CN115308346A - Method for extracting and detecting content of multiple vitamins in blood - Google Patents
Method for extracting and detecting content of multiple vitamins in blood Download PDFInfo
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- CN115308346A CN115308346A CN202211070815.8A CN202211070815A CN115308346A CN 115308346 A CN115308346 A CN 115308346A CN 202211070815 A CN202211070815 A CN 202211070815A CN 115308346 A CN115308346 A CN 115308346A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
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Abstract
The invention discloses a method for extracting and detecting the content of multiple vitamins in blood, which comprises the following steps: quantitatively obtaining blood by using a dry blood collecting system, and drying to prepare a dry blood sample; extracting and purifying a substance to be detected in the dried blood sample by using an extraction solvent to obtain a vitamin sample to be detected; analyzing by a liquid chromatogram-mass spectrum combined instrument, and quantifying by adopting an internal standard method. The method has high extraction efficiency on various water-soluble and fat-soluble vitamins, is combined with a liquid chromatography-tandem mass spectrometry (LC-MS) combined instrument for content detection, is simple, quick and accurate, and provides scientific basis for research on disease diagnosis and health monitoring of large-sample crowds.
Description
Technical Field
The invention belongs to the technical field of blood detection, and particularly relates to a method for extracting and detecting the content of multiple vitamins in blood.
Background
The B vitamins in the blood are water-soluble vitamins with various biological activities. Water-soluble B vitamins are involved in many important metabolic reactions and are critical to maintaining our health. B vitamin deficiencies can lead to many different types of diseases such as dementia, anemia, cardiovascular disease, neural tube defects, crohn's disease, celiac disease, and hiv. VB1 is an important coenzyme, involved in carbohydrate metabolism and directly involved in neural functions. VB2 is a cofactor for several reductases of the oxidase in energy metabolism. VB2 also acts as a coenzyme for folate, VB12, VB6 and homocysteine metabolism. Both VB3 and VB5 are involved in neurotransmission and fatty acid synthesis, oxidation/reduction reactions or one-carbon metabolism. Due to the importance of the B vitamins, the optimal nutritional status of these vitamins must be maintained.
Fat-soluble vitamins are a variety of biochemical micronutrients essential for healthy development, growth, metabolism, and cell regulation. We were unable to synthesize the fat-soluble vitamins completely, nor to achieve the desired concentrations. Vitamin a is vital to human life and together with its derivatives regulates a variety of processes including reproduction, embryogenesis, vision, growth, cell differentiation and proliferation, maintenance of epithelial cell integrity and immune function, and has been shown to be associated with mental and developmental disorders that disturb cognition. Vitamin E regulates the redox balance in the body due to its high concentration in the fat-soluble vitamin group and is ubiquitous throughout the body. Fat-soluble vitamin deficiencies or excesses can cause many health problems.
Plasma is the most readily available sample matrix for quantification of B vitamins and fat soluble vitamins. However, due to its complexity and other analytical challenges (e.g., sensitivity of fat-soluble vitamins to light, oxygen, heat, pH, chemical heterogeneity, availability of standards), it has been challenging to develop a method to simultaneously quantify multiple water-soluble and fat-soluble vitamins at physiological concentrations.
Currently, the methods for measuring B vitamins and fat-soluble vitamins in blood in the industry include: high performance liquid chromatography and liquid chromatography-mass spectrometry are combined. At present, the commonly used B vitamins and fat-soluble vitamins for detecting human bodies are detected by adopting blood plasma, blood plasma samples need professionals to collect venous blood, blood pollution is easily caused, and samples need cold chain transportation and are quite inconvenient in the process. The pretreatment of the serum by adopting a detection method is complex, and water-soluble vitamins and fat-soluble vitamins in the serum need to be respectively extracted for detection. The existing detection method for detecting multiple vitamins by manually extracting the dry blood spots comprises the steps of adopting ten dry blood spot samples, simultaneously extracting water-soluble vitamins and fat-soluble vitamins in the dry blood spot samples by using a mixed solvent of methanol, formic acid and water, wherein the amount of the dry blood spot samples required by the method is large, the difficulty of collecting ten dry blood spots of one subject at a time in practical application is high, the method is difficult to meet the requirements of disease diagnosis and health monitoring of the crowd with large samples, and the extraction efficiency of the water-soluble vitamin B3 and the fat-soluble vitamin A extracted by the method is low, so that the accuracy of vitamin quantification is influenced.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for extracting and detecting the content of multiple vitamins (including water-soluble vitamins and fat-soluble vitamins) in blood, which has high extraction efficiency on the multiple water-soluble vitamins and the fat-soluble vitamins in a sample and has more accurate quantitative result.
The invention provides a method for extracting multiple vitamins in blood, which comprises the following steps:
(1) Obtaining blood by using a dry blood collecting system, and drying to prepare a dry blood sample;
(2) Extracting and purifying a substance to be detected in the dried blood sample by using an extraction solvent to obtain a vitamin sample to be detected;
wherein the extraction solvent is a mixed extraction solvent of water and a methanol-acetonitrile solution.
Preferably, in step (1), the dry blood collection system is a dry blood spot collection card (DBS) or a volume absorption micro-sampler (VAMS).
Preferably, in the step (1), the drying time is 0.5 to 4 hours.
The invention can realize the extraction of water-soluble and fat-soluble multivitamins in the dry blood sample by optimizing the extraction solvent and the adding sequence, has high extraction efficiency and more accurate quantitative result.
Preferably, in the step (2), the volume ratio of methanol to acetonitrile in the methanol-acetonitrile solution is 1:2-2:1.
In some embodiments provided herein, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile solution is preferably 1.5 to 1.5.
Preferably, in the step (2), the volume ratio of water to the methanol-acetonitrile solution in the mixed extraction solvent is 1-2:2-1.
In order to ensure sufficient extraction, preferably, in the step (2), the order of adding the extraction solvent to the sample is that water is added firstly, and then methanol-acetonitrile solution is added;
preferably, the sample is added with water and then extracted for 5-30min, and then a methanol-acetonitrile solution is added for extraction for 5-30min;
preferably, the extraction solvent extracts more than one dried blood sample simultaneously.
Preferably, the purification mode in the step (2) is centrifugation, and the centrifugation time is 3-10min.
The invention also provides a method for detecting the content of multiple vitamins in blood, which comprises the following steps:
(1) Preparing a vitamin sample to be detected by adopting the method for extracting the multiple vitamins in the blood;
(2) Analyzing a vitamin sample to be detected by using a liquid chromatography-mass spectrometer, and quantifying by adopting an internal standard method;
wherein the conditions of the liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the aqueous solution of heptafluorobutyric acid-formic acid is taken as a mobile phase A, the acetonitrile solution is taken as a mobile phase B, and the gradient elution procedure is as follows:
preferably, in the mobile phase A, the mass concentration of the heptafluorobutyric acid is 0.05-0.1%, and the mass concentration of the formic acid is 0.05-0.1%.
In some embodiments of the present invention, in the mobile phase a, the mass concentration of the heptafluorobutyric acid is 0.05%, and the mass concentration of the formic acid is 0.1%.
Preferably, the flow rate is 0.4-0.6mL/min; the column temperature is 25-45 ℃.
Preferably, the mass spectrometry conditions are: an ionization mode: electrospray positive ion mode; the detection mode is as follows: multiple reaction monitoring, MRM; air curtain pressure: 20-50psi; ion spray voltage: 3000-5500V; temperature: 300-550 ℃; atomizing: 30-60psi; auxiliary heating gas: 30-60psi.
Compared with the prior art, the invention has the following advantages:
the invention provides a method for extracting and detecting the content of multiple vitamins in blood, which collects a dry blood sample by a dry blood collecting system, has simple sampling process and is convenient to transport and store.
The extraction method adopted by the invention has high extraction efficiency on various water-soluble and fat-soluble vitamins in the dried blood sample, and content detection is carried out by combining a liquid chromatography-tandem mass spectrometry (LC-MS) combined instrument.
The content determination method is scientific and effective by performing linear, precision and durability tests (stability), specificity tests (blank), detection limit, quantification limit, accuracy (recovery rate) tests and actual sample determination on the content determination method of various water-soluble and fat-soluble vitamins in the dry blood collection system, and can achieve the purpose of quality control on the content of various water-soluble and fat-soluble vitamins in the dry blood collection system.
Drawings
FIG. 1 is a control chromatogram;
fig. 2 is a chromatographic column: phenomenex, kinetex PFP, 100X 4.6mm,2.6 μm; column temperature: at 40 ℃; flow rate: 0.6mL/min; mobile phase: a is 0.1% formic acid water solution, B is chromatogram of 0.1% formic acid acetonitrile solution;
fig. 3 is a chromatographic column: phenomenex, kinetex PFP, 100X 4.6mm,2.6 μm; column temperature: at 40 ℃; flow rate: 0.6mL/min; mobile phase: a is 0.1 percent formic acid and 0.05 percent heptafluorobutyric acid aqueous solution, and B is a chromatogram of acetonitrile solution;
fig. 4 is a chromatographic column: waters, HSS T3, 100X 2.1mm,2.5 μm; column temperature: at 40 ℃; flow rate: 0.2mL/min; mobile phase: a is 0.1 percent formic acid and 0.05 percent heptafluorobutyric acid aqueous solution, and B is a chromatogram of acetonitrile solution;
FIG. 5 is a chromatogram corresponding to comparative example 1;
FIG. 6 is a chromatogram corresponding to comparative example 2;
FIG. 7 is a chromatogram corresponding to comparative example 3;
FIG. 8 is a chromatogram corresponding to comparative example 4;
fig. 9 is a chromatogram corresponding to comparative example 6.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the embodiments of the present invention, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions and alterations are intended to be included in the scope of the present invention.
Example 1:
1 apparatus
Liquid chromatography-mass spectrometer: LC-30A liquid chromatograph (SHIMADZU, japan) connected in series to a mass spectrometer of the TRIPLE QUAD 6500+ type (AB SCIEX, USA); XSE205DU model electronic balance (Mettler Toledo, switzerland).
2 reagent
Reagent: first-stage water consumption; acetonitrile (chromatographically pure); methanol (chromatographically pure); isopropanol (chromatographically pure); heptafluorobutyric acid (LC-MS); formic acid (LC-MS).
Control (see table 1):
TABLE 1
0.1% formic acid 0.05% aqueous heptafluorobutyric acid: 1L of water is taken, and 1mL of formic acid and 0.5mL of heptafluorobutyric acid are added and mixed evenly.
70% methanol solution: weighing 700mL of methanol and 300mL of water, and mixing uniformly.
3 analytical method
3.1 Instrument conditions and parameters:
3.1.1 liquid phase conditions
The invention optimizes the liquid chromatogram conditions:
the invention uses a liquid chromatography-mass spectrometer, a detected target object contains fat-soluble vitamins and water-soluble vitamins, the properties of the target object are different, and the key of optimizing the liquid phase condition is to keep and separate a target compound on a chromatographic column, so that the invention optimizes the liquid phase condition as follows:
(1) Attempts were made to use a chromatographic column: phenomenex, kinetex PFP, 100X 4.6mm,2.6 μm; column temperature: 40 ℃; flow rate: 0.6mL/min; mobile phase: a is 0.1% formic acid water solution, B is 0.1% formic acid acetonitrile solution, as shown in figure 2, thiamine and nicotinamide are not retained on the chromatographic column;
(2) Attempts were made to use a chromatographic column: phenomenex, kinetex PFP, 100X 4.6mm,2.6 μm; column temperature: at 40 ℃; flow rate: 0.6mL/min; mobile phase: a is 0.1% formic acid 0.05% heptafluorobutyric acid aqueous solution, B is acetonitrile solution, as shown in figure 3, target thing retinol, alpha-tocopherol, thiamine, riboflavin, nicotinamide and pantothenic acid are all kept on the chromatographic column, the separation is effectual.
(3) Attempts were made to use a chromatographic column: waters, HSS T3, 100X 2.1mm,2.5 μm; column temperature: 40 ℃; flow rate: 0.2mL/min; mobile phase: a is 0.1% formic acid 0.05% heptafluorobutyric acid aqueous solution, B is acetonitrile solution, as shown in figure 4, alpha-tocopherol, thiamine, riboflavin are not retained on the chromatographic column;
thus, the present invention identifies preferred liquid phase conditions as: and (3) chromatographic column: phenomenex, kinetex PFP, 100X 4.6mm,2.6 μm; column temperature: flow rate at 40 ℃:0.6mL/min; mobile phase: a was 0.1% formic acid 0.05% heptafluorobutyric acid in water and B was acetonitrile, and the elution was carried out in the following gradient 2:
TABLE 2
3.1.2 Mass Spectrometry conditions
MRM mode (see Table 3)
TABLE 3
| CUR | CAD | IS | TEM | GS1 | | EP | CXP | |
| 40 | 9 | 5500 | 350 | 50 | 50 | 10 | 10 |
* And (4) quantifying ion pairs.
3.2 preparation of control solutions
Precisely weighing a proper amount of reference substances of retinol, alpha-tocopherol, thiamine hydrochloride, riboflavin, nicotinamide and pantothenic acid, dissolving the retinol and the alpha-tocopherol in absolute ethyl alcohol, dissolving the thiamine hydrochloride, the nicotinamide and sodium pantothenate in water, dissolving the riboflavin in a proper amount of hydrochloric acid, diluting with water, fixing the volume, and shaking up to obtain a stock standard solution.
Precisely transferring a proper amount of the stock standard solutions respectively, diluting the stock standard solutions to a proper concentration by using 60% methanol, precisely transferring 20 mu L of mixed standard solutions, adding 80 mu L of bovine serum albumin solution (2%), and uniformly mixing by vortexes to obtain working standard solutions, which are shown in Table 4 below. Working standard solution with proper concentration is prepared according to the response and actual conditions of the instrument.
TABLE 4
3.3 preparation of internal standard solution
Preparing an internal standard solution: an appropriate amount of internal standard solution is precisely transferred and diluted to an appropriate concentration by adding acetonitrile, which is shown in the following table 5
TABLE 5
3.4 Standard working Curve plotting
Precisely sucking 40 mu L of the working standard solution into a 1.5mL brown centrifuge tube, pretreating according to the steps of 3.5, injecting into a liquid chromatography-mass spectrometer, and establishing a standard curve equation.
3.5 preparation of test articles
Respectively collecting 20 mu L of whole blood by using a dry blood collection system (DBS and VAMS), drying for about 0.5h-4h, putting two dry blood samples into a 1.5mL light-proof centrifuge tube (equivalent to 40 mu L of blood), adding 200 mu L deionized water, carrying out vortex flow for 1min, carrying out ultrasound treatment for 10min (30 KHz, normal temperature), adding 400 mu L organic solvent containing an isotope internal standard (prepared by methanol/acetonitrile (1:1)), carrying out vortex flow for 1min, carrying out ultrasound treatment for 10min (30 KHz, normal temperature), centrifuging for 5 min (20000 r/min, normal temperature), taking 400 mu L supernatant, carrying out nitrogen blow-drying (1h, 40 ℃), adding 80 mu L methanol/water (1:1) for redissolution, carrying out vortex flow for 1min, and centrifuging for 5 min (20000 r/min, normal temperature), thus obtaining a sample solution. Precisely absorbing 5 mu L of sample solution, injecting the sample solution into a liquid chromatography-mass spectrometer, and measuring the content of 6 vitamins.
3.6 results calculation:
and calculating the content of each vitamin extracted from the VAMS through a standard curve equation.
4 methodological validation
4.1 specificity test (blank)
4.1.1 test methods
A blank is processed according to a 3.5 sample preparation method without adding a sample, and the blank is measured according to a 3.1 condition and compared with the peak-out time of a working standard solution of retinol, alpha-tocopherol, thiamine hydrochloride, riboflavin, nicotinamide and pantothenic acid.
4.1.2 conclusions of the experiment:
the blank has no peak at the peak-producing time of retinol, alpha-tocopherol, thiamine hydrochloride, riboflavin, nicotinamide and pantothenic acid, which indicates that the blank has no interference to the determination result.
4.2 Linear Range confirmation
4.2.1 Standard Curve and Linear relationship
Retinol, α -tocopherol, thiamine hydrochloride, riboflavin, niacinamide, pantothenic acid had good linear correlations within the respective linear ranges, and the results are shown in table 6.
TABLE 6
4.2.2 line test conclusion
And (3) linear evaluation: the correlation coefficients R of the retinol, the alpha-tocopherol, the thiamine, the riboflavin, the nicotinamide and the pantothenic acid are respectively 0.996, 0.997, 0.995, 0.994, 0.997 and 0.995, so that the retinol, the alpha-tocopherol, the thiamine, the riboflavin, the nicotinamide and the pantothenic acid measured by the method have good linearity and meet the requirement of GB/T27404-2008 laboratory quality control Specification [ GB/T27404-2008 requires that the correlation coefficient R is more than or equal to 0.99 ].
4.3 detection and quantitation limits
The detection limit DL and the quantification limit QL of the analytical method are calculated from the signal-to-noise ratio (S/N). DL is defined as the concentration to be analyzed corresponding to S/N =3, and QL is defined as the concentration to be analyzed corresponding to S/N = 10.
Retinol: the detection limit is 5ng/mL, and the quantification limit is 15ng/mL;
α -tocopherol: the detection limit is 30ng/mL, and the quantification limit is 100ng/mL;
thiamine: the detection limit is 0.06ng/mL, and the quantification limit is 0.2ng/mL;
riboflavin: the detection limit is 0.3ng/mL, and the quantification limit is 1ng/mL;
nicotinamide: the detection limit is 0.04ng/mL, and the quantification limit is 0.12ng/mL;
pantothenic acid: the detection limit is 10ng/mL, and the quantification limit is 30ng/mL.
4.4 precision test
4.4.1 test methods
And respectively taking 6 parts of VAMS and DBS samples, processing the samples according to a 3.5 sample preparation method, detecting the content of the samples, and calculating the RSD (%).
4.4.2 test data (see Table 7 below) [ test samples: extracted dried blood sample ].
TABLE 7
4.4.3 conclusion of the test
The RSD of the content of the retinol, the alpha-tocopherol, the thiamine, the riboflavin, the nicotinamide and the pantothenic acid in the test sample is 2.33-14.44 percent, which shows that the method has better precision in the day.
4.5 day precision
4.5.1 test methods:
taking 6 parts of VAMS and DBS samples respectively, dividing the samples into 3 groups, each group comprises 2 parts, detecting one group in one day, processing the samples according to the preparation method of 3.5 samples, detecting the content of the samples, and calculating the RSD (%).
4.5.2 test data: (see table 8 below) [ test samples are: extracted dried blood samples ]
TABLE 8
4.5.3 conclusion of the test
The RSD of the content of the retinol, the alpha-tocopherol, the thiamine, the riboflavin, the nicotinamide and the pantothenic acid in the test sample is 3.01-14.47%, which shows that the method has better daytime precision.
4.6 durability test (stability)
4.6.1 test methods:
processing the samples according to the preparation method of the 3.5 samples, placing VAMS and DBS samples into an aluminum foil bag for sealing, respectively placing the samples at room temperature, 4 ℃, 18 ℃ and 80 ℃ for 0 day, 5 days and 20 days, and then detecting the content of the samples according to the 3.1 condition.
4.6.2 test data: (see table 9 below) [ test samples are: extracted dried blood sample ]
TABLE 9
4.6.3 test conclusion
After the VAMS and DBS samples are respectively placed at room temperature, 4 ℃, 18 ℃ and 80 ℃ for 0 day, 5 days and 20 days, the content stability of the retinol, alpha-tocopherol, thiamine, riboflavin, nicotinamide and pantothenic acid is good, which indicates that the retinol, alpha-tocopherol, thiamine, riboflavin, nicotinamide and pantothenic acid of the dried blood samples are good in stability within 0 day, 5 days and 20 days when the retinol, alpha-tocopherol, thiamine, riboflavin, nicotinamide and pantothenic acid are placed at the room temperature, 4 ℃, 18 ℃ and 80 ℃.
4.7 accuracy test (recovery)
4.7.1 test methods
Adding a standard: precisely transferring 1.00mL of whole blood, precisely adding a proper amount of standard solution, and uniformly mixing by vortex. The samples were processed according to the 3.5 sample preparation method. And (3) evaluating the extraction recovery rates of the retinol, the alpha-tocopherol, the thiamine, the riboflavin, the nicotinamide and the pantothenic acid by adopting three concentrations of LQC, MQC and HQC and adopting quality control samples of 6 parallel samples of each concentration.
4.7.2 test data (see Table 10 below) [ test samples: extracted dried blood sample ]
Table 10 recovery test results table (n = 6)
Recovery (%) = (measured-background)/spiked value × 100%
4.7.3 conclusion of the test
The average recovery rates of retinol, alpha-tocopherol, thiamine, riboflavin, nicotinamide and pantothenic acid in the sample are all within an acceptable range, which shows that the method has better recovery rate and good accuracy.
Comparative example 1: comparative testing of the literature methods with the methods of the invention
Literature methods
The method for detecting water-soluble and fat-soluble vitamins in dried blood spots (CN 106770802A) comprises the following steps: adopting a dry blood filter paper specimen, using a perforating pliers to punch 10-20 blood spots with the diameter of 3.2 mm, putting the blood spots into a centrifuge tube, adding 200-500 mu L of extracting solution (the extracting solution is 84.75-98.95% of methanol, 1-15% of water and 0.05-0.25% of formic acid, or the extracting solution comprises the following components by volume percentage, namely 20-79% of ethanol, 20-70% of methanol and 1-10% of water), and incubating for 45 minutes at the temperature of 27-30 ℃ with vibration frequency oscillation of 650-750 rpm; centrifuging, adding all the supernatant into a 96-well plate, blow-drying with a nitrogen blow-drying instrument, re-dissolving another 100 μ L of mobile phase, adding 50 μ L of mixed internal standard working solution, covering with an aluminum foil sealing film to reduce volatilization, and oscillating and mixing uniformly; and (3) putting the 96-well plate into an automatic sample injector of a tandem mass spectrometry system, starting application software, establishing a sample list, and selecting a correct internal standard concentration file and a collection method to detect the content of multiple vitamins in the dry blood filter paper sheet.
2 comparative test results (see Table 11 and FIG. 5)
TABLE 11
3 conclusion of the test
By comparing the content of water-soluble and fat-soluble vitamins in the dried blood samples (dried blood spots and VAMS) measured by the literature method and the method of the invention, the relative standard deviation of the results of the retinol and the nicotinamide of the two methods is more than 30 percent, and the relative deviation of the results of the alpha-tocopherol, the thiamine, the riboflavin and the pantothenic acid is less than 10 percent, which proves that the method for measuring the content of the water-soluble and fat-soluble vitamins in the dried blood has higher extraction efficiency and more accurate quantitative result.
Comparative example 2: comparative test of adding different extraction solvents in the invention
The added extraction solvents are water and n-hexane: two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL organic solvent (normal hexane) containing isotope internal standard is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 400 muL supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 muL methanol/water (1:1) is added for redissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), so that a sample solution is obtained.
The extraction solvent added was water and 200g/L trichloroacetic acid (prepared with methanol-acetonitrile (1:1)): two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL trichloroacetic acid (methanol-acetonitrile (1:1)) containing isotope internal standards is added for preparation, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 400 muL supernatant is taken and is dried by nitrogen gas (1h, 40 ℃), 80 muL methanol-water (1:1) is added for redissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining a sample solution.
The extraction solvents added were water and methanol-acetonitrile (1:1): two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugal treatment is carried out for 5 minutes (20000 r/min, normal temperature), 400 muL supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 muL methanol-water (1:1) is added for re-dissolution, vortex flow is carried out for 1 minute, and centrifugal treatment is carried out for 5 minutes (20000 r/min, normal temperature), and then sample solution is obtained.
2 comparative test results (see Table 12 and FIG. 6)
TABLE 12
3 conclusion of the test
Through the comparison of experiments of adding different extraction solvents in the invention, the relative standard deviation of the results of retinol and nicotinamide is more than 40%, and the relative standard deviation of the results of alpha-tocopherol, thiamine, riboflavin and pantothenic acid is less than 10%, so that the extraction efficiency is higher and the quantitative result is more accurate when the extraction solvent which is water and methanol-acetonitrile (1:1) solution is added in the pretreatment of a dried blood sample.
Comparative example 3: comparative test of adding organic solvents with different proportions in the invention
The extraction solvent added was water, and an organic solvent methanol-acetonitrile (1:2) solution: two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL organic solvent containing isotope internal standard (methanol-acetonitrile solution (1:2)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 400 muL supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 muL methanol-water (1:1) is added for re-dissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining sample solution.
The extraction solvent added was water, and the organic solvent methanol-acetonitrile (2:1) solution: two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL organic solvent containing isotope internal standard (methanol-acetonitrile solution (2:1)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 400 muL supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 muL methanol-water (1:1) is added for re-dissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining sample solution.
The extraction solvent added was water, and an organic solvent methanol-acetonitrile (1.5) solution: two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL organic solvent containing isotope internal standard (methanol-acetonitrile solution (1.5)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 400 muL supernatant is taken, nitrogen is used for blow drying (1 h,40 ℃) and 80 muL methanol-water (1:1) is added for redissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining sample solution.
The extraction solvent added was water, and an organic solvent methanol-acetonitrile (1.5: two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL organic solvent containing isotope internal standard (methanol-acetonitrile solution (1.5.
The extraction solvent added was water, and an organic solvent methanol-acetonitrile (1:1) solution: two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL organic solvent containing isotope internal standard (methanol-acetonitrile solution (1:1)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 400 muL supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 muL methanol-water (1:1) is added for re-dissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining sample solution.
2 comparative test results (see Table 13 and FIG. 7)
Watch 13
3 conclusion of the test
Through the comparison of experiments of adding different proportions of organic solvents in the invention, when the volumes of the organic solvent methanol-acetonitrile solutions added are 1:2, 2:1 and 1:1 respectively, the relative standard deviation of the results of alpha-tocopherol is >20%, and the relative standard deviation of the results of retinol, thiamine, riboflavin, nicotinamide and pantothenic acid is < 10%; whereas when the organic solvents methanol-acetonitrile solution were added at volumes of 1.5, 1.5. The method proves that when the extraction solvent is added into the dried blood sample for pretreatment, the extraction efficiency is higher and the quantitative result is more accurate when the methanol-acetonitrile (1.5-1.5.
Comparative example 4: comparative experiment of adding mixed extraction solvent and adding extraction solvent respectively in the invention
A mixed extraction solvent (water: methanol: acetonitrile (1): two dried blood samples (VAMS and DBS) were placed in 1.5mL light-resistant centrifuge tubes (equivalent to 40. Mu.L blood), water methanol acetonitrile (1: acetonitrile 1).
The extraction solvents water, and methanol-acetonitrile (1:1) solution were added separately: two dried blood samples (VAMS and DBS) are put into a 1.5mL light-proof centrifuge tube (equivalent to 40 muL blood), 200 muL deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 muL organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugal treatment is carried out for 5 minutes (20000 r/min, normal temperature), 400 muL supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 muL methanol-water (1:1) is added for re-dissolution, vortex flow is carried out for 1 minute, and centrifugal treatment is carried out for 5 minutes (20000 r/min, normal temperature), and then sample solution is obtained.
2 comparative test results (see Table 14 and FIG. 8)
TABLE 14
3 conclusion of the test
Through experimental comparison of different modes of adding the extraction solvent in the invention, the relative deviation of the results of the retinol and the nicotinamide in the two methods is more than 50%, and the relative deviation of the results of the alpha-tocopherol, the thiamine, the riboflavin and the pantothenic acid is less than 10%, so that the extraction efficiency is higher and the quantitative result is more accurate when the extraction solvent which is water or a methanol-acetonitrile (1:1) solution is added in the pretreatment of a dried blood sample.
Comparative example 5: comparative experiment with addition of two extraction solvents (water and organic solvent) in different proportions in the present invention
The extraction solvent added was 200. Mu.L of water, and 100. Mu.L of methanol-acetonitrile (1:1) solution: two dried blood samples (VAMS and DBS) are put into a 1.5mL lightproof centrifuge tube (equivalent to 40 μ L of blood), 200 μ L of deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 100 μ L of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 200 μ L of supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 μ L of methanol-water (1:1) is added for redissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining sample solution.
The extraction solvent added was 200. Mu.L of water, and 200. Mu.L of methanol-acetonitrile (1:1) solution: two dried blood samples (VAMS and DBS) are put into a 1.5mL lightproof centrifuge tube (equivalent to 40 μ L of blood), 200 μ L of deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 200 μ L of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 266 μ L of supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 μ L of methanol-water (1:1) is added for redissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining sample solution.
The extraction solvent added was 200. Mu.L of water, and 400. Mu.L of methanol-acetonitrile (1:1) solution: two dried blood samples (VAMS and DBS) are put into a 1.5mL lightproof centrifuge tube (equivalent to 40 μ L of blood), 200 μ L of deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 μ L of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 400 μ L of supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 μ L of methanol-water (1:1) is added for redissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining sample solution.
2 comparative test results (see Table 15)
Conclusion of the experiment
Through the comparison of experiments of adding two extraction solvents (water and an organic solvent) in different proportions, the relative standard deviation of the results of retinol, alpha-tocopherol, thiamine, riboflavin, nicotinamide and pantothenic acid is less than 10%, and the volume ratio of the water added with the extraction solvent to the methanol-acetonitrile (1:1) solution can be 1-2:2-1 when a dried blood sample is pretreated.
Comparative example 6: comparative test of different addition sequences of two extraction solvents (water and organic solvent) in the present invention
The solvent was added in the order of 200. Mu.L water followed by 400. Mu.L methanol-acetonitrile (1:1): two dried blood samples (VAMS and DBS) are put into a 1.5mL lightproof centrifuge tube (equivalent to 40 μ L of blood), 200 μ L of deionized water is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), 400 μ L of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortex flow is carried out for 1 minute, ultrasonic treatment is carried out for 10 minutes (30 KHz, normal temperature), centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), 400 μ L of supernatant is taken, nitrogen is used for blow drying (1h, 40 ℃), 80 μ L of methanol-water (1:1) is added for redissolution, vortex flow is carried out for 1 minute, and centrifugation is carried out for 5 minutes (20000 r/min, normal temperature), thus obtaining sample solution.
The solvent was added in the order of 400. Mu.L methanol-acetonitrile (1:1) solution, followed by 200. Mu.L water: two dried blood samples (VAMS and DBS) are put into a 1.5mL lightproof centrifuge tube (equivalent to 40 mu L of blood), 400 mu L of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1) solution) is added, vortex is carried out for 1min, ultrasonic treatment is carried out for 10min (30 KHz, normal temperature), 200 mu L of deionized water is added, vortex is carried out for 1min, ultrasonic treatment is carried out for 10min (30 KHz, normal temperature), centrifugation is carried out for 5 min (20000 r/min, normal temperature), 400 mu L of supernatant is taken and dried by nitrogen (1h, 40 ℃), 80 mu L of methanol-water (1:1) is added for redissolution, vortex is carried out for 1min, and centrifugation is carried out for 5 min (20000 r/min, normal temperature), thus obtaining sample solution.
2 comparative test results (see Table 16 and FIG. 9)
TABLE 16
3 conclusion of the test
Through the test comparison of different modes of adding the extraction solvent in the invention, the relative standard deviation of the results of the retinol and the nicotinamide of the two methods is more than 50 percent, and the relative standard deviation of the results of the alpha-tocopherol, the thiamine, the riboflavin and the pantothenic acid is less than 10 percent, which proves that when the pretreatment of a dried blood sample is carried out, the order of adding the extraction solvent is firstly adding 200 mu L of water and then adding 400 mu L of methanol-acetonitrile (1:1) solution, the extraction efficiency is higher, and the quantitative result is more accurate.
In conclusion, the special dry blood collecting system is adopted to collect the dry blood sample, and the sampling process is simple and easy to operate; by optimizing the proportion of the extraction solvent and the sequence of adding the solvent, the extraction of various fat-soluble and water-soluble vitamins in dry blood samples (VAMS and DBS) can be realized, the extraction efficiency is high, the required sample amount is small, and the detection result of the vitamins has higher precision and accuracy.
Claims (10)
1. A method for extracting multivitamins in blood is characterized by comprising the following steps:
(1) Quantitatively acquiring blood by using a dry blood collecting system, and drying to prepare a dry blood sample;
(2) Extracting and purifying a substance to be detected in the dried blood sample by using an extraction solvent to obtain a vitamin sample to be detected;
wherein the extraction solvent is a mixed extraction solvent of water and a methanol-acetonitrile solution.
2. The method for extracting multivitamins from blood according to claim 1, wherein in step (1), the dry blood collection system is a dry blood spot collection card (DBS) or a volume absorption micro-sampler (VAMS).
3. The method for extracting multivitamins from blood according to claim 1, wherein the drying time in step (1) is 0.5-4 hours.
4. The method for extracting multivitamins from blood according to claim 1, wherein in the step (2), the volume ratio of methanol to acetonitrile in the methanol-acetonitrile solution is 1:2-2:1, preferably 1.5-1.5.
5. The method for extracting multivitamins from blood according to claim 1, wherein in the step (2), the volume ratio of water to the methanol-acetonitrile solution in the extraction solvent is 1-2:2-1.
6. The method for extracting multivitamins from blood according to claim 1, wherein in the step (2), the extraction solvent is added to the sample in the order of adding water and then adding methanol-acetonitrile solution, preferably, the extraction solvent is added to water for 5-30min and then adding methanol-acetonitrile solution for extraction for 5-30min.
7. The method according to claim 1, wherein in the step (2), the extraction solvent simultaneously extracts more than one dried blood sample; the purification mode is centrifugation, and the centrifugation time is 3-10min.
8. A method for detecting the content of multiple vitamins in blood is characterized by comprising the following steps:
(1) Preparing a vitamin sample to be detected by adopting the method for extracting the multivitamins in the blood as set forth in any one of claims 1 to 7;
(2) Analyzing a vitamin sample to be detected by using a liquid chromatography-mass spectrometer, and quantifying by adopting an internal standard method;
wherein the conditions of the liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the aqueous solution of heptafluorobutyric acid-formic acid is taken as a mobile phase A, the acetonitrile solution is taken as a mobile phase B, and the gradient elution procedure is as follows:
。
9. The method according to claim 8, wherein in the mobile phase A, the concentration by mass of the heptafluorobutyric acid is 0.05-0.1%, and the concentration by mass of the formic acid is 0.05-0.1%; the flow rate is 0.4-0.8mL/min; the column temperature is 25-45 ℃.
10. The method according to claim 8, wherein the mass spectrometry conditions are as follows: an ionization mode: electrospray positive ion mode; the detection mode is as follows: multiple reaction monitoring, MRM; air curtain pressure: 20-50psi; ion spray voltage: 3000-5500V; temperature: 300-550 ℃; atomizing: 30-60psi; auxiliary heating gas: 30-60psi.
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