detection is carried out, components such as erythrocytes and the like in a whole blood sample can form a serious matrix effect on the detection of the fat-soluble vitamin, and great difficulty is caused to the development of a whole blood fat-soluble vitamin detection method. In the prior art for detecting soluble vitamins in peripheral blood fat disclosed at present, each scheme can only detect one or two of fat-soluble vitamins, and cannot realize synchronous detection on more than two fat-soluble vitamins. In the prior art for detecting the total blood lipid and vitamin solubility of peripheral blood by using the liquid chromatography-tandem mass spectrometry, the used sample is complicated in pretreatment steps, comprises the steps of protein precipitation, liquid-liquid extraction, drying of extract liquor, redissolving of an object to be detected and the like, is complicated in operation, consumes time and labor, is difficult to realize high-throughput automatic operation, and cannot realize synchronous detection of more than two lipid-soluble vitamins.
Disclosure of Invention
The invention aims to provide a LC-MS detection method which is simple and convenient in pretreatment, high in detection efficiency and low in cost aiming at the defects of the prior art and the clinical detection requirement of peripheral blood fat soluble vitamins, and simultaneously detects vitamin A, vitamin D3, vitamin D2 and vitamin E in peripheral blood. In order to realize the purpose of the invention, the invention provides the following technical scheme:
the invention relates to a method for quantitatively detecting the content of liposoluble vitamin in a peripheral blood sample, wherein the liposoluble vitamin is at least one selected from vitamin A, vitamin D3, vitamin D2 and vitamin E; the method comprises the following steps:
mixing the peripheral blood sample with a protein precipitation solution containing an internal standard, centrifuging after vortexing, and taking supernatant liquid to be tested on a computer;
detecting and analyzing the liquid to be detected by adopting a high performance liquid chromatography-tandem mass spectrometry system;
the protein sedimentation agent containing the internal standard is prepared from an organic solvent and an isotope internal standard, wherein the organic solvent comprises alcohol solvents such as methanol, ethanol and propanol, the isotope internal standard is selected from at least one of a vitamin A isotope internal standard, a vitamin D3 isotope internal standard and a vitamin E isotope internal standard, and the isotope internal standard optionally contains a vitamin D2 isotope internal standard.
Based on the technology, the invention can accurately and efficiently detect the concentrations of vitamin A, vitamin D2, vitamin D3 and vitamin E in a human peripheral blood sample through a simple pretreatment method, and provides a basis for nutrition supplement, clinical diagnosis and treatment.
In a preferred embodiment of the invention, the selected internal standards are vitamin A isotope internal standard, vitamin D3 isotope internal standard and vitamin E isotope internal standard, and the specific forms are vitamin A-D6, vitamin D3-D6 and vitamin E-D6 respectively.
In a preferred embodiment of the present invention, the peripheral blood sample is used in an amount of not more than 50 μ L.
In a preferred embodiment of the present invention, the volume ratio of the peripheral blood sample to be tested to the protein sedimentation agent containing the internal standard is 1:2-1:5, preferably 1: 3. The invention is beneficial to extracting fat-soluble vitamin by reasonably setting the proportion of the two.
In a preferred embodiment of the present invention, the time for analyzing the liquid quality of each peripheral blood sample to be tested does not exceed 5 minutes.
In a preferred embodiment of the present invention, the high performance liquid chromatography conditions are:
the conditions of the high performance liquid chromatography are as follows:
mobile phase A: aqueous phase containing ammonium salt
Mobile phase B: organic phase containing ammonium salt
A chromatographic column: modified C18 chromatographic column
The mobile phase gradient parameters are shown in table 1.
Table 1: high performance liquid chromatography mobile phase gradient
Time (min) Flow rate (mL/min) Mobile phase A (%) Mobile phase B (%)
0 0.7 90 10
0.4 0.7 90 10
1.2 0.7 25 75
3 0.7 2 98
4 0.7 2 98
4.05 0.7 90 10
4.4 0.7 90 10
By adopting the liquid chromatography conditions, the method can effectively separate all lipid-soluble vitamins and control the liquid quality analysis time within 5 minutes.
In a preferred embodiment of the present invention, the mass spectrum conditions of the mass spectrum are:
an ion source: atmospheric pressure chemical ionization source
Mode (2): and positive and negative ion segmentation mode, wherein the detection mode of vitamin E is negative ions, and the detection modes of vitamin D2, vitamin D3 and vitamin A are positive ions.
The mass spectrum condition of the invention can effectively reduce the matrix effect of the complex matrix and ensure the accuracy of the detection result.
In a preferred embodiment of the invention, the method of the invention is for diagnostic purposes or non-diagnostic purposes.
Compared with the prior art, the invention has one or more or all of the following advantages:
(1) the required sample amount is small, and the concentration of vitamin A, vitamin D2, vitamin D3 and vitamin E can be detected by 50uL of peripheral blood samples, so the requirement on the sample amount is low;
(2) according to the invention, the internal standard component and the protein precipitator are prepared together, so that the purposes of protein sedimentation and internal standard correction can be achieved by one-step operation, and the pretreatment process is simplified;
(3) in the pretreatment process, only the sample and the protein sedimentation agent are mixed, vortexed and centrifuged, so that the detection cost is low, the pretreatment time is short, the automatic high-flux sample pretreatment is easy to realize, and the technical requirement on operators is low;
(4) according to the invention, by reasonably setting chromatographic conditions, the liquid quality analysis time of each sample is only 4.4min, and the detection efficiency is high;
(5) in the mass spectrometry condition, the vitamin E is detected in a negative ion mode, so that the complex matrix effect in the peripheral blood sample can be effectively reduced, the matrix interference possibly generated by lipid substances in red blood cells can be effectively reduced, and the accuracy of the detection result can be ensured.
Drawings
FIG. 1 ion extraction chromatograms of vitamin D3 (top panel) and its internal standard (bottom panel);
FIG. 2 ion extraction chromatograms of vitamin D2 (top panel) and its internal standard (bottom panel);
FIG. 3: ion extraction chromatograms of vitamin a (top panel) and its internal standard (bottom panel);
FIG. 4: ion extraction chromatograms of vitamin E (top panel) and its internal standard (bottom panel).
The specific implementation mode is as follows:
the present invention is further illustrated by the following examples, which are intended to be illustrative of the present invention and are not to be construed as limiting the invention, and any modifications and variations of the present invention are intended to fall within the spirit and scope of the appended claims.
Example 1:
(1) configuration of the standard curve: accurately weighed standard substances of vitamin A, vitamin D2, vitamin D3 and vitamin E were dissolved in ethanol to obtain corresponding stock solutions of the standard substances, and then the stock solutions were diluted with PBS to obtain standard curves of the concentrations shown in the following Table 2.
Table 2: concentration of standard curve
Analyte Linear range (ng/mL) SD1 SD2 SD3 SD4 SD5 SD6
Vitamin D3 5～250 5 10 25 50 100 250
Vitamin D2 2～100 2 4 10 20 40 100
Vitamin A 40～2000 40 80 200 400 1000 2000
Vitamin E 500～25000 500 1000 2500 5000 10000 25000
(2) Preparation of a protein settling agent: accurately weighed vitamin A-D6, vitamin D3-D6 and vitamin E-D6 are dissolved by ethanol to obtain corresponding isotope internal standard stock solutions, and then the isotope internal standard stock solutions are mixed and diluted by ethanol to form protein sedimentation agent solutions with certain concentrations, as shown in Table 3.
Table 3: concentration of internal standard
Isotope internal standard Isotope internal standard concentration (ng/mL)
Vitamin D3-D6(VD3-D6) 10
Vitamin A-d6(VA-d6) 80
Vitamin E-d6(VE-d6) 1,000
(3) And (3) configuring a standard quality control sample: and (3) uniformly mixing a plurality of peripheral blood whole blood samples to be used as low quality control for detecting the dissolved vitamins in the peripheral blood fat, and accurately adding the standard substance stock solutions with medium concentration and high concentration respectively to obtain medium quality control and high quality control.
(4) Processing of the sample: 50 mu L of standard curve solution, quality control sample and sample to be detected are respectively put into a 96-well plate of 1.2mL, then 150 mu L of protein sedimentation agent is added into each well, and the mixture is centrifuged after being fully vortexed. Taking 100uL of supernatant, and detecting on a computer.
(5) Detection on machine
The conditions of the high performance liquid chromatography are as follows:
mobile phase A: aqueous solution containing ammonium acetate
Mobile phase B: formic acid solution containing ammonium acetate
A chromatographic column: modified C18 chromatographic column
The mobile phase gradient parameters are shown in table 4.
Table 4: high performance liquid chromatography mobile phase gradient
Time (min) Flow rate (mL/min) Mobile phase A (%) Mobile phase B (%)
0 0.7 90 10
0.4 0.7 90 10
1.2 0.7 25 75
3 0.7 2 98
4 0.7 2 98
4.05 0.7 90 10
4.4 0.7 90 10
The mass spectrum conditions are as follows:
an ion source: APCI (APCI is an atmospheric pressure chemical ionization source, the sample is first formed into a mist, then a corona discharge discharges against it, in a high voltage arc, the sample is ionized, then desolvated to form ions, and finally detected.)
Mode (2): positive and negative ion segment mode (positive and negative ion detection segment time: 3.3 minutes)
The mass spectrometric detection parameters are shown in table 5.
Table 5: mass spectrometric detection parameters
Figure RE-GDA0003058010880000071
Note: the analytes-1 are all quantitative ion pairs, and the analyte-2 is a qualitative ion pair.
The retention times of the compounds are shown in table 6.
Table 6: fat soluble vitamin retention time
Name of Compound Retention time (min)
Vitamin D3 2.75
Vitamin D2 2.81
Vitamin A 2.82
Vitamin E 3.86
(6) Data processing
Data were collected using Sciex Analyst software and processed using multisquant software. And (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation. The measured concentration of the analyte in the sample is calculated from the following regression equation:
y＝ax+b
where y is the peak area ratio of analyte to internal standard
b is intercept
a slope of
x is the concentration of the analyte
The results are shown in Table 7.
Table 7: the result of the detection
Vitamin A (ng/mL) Vitamin D2(ng/mL) Vitamin VD3(ng/mL) Vitamin E (ng/mL)
Sample 1 456.12 2.18 7.05 5891.11
Sample 2 415.75 1.81 7.48 7234.35
Sample 3 370.63 2.01 5.42 5276.21
Sample 4 441.25 1.27 7.93 6936.17
Sample 5 640.14 1.13 11.77 10100.58
Sample 6 373.06 1.35 5.43 4398.84
Sample 7 433.19 1.49 8.77 5293.34
Sample 8 564.55 1.42 8.39 6224.19
Sample 9 493.69 1.17 8.2 5257.36
Sample 10 342.22 1.84 7.85 5333
Sample 11 588.98 2.87 7.74 8920.18
Sample 12 386.48 1.76 14.32 6660.46
Sample 13 578.9 1.83 9.71 5226.35
Sample 14 361.62 1.52 7.65 7927.09
Sample 15 460.67 1.14 21.86 7359.72
Sample 16 387.38 1.19 7.72 4695.4
Sample 17 474.81 1.79 6.6 4637.85
Sample 18 397.49 1.49 21.51 7453.07
Sample 19 531.34 1.09 12.97 7578.74
Sample 20 551.5 1.07 4.97 6165.21
According to the analysis of the results of the above examples, the present invention can simultaneously detect vitamin A, vitamin D2, vitamin D3 and vitamin E in peripheral blood samples. The method has simple sample pretreatment and is easy to carry out automatic high-flux sample treatment; only 50uL of peripheral blood samples are needed in each detection, and the kit is particularly suitable for infant groups with nutrition detection requirements and blood sampling difficulty; the liquid quality analysis time is only 4.5min, and the detection efficiency is high; the detection mode of the vitamin E is negative ions, so that the matrix effect of a complex matrix can be effectively reduced, and the accuracy of a detection result is ensured. Therefore, the invention provides a method for simultaneously detecting vitamin A, vitamin D2, vitamin D3 and vitamin E in a peripheral blood sample, and the method has the advantages of simple pretreatment, low detection cost and high detection efficiency.
The fat-soluble vitamins in the examples were subjected to a methodological validation test. The accuracy of the methodology of the present invention is determined by performing a standard recovery experiment on clinical samples, the precision of the methodology of the present invention is determined by day precision experiments, the spectrograms of each analyte and its internal standard are shown in fig. 1-4, and it can be known from the results of the spectrograms that the method of the present invention can well separate each lipo-vitamin. The results of verifying the accuracy and precision of each analyte are shown in tables 8-11 below.
Table 8: verification of accuracy and precision of vitamin D3
Figure RE-GDA0003058010880000091
Table 9: verification of accuracy and precision of vitamin D2
Figure RE-GDA0003058010880000101
Table 10: verification of accuracy and precision of vitamin A
Figure RE-GDA0003058010880000111
Table 11: verification of accuracy and precision of vitamin E
Figure RE-GDA0003058010880000121
The verification result shows that the technical scheme of the invention can simultaneously detect a plurality of fat-soluble vitamins in peripheral blood, including vitamin A, vitamin D3, vitamin D2 and vitamin E. The method has simple sample pretreatment, and only needs to add the protein settling agent and then carry out vortex centrifugation; the result has high accuracy and good precision, and the coefficient of variation (CV%) of each analyte is less than 15%; the deviation (bias) of the recovery rate of each analyte in the standard sample is less than 15 percent, and the method meets the verification requirement.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims (6)

1. A method for quantitatively detecting the content of liposoluble vitamins in a peripheral blood sample, wherein the liposoluble vitamins are at least one selected from vitamin A, vitamin D3, vitamin D2 and vitamin E; the method comprises the following steps:
mixing the peripheral blood sample with a protein precipitation solution containing an internal standard, centrifuging after vortexing, and taking supernatant liquid to be tested on a computer;
detecting and analyzing the liquid to be detected by adopting a high performance liquid chromatography-tandem mass spectrometry system;
the protein sedimentation agent containing the internal standard is prepared from an organic solvent and an isotope internal standard, wherein the organic solvent is selected from one or more alcohol solvents of methanol, ethanol and isopropanol, and the isotope internal standard is selected from at least one of a vitamin A isotope internal standard, a vitamin D3 isotope internal standard and a vitamin E isotope internal standard; the isotopic internal standard optionally comprises a vitamin D2 isotopic internal standard.
2. The method of claim 1, wherein the vitamin a isotope internal standard, the vitamin D3 isotope internal standard, and the vitamin E isotope internal standard are vitamin a-D6, vitamin D3-D6, and vitamin E-D6, respectively.
3. The method of claim 1, wherein the volume ratio of the peripheral blood sample to the protein sedimentation agent containing the internal standard is 1:2-1: 5.
4. The method of claim 1, wherein the time for analyzing the fluid quality of each peripheral blood test sample is no more than 5 minutes.
5. The method of claim 1, wherein the high performance liquid chromatography conditions are:
mobile phase A: aqueous phase containing ammonium salt
Mobile phase B: organic phase containing ammonium salt
A chromatographic column: modified C18 chromatographic column
The content of the mobile phase A is 2-90%, the content of the mobile phase B is 10-98%, and the ammonium salt in the mobile phase is selected from one or more of ammonium formate, ammonium acetate and ammonium fluoride.
6. The method of claim 1, the mass spectrometry conditions of the mass spectrometer being:
an ion source: atmospheric pressure chemical ionization source (APCI) or electrospray ion source (ESI);
mode (2): and positive and negative ion segmentation modes, wherein the detection mode of vitamin E is negative ions, and the detection modes of vitamins D3, D2 and vitamin A are positive ions.
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