Extraction and content detection method of multiple vitamins in blood

Abstract

The present invention discloses a method for extracting and detecting the content of multiple vitamins in blood, including the steps of: using a dry blood collection system to quantitatively obtain blood, drying to prepare a dry blood sample; extracting and purifying the analyte in the dry blood sample with an extraction solvent, thereby obtaining a vitamin sample to be detected; analyzing with a liquid chromatography-mass spectrometer, and using an internal standard method for quantification. The method of the present invention has high extraction efficiency for multiple water-soluble and fat-soluble vitamins, and is combined with a liquid chromatography-tandem mass spectrometer (LC-MS) to detect the content. The detection method is simple, rapid, and accurate, in order to provide a scientific basis for the research on disease diagnosis and health monitoring needs of a large sample of people.

Description

Technical Field

[0001] The invention belongs to the technical field of blood detection, and in particular relates to a method for extracting and detecting the contents of multiple vitamins in blood. Background Art

[0002] The B vitamins in the blood are water-soluble vitamins with a variety of biological activities. Water-soluble B vitamins are involved in many important metabolic reactions and are essential for maintaining our health. B vitamin deficiency 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 that participates in carbohydrate metabolism and is directly involved in nerve function. VB2 is a cofactor for several reductase enzymes in energy metabolism. VB2 also serves as a coenzyme for folic acid, VB12, VB6, and homocysteine ​​metabolism. VB3 and VB5 are both involved in neurotransmission and fatty acid synthesis, oxidation / reduction reactions, or one-carbon metabolism. Due to the importance of B vitamins, optimal nutritional status of these vitamins must be maintained.

[0003] Fat-soluble vitamins are a variety of biochemical micronutrients necessary for healthy development, growth, metabolism, and cell regulation. We cannot fully synthesize fat-soluble vitamins, nor can we achieve the required concentrations. Vitamin A is essential throughout human life. Together with its derivatives, it regulates a variety of processes, including reproduction, embryogenesis, vision, growth, cell differentiation and proliferation, maintenance of epithelial cell integrity, and immune function. Studies have shown that vitamin A is also associated with mental and developmental disorders that disrupt cognition. Vitamin E regulates redox balance in the body due to its high concentration in the fat-soluble vitamin group and is ubiquitously present throughout the body. Deficiency or excess of fat-soluble vitamins can lead to many health problems.

[0004] Plasma is the most accessible 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 for simultaneous quantification of multiple water-soluble and fat-soluble vitamins at physiological concentrations.

[0005] At present, the methods for determining B vitamins and fat-soluble vitamins in blood in the industry include: high performance liquid chromatography and liquid chromatography-mass spectrometry. At present, the commonly used method for detecting B vitamins and fat-soluble vitamins in the human body is plasma detection. Plasma samples require professionals to collect venous blood, which is easy to cause blood contamination, and the samples need to be transported in a cold chain, which is very inconvenient on the way. The pre-treatment of the detection method using serum is more complicated, and it is necessary to extract the water-soluble vitamins and fat-soluble vitamins in the serum separately for detection. The current detection method for detecting multiple vitamins by manually extracting dry blood spots is to use ten dry blood spot samples, and extract the water-soluble and fat-soluble vitamins in the dry blood spot samples simultaneously through a mixed solvent of methanol, formic acid and water. The amount of dry blood spot samples required for this method is large. In practical applications, it is difficult to collect ten dry blood spots from the same subject at one time, which is difficult to apply to the disease diagnosis and health monitoring needs of a large sample population. In addition, the extraction efficiency of water-soluble vitamin B3 and fat-soluble vitamin A extracted by this method is low, which affects the accuracy of vitamin quantification. Summary of the invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a method for extracting and detecting the content of multiple vitamins (including water-soluble vitamins and fat-soluble vitamins) in blood. The method has high extraction efficiency for multiple water-soluble and fat-soluble vitamins in samples and more accurate quantitative results.

[0007] The present invention provides a method for extracting multiple vitamins from blood, comprising the following steps:

[0008] (1) obtaining blood using a dry blood collection system and drying the blood to prepare a dry blood sample;

[0009] (2) extracting and purifying the substance to be tested in the dried blood sample with an extraction solvent to obtain a vitamin sample to be tested;

[0010] Wherein, the extraction solvent is a mixed extraction solvent of water and methanol-acetonitrile solution.

[0011] Preferably, in step (1), the dried blood collection system is a dried blood spot collection card (DBS) or a volume absorption microsampler (VAMS).

[0012] Preferably, in step (1), the drying time is 0.5-4 hours.

[0013] The present invention can realize the extraction of water-soluble and fat-soluble multivitamins in a dried blood sample by optimizing the extraction solvent and the addition sequence, with high extraction efficiency and more accurate quantitative results.

[0014] Preferably, in step (2), in the methanol-acetonitrile solution, the volume ratio of methanol to acetonitrile is 1:2-2:1.

[0015] In some embodiments provided by the present invention, in the methanol-acetonitrile solution, the volume ratio of methanol to acetonitrile is preferably 1:1.5-1.5:1.

[0016] Preferably, in step (2), the volume ratio of water to methanol-acetonitrile solution in the mixed extraction solvent is 1-2:2-1.

[0017] To ensure sufficient extraction, preferably, in step (2), the order of adding the extraction solvent to the sample is to first add water and then add the methanol-acetonitrile solution;

[0018] Preferably, water is added to the sample and then extracted for 5-30 minutes, and then methanol-acetonitrile solution is added and extracted for 5-30 minutes;

[0019] Preferably, the extraction solvent extracts more than one dried blood sample simultaneously.

[0020] Preferably, the purification method in step (2) is centrifugation, and the centrifugation time is 3-10 min.

[0021] Another aspect of the present invention provides a method for detecting the content of multiple vitamins in blood, comprising the steps of:

[0022] (1) preparing a vitamin sample to be tested by using the above-mentioned method for extracting multiple vitamins from blood;

[0023] (2) analyzing the vitamin sample to be tested by liquid chromatography-mass spectrometry and quantifying by internal standard method;

[0024] The conditions of the liquid chromatography are as follows: using octadecylsilane bonded silica gel as the filler; using heptafluorobutyric acid-formic acid aqueous solution as the mobile phase A, using acetonitrile solution as the mobile phase B, and the gradient elution procedure is as follows:

[0025]

[0026]

[0027] Preferably, in the mobile phase A, the mass concentration of heptafluorobutyric acid is 0.05-0.1%, and the mass concentration of formic acid is 0.05-0.1%.

[0028] In some embodiments provided by the present invention, in the mobile phase A, the mass concentration of heptafluorobutyric acid is 0.05%, and the mass concentration of formic acid is 0.1%.

[0029] Preferably, the flow rate is 0.4-0.6 mL / min; and the column temperature is 25-45°C.

[0030] Preferably, the mass spectrometry conditions are: ionization mode: electrospray positive ion mode; detection mode: multiple reaction monitoring MRM; curtain gas pressure: 20-50psi; ion spray voltage: 3000-5500V; temperature: 300-550°C; nebulizing gas: 30-60psi; auxiliary heating gas: 30-60psi.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] The present invention provides a method for extracting and detecting the content of multiple vitamins in blood. A dry blood collection system is used to collect dry blood samples. The sampling process is simple and convenient for transportation and storage.

[0033] The extraction method adopted by the present invention has high extraction efficiency for multiple water-soluble and fat-soluble vitamins in dried blood samples, and is combined with liquid chromatography-tandem mass spectrometry (LC-MS) to detect the content. The detection method is simple, rapid and accurate, and is expected to provide a scientific basis for the research needs of disease diagnosis and health monitoring of large sample populations.

[0034] The present invention conducts linearity, precision, durability test (stability), specificity test (blank), detection limit, quantitative limit, accuracy (recovery rate) test, and actual sample measurement on the content determination method of various water-soluble and fat-soluble vitamins in the dry blood collection system, thereby proving that the content determination method is scientific and effective, and can achieve the purpose of quality control of the content of various water-soluble and fat-soluble vitamins in the dry blood collection system. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the control chromatogram;

[0036] Figure 2 The chromatographic column is Phenomenex, Kinetex PFP, 100×4.6 mm, 2.6 μm; the column temperature is 40°C; the flow rate is 0.6 mL / min; the mobile phase is A is 0.1% formic acid aqueous solution, and B is 0.1% formic acid acetonitrile solution;

[0037] Figure 3 Chromatographic column: Phenomenex, Kinetex PFP, 100×4.6 mm, 2.6 μm; column temperature: 40°C; flow rate: 0.6 mL / min; mobile phase: A is 0.1% formic acid 0.05% heptafluorobutyric acid aqueous solution, B is chromatogram of acetonitrile solution;

[0038] Figure 4Chromatographic column: Waters, HSS T3, 100×2.1 mm, 2.5 μm; column temperature: 40°C; flow rate: 0.2 mL / min; mobile phase: A is 0.1% formic acid 0.05% heptafluorobutyric acid aqueous solution, B is chromatogram of acetonitrile solution;

[0039] Figure 5 It is the chromatogram corresponding to Comparative Example 1;

[0040] Figure 6 It is the chromatogram corresponding to Comparative Example 2;

[0041] Figure 7 It is the chromatogram corresponding to Comparative Example 3;

[0042] Figure 8 It is the chromatogram corresponding to Comparative Example 4;

[0043] Fig. 9 It is the chromatogram corresponding to Comparative Example 6. DETAILED DESCRIPTION

[0044] The present invention is further illustrated by specific implementation methods below. The following examples are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0045] Embodiment 1:

[0046] 1 Instrument

[0047] Liquid chromatography-mass spectrometry: LC-30A liquid chromatograph (SHIMADZU, Japan) connected in series with TRIPLE QUAD6500+ mass spectrometer (AB SCIEX, USA); XSE205DU electronic balance (Mettler Toledo, Switzerland).

[0048] 2. Reagents

[0049] Reagents: primary water; acetonitrile (chromatographic grade); methanol (chromatographic grade); isopropanol (chromatographic grade); heptafluorobutyric acid (LC-MS); formic acid (LC-MS).

[0050] Reference substances (see Table 1):

[0051] Table 1

[0052]

[0053] 0.1% formic acid 0.05% heptafluorobutyric acid aqueous solution: Take 1L of water, add 1mL of formic acid and 0.5mL of heptafluorobutyric acid, and mix well.

[0054] 70% methanol solution: Measure 700 mL of methanol and 300 mL of water and mix well.

[0055] 3 Analysis methods

[0056] 3.1 Instrument conditions and parameters:

[0057] 3.1.1 Liquid phase conditions

[0058] The present invention optimizes the liquid chromatography conditions:

[0059] The present invention uses a liquid chromatography-mass spectrometer. The target substances to be detected include fat-soluble vitamins and water-soluble vitamins. The properties of the target substances are different. The key to optimizing the liquid phase conditions is to retain the target compounds on the chromatographic column and achieve separation. For this purpose, the present invention optimizes the liquid phase conditions as follows:

[0060] (1) Try using the chromatographic column: Phenomenex, Kinetex PFP, 100×4.6 mm, 2.6 μm; column temperature: 40°C; flow rate: 0.6 mL / min; mobile phase: A is 0.1% formic acid in water, B is 0.1% formic acid in acetonitrile, such as Figure 2 As shown, thiamine and nicotinamide were not retained on the column;

[0061] (2) Try using the chromatographic column: Phenomenex, Kinetex PFP, 100×4.6 mm, 2.6 μm; column temperature: 40°C; flow rate: 0.6 mL / min; mobile phase: A is 0.1% formic acid and 0.05% heptafluorobutyric acid aqueous solution, B is acetonitrile solution, such as Figure 3 As shown in the figure, the target substances retinol, α-tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid are all retained on the chromatographic column, and the separation effect is good.

[0062] (3) Try using the chromatographic column: Waters, HSS T3, 100×2.1 mm, 2.5 μm; column temperature: 40°C; flow rate: 0.2 mL / min; mobile phase: A is 0.1% formic acid and 0.05% heptafluorobutyric acid aqueous solution, B is acetonitrile solution, such as Figure 4 As shown, α-tocopherol, thiamine, and riboflavin were not retained on the column;

[0063] Therefore, the preferred liquid phase conditions determined in the present invention are: chromatographic column: Phenomenex, Kinetex PFP, 100×4.6 mm, 2.6 μm; column temperature: 40° C. flow rate: 0.6 mL / min; mobile phase: A is 0.1% formic acid 0.05% heptafluorobutyric acid aqueous solution, B is acetonitrile, and gradient elution is performed according to Table 2 below:

[0064] Table 2

[0065]

[0066] 3.1.2 Mass spectrometry conditions

[0067] MRM mode (see Table 3)

[0068] Table 3

[0069] CUR CAD IS TEM GS1 GS2 EP CXP 40 9 5500 350 50 50 10 10

[0070]

[0071] *Quantitative ion pairs.

[0072] 3.2 Preparation of reference solution

[0073] Accurately weigh appropriate amounts of retinol, α-tocopherol, thiamine hydrochloride, riboflavin, niacinamide, and pantothenic acid reference substances; dissolve retinol and α-tocopherol in anhydrous ethanol; dissolve thiamine hydrochloride, niacinamide, and sodium pantothenate in water; dissolve riboflavin in appropriate amount of hydrochloric acid and then dilute with water, make up to volume, and shake well to obtain the stock standard solution.

[0074] Accurately pipette appropriate amount of reserve standard solution, dilute to appropriate concentration with 60% methanol, then accurately pipette 20 μL of mixed standard solution, add 80 μL of bovine serum albumin solution (2%), vortex mix, and obtain working standard solution, see Table 4 below. Prepare working standard solution of appropriate concentration according to instrument response and actual situation.

[0075] Table 4

[0076]

[0077] 3.3 Preparation of internal standard solution

[0078] Preparation of internal standard solution: Accurately pipette an appropriate amount of internal standard solution and dilute it to an appropriate concentration with acetonitrile, see Table 5 below

[0079] Table 5

[0080]

[0081] 3.4 Drawing of standard working curve

[0082] Accurately pipette 40 μL of the working standard solution into a 1.5 mL brown centrifuge tube, perform pretreatment according to the steps described in 3.5, inject into the liquid chromatography-mass spectrometer, and establish the standard curve equation.

[0083] 3.5 Preparation of test samples

[0084] 20 μL of whole blood was collected by using the dry blood collection system (DBS and VAMS), dried for about 0.5h-4h, and two dried blood samples were placed in a 1.5mL light-proof centrifuge tube (equivalent to 40 μL of blood), 200 μL of deionized water was added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), 400 μL of organic solvent containing isotope internal standard (prepared with methanol / acetonitrile (1:1)) was added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), centrifuged for 5 minutes (20000r / min, room temperature), 400 μL of supernatant was taken, dried with nitrogen (1h, 40℃), and 80 μL of methanol / water (1:1) was added for re-dissolution, vortexed for 1 minute, and centrifuged for 5 minutes (20000r / min, room temperature) to obtain the sample solution. 5 μL of the sample solution was accurately aspirated and injected into the liquid chromatography-mass spectrometer to determine the content of 6 vitamins.

[0085] 3.6 Calculation results:

[0086] The content of each vitamin extracted from VAMS was calculated using the standard curve equation.

[0087] 4 Methodological validation

[0088] 4.1 Specificity test (blank)

[0089] 4.1.1 Test methods

[0090] Without adding sample, treat blank according to the sample preparation method in 3.5, measure blank according to the conditions in 3.1, and compare the peak time with that of retinol, α-tocopherol, thiamine hydrochloride, riboflavin, niacinamide, and pantothenic acid working standard solutions.

[0091] 4.1.2 Test conclusion:

[0092] The blank had no peaks at the peak times of retinol, α-tocopherol, thiamine hydrochloride, riboflavin, niacinamide, and pantothenic acid, indicating that the blank had little interference with the determination results.

[0093] 4.2 Confirmation of linear range

[0094] 4.2.1 Standard curve and linear relationship

[0095] Retinol, α-tocopherol, thiamine hydrochloride, riboflavin, niacinamide, and pantothenic acid have good linear correlation within their respective linear ranges. The results are shown in Table 6.

[0096] Table 6

[0097]

[0098] 4.2.2 Conclusion of linear test

[0099] Linear evaluation: The correlation coefficients R of retinol, α-tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid are 0.996, 0.997, 0.995, 0.994, 0.997 and 0.995 respectively. Therefore, the method for determining retinol, α-tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid shows good linearity, which meets the requirements of GB / T27404-2008 "Laboratory Quality Control Specifications" [GB / T27404-2008 requires a correlation coefficient R ≥ 0.99].

[0100] 4.3 Limit of detection and limit of quantification

[0101] The detection limit DL and quantification limit QL of the analytical method were calculated from the signal-to-noise ratio (S / N). DL was defined as the concentration of the analyte corresponding to S / N=3, and QL was defined as the concentration of the analyte corresponding to S / N=10.

[0102] Retinol: detection limit is 5ng / mL, quantification limit is 15ng / mL;

[0103] α-Tocopherol: detection limit is 30 ng / mL, quantification limit is 100 ng / mL;

[0104] Thiamine: detection limit is 0.06ng / mL, quantification limit is 0.2ng / mL;

[0105] Riboflavin: detection limit is 0.3 ng / mL, quantification limit is 1 ng / mL;

[0106] Nicotinamide: detection limit is 0.04ng / mL, quantification limit is 0.12ng / mL;

[0107] Pantothenic acid: The detection limit is 10 ng / mL and the quantification limit is 30 ng / mL.

[0108] 4.4 Precision test

[0109] 4.4.1 Test methods

[0110] Take 6 VAMS and DBS samples respectively, process the samples according to the sample preparation method in 3.5, detect the sample content, and calculate its RSD (%).

[0111] 4.4.2 Test data (see Table 7 below) [Test sample: extracted dried blood sample].

[0112] Table 7

[0113]

[0114] 4.4.3 Test conclusion

[0115] The RSDs of retinol, α-tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid in the samples were between 2.33% and 14.44%, indicating that the method had good intra-day precision.

[0116] 4.5 Inter-day precision

[0117] 4.5.1 Test method:

[0118] Take 6 VAMS and DBS samples respectively and divide them into 3 groups, 2 samples in each group, and test one group every day. Process the samples according to the sample preparation method in 3.5, test the sample content, and calculate its RSD (%).

[0119] 4.5.2 Test data: (see Table 8 below) [Test sample: extracted dried blood sample]

[0120] Table 8

[0121]

[0122] 4.5.3 Test conclusion

[0123] The RSDs of retinol, α-tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid in the samples were between 3.01 and 14.47%, indicating that the method had good inter-day precision.

[0124] 4.6 Durability test (stability)

[0125] 4.6.1 Test method:

[0126] Treat the samples according to the sample preparation method in 3.5. Put the VAMS and DBS samples into aluminum foil bags and seal them. After placing them at room temperature, 4°C, -18°C, and -80°C for 0 days, 5 days, and 20 days, respectively, the sample content was detected according to the conditions in 3.1.

[0127] 4.6.2 Test data: (see Table 9 below) [Test sample: extracted dried blood sample]

[0128] Table 9

[0129]

[0130]

[0131] 4.6.3 Test conclusion

[0132] After the VAMS and DBS samples were placed at room temperature, 4°C, -18°C and -80°C for 0 day, 5 days and 20 days, respectively, the retinol, α-tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid contents were stable, indicating that the retinol, α-tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid in the dried blood samples were stable at room temperature, 4°C, -18°C and -80°C for 0 day, 5 days and 20 days.

[0133] 4.7 Accuracy test (recovery rate)

[0134] 4.7.1 Test methods

[0135] Spiking: Accurately pipette 1.00 mL of whole blood, accurately add appropriate amount of standard spike solution, and vortex to mix. Process the sample according to the sample preparation method in 3.5. Use three concentrations of LQC, MQC and HQC, and 6 parallel samples of each concentration to evaluate the extraction recovery of retinol, α-tocopherol, thiamine, riboflavin, niacinamide, and pantothenic acid.

[0136] 4.7.2 Test data (see Table 10 below) [Test sample: extracted dried blood sample]

[0137] Table 10 Recovery test results (n = 6)

[0138]

[0139]

[0140] Recovery rate (%) = (measured value - background value) / spiked value × 100%

[0141] 4.7.3 Test conclusion

[0142] The average recoveries of retinol, α-tocopherol, thiamine, riboflavin, niacinamide, and pantothenic acid in the samples were all within the acceptable range, indicating that the method had a good recovery rate and proved good accuracy.

[0143] Comparative Example 1: Comparative test between the literature method and the method of the present invention

[0144] 1 Literature Methods

[0145] Literature on water-soluble and fat-soluble vitamins in dried blood spots (CN106770802A) Detection method: Use dried blood filter paper specimens, use punching forceps to take 10 to 20 blood spots with a diameter of 3.2 mm and put them into a centrifuge tube, add extract (the extract is 84.75 to 98.95% methanol, 1 to 15% water and 0.05 to 0.25% formic acid; or the extract includes the following components in volume percentage: 20 to 79% ethanol, 20 to 70% methanol and 1 to 10% water) 200 to 500 μg / ml of water, add 1% to 2% ethanol, ... 00μL, incubate at 650-750rpm for 45 minutes at 27-30℃; after centrifugation, take all the supernatant and add it to a 96-well plate, blow dry it with a nitrogen blow dryer, take another 100μL of mobile phase to reconstitute, and add 50μL of mixed internal standard working solution, cover it with aluminum foil to reduce volatility, and oscillate to mix; put the 96-well plate into the automatic sampler of the tandem mass spectrometry system, enable the application software, create a sample list, select the correct internal standard concentration file and acquisition method to detect the content of multiple vitamins in the dried blood filter paper.

[0146] 2 Comparative test results (see Table 11 and Figure 5 )

[0147] Table 11

[0148]

[0149]

[0150] 3 Experimental conclusion

[0151] By comparing the contents of water-soluble and fat-soluble vitamins in dried blood samples (dried blood spots and VAMS) determined by the literature method and the method of the present invention, the relative standard deviations of the retinol and niacinamide results of the two methods were >30%, and the relative deviations of the α-tocopherol, thiamine, riboflavin and pantothenic acid results were <10%, proving that the method of the present invention for determining the contents of water-soluble and fat-soluble vitamins in dried blood has higher extraction efficiency and more accurate quantitative results.

[0152] Comparative Example 2: Comparative test of adding different extraction solvents in the present invention

[0153] 1 Test method

[0154] The added extraction solvents are water and n-hexane: two dried blood samples (VAMS and DBS) are placed in a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL of blood), 200 μL of deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), 400 μL of organic solvent containing isotope internal standard (n-hexane) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), centrifuged for 5 minutes (20000 r / min, room temperature), 400 μL of supernatant is taken, blown dry with nitrogen (1 h, 40 ° C), re-dissolved with 80 μL of methanol / water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000 r / min, room temperature) to obtain the sample solution.

[0155] The added extraction solvents are water and 200 g / L trichloroacetic acid (prepared with methanol-acetonitrile (1:1)): two dried blood samples (VAMS and DBS) are placed in a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL of blood), 200 μL of deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), 400 μL of trichloroacetic acid containing isotope internal standard (prepared with methanol-acetonitrile (1:1)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), centrifuged for 5 minutes (20000 r / min, room temperature), 400 μL of supernatant is taken, blown dry with nitrogen (1 h, 40°C), re-dissolved with 80 μL of methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000 r / min, room temperature) to obtain the sample solution.

[0156] The added extraction solvents were water and methanol-acetonitrile (1:1): two dried blood samples (VAMS and DBS) were placed in a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL of blood), 200 μL of deionized water was added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), 400 μL of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) was added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), centrifuged for 5 minutes (20000 r / min, room temperature), 400 μL of supernatant was taken, dried with nitrogen (1 h, 40 ° C), 80 μL of methanol-water (1:1) was added for re-dissolution, vortexed for 1 minute, and centrifuged for 5 minutes (20000 r / min, room temperature) to obtain the sample solution.

[0157] 2 Comparative test results (see Table 12 and Figure 6 )

[0158] Table 12

[0159]

[0160] 3 Experimental conclusion

[0161] By comparing the experiments of adding different extraction solvents in the present invention, the relative standard deviations of the results of retinol and niacinamide of the two methods are>40%, and the relative standard deviations of the results of α-tocopherol, thiamine, riboflavin and pantothenic acid are<10%, which proves that when the extraction solvents of water and methanol-acetonitrile (1:1) solution are added during the pretreatment of the dried blood sample, the extraction efficiency is higher and the quantitative results are more accurate.

[0162] Comparative Example 3: Comparative test of adding different proportions of organic solvents in the present invention

[0163] 1 Test method

[0164] The added extraction solvents are water and organic solvent methanol-acetonitrile (1:2) solution: two dried blood samples (VAMS and DBS) are placed in a 1.5mL light-proof centrifuge tube (equivalent to 40μL blood), 200μL deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), 400μL of organic solvent containing isotope internal standard (methanol-acetonitrile solution (1:2)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), centrifuged for 5 minutes (20000r / min, room temperature), 400μL of supernatant is taken, blown dry with nitrogen (1h, 40℃), re-dissolved with 80μL methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000r / min, room temperature) to obtain the sample solution.

[0165] The added extraction solvents are water and organic solvent methanol-acetonitrile (2:1) solution: two dried blood samples (VAMS and DBS) are placed in a 1.5mL light-proof centrifuge tube (equivalent to 40μL blood), 200μL deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), 400μL of organic solvent containing isotope internal standard (methanol-acetonitrile solution (2:1)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), centrifuged for 5 minutes (20000r / min, room temperature), 400μL of supernatant is taken, blown dry with nitrogen (1h, 40℃), re-dissolved with 80μL methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000r / min, room temperature) to obtain the sample solution.

[0166] The added extraction solvents are water and organic solvent methanol-acetonitrile (1:1.5) solution: two dried blood samples (VAMS and DBS) are placed in a 1.5mL light-proof centrifuge tube (equivalent to 40μL blood), 200μL deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), 400μL of organic solvent containing isotope internal standard (methanol-acetonitrile solution (1:1.5)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), centrifuged for 5 minutes (20000r / min, room temperature), 400μL of supernatant is taken, blown dry with nitrogen (1h, 40℃), re-dissolved with 80μL methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000r / min, room temperature) to obtain the sample solution.

[0167] The added extraction solvents are water and organic solvent methanol-acetonitrile (1.5:1) solution: two dried blood samples (VAMS and DBS) are placed in a 1.5mL light-proof centrifuge tube (equivalent to 40μL blood), 200μL deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), 400μL of organic solvent containing isotope internal standard (methanol-acetonitrile solution (1.5:1)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), centrifuged for 5 minutes (20000r / min, room temperature), 400μL of supernatant is taken, blown dry with nitrogen (1h, 40℃), re-dissolved with 80μL methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000r / min, room temperature) to obtain the sample solution.

[0168] The added extraction solvents are water and organic solvent methanol-acetonitrile (1:1) solution: two dried blood samples (VAMS and DBS) are placed in a 1.5mL light-proof centrifuge tube (equivalent to 40μL blood), 200μL deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), 400μL of organic solvent containing isotope internal standard (methanol-acetonitrile solution (1:1)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30KHz, room temperature), centrifuged for 5 minutes (20000r / min, room temperature), 400μL of supernatant is taken, blown dry with nitrogen (1h, 40℃), re-dissolved with 80μL methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000r / min, room temperature) to obtain the sample solution.

[0169] 2 Comparative test results (see Table 13 and Figure 7 )

[0170] Table 13

[0171]

[0172]

[0173]

[0174] 3 Experimental conclusion

[0175] By comparing the experiments of adding different proportions of organic solvent in the present invention, it is found that when the volume of the organic solvent methanol-acetonitrile solution is 1:2, 2:1 and 1:1, the relative standard deviation of the result of α-tocopherol is >20%, and the relative standard deviation of the result of retinol, thiamine, riboflavin, nicotinamide and pantothenic acid is <10%; and when the volume of the organic solvent methanol-acetonitrile solution is 1:1.5, 1.5:1 and 1:1, the relative standard deviation of the result of α-tocopherol is <10%, and the relative standard deviation of the result of retinol, thiamine, riboflavin, nicotinamide and pantothenic acid is <10%. It is proved that when the extraction solvent of the present invention is water, methanol-acetonitrile (1:1.5-1.5:1) when pre-treating the dried blood sample, the extraction efficiency is higher and the quantitative result is more accurate.

[0176] Comparative Example 4: Comparative test of adding mixed extraction solvent and adding extraction solvent separately in the present invention

[0177] 1 Test method

[0178] Add mixed extraction solvent (water: methanol: acetonitrile (1:1:1)): Place two dried blood samples (VAMS and DBS) in a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL blood), add water: methanol: acetonitrile (1:1:1), vortex for 1 minute, ultrasonicate for 10 minutes (30 KHz, room temperature), centrifuge for 5 minutes (20000 r / min, room temperature), take 400 μL of supernatant, blow dry with nitrogen (1 h, 40 ° C), add 80 μL methanol-water (1:1) for re-dissolution, vortex for 1 minute, and centrifuge for 5 minutes (20000 r / min, room temperature) to obtain the sample solution.

[0179] Add extraction solvent water and methanol-acetonitrile (1:1) solution respectively: put two dried blood samples (VAMS and DBS) into a 1.5mL light-proof centrifuge tube (equivalent to 40μL blood), add 200μL deionized water, vortex for 1 minute, ultrasonic for 10 minutes (30KHz, room temperature), add 400μL of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)), vortex for 1 minute, ultrasonic for 10 minutes (30KHz, room temperature), centrifuge for 5 minutes (20000r / min, room temperature), take 400μL supernatant, blow dry with nitrogen (1h, 40℃), add 80μL methanol-water (1:1) for re-dissolution, vortex for 1 minute, and centrifuge for 5 minutes (20000r / min, room temperature) to obtain the sample solution.

[0180] 2 Comparative test results (see Table 14 and Figure 8 )

[0181] Table 14

[0182]

[0183]

[0184] 3 Experimental conclusion

[0185] By comparing the different methods of adding the extraction solvent in the present invention, the relative deviation of the results of retinol and niacinamide of the two methods is greater than 50%, and the relative deviation of the results of α-tocopherol, thiamine, riboflavin and pantothenic acid is less than 10%, which proves that when the extraction solvent of the present invention is water and methanol-acetonitrile (1:1) solution added during the pretreatment of the dried blood sample, the extraction efficiency is higher and the quantitative result is more accurate.

[0186] Comparative Example 5: Comparative test of adding two extraction solvents (water and organic solvent) in different proportions in the present invention

[0187] 1 Test method

[0188] The added extraction solvent is 200 μL water and 100 μL methanol-acetonitrile (1:1) solution: two dried blood samples (VAMS and DBS) are placed in a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL blood), 200 μL deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), 100 μL of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), centrifuged for 5 minutes (20000 r / min, room temperature), 200 μL of supernatant is taken, blown dry with nitrogen (1h, 40°C), re-dissolved with 80 μL methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000 r / min, room temperature) to obtain the sample solution.

[0189] The added extraction solvent is 200 μL water and 200 μL methanol-acetonitrile (1:1) solution: two dried blood samples (VAMS and DBS) are placed in a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL blood), 200 μL deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), 200 μL of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), centrifuged for 5 minutes (20000 r / min, room temperature), 266 μL of supernatant is taken, blown dry with nitrogen (1h, 40°C), re-dissolved with 80 μL methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000 r / min, room temperature) to obtain the sample solution.

[0190] The added extraction solvent is 200 μL water and 400 μL methanol-acetonitrile (1:1) solution: two dried blood samples (VAMS and DBS) are placed in a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL blood), 200 μL deionized water is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), 400 μL of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)) is added, vortexed for 1 minute, ultrasonicated for 10 minutes (30 KHz, room temperature), centrifuged for 5 minutes (20000 r / min, room temperature), 400 μL of supernatant is taken, blown dry with nitrogen (1h, 40°C), re-dissolved with 80 μL methanol-water (1:1), vortexed for 1 minute, and centrifuged for 5 minutes (20000 r / min, room temperature) to obtain the sample solution.

[0191] 2 Comparative test results (see Table 15)

[0192] Table 15

[0193]

[0194] 3 Experimental conclusion

[0195] By comparing the experiments of adding two extraction solvents (water and organic solvent) in different proportions in the present invention, the relative standard deviations of the results of retinol, α-tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid were all less than 10%, proving that when the present invention performs pretreatment on the dried blood sample, the volume ratio of the added extraction solvent water to the methanol-acetonitrile (1:1) solution can be 1-2:2-1.

[0196] Comparative Example 6: Comparative test of different addition orders of two extraction solvents (water and organic solvent) in the present invention

[0197] 1 Test method

[0198] The order of adding solvents is to add 200 μL of water first, and then add 400 μL of methanol-acetonitrile (1:1) solution: put two dried blood samples (VAMS and DBS) into a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL of blood), add 200 μL of deionized water, vortex for 1 minute, and ultrasonicate for 10 minutes (30 KHz, room temperature), add 400 μL of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1)), vortex for 1 minute, ultrasonicate for 10 minutes (30 KHz, room temperature), centrifuge for 5 minutes (20000 r / min, room temperature), take 400 μL of supernatant, blow dry with nitrogen (1h, 40°C), add 80 μL of methanol-water (1:1) for re-dissolution, vortex for 1 minute, and centrifuge for 5 minutes (20000 r / min, room temperature) to obtain the sample solution.

[0199] The order of adding solvents is to first add 400 μL of methanol-acetonitrile (1:1) solution, and then add 200 μL of water: put two dried blood samples (VAMS and DBS) into a 1.5 mL light-proof centrifuge tube (equivalent to 40 μL of blood), add 400 μL of organic solvent containing isotope internal standard (methanol-acetonitrile (1:1) solution), vortex for 1 min, ultrasonicate for 10 min (30 KHz, room temperature), add 200 μL of deionized water, vortex for 1 min, ultrasonicate for 10 min (30 KHz, room temperature), centrifuge for 5 min (20000 r / min, room temperature), take 400 μL of supernatant, blow dry with nitrogen (1 h, 40°C), add 80 μL of methanol-water (1:1) to dissolve, vortex for 1 min, and centrifuge for 5 min (20000 r / min, room temperature) to obtain the sample solution.

[0200] 2 Comparative test results (see Table 16 and Fig. 9 )

[0201] Table 16

[0202]

[0203] 3 Experimental conclusion

[0204] By comparing the different methods of adding the extraction solvent in the present invention, the relative standard deviations of the results of retinol and niacinamide of the two methods were >50%, and the relative standard deviations of the results of α-tocopherol, thiamine, riboflavin and pantothenic acid were <10%, proving that when the order of adding the extraction solvent during the pretreatment of the dried blood sample in the present invention is first adding 200 μL of water and then adding 400 μL of methanol-acetonitrile (1:1) solution, the extraction efficiency is higher and the quantitative results are more accurate.

[0205] In summary, the present invention uses a dedicated dry blood collection system to collect dry blood samples, and the sampling process is simple and easy to operate; by optimizing the extraction solvent ratio and the order of adding solvents, the extraction of various fat-soluble and water-soluble vitamins in dry blood samples (VAMS and DBS) can be achieved, the extraction efficiency is high, the required sample amount is small, and the detection results of vitamins have higher precision and accuracy.

Claims

1. A method for detecting the content of multiple vitamins in blood, characterized in that: The multivitamins include retinol, α - tocopherol, thiamine, riboflavin, niacinamide and pantothenic acid, the method comprising the steps of: (1) using a dry blood collection system to quantitatively obtain blood and drying it to produce a dry blood sample; (2) extracting and purifying the substance to be tested in the dried blood sample with an extraction solvent to obtain a vitamin sample to be tested; (3) analyzing the vitamin sample to be tested by liquid chromatography-mass spectrometry and quantifying by internal standard method; The extraction solvent is a mixed extraction solvent of water and methanol-acetonitrile solution, and the volume ratio of water to methanol-acetonitrile solution is 1-2:2-1; in the methanol-acetonitrile solution, the volume ratio of methanol to acetonitrile is 1:1.5-1.5:1; the order of adding the extraction solvent to the sample is first adding water to extract for 5-30 minutes, and then adding methanol-acetonitrile solution to extract for 5-30 minutes; the conditions of the liquid chromatography are: octadecylsilane bonded silica gel is used as a filler, and the chromatographic column is Phenomenex, Kinetex PFP; heptafluorobutyric acid-formic acid aqueous solution is used as mobile phase A, and acetonitrile solution is used as mobile phase B, the mass concentration of heptafluorobutyric acid in mobile phase A is 0.05-0.1%, and the mass concentration of formic acid is 0.05-0.1%; the flow rate of the mobile phase is 0.4-0.8mL / min; the column temperature is 25-45°C; the gradient elution program is as follows: 。 2. The method for detecting the content of multiple vitamins in blood according to claim 1, characterized in that: In step (1), the dried blood collection system is a dried blood spot collection card DBS or a volume absorption microsampler VAMS.

3. The method for detecting the content of multiple vitamins in blood according to claim 1, characterized in that: In step (1), the drying time is 0.5-4 hours.

4. The method for detecting the content of multiple vitamins in blood according to claim 1, characterized in that: In step (2), the extraction solvent extracts more than one dried blood sample at the same time; the purification method is centrifugation, and the centrifugation time is 3-10 minutes.

5. The method for detecting the content of multiple vitamins in blood according to claim 1, characterized in that: The mass spectrometry conditions were as follows: ionization mode: electrospray positive ion mode; detection mode: multiple reaction monitoring MRM; curtain gas pressure: 20-50 psi; ion spray voltage: 3000-5500 V; temperature: 300-550°C; Atomizing gas: 30-60psi; auxiliary heating gas: 30-60psi.

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