CN113917015A

CN113917015A - Detection method for simultaneously detecting multiple vitamins in human serum

Info

Publication number: CN113917015A
Application number: CN202111162487.XA
Authority: CN
Inventors: 谢永明; 刘喜艳; 王芳
Original assignee: Shanghai Applied Protein Technology Co Ltd
Current assignee: Shanghai Applied Protein Technology Co Ltd
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2022-01-11

Abstract

The invention provides a detection method for simultaneously detecting multiple vitamins in human serum, which belongs to the technical field of vitamin detection and can simultaneously detect vitamin A, vitamin E, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum.

Description

Detection method for simultaneously detecting multiple vitamins in human serum

Technical Field

The invention belongs to the technical field of vitamin detection, and particularly relates to a method for simultaneously and quantitatively analyzing the concentrations of vitamin A, vitamin E and 25-hydroxyvitamin D in human serum based on an LC-MS/MS technology.

Background

Vitamins are essential nutrients for the body and are involved in the metabolism of substances and energy in the body mainly by constituting coenzymes or prosthetic groups. Vitamins are classified into water-soluble vitamins and fat-soluble vitamins 2 according to their solubility differences, wherein the fat-soluble vitamins include vitamin A, D, E, K, etc.

Vitamin A (VA), a fat-soluble micronutrient essential to the human body, exerts a variety of physiological functions, including vision, affecting immune response, iron metabolism, bone metabolism, sexual reproduction, and the like. With the progress of research in recent years, it has been found that the polypeptide plays an important role not only in the morphological construction of embryonic heart, nervous system, lung and other organ systems, but also in enhancing the immune function of newborn, protecting against oxidation, reducing the infant mortality, and the like. VE has the functions of resisting oxidation and removing free radicals, is closely related to the generation and reproductive functions of sperms, and the low content of VE in the bodies of pregnant women can increase the occurrence risk of hypertensive diseases in pregnancy and poor pregnancy outcome. Vitamin E comprises 8 compounds including two types of tocopherol and tocotrienol, wherein alpha-tocopherol is distributed most widely and has highest biological activity. Vitamin D is also known as a vitamin to combat rickets and is also a steroid hormone. Closely related to the human body are two forms of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). VD3 was converted to 25- (OH) D3 in the liver and VD2 was converted to 25- (OH) D2, the two VD metabolites were referred to as 25- (OH) D and were stable storage forms of VD in vivo. Vitamin D deficiency can lead to rickets in children and osteomalacia in adults, and recent studies show that vitamin D deficiency is also related to tumors, diabetes, cardiovascular diseases and the like. Therefore, the accurate determination of vitamin A, vitamin E and vitamin D in the serum has important significance for preventing and treating related diseases.

The fat-soluble vitamin is mainly determined by enzyme immunoassay, high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, on-line two-dimensional liquid chromatography, microemulsion liquid chromatography and the like. Enzyme immunoassays are relatively inexpensive and detect vitamins quickly, but with little specificity and sensitivity (e.g., retinol binding protein, rather than retinol itself, is measured by enzyme immunoassay when VA is measured). HPLC methods have high specificity and accuracy, but most HPLC methods require large volumes of sample, time-consuming and complex sample preparation procedures prior to quantitation, and the instruments are expensive, costly, and have high technical requirements on operators, which all restrict the application of HPLC. Compared with other methods, the liquid chromatography tandem mass spectrometry has the advantages of high separation capacity, short analysis time, high resolution and sensitivity and the like.

The kit approved by MPA for measuring the concentration of fat-soluble vitamins mainly adopts a chemiluminescence method, a colloidal gold method and a fluorescence immunochromatography method, can only detect the content of 25-hydroxyvitamin D, cannot realize the simultaneous measurement of a plurality of fat-soluble vitamins, has insufficient accuracy and poor specificity, and is easy to generate cross reaction with other metabolites with similar structures in blood. The clinical test method for simultaneously detecting multiple fat-soluble vitamins by the liquid chromatography-tandem mass spectrometry method is mostly established by the laboratory, and most of the adopted reagents are non-kit products, so that errors of reagent batches and manual sample configuration are easily introduced, and the detection inaccuracy is caused. In recent years, liquid chromatography-tandem mass spectrometry is gradually increased in NMPA approved fat-soluble vitamin detection kits, but single detection of 25-hydroxy vitamin D is mainly used, so that high flux cannot be realized, the clinical detection requirements are met, the detection complexity is increased, and most products are freeze-dried powder.

Disclosure of Invention

Based on the problems in the prior art, the invention provides a novel detection method for simultaneously determining vitamin A, vitamin E, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum, the adopted detection method is a liquid chromatography tandem mass spectrometry method, and blank bovine serum is used as a matrix of a calibrator and a quality control product to replace human serum, so that the method has the characteristics of simplicity and convenience in use, low cost, small required sample volume, rapidness in pretreatment, high accuracy, high sensitivity, high precision, short analysis time and the like.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a detection method for simultaneously detecting multiple vitamins in human serum comprises the steps of carrying out pretreatment of liquid chromatography tandem mass spectrometry on samples to be detected, respectively carrying out liquid chromatography tandem mass spectrometry on each pretreated sample, and simultaneously accurately and quantitatively analyzing vitamin A, vitamin E and 25-hydroxyvitamin D in human serum; the quality control product and the standard product used in the liquid chromatography tandem mass spectrometry detection adopt a blank matrix as a solvent, and the blank matrix comprises blank bovine serum, an antioxidant, an excipient and a preservative which are washed by activated carbon.

According to the scheme, the preparation of the bovine serum cleaned by the activated carbon comprises the following steps:

step S11 activated carbon cleaning: weighing a certain mass of activated carbon, and mixing the activated carbon and ultrapure water according to a mass ratio of 1: 5-15, uniformly mixing, placing in a shaking table at 225rpm and 25 ℃, shaking for 10 minutes, centrifuging at 7800rpm for 10-40 minutes, removing supernatant, and repeating the step once again;

step S12 primary treatment of newborn bovine serum activated carbon: mixing newborn calf serum and the activated carbon cleaned in the step S11 according to the volume ratio of 1:0.5-2, placing the mixture in a shaking table at 225rpm and 37 ℃, shaking and uniformly mixing the mixture for 15-24 hours, centrifuging the mixture at 7800rpm for 10-40 minutes, and keeping the supernatant;

step S13 secondary treatment of newborn bovine serum activated carbon: mixing the supernatant obtained at the end of the step S12 with newly cleaned activated carbon according to the volume ratio of 1:0.5-2, placing the mixture in a shaking table at 225rpm and 37 ℃, shaking and uniformly mixing the mixture for 15-24 hours, centrifuging the mixture for 10-40 minutes at 7800rpm, and keeping the supernatant;

step S14 activated carbon removal: centrifuging the supernatant obtained at the end of the step S13 at 12000rpm for 30-90 minutes, discarding the precipitate, repeating the step for 2-5 times again on the supernatant, and retaining the supernatant to obtain blank bovine serum.

According to the scheme, the antioxidant is 2, 6-di-tert-butyl-4-methylphenol, butyl hydroxy anisole and sodium metabisulfite, and the final concentration of the three components is 0.01-0.5%; the excipient is bovine serum albumin with the final concentration of 0.1% -1%; the preservative is PC-300 with the final concentration of 0.01-0.1%.

According to the scheme, the detection method for simultaneously detecting the multiple vitamins in human serum comprises the following detailed steps:

step S1, preparing blank bovine serum cleaned by activated carbon, adding an antioxidant, an excipient and a preservative into the blank bovine serum, and carrying out ultrasonic treatment for 10 minutes to make the matrix in a uniform state;

step S2, preparing a calibrator solution, a quality control solution and an internal standard working solution with series concentrations;

step S3, adding internal standard working solution with the volume of 1-3 times of that of the sample to be detected, and uniformly mixing; and then adding n-hexane into the sample to be detected, wherein the volume ratio of the n-hexane: sample to be tested ═ (5-15): 1, mixing evenly by vortex, and centrifuging for 5-15 minutes at 12000 rpm;

step S4, drying the upper layer liquid finally obtained in the step S3 by using nitrogen, redissolving the upper layer liquid by using redissolving liquid with the volume 1-3 times that of the sample solution to be detected, and then performing LC-MS/MS analysis;

the redissolution is a methanol aqueous solution, and the volume mixing ratio is as follows: ultrapure water ═ 1-9: 1.

according to the above scheme, the conditions of the liquid chromatography are as follows:

and (3) analyzing the column: phenomenex Kinetex C18, 2.6 μm, 3.0 x 50 mm;

mobile phase: phase A: 0.1% aqueous formic acid solution containing 2mM ammonium formate; phase B: 0.1% formic acid in methanol;

elution gradient:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	25	75
1	2	98
			2.8	2	98
2.81	25	75
			3.5	25	75

Flow rate: 0.8 mL/min; column temperature: 45 ℃; detection duration: 3.5 min; sample introduction amount: 10 μ L.

According to the above scheme, the mass spectrum conditions are as follows:

ion source temperature: at 450 ℃; ion source atomization gas: 50 psi; heating auxiliary gas by an ion source: 50 psi; air curtain air: 30 psi; spray capillary voltage: 5500V; collision gas: 9; compound MRM parameters using ESI ion source, positive ion mode segmented scan analysis are shown in the following table:

dwell Time is the scan Time, CE is the collision voltage, DP is the declustering voltage, EP is the entrance voltage, and CXP is the collision cell exit voltage.

The invention has the beneficial effects that:

1. the liquid chromatography tandem mass spectrometry detection method for simultaneously detecting vitamin A, vitamin E and 25-hydroxyvitamin D in serum is provided, a pair of quantitative ion pairs is respectively selected for each substance to be detected, the corresponding retention time of the pair is taken as a qualitative basis, and a standard curve is made for quantification by using a standard substance; and the accuracy and the effectiveness of the quality control product inspection method at three levels of low, medium and high are applied.

2. The method provided by the invention can be used for simultaneously detecting the vitamin A, the vitamin E and the vitamin D, achieves the function of single-sample multi-index synchronous detection, has the characteristics of high accuracy, short detection time, less reagent consumption, convenience in operation, low cost and the like, and can be used for accurately determining the concentrations of the vitamin A, the vitamin E and the vitamin D in human serum.

3. The method provided by the invention adopts the blank matrix solution of the newborn bovine serum as the simulated serum, is similar to the matrix of the clinical serum sample, has low requirement on operators and good repeatability, and can be more applied to the inspection of the clinical sample.

Drawings

FIG. 1 is a total chromatogram of all the mixed standards of the analytes in the examples.

FIG. 2 is a graph showing a standard curve of vitamin A measured by the method of the present invention in examples.

FIG. 3 is a chromatogram of vitamin A measured by the method of the present invention in examples.

FIG. 4 is a graph showing the standard curve of vitamin E measured by the method of the present invention in examples.

FIG. 5 is a representative chromatogram of vitamin E measured by the method of the present invention in the examples.

FIG. 6 is a standard graph of the determination of 25-hydroxyvitamin D2 according to the method of the present invention in examples.

FIG. 7 is a chromatogram of 25-hydroxyvitamin D2 determined by the method of the invention in examples.

FIG. 8 is a standard graph of the determination of 25-hydroxyvitamin D3 according to the method of the present invention in examples.

FIG. 9 is a chromatogram of 25-hydroxyvitamin D3 determined by the method of the invention in examples.

Fig. 3, 5, 7 and 9 are graphs of results of LC-MS/MS detection and analysis, which are results of examples, and characters in the graphs are results of the examples, and will change according to the result of each detection and analysis, that is, the characters in the graphs are irrelevant to whether the detection method provided by the present invention can be repeatedly implemented, and the characters in the graphs are unclear and do not affect the detection method provided by the present invention repeatedly implemented by those skilled in the art.

Detailed Description

The technical solution of the present invention will be described below with reference to the specific embodiments and the accompanying drawings.

A detection method for simultaneously detecting multiple vitamins in human serum comprises the steps of carrying out pretreatment of liquid chromatography tandem mass spectrometry on samples to be detected, respectively carrying out liquid chromatography tandem mass spectrometry on each pretreated sample, and simultaneously accurately and quantitatively analyzing vitamin A, vitamin E and 25-hydroxyvitamin D in human serum; the quality control product and the standard product used in the liquid chromatography tandem mass spectrometry detection adopt a blank matrix as a solvent, the blank matrix comprises blank bovine serum cleaned by activated carbon, an antioxidant, an excipient and a preservative, the antioxidant is 2, 6-di-tert-butyl-4-methylphenol, butyl hydroxy anisole and sodium metabisulfite, and the final concentration of the three components is 0.2%; the excipient is bovine serum albumin with the final concentration of 0.2%; the preservative is PC-300 with the final concentration of 0.02%, and the detection method comprises the following detailed steps:

step S1, preparing blank bovine serum cleaned by activated carbon, and adding an antioxidant, an excipient and a preservative into the blank bovine serum to obtain a blank matrix.

Step S11 activated carbon cleaning:

weighing a certain mass of activated carbon by using a 50mL centrifuge tube, and mixing the activated carbon and ultrapure water according to a mass ratio of 1: 10, uniformly mixing, placing in a shaking table, shaking at 225rpm for 10 minutes, placing the centrifugal tube on a centrifugal machine, centrifuging at 7800rpm for 30 minutes, removing supernatant, and repeating the step once again;

step S12 primary treatment of newborn bovine serum activated carbon:

mixing newborn calf serum and the activated carbon cleaned in the step S11 according to the volume ratio of 1:1.2, placing the mixture in a shaking table, shaking and uniformly mixing the mixture at the speed of 225rpm and the temperature of 37 ℃ for 18 hours, placing a centrifugal tube on a centrifugal machine, centrifuging the centrifugal tube at the speed of 7800rpm for 30 minutes, and keeping a supernatant;

step S13 secondary treatment of newborn bovine serum activated carbon:

mixing the supernatant obtained at the end of the step S12 with the newly cleaned activated carbon according to the volume ratio of 1:1.2, placing the mixture in a shaking table, shaking and uniformly mixing the mixture for 18 hours at the speed of 225rpm and the temperature of 37 ℃, placing the centrifugal tube on a centrifugal machine, centrifuging the centrifugal tube at the speed of 7800rpm for 30 minutes, and keeping the supernatant;

step S14 activated carbon removal:

transferring the supernatant obtained finally in the step S13 to a 2mL centrifuge tube, centrifuging at 12000rpm for 60 minutes, transferring the supernatant to a new centrifuge tube, repeating the step 3 times again, and reserving the supernatant to obtain blank bovine serum;

step S15, adding an antioxidant, an excipient and a preservative into the processed blank bovine serum, and stirring the mixture to be in a uniform state to obtain a blank matrix;

step S21, preparing a stable isotope internal standard stock solution:

accurately weighing appropriate amount of Vitamin A-D6(Vitamin A-D6), Vitamin E-D6(Vitamin E-D6), 25-hydroxyvitamin D2-D3 (25-hydroxyvitamine D2-D3), 25-hydroxyvitamin D3-D3 (25-hydroxyvitamine D3-D3), respectively, dissolving with pure methanol, and preparing stable isotope internal standard stock solutions respectively at the following concentrations: vitamin A-d6, 1000. mu.g/mL; vitamin E-d6, 1000. mu.g/mL; 25-hydroxyvitamin D2-D3, 100 μ g/mL; 25-hydroxyvitamin D3-D3, 1000. mu.g/mL;

step S22, preparing a stable isotope internal standard intermediate working solution:

accurately transferring a proper amount of isotope internal standard stock solution prepared in the step S21, and using methanol as diluent to respectively dilute into internal standard intermediate working solution with the mass concentration of 8-500 mug/mL;

step S23, preparing stable isotope internal standard working solution:

accurately transferring a proper amount of stable isotope internal standard intermediate working solution prepared in the step S22, mixing and diluting, wherein the diluted solution is acetonitrile, and preparing to obtain stable isotope internal standard working solution; the stable isotope internal standard working solution simultaneously comprises: the vitamin A-D6500 ng/mL, the vitamin E-D6100 ng/mL, the 25-hydroxy vitamin D2-D38 ng/mL and the 25-hydroxy vitamin D3-D38 ng/mL.

Step S24, preparing a standard substance stock solution:

accurately weighing appropriate amounts of vitamin A (vitamin A), vitamin E (vitamin E), 25-hydroxyvitamin D2 (25-hydroxyvitamine D2) and 25-hydroxyvitamin D3 (25-hydroxyvitamine D3) standard substances, respectively, dissolving with methanol, and preparing into standard substance stock solution with the following concentrations: vitamin A, 2.5 mg/mL; vitamin E, 6 mg/mL; 25-hydroxyvitamin D2, 0.5 mg/mL; 25-hydroxy vitamin D3, 0.5 mg/mL.

Step S25, preparing intermediate working solution of the mixed standard product:

accurately transferring appropriate amount of vitamin A, vitamin E, 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 standard substance stock solution prepared in the step S24 in sequence, mixing and using pure methanol as diluent to prepare a mixed standard substance intermediate working solution, wherein the concentration is as follows: vitamin A, 62.5 mug/mL; vitamin E, 1500. mu.g/mL; 25-hydroxyvitamin D2, 3.75 μ g/mL; 25-hydroxy vitamin D3, 6.25. mu.g/mL.

Step S26, preparing a mixed standard working solution:

accurately transferring a proper amount of the intermediate working solution of the mixed standard product prepared in the step S25, and using the blank matrix prepared in the step S15 as diluent to respectively prepare 6 gradient working solutions of the mixed standard product, wherein the concentration is shown in the following table;

step S27, preparing a mixed quality control working solution:

accurately transferring a proper amount of the intermediate working solution of the mixed standard product prepared in the step S25, and using the blank matrix prepared in the step S15 as a diluent to prepare mixed quality control working solutions with low, medium and high concentration levels respectively, wherein the concentrations are shown in the following table;

serial number	Name of Compound	LQC	MQC	HQC
						1	Vitamin A	75	500	1000
2	Vitamin E	1.8	12	24
					3	25-hydroxy vitamin D2	4.5	30	60
4	25-hydroxy vitamin D3	7.5	50	100

Step S3, adding 2 times volume of internal standard working solution into the sample to be detected, and mixing uniformly; and then adding n-hexane into the sample to be detected, wherein the volume ratio of the n-hexane: the sample to be tested is 9: 1, vortex and mix evenly;

step S4, putting 800 mu L of the liquid finally obtained in the step S3 into a 96-well plate, drying the liquid by using nitrogen, and then mixing the liquid and the nitrogen according to the volume ratio of the sample solution to be detected being 1: adding the compound solution with the dosage of 1 for redissolution, and then performing LC-MS/MS analysis;

the redissolution is a methanol water solution, and the mixing ratio is that methanol: ultrapure water 9: 1.

the conditions of the liquid chromatography are as follows:

and (3) analyzing the column: phenomenex Kinetex C18, 2.6 μm, 3.0 x 50 mm;

elution gradient:

The mass spectrum conditions were as follows:

Detecting the mixed standard working solution with the 7 gradients by using a high performance liquid chromatography-mass spectrometer to obtain chromatographic peak areas of 7 standard solutions with different concentrations, respectively taking the ratio of the chromatographic peak areas of the 7 standard solutions with different concentrations to the chromatographic peak area of a corresponding isotope internal standard as a vertical coordinate y of a standard curve equation, taking the concentrations of the 7 standard solutions with different concentrations as horizontal coordinates x of a standard curve, performing linear regression on the detected data with 7 different concentrations, and fitting to obtain standard curve equations corresponding to all the standards: y-a x + b, the results are shown in fig. 1-9.

The method provided by the invention is further subjected to in-batch and inter-batch precision analysis, and the process and the result are as follows:

precision describes how closely an analyte is determined repeatedly, and the relative standard deviation (coefficient of variation, CV%) defined as the measured value should be obtained using the results of analyzing batch samples with the same proof of accuracy as that of the proof of accuracy, to obtain precision of quality control samples of low, medium and high concentrations, both within the same batch (intra-batch precision) and between different batches (inter-batch precision).

Internal precision: the invention adopts blank bovine serum to prepare low, medium and high concentration mixed quality control working solution; each concentration was tested in 6 replicates and assayed for 1 day.

Batch precision: the invention adopts blank bovine serum to prepare low, medium and high concentration mixed quality control working solution; each concentration was tested in 6 replicates for 3 consecutive days.

The detection result shows that the variation coefficient (CV%) in batch of all the analytes to be detected is less than or equal to 15%, and the variation coefficient (CV%) in batch is less than or equal to 15%.

And (3) residual test:

the residue should be estimated by injecting a blank sample after injecting the high concentration sample or the calibration standard to the extent that the residue in the blank sample after injecting the residual high concentration sample should not exceed 20% of the lower limit of quantitation.

The method adopts blank bovine serum to prepare a calibration standard sample ULOQ, and evaluates the calibration standard sample ULOQ by continuously injecting 3 blank samples after injecting the calibration standard sample ULOQ; the assay was continued for 3 days:

the test results show that the residues in the blank sample after ULOQ of the analyte to be tested are all less than 20% LLOQ.

The present invention is provided by the above embodiments only for illustrating and not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.

Claims

1. A detection method for simultaneously detecting multiple vitamins in human serum is characterized in that a sample to be detected is subjected to pretreatment of liquid chromatography tandem mass spectrometry, each pretreated sample is respectively subjected to liquid chromatography tandem mass spectrometry, and simultaneously vitamin A, vitamin E and 25-hydroxyvitamin D in human serum are accurately and quantitatively analyzed; the quality control product and the standard product used in the liquid chromatography tandem mass spectrometry detection adopt a blank matrix as a solvent, and the blank matrix comprises blank bovine serum, an antioxidant, an excipient and a preservative which are washed by activated carbon.

2. The method for simultaneously detecting multiple vitamins in human serum according to claim 1, wherein the preparation of the bovine serum subjected to activated carbon cleaning treatment comprises the following steps:

3. The method of claim 1, wherein the antioxidant is 2, 6-di-tert-butyl-4-methylphenol, butyl hydroxy anisole and sodium metabisulfite, and the final concentration of each of the three components is 0.01-0.5%; the excipient is bovine serum albumin with the final concentration of 0.1% -1%; the preservative is PC-300 with the final concentration of 0.01-0.1%.

4. The method for simultaneously detecting multivitamins in human serum according to claim 1, comprising the following detailed steps:

5. the method for simultaneously detecting multivitamins in human serum according to claim 4, wherein the conditions of the liquid chromatography are as follows:

and (3) analyzing the column: phenomenex Kinetex C18, 2.6 μm, 3.0 x 50 mm;

elution gradient:

6. The method according to claim 5, wherein the mass spectrometry conditions are as follows: