CN111239292A

CN111239292A - Method for simultaneously detecting multiple fat-soluble vitamins and homocysteine

Info

Publication number: CN111239292A
Application number: CN202010129295.8A
Authority: CN
Inventors: 丁芬
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-10-24
Filing date: 2020-02-28
Publication date: 2020-06-05
Anticipated expiration: 2040-02-28
Also published as: CN111239292B

Abstract

The invention discloses a method for simultaneously detecting multiple fat-soluble vitamins and homocysteine, which comprises the following steps: the plasma sample is pretreated, and the pretreatment method comprises the following steps: adding 1, 4-dithiothreitol into the serum sample, uniformly mixing, and then adding a protein precipitator for treatment; extracting fat-soluble vitamins and homocysteine from the blood plasma sample after the pretreatment; and drying and redissolving the extract, and then carrying out sample injection analysis on the extract by using a liquid chromatography-mass spectrometry system, wherein the extracting agent is n-hexane.

Description

Method for simultaneously detecting multiple fat-soluble vitamins and homocysteine

Technical Field

The invention relates to the technical field of analysis and detection, in particular to a method for simultaneously detecting multiple fat-soluble vitamins and homocysteine.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Vitamin a is a fat-soluble vitamin that can be divided into two categories: retinol and carotene. Vitamin a alcohol is only present in animal foods; carotene can be taken from plant and animal food. Vitamin a has multiple physiological functions and is essential for vision, growth, development of epithelial tissues and bones, and growth and development of fetuses. The effects of the method comprise the following aspects: 1. maintain normal visual function, prevent nyctalopia and hypopsia, and is helpful for treating various eye diseases; 2. the immune system can be normally operated, and the function of resisting respiratory system infection is achieved; 3. maintaining normal growth and development of bones and health of skin, hair, teeth and gums; if the pregnant women lack the vitamin A, the development of the fetus can be directly influenced, and even dead fetus can occur.

Vitamin D is also known as a vitamin for rickets, of which the most important are D2 and D3. The vitamin is a sterol derivative, and belongs to fat-soluble vitamins. The vitamin D is a derivative of provitamin D after ultraviolet irradiation, and provitamin D exists in animals and plants. Human skin contains a substance called 7-dehydrocholesterol which is converted to vitamin D under UV irradiation. Vitamin D can help to absorb calcium and phosphorus, and prevent rickets in children and osteomalacia in adults. In addition, vitamin D also has an enhancing effect on the immune system.

Vitamin E, also known as vitamin E, known as tocopherol or gestagen, is a fat-soluble vitamin that is one of the most important antioxidants. The vitamin is a fat-soluble vitamin with 8 forms, is dissolved in organic solvents such as fat, ethanol and the like, is insoluble in water, is stable to heat and acid, is unstable to alkali, is sensitive to oxygen and is insensitive to heat. The effects and actions of vitamin E are: 1. delaying aging, effectively reducing wrinkle generation, and keeping clear appearance; 2. the oxygen consumption of cells is reduced, so that the human body has better endurance, and the conditions of leg cramps and hand and foot stiffness are favorably alleviated; 3. antioxidation protects body cells from poisoning by free radicals; 4. improving lipid metabolism, and preventing coronary heart disease and atherosclerosis; 5. preventing cancer, preventing multiple chronic diseases; preventing inflammatory dermatoses and alopecia; preventing hemolytic anemia, protecting red blood cells from easy rupture; preventing and treating thyroid diseases; improving blood circulation, protecting tissue, reducing cholesterol, and preventing hypertension; 6. vitamin E is an important vasodilator and anticoagulant; preventing and treating varicose veins; prevent blood coagulation, reduce the production of speckle tissue; 7. the liver cell membrane is strengthened, the alveolar cells are protected, and the probability of infection of the lung and the respiratory system is reduced; 8. protecting skin from ultraviolet rays and pollution, and reducing scar and pigment deposition; accelerating the healing of the wound; 9. promoting sex hormone secretion, and increasing sperm motility and number of male; increase female estrogen concentration, improve fertility, and prevent abortion. 10. Recently, vitamin E has also been found to inhibit the lipid peroxidation in the lens of the eye, dilate peripheral blood vessels, improve blood circulation, and prevent myopia from occurring and developing.

Vitamin K (vitamin K) is a blood coagulation vitamin, has phylloquinone bioactivity, is dissolved in lipid of mitochondrial membrane, plays an electron transfer role, can increase intestinal tract peristalsis and secretion function, and can affect metabolism of some hormones due to decrease of smooth muscle tension and contraction in absence of vitamin K. For example, the decomposition of glucocorticoid in liver is delayed, the medicine has the function similar to hydrocortisone, and the endocrine activity of thyroid gland can be increased by long-term injection of vitamin K. The efficacy and the effect of the vitamin K are as follows: 1. can promote blood coagulation. Although the human body needs a small amount, the newborn is extremely deficient. Lack of it may result in reduced coagulation function, and in severe cases, bleeding may not be continuous, and even death may occur. 2. Can promote bone metabolism and effectively reduce fracture risk. 3. Can reduce the massive hemorrhage of women in physiological period, and prevent internal hemorrhage, hemorrhoid, etc. The middle-aged and the elderly women, people who prefer meat dishes and frequent nosebleed and people who take antibiotics for a long time need to pay special attention to the fact that vitamin K is often taken from food. 4. Can promote calcium absorption.

Homocysteine (HCY), a sulfur-containing amino acid formed after the removal of the methyl group of methionine, is an intermediate product in the methionine cycle. The first reports on their metabolic disturbances came from the observation that congenital cystathionine synthase deficiency leads to the appearance of homocysteinuria in patients. Thereafter, metabolic disorders caused by changes in several other enzymes or coenzymes involved in the metabolism of HCY have been discovered. The clinical significance of homocysteine: 1. relationship to cardiovascular and cerebrovascular diseases: elevated HCY levels are independent risk factors for cardiovascular and cerebrovascular disease, with the risk increasing with increasing total plasma HCY levels [1 ]. It has been found that hyperHCY is an independent risk factor for atherosclerosis, HCY is more closely related to heart failure, and for those who do not yet have heart disease symptoms, the risk of heart failure in HCY-elevated patients is doubled, and the risk of women is increased by 3 times. 2. Relationship to neurological diseases: cerebrovascular diseases are the main diseases seriously harming human health at present, and along with the deep development of subbiology, the function of HCY in cerebral apoplexy is increasingly emphasized [2 ]; studies have shown that elevated HCY is a risk factor that is significantly associated with stroke; high HCY is an important factor causing senile dementia and parkinson. 3. Relationship to hypertension: studies have shown a strong synergy between hyperhomocysteinemia and hypertension in young women with peripheral arterial occlusive disease. 4. Relationship to diabetes: plasma HCY levels can be an independent risk factor for macrovascular disease in type 2 diabetic patients [3 ]; monitoring of HCY levels in diabetic patients is useful for the assessment of prognosis, with elevated HCY being more severe in patients with diabetes with renal, retinal and vascular complications. 5. Relationship to Chronic Renal Failure (CRF): CRF is commonly associated with hyperHCY, the incidence rate of which is 33 times that of normal people; HCY is elevated in uremia. 6. Relationship to thromboembolism: the incidence of deep vein thrombosis can be increased by 4 times by the high HCY blood disease (> 20 mu mol/L); high HCY is an independent risk factor for the Pulmonary Thromboembolism (PTE) attack of Chinese Han population, and the reduction of HCY can reduce the risk of Venous Thromboembolism (VTE) of Chinese. 7. Relationship to monitoring liver injury: the detection of HCY concentration in blood plasma can also be applied to the screening of chronic liver diseases of common people. 8. And bearing and rearing better children: the detection of HCY by pregnant women is of great significance for preventing pregnancy complications and prenatal and postnatal care (preventing premature delivery, abortion, weight loss of newborn, fetal neural tube malformation and the like).

The inventor finds that in certain cardiovascular diseases, the plasma concentrations of fat-soluble vitamins and homocysteine are changed or abnormal and have certain relevance, but in the prior art, the blood plasma concentrations of various fat-soluble vitamins are generally detected simultaneously and homocysteine is detected separately, on one hand, the detection methods are different, the errors of the detection results are greatly different, and the relevance between data is interfered to a certain degree; on the other hand, the analysis time is prolonged seriously and the detection efficiency is reduced by separate detection.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a method for simultaneously detecting multiple fat-soluble vitamins and homocysteine.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the method for simultaneously detecting a plurality of fat-soluble vitamins and homocysteine comprises the following steps:

the plasma sample is pretreated, and the pretreatment method comprises the following steps: adding 1, 4-dithiothreitol into the serum sample, uniformly mixing, and then adding a protein precipitator for treatment;

extracting fat-soluble vitamins and homocysteine from the blood plasma sample after the pretreatment;

drying the extract, redissolving, and performing sample injection analysis by using a liquid chromatography-mass spectrometry system;

the extracting agent is n-hexane.

The beneficial effects of the above one or more embodiments of the invention are as follows:

when 1, 4-dithiothreitol is added into a serum sample and then protein is precipitated, the adsorption of the protein to fat-soluble vitamins and homocysteine can be effectively prevented, the loss of substances to be detected is avoided, and the detection accuracy is improved.

In addition, after the 1, 4-dithiothreitol is deproteinized, when extraction is carried out, the distribution coefficient of fat-soluble vitamin and homocysteine between blood plasma and normal hexane can be improved simultaneously, so that the normal hexane can more completely extract the fat-soluble vitamin and homocysteine, and the method is favorable for improving the detection accuracy of the two substances.

The 1, 4-dithiothreitol can also improve the extraction selectivity of n-hexane to fat-soluble vitamins and homocysteine, and prevent the n-hexane from excessively extracting other substances in blood plasma and interfering the detection of the fat-soluble vitamins and homocysteine.

The fat-soluble vitamin and the homocysteine are simultaneously detected by the same method, so that the fat-soluble vitamin and the homocysteine have similar loss and similar error, the detected result has better synchronism, and the influence of the relation of the plasma concentrations of the fat-soluble vitamin and the homocysteine on the cardiovascular diseases is conveniently researched.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a liquid chromatography separation spectrum of a mixed standard sample of four fat-soluble vitamins and homocysteine in the example of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

the extracting agent is n-hexane.

In some embodiments, the concentration of the 1, 4-dithiothreitol solution is 70-120. mu.g/ml, and further, the concentration of the 1, 4-dithiothreitol solution is 100. mu.g/ml.

Further, the volume ratio of the serum sample to the 1, 4-dithiothreitol solution is 3.5-4.5: 1.

When the concentration of the 1, 4-dithiothreitol in plasma is the concentration, the adsorption of a target substance on protein can be better avoided, the concentration of the 1, 4-dithiothreitol is low, the sample cannot interfere with a substance to be detected after being extracted, and the improvement of the detection accuracy is more favorable.

In some embodiments, the protein precipitant is a mixture of methanol and water, wherein the mass percent of the water is 1% to 10%, and further, the mass percent of the water in the protein precipitant is 2%.

Furthermore, the volume ratio of the serum to the protein precipitator is 1: 1.2-2.

In some embodiments, the extract is blown dry with nitrogen.

Further, the redissolved cosolvent is a 95% methanol aqueous solution.

In some embodiments, the liquid chromatography detection conditions are: phenomenex kinex C18, 30 x 3mm, 2.6 μm; flow rate: 0.7 ml/min; column temperature: 40 ℃; mobile phase: a: 0.1% formic acid + water; b, 0.1 percent of formic acid, acetonitrile and isopropanol, wherein the volume ratio of the acetonitrile to the isopropanol is 2-3: 10-15.

The mobile phase can smoothly and completely separate six fat-soluble vitamins from homocysteine, and is favorable for improving the detection accuracy.

In some embodiments, the gradient elution procedure for liquid chromatography is: 0-0.4min, the volume percentage of the mobile phase A is always 60%; the volume percentage of the mobile phase A is gradually reduced from 60 percent to 40 percent in 0.4-1.5 min; 1.5-2.5min, the volume percentage of the mobile phase A is gradually reduced from 40% to 0; 2.5-4.5min, the volume percentage of the mobile phase A is always 0; 4.5-4.51min, the volume percentage of the mobile phase A rises to 60%; 4.51-5.0min, the volume percentage of mobile phase A was always 60%.

The invention is further illustrated by the following examples:

example 1

A method for simultaneously detecting a plurality of fat-soluble vitamins and homocysteine in serum adopts liquid chromatography tandem mass spectrometry to detect, and comprises the following steps:

centrifuging the whole blood at 3400r/min for 10min, collecting supernatant to obtain serum, and storing the serum at 4 deg.C in a dark condition (within 24 h) until the serum is ready for analysis, or storing at-20 deg.C in a dark condition (within 72 h), or storing at-80 deg.C in a dark condition (within 30 days).

(II) preparation of various solutions

1) Preparing an internal standard stock solution (the solvent is methanol water, and the mass percentage of water is 50%), wherein the concentrations of all substances in the internal standard stock solution are respectively as follows:

vitamin a-d 6: 10 mu g/mL;

25-hydroxy vitamin D3-D6: 50 mu g/mL;

50 mug/mL of homocysteine Hcy-d 8;

vitamin E-d 6: 100 mu g/mL;

vitamin K-d 4: 10. mu.g/mL.

2) Taking an internal standard stock solution with a certain volume, and then diluting the internal standard stock solution by adopting methanol water (the mass percent of water is 50%) to obtain internal standard working solutions, wherein the concentrations of various substances in the internal standard working solutions are respectively as follows:

vitamin a-d 6: 200 ng/mL;

25-hydroxy vitamin D3-D6: 200 ng/mL;

homocysteine Hcy-d8:2000 ng/mL;

vitamin E-d 6: 2000 ng/mL;

vitamin K-d 4: 40 ng/mL.

3) Preparing stock solutions of fat-soluble vitamins (vitamin A, 25OH-VD2, 25OH-VD3, vitamin E and vitamin K) and homocysteine, wherein the concentration of the stock solutions is 100 mu g/ml.

Then, 6 gradients of mixed standard solutions were prepared in 5% BSAm/v (bovine serum albumin) methanol and dispensed into 1.5ml brown bottles, which were stored at-20 ℃ until use.

(III) sample pretreatment and detection

Respectively taking 200 mu L of a serum sample, putting the serum sample into a 2mL EP (epoxy resin) tube, adding 50 mu L of internal standard working solution and 50 mu L of reducing agent (1, 4-Dithiothreitol (DTT) solution with the concentration of 100 mu g/mL), and uniformly mixing by vortex; add 350 μ L protein precipitant (aqueous methanol, 2% water) to it, vortex for 10 seconds; then adding 1.0mL of extracting agent (n-hexane) for liquid-liquid extraction, vortexing for 10min, and then centrifuging at 10000rpm for 10 min; taking 900 mu L of centrifuged supernatant, drying the supernatant under nitrogen at room temperature, redissolving the residue by using 100 mu L of double solvent, vortexing, and feeding 20 mu L of sample to an upper liquid chromatography-mass spectrometry system for analysis.

The liquid chromatography conditions were: a chromatographic column: phenomenex kinex C18, 30 x 3mm, 2.6 μm; flow rate: 0.7 ml/min; temperature of the chromatographic column: at 40 ℃. Mobile phase: a: 0.1% formic acid + water; b, 0.1% formic acid, acetonitrile and isopropanol, wherein the volume ratio of the acetonitrile to the isopropanol is 2: 10. Gradient elution procedure for mobile phase, as shown in table 1:

table 1 gradient elution procedure for mobile phase

Time(min)	A％	B％
				0	60	40
0.4	60	40
			1.5	40	60
2.5	0	100
			4.5	0	100
4.51	60	40
			5.0	60	40

Mass spectrum conditions:

1. an ion source: an APCI source; NC is 5.0; 450 parts of TEM; 6, CAD; CUR of 25; GS1: 50.

2. Mass spectrometry ion pairs as shown in table 2.

TABLE 2

The data acquisition mode is as follows: (multiple reaction monitoring mode) MRM;

temperature of atomized gas: at 450 ℃;

atomizing airflow: 5.0L/min;

capillary voltage: 4500V;

an ion source: atmospheric pressure ionization source (APCI);

ion mode: positive ion mode.

(IV) the method in this example is demonstrated as follows:

1) transferring 10 μ L of stock solutions of five liposoluble vitamins and homocysteine into 10ml volumetric flasks, mixing, adding methanol water to desired volume, and detecting by the above method, as shown in FIG. 1, completely separating six substances.

2) Linear Range and sensitivity test

The concentration range of a standard curve is determined according to the content range of a target substance in a human body, 6 calibration curve points with different concentrations are prepared by using a serum sample (human serum matrix) without the target substance in the test, the concentration of VA is respectively 40, 80, 160, 400, 800 and 1600ng/mL, the concentration of α -VE is respectively 500, 1000, 2000, 5000, 10000 and 20000ng/mL, the concentration of 25OH-VD2 is respectively 2, 4, 8, 20, 40 and 80ng/mL, the concentration of 25OH-VD3 is respectively 5, 10, 20, 50, 100 and 200ng/mL, the concentration of VK is respectively 0.5, 1, 2, 5, 10 and 20ng/mL, the concentration of Hcy is respectively 500, 1000, 2000, 3000, 5000 and 8000ng/mL, the obtained sample is purified and detected by adopting the sample pretreatment method, and the result is shown in Table 3.

TABLE 3 Linear Range and sensitivity

3) Test of method precision and accuracy

The 16ml serum samples were mixed well, divided into 4 tubes, each tube had a serum volume of 4ml, one tube was taken as the background value test (sera), and the other 3 tubes were added with VE, VD3, VD2, VA, VK and Hcy standards as L, M, H three gradient samples (standard values were added according to standard volume/serum volume < 2%). From the above 4 tubes, 3 portions of each tube were sampled and the VE, VD3, VD2, VA, Vk, Hcy values were measured using a 96-well plate, and the pretreatment method and the measurement method of the sample were the same as above. L, M, H the error of recovery rate of sample addition is within + -15%, and the CV of sample precision is less than 15%. For each concentration, 6 samples were processed in parallel and the data obtained are shown in table 4.

TABLE 4 precision and accuracy

(V) measurement of actual sample

The blood samples 1 to 5 were tested by the above methods, and the test results are shown in Table 5.

TABLE 5

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The method for simultaneously detecting a plurality of fat-soluble vitamins and homocysteine is characterized by comprising the following steps: the method comprises the following steps:

the extracting agent is n-hexane.

2. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 1, which is characterized in that: the concentration of the 1, 4-dithiothreitol solution is 70-120 mug/ml, and further, the concentration of the 1, 4-dithiothreitol solution is 100 mug/ml.

3. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 2, characterized in that: the volume ratio of the serum sample to the 1, 4-dithiothreitol solution is 3.5-4.5: 1.

4. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 1, which is characterized in that: the protein precipitator is a mixed solution of methanol and water, wherein the mass percent of the water is 1-10%.

5. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 1, which is characterized in that: in the protein precipitant, the mass percentage of water is 2%.

6. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 1, which is characterized in that: the volume ratio of the serum to the protein precipitator is 1: 1.2-2.

7. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 1, which is characterized in that: the extract was blown dry with nitrogen.

8. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 1, which is characterized in that: the redissolved cosolvent is a 95% methanol aqueous solution.

9. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 1, which is characterized in that: the detection conditions of the liquid chromatogram are as follows: phenomenex kinex C18, 30 x 3mm, 2.6 μm; flow rate: 0.7 ml/min; column temperature: 40 ℃; mobile phase: a: 0.1% formic acid + water; b, 0.1 percent of formic acid, acetonitrile and isopropanol, wherein the volume ratio of the acetonitrile to the isopropanol is 2-3: 10-15.

10. The method for simultaneously detecting multiple fat-soluble vitamins and homocysteine according to claim 1, which is characterized in that: the gradient elution procedure for liquid chromatography was: 0-0.4min, the volume percentage of the mobile phase A is always 60%; the volume percentage of the mobile phase A is gradually reduced from 60 percent to 40 percent in 0.4-1.5 min; 1.5-2.5min, the volume percentage of the mobile phase A is gradually reduced from 40% to 0; 2.5-4.5min, the volume percentage of the mobile phase A is always 0; 4.5-4.51min, the volume percentage of the mobile phase A rises to 60%; 4.51-5.0min, the volume percentage of mobile phase A was always 60%.