CN111198238A - Extraction method and detection method of vitamin D metabolite-25 (OH) D3 in serum - Google Patents
Extraction method and detection method of vitamin D metabolite-25 (OH) D3 in serum Download PDFInfo
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Abstract
The invention discloses an extraction method and a detection method of a vitamin D metabolite-25 (OH) D3 in serum, wherein the extraction method comprises the following steps: 1) releasing 25(OH) D3 from the precipitated protein and transiently dissociating at least once to obtain a sample mixture comprising 25(OH) D3, phospholipids and proteins; 2) adsorbing at least phospholipid and protein in the mixture sample by using an activated carbon extraction plate; 3) eluting the activated carbon extraction plate, and then pressing the activated carbon extraction plate and the collection plate together to collect the eluent containing 25(OH) D3; 4) drying the collecting plate and redissolving; the detection method comprises the steps of taking a re-solution of 25(OH) D3 obtained by the extraction method of a vitamin D metabolite-25 (OH) D3 in serum as an object to be detected; and (4) loading the substance to be detected to a liquid chromatogram tandem mass spectrometer for detection. The extraction method provided by the invention can simultaneously complete sample enrichment and purification, has higher extraction rate, and can greatly improve the sensitivity of subsequent detection.
Description
Technical Field
The invention relates to a biochemical detection method, in particular to an extraction method and a detection method of a vitamin D metabolite-25 (OH) D3 in serum.
Background
Vitamin D (vitamind) is a fat-soluble steroid, abbreviated VD, which is also currently considered a steroid hormone, the most important members of the vitamin D family being VD2 (ergocalciferol) and VD3 (cholecalciferol). The chemical structures of VD2 and VD3 are different, and no clear evidence proves that the physiological functions of the two are different. Natural foods containing vitamin D are rare in nature, and wild salmon and mushrooms irradiated with ultraviolet rays contain VD. VD can also be ingested by VD fortified foods, most commonly milk, and secondly some grains, juices, margarines, and the like.
In the process of VD metabolism, VD precursors exist in the form of 7-dehydrocholesterol (7-dehydrocholestio 1) and ergosterol, respectively, in the skin and plants of all terrestrial vertebrates (including humans), and are converted into VD after being excited by high-energy B-type ultraviolet irradiation in sunlight, and then 25 hydroxy vitamin D (25 (OH) D3) and l, 25 dihydroxy vitamin D (1,25[ OH ]2D) are formed in sequence under the catalysis of VD hydroxylases of human or animals liver and kidney, while 1,25(OH)2D is an active form which produces biological effects in vivo, so that the synthesis of the so-called active VD 1,25 dihydroxy vitamin D is regulated by serum calcium, phosphorus concentration and parathyroid hormone (parathyroid hormone-PTH), when the blood calcium concentration is reduced, parathyroid hormone secretion is stimulated, and the activity of kidney l α hydroxylase is enhanced, so that the activity synthesis is increased, the latter inhibits the secretion of parathyroid hormone by a feedback regulation mechanism, and the formation of parathyroid hormone is coordinated with the metabolic mechanisms of calcium and phosphorus, thus, a large number of observed metabolic data of metabolic diseases in vitro and chronic diseases of vitamin D (3) are concerned with the development of cardiovascular diseases.
For Chinese adult bone health, the recommended vitamin D reference value adopts the standards of American health institute, English society for osteoporosis and Australian society for osteoporosis (Osteoporiosis Australia), namely 25(OH) D3 is more than or equal to 50nmol/L and less than or equal to 150nmol/L, and vitamin D can be considered as sufficient;
china is a wide multi-national country with a large audience, the condition of vitamin D is gradually concerned in recent years, the vitamin D condition of different regions and people is reported, and the results show great difference. It can be seen that vitamin D deficiency is common among chinese populations, even in southern regions with abundant sunlight; meanwhile, the old and pregnant women are high risk groups for vitamin D deficiency. If 25(OH) D3<50nmol/L (20 ng/mL) is used to define vitamin D deficiency, most regions report a vitamin D deficiency incidence of greater than 60%; if 25(OH) D3<75nmol/L (30 ng/mL) is used to define vitamin D deficiency, then the incidence of vitamin D deficiency is reported to be greater than 90% in most regions.
The recommended supplemental dose and upper tolerance limit for vitamin D will vary with age and the particular circumstances, and a daily dose, weekly dose, monthly dose, or once every 4 months may be used to maintain vitamin D.
For pregnant women, 25(OH) D3 passes through the placenta and into the fetal blood. Since 25(OH) D3 has a half-life of 2-3 weeks, vitamin D levels will be maintained within a normal level for several weeks after birth of the fetus in the presence of sufficient maternal 25(OH) D3. However, many studies have shown that vitamin D deficiency exists in most pregnant women and newborns, and supplementation with vitamin D at doses of 2000-4000IU/D during pregnancy improves the 25(OH) D3 levels in pregnant women and newborns.
In special cases, such as obesity, 2-5 times the normal dose is required to treat or prevent vitamin D deficiency; patients taking anticonvulsants, aids medications and glucocorticoids often need to take higher doses of vitamin D; granulomatous diseases such as sarcoidosis and tuberculosis are prone to hypercalcuria and hypercalcemia at levels of 25(OH) D3 in excess of 30ng/mL (75 nmol/L) due to elevated levels of active vitamin D (1,25[ OH ]2D) produced by macrophages and granulomas in patients whose administration of vitamin D is therefore monitored.
The vitamin D level is reduced due to various factors, and the synthesis amount of the vitamin D3 can be reduced by 95-99% by adopting sun-screening measures for human body self synthesis; studies have also shown that dark skinned people have a lower capacity for vitamin D synthesis than whiter people. In addition, air pollution causes ozone and nitrogen dioxide to rise and thus absorb ultraviolet radiation to cause vitamin D deficiency. Seasonal variations may also affect vitamin D levels. The prevalence of vitamin D deficiency or insufficiency in winter and spring is greater than in summer.
Elderly people in particular are at risk for clinical symptoms associated with vitamin D deficiency, and with age, changes in wear patterns, decreased outdoor activity, etc. often result in decreased vitamin D synthesis due to decreased sun exposure. Studies have shown that elderly people aged 77-82 years have a two-fold decrease in vitamin D synthesis capacity compared to adolescents aged 8-18 years, and thus elderly people are often at risk for vitamin D deficiency.
Since the mother is the only source of vitamin D demand for the newborn, the vitamin D deficiency of pregnant women during pregnancy usually leads to vitamin D deficiency of the newborn and increases the risk of rickets in children. Statistically, 27% -91%, 39% -65%, 19% -96% and 25% -87% of pregnant women in the United states, Canada, Europe and New Zealand, respectively, are vitamin D deficient or deficient.
Obese people, due to their body fat, retain vitamin D and require 2-5 times the normal dose to treat or prevent vitamin D deficiency. Patients taking antiepileptic, anticonvulsant, AIDS drug and glucocorticoid.
Relationship of vitamin D to health: 1. osteoporosis, VD is named as vitamin for resisting rickets for preventing rickets and osteomalacia at the earliest time, and the principle is that VD plays roles in calcium-phosphorus metabolism and bone calcification; 2. muscle weakness and myalgia, and fracture caused by falling is a common disease of the old, and the reasons are mainly three: osteoporosis, muscle weakness, ataxia, all three factors associated with VD deficiency. The icelandic carries out follow-up survey on 5461 66-96-year-old people for 5.4 years, and the risk of femoral fracture is increased by 2.08 times compared with the femoral fracture in a group with the serum VD level less than 30nmol/L (12 ng/mL) compared with a group with the serum VD level of 50-75nmol/L (20-30 ng/mL); cancer, the relationship between VD and cancer is the most interesting and one of the most controversial focus, and a great deal of epidemiological studies show that the risk of various cancers is increased when VD is absent or insufficient, i.e. 25(OH) D3 is lower than 20ng/mL (50nmol/L), and in vitro experiments, 1,25(OH) D3 is also determined to inhibit the growth of cancer cells and promote the differentiation and apoptosis of the cancer cells; 4. other diseases A number of epidemiological and clinical data indicate that low blood 25(OH) D3 levels can significantly increase mortality from heart disease (myocardial infarction, heart failure, etc.), that diabetic patients also commonly lack VD (up to 91%), and that exacerbations are associated with a decrease in blood 25(OH) D3. By comprehensively analyzing the epidemiological research data of various countries in the world, the patients with low 25(OH) D3 are also found to be susceptible to various autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, lupus erythematosus and the like. In animal experiments and a few clinical preventive trials, VD can prevent the occurrence of auto-allergic diseases.
Vitamin D plays a role not only in maintaining bone health, but also has a potential role in non-bone diseases, such as autoimmune diseases, cancer, mental health problems, cardiovascular diseases, and the like. When there is a need to rapidly correct vitamin D deficiency, such as a patient with disease symptoms, or to prepare to begin high-potency anti-resorptive medication (Zoledronate or dinocap Denosumab), the recommended treatment regimen is based on a fixed loading dose followed by regular maintenance therapy.
In China, vitamin D deficiency rickets are still common diseases of infants at present, and the bones are diseased due to the fact that calcium and phosphorus metabolism in vivo is abnormal due to vitamin D deficiency, and long metaphysis and bone tissue mineralization are incomplete. Vitamin D deficiency can also affect the functions of tissues and organs such as nerves, muscles, hematopoiesis, immunity and the like, and has great harm to the health of children. Therefore, the active prevention of vitamin D deficiency and vitamin D deficient rickets is an important task for pediatric health care workers. In order to better develop the prevention and treatment work of vitamin D deficiency and vitamin D deficiency rickets, the national cooperation group for prevention and treatment of rickets and the infant nutrition professional committee of the China Association for science and technology, the suggestions for prevention and treatment of vitamin D deficiency and vitamin D deficiency rickets are drawn up, and the treatment scheme is as follows: a. general therapy strengthens nursing, reasonable diet, insists on frequent sun exposure (avoid direct exposure for less than 6 months); b. a pharmaceutical therapy; c. other treatments are as follows: 1, calcium supplement: a suitable amount of calcium supplement preparation, in particular dietary calcium, would be beneficial for bone health, 2, micronutrient supplement: vitamin D deficient rickets are mostly accompanied by zinc and iron reduction, and timely supplement is beneficial to bone growth, 3, surgical operation.
Potential side effects may occur when vitamin D continues to reach high levels, such as 25(OH) D3>150nmol/L (> 60 ng/mL); persistent serum 25(OH) D3>500nmol/L (> 200 ng/mL) can be considered as a potential vitamin D intoxication. Vitamin D poisoning may cause non-specific symptoms such as loss of appetite, weight loss, polyuria, arrhythmia, etc.; also, overt symptoms include calcification of blood vessels and tissues due to chronic persistent elevated blood calcium levels, affecting the heart, blood vessels, and kidneys. Vitamin D intoxication can result from improper high dose treatment or accidental overdose. Excessive intake of vitamin D2 and vitamin D3 can both cause toxicity due to hypercalcemia. Serum hypercalcemia can lead to soft tissue calcification and thus renal and cardiovascular damage.
The food and nutrition Commission of the American medical research institute summarized some evidence of vitamin D supplementation over various dose ranges (800-; while doses equal to or higher than 50000 IU/day, for weeks or months, often lead to toxicities, including hypercalcemia.
The European Food Safety Agency (EFSA) reviewed the relevant evidence and concluded: adults and children over the age of 11 years are safe to ingest vitamin D up to 4000 IU/day (100 mug/day).
25-hydroxyvitamin D (25 (OH) D3) is the best indicator of vitamin D status in humans, but the large difference in results between assays has affected the clinical diagnosis and treatment of vitamin D deficiency. Standardization of clinical and scientific laboratory testing methods is a necessary basis for establishing evidence-based medical guidelines. In 2010, the national institutes of health and nutrition (NIH-ODS) initiated the vitamin D standardization program.
There are many different analytical techniques and methods available for analyzing 25(OH) D3 concentration in human body, among which immunoassay has been the most important method for detecting 25(OH) D3 concentration in serum and is still widely used up to now, and includes Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), full-automatic biochemical assay, etc. The main drawback of immunoassays is the lack of process specificity due to cross-reactivity between antibodies, whereas chromatography allows efficient separation of these analytes, thus obtaining sufficient specificity. In this regard, the liquid chromatography-mass spectrometry (LC-MS/MS) method has great advantages, and the analysis technique is now increasingly used for the analysis of 25(OH) D3 in clinical laboratory because of the high selectivity, specificity and sensitivity of the LC-MS/MS method. The liquid chromatography-mass spectrometry (LC-MS/MS) method is currently recognized by the American institute of medicine (IOM) as the "gold standard" for vitamin D detection.
At present, the extraction method of 25(OH) D3 in serum is mostly a liquid-liquid extraction method, and the method comprises the following steps: 1. mixing: pouring the solution and the extract into a separating funnel respectively; 2. oscillating: inverting the separating funnel and oscillating forcefully; 3. standing: placing the separating funnel on an iron support and standing; 4. liquid separation: after the liquids are layered, the glass plug at the upper end of the funnel is opened, the lower layer of liquid is discharged from the lower end, and the upper layer of liquid (different types of extracting agents, 25(OH) D3 or the upper layer or the lower layer) is discharged from the upper end, however, the liquid-liquid extraction method has certain defects: 1. the operation is complicated and time-consuming. 2. A large amount of organic solvent is required to be consumed, which results in high cost and environmental pollution; 3. it is difficult to extract highly water-soluble substances from water.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention discloses an extraction method and a detection method of a vitamin D metabolite-25 (OH) D3 in serum.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention discloses an extraction method of a vitamin D metabolite-25 (OH) D3 in serum, which comprises the following steps:
step 1) sample extraction: placing a set amount of serum into a centrifuge tube, adding a precipitated protein breaking agent into the centrifuge tube to break precipitated protein in the serum so as to release 25(OH) D3 from the precipitated protein, uniformly mixing the serum sample with the precipitated protein breaking agent, and then performing instant separation at least once to obtain a mixture sample containing 25(OH) D3, phospholipid and protein;
step 2), loading: loading the mixture sample obtained in the step 1) onto an activated carbon extraction plate, standing after all the mixture sample is immersed into the activated carbon extraction plate, and adsorbing at least phospholipid and protein in the mixture sample by the activated carbon extraction plate;
step 3) elution: carrying out elution treatment on the activated carbon extraction plate at least once, after elution is finished, superposing the activated carbon extraction plate and the collection plate integrally from top to bottom, and then pressing the activated carbon extraction plate and the collection plate together so that the collection plate collects an eluent containing 25(OH) D3;
step 4), re-dissolving: the collection plate was blown with gas until the solvent of the eluent evaporated and the 25(OH) D3 extract remained in the wells of the collection plate, and a re-solvent was added to the wells of the collection plate to re-dissolve the extract, thereby obtaining a re-solution of 25(OH) D3.
As one of the preferable schemes of the invention, in the step 1), 100-;
further preferably, after the first transient separation is finished, at least 100-300 μ L50:50 water/isopropanol mixed solution is added into the centrifugal tube, and the centrifugal tube is vortexed at the rotating speed of 2500-3000rpm for at least 1min, and then is subjected to transient separation at the rotating speed of 7000-12000rpm for 10-50s after being uniformly mixed.
As one of the preferable schemes of the invention, in the step 2), the mixture sample obtained in the step 1) is loaded on an activated carbon extraction plate of 400-500 μ L/mL, and the mixture sample is left standing for 3-6min after being immersed in the activated carbon extraction plate.
As one preferable scheme of the present invention, in the step 3), the activated carbon extraction plate is eluted with isooctane or n-hexane for three times, and after the last elution is finished, the activated carbon extraction plate and the collection plate which are overlapped up and down are pressed together by a positive pressure device.
Further preferably, the opening pressure of the positive pressure device is 10-15psi, and the pressing time is 5-8 s.
More preferably, the activated carbon extraction plate is eluted with 1.2-2.1 mL of isooctane or n-hexane.
In a preferred embodiment of the present invention, in the step 4), a nitrogen blow-drying collecting plate is manufactured by using a nitrogen instrument.
As one of the preferable embodiments of the present invention, 100-200. mu.L of a methanol/water mixed solution with a volume ratio of 70:30 is used as a double solvent for dissolving vitamin D.
Further preferably, the vitamin D compound solution is vortexed at 2000-4000rpm for at least 3min and then centrifuged at 2500-3000rpm for at least 1min at a temperature of 4-8 ℃.
As one of the preferable schemes of the invention, the activated carbon extraction plate adopts a porous plate, each hole of the porous plate is filled with activated carbon, the collection plate adopts a porous plastic plate, and the collection plate has the same number of holes as the activated carbon extraction plate; preferably, the plastic plate is a polyethylene plate.
As one of the preferable schemes of the invention, the preparation method of the activated carbon comprises the following steps:
carbonizing an organic raw material at least comprising shells, fruit stones, coal and wood at a high temperature of 600-900 ℃ under the condition of air isolation to obtain a carbide; the carbide is reacted with air such that its surface is eroded to produce a microporous structure.
The invention also discloses a detection method of the vitamin D metabolite in the serum, which comprises the following steps: taking the re-solution of 25(OH) D3 obtained by the extraction method of the vitamin D metabolite-25 (OH) D3 in the serum as a substance to be detected; and loading the object to be detected to a liquid chromatogram tandem mass spectrometer, and detecting by adopting the liquid chromatogram tandem mass spectrometer.
Compared with the prior art, the invention has at least the following advantages:
1) the extraction method of the vitamin D metabolite-25 (OH) D3 in the serum provided by the invention can complete sample enrichment and purification simultaneously, and can greatly improve the detection sensitivity of subsequent detection.
2) The extraction method of the vitamin D metabolite-25 (OH) D3 in the serum, which is provided by the invention, is a solid phase extraction method, and has higher extraction efficiency and recovery rate.
3) The extraction method of the vitamin D metabolite-25 (OH) D3 in the serum provided by the invention is a solid phase extraction method, is simple to operate, saves time and labor, and shortens the extraction time by more than half compared with the traditional liquid-liquid extraction method.
4) Compared with the traditional liquid-liquid extraction method, the extraction method of the serum vitamin D metabolite-25 (OH) D3 provided by the invention saves solvent and time, is easy to realize automation, and can realize automatic batch treatment.
5) The extraction method of the vitamin D metabolite-25 (OH) D3 in the serum provided by the invention has good reproducibility.
6) According to the invention, the solid phase column (activated carbon filler) has a developed microporous structure, so that the filler can absorb a water-based sample more quickly and completely, and the waiting time after sample loading is reduced;
7) the extraction volume of the biological fluid can be as low as 100 mu L, and the extraction process is a purification, concentration and enrichment process, so the smaller the extraction volume, the better.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below.
FIG. 1 is a chromatogram of 25(OH) D3 in serum measured by the method for measuring vitamin D metabolite-25 (OH) D3 in serum as disclosed in the examples of the present invention;
FIG. 2 is an ion spectrum of 25(OH) D3 in serum detected by the method for detecting vitamin D metabolite-25 (OH) D3 in serum disclosed in the embodiments of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.
The invention discloses an extraction method of a vitamin D metabolite-25 (OH) D3 in serum, which comprises the following steps:
step 1) sample extraction: placing a set amount of serum into a centrifuge tube, adding a precipitated protein breaking agent into the centrifuge tube to break precipitated protein in the serum so as to release 25(OH) D3 from the precipitated protein, uniformly mixing the serum sample with the precipitated protein breaking agent, and then performing instant separation at least once to obtain a mixture sample containing 25(OH) D3, phospholipid and protein;
step 2), loading: loading the mixture sample obtained in the step 1) onto an activated carbon extraction plate, standing after all the mixture sample is immersed into the activated carbon extraction plate, and adsorbing at least phospholipid and protein in the mixture sample by the activated carbon extraction plate;
step 3) elution: carrying out elution treatment on the activated carbon extraction plate at least once, after elution is finished, superposing the activated carbon extraction plate and the collection plate integrally from top to bottom, and then pressing the activated carbon extraction plate and the collection plate together so that the collection plate collects an eluent containing 25(OH) D3;
step 4), re-dissolving: the collection plate was blown with gas until the solvent of the eluent evaporated and the 25(OH) D3 extract remained in the wells of the collection plate, and a re-solvent was added to the wells of the collection plate to re-dissolve the extract, thereby obtaining a re-solution of 25(OH) D3.
As one of the preferable schemes of the invention, the invention discloses a method for extracting a vitamin D metabolite-25 (OH) D3 in serum, which comprises the following steps:
step 1): adding 100-;
after the first transient separation is finished, at least 100-300 mu L of 50:50 water/isopropanol mixed solution is added into the centrifugal tube, and the centrifugal tube is vortexed at the rotating speed of 2500-3000rpm for at least 1min, and then is instantaneously separated at the rotating speed of 7000-12000rpm for 10-50s after being uniformly mixed.
Step 2): and (2) loading the mixture sample obtained in the step 1) into a 400-fold 500-microliter/mL activated carbon extraction plate, and standing for 3-6min after all the mixture sample is immersed into the activated carbon extraction plate.
Step 3): and (3) eluting the activated carbon extraction plate by using 1.2-2.1 mL of isooctane or n-hexane for three times, laminating the activated carbon extraction plate and the collection plate which are overlapped up and down by using a positive pressure device after the last elution is finished, and collecting the eluent containing 25(OH) D3 by using the collection plate, wherein the opening pressure of the positive pressure device is 10-15psi, and the laminating time is 5-8 s.
Step 4): nitrogen was blown through the collection plate using a nitrogen instrument until the solvent of the eluent was evaporated to leave the extract of 25(OH) D3 remaining in the wells of the collection plate, and then 100-200. mu.L of a methanol/water mixture at a volume ratio of 70:30 was used as a re-solvent for dissolving 25(OH) D3; the reconstituted solution of 25(OH) D3 was vortexed at 2000-4000rpm for at least 3min and then centrifuged at 3000rpm at 2500-8 ℃ for at least 1min (optionally without centrifugation, where impurities in the reconstituted solution are visible to the naked eye, centrifugation is required, otherwise centrifugation is not required, and the impurities are almost removed because they have been centrifuged several times in step (1)).
Wherein, the active carbon extraction board adopts the perforated plate (if adopt 96 orifice plates), every downthehole active carbon that is filled with of perforated plate to form 96 extraction columns, the collecting plate adopts porous plastic slab (if adopt 96 orifice plates), the collecting plate have with the same quantity of hole of active carbon extraction board, the plastic slab adopts the polyethylene board. Correspondingly, the positive pressure device adopts a CommaVac 96 hole positive pressure extraction device stably combined with the 96 extraction columns. When the extraction liquid is collected, a 96-hole collection plate is placed under a 96-hole activated carbon extraction plate, the 96-hole activated carbon extraction plate is pressed by a positive pressure device, and the extraction liquid containing 25(OH) D3 is collected by the collection plate.
The preparation method of the activated carbon comprises the following steps:
carbonizing an organic raw material at least comprising a fruit shell, a fruit core, coal and wood at a high temperature of 600-900 ℃ under the condition of air isolation to obtain a carbide so as to reduce non-carbon components and oxygen elements such as H2O, CO2 and other gases (the process is called carbonization); the char is reacted with air such that its surface is eroded, creating a microporous structure. Activated carbon is commercially available or is available as a specialty product from a commercial vendor.
The activated carbon can effectively adsorb or remove phospholipid in a serum sample, and in addition, the activated carbon can adsorb many other substances due to the characteristics of developed microporous structure, light weight, high porosity, strong liquid adsorption capacity and the like. In the present application, it is emphasized that activated carbon can more effectively adsorb phospholipids in serum samples, because phospholipids in serum are the main source of matrix effect, which can cause the difference of analysis results and result in unpredictable mass spectrum results. The activated carbon can selectively capture phospholipid interferents causing ion inhibition without influencing the loss of a target substance (25 (OH) D3), thereby providing excellent purification effect for a lipid-containing sample, and effectively improving the sensitivity of detecting the 25(OH) D3 by an instrument.
The technical solution of the present invention is further explained below with reference to specific examples 1 to 3.
Example 1:
the embodiment 1 of the invention discloses a method for extracting a vitamin D metabolite-25 (OH) D3 in serum, which comprises the following steps:
step 1) sample extraction: adding 100 mu L of serum sample into a centrifuge tube (1.5mL sterile centrifuge tube), adding 100 mu L of water/isopropanol mixed solution with the volume ratio of 50:50 as a precipitated protein breaking agent into the centrifuge tube to break precipitated protein in the serum, so that 25(OH) D3 is released from the precipitated protein, uniformly mixing the serum sample and the precipitated protein breaking agent by vortex at the rotating speed of 2500rpm, and instantaneously separating at the rotating speed of 7000rpm for 10 s;
after the first transient separation is finished, at least adding 100 mu L of 50:50 water/isopropanol mixed solution into the centrifugal tube, vortexing at the rotating speed of 2500rpm for 1min, then uniformly mixing, and then transiently separating at the rotating speed of 7000rpm for 10s, thereby finally obtaining a mixture sample containing 25(OH) D3, phospholipid and protein;
step 2), loading: loading the mixture sample obtained in the step 1) onto an activated carbon extraction plate of 400 mu L/mL, standing for 3min after all the mixture sample is immersed into the activated carbon extraction plate, and adsorbing at least phospholipid and protein in the mixture sample by the activated carbon extraction plate;
step 3) elution: and (3) eluting the active carbon extraction plate by adopting 1.2 mL of isooctane or n-hexane for three times, and laminating the active carbon extraction plate and the collection plate which are overlapped up and down by using a positive pressure device after the last elution is finished, so that the collection plate collects the eluent containing 25(OH) D3, wherein the opening pressure of the positive pressure device is 10psi, and the laminating time is 5 s.
Step 4), re-dissolving: nitrogen was blown to the collection plate by a nitrogen analyzer until the solvent of the eluate was evaporated to leave an extract of 25(OH) D3 remaining in the pores of the collection plate, and then the extract was redissolved using 100 μ L of a methanol/water mixed solution at a volume ratio of 70:30 as a redissolution to dissolve 25(OH) D3, thereby obtaining a redissolution of 25(OH) D3; the reconstituted solution of 25(OH) D3 was vortexed at 2000rpm for 3min and then centrifuged at 2500rpm for 1min at 4 ℃ (optionally without centrifugation, where centrifugation is required if impurities are visible to the naked eye, otherwise centrifugation is not required, and the impurities are almost removed by centrifugation multiple times in step (1)).
Wherein, the active carbon extraction board adopts the perforated plate (if adopt 96 orifice plates), every downthehole active carbon that is filled with of perforated plate to form 96 extraction columns, the collecting plate adopts porous plastic slab (if adopt 96 orifice plates), the collecting plate have with the same quantity of hole of active carbon extraction board, the plastic slab adopts the polyethylene board. Correspondingly, the positive pressure device adopts a CommaVac 96 hole positive pressure extraction device stably combined with the 96 extraction columns. When the extraction liquid is collected, a 96-hole collection plate is placed under a 96-hole activated carbon extraction plate, the 96-hole activated carbon extraction plate is pressed by a positive pressure device, and the extraction liquid containing 25(OH) D3 is collected by the collection plate.
Example 2:
the embodiment 2 of the invention discloses a method for extracting a vitamin D metabolite-25 (OH) D3 in serum, which comprises the following steps:
step 1) sample extraction: adding 150 mu L of serum sample into a centrifuge tube (1.5mL of sterile centrifuge tube), adding 200 mu L of water/isopropanol mixed solution with the volume ratio of 50:50 as a precipitated protein breaking agent into the centrifuge tube to break precipitated protein in the serum, so that 25(OH) D3 is released from the precipitated protein, uniformly mixing the serum sample and the precipitated protein breaking agent by vortex at the rotating speed of 2800rpm, and instantaneously separating at the rotating speed of 10000rpm for 30 s;
after the first flash separation is finished, at least adding 200 mu L of 50:50 water/isopropanol mixed solution into the centrifugal tube, vortexing at 2800rpm for 2min, then uniformly mixing, and then flash separating at 10000rpm for 30s, thereby finally obtaining a mixture sample containing 25(OH) D3, phospholipid and protein;
step 2), loading: loading the mixture sample obtained in the step 1) onto an activated carbon extraction plate with the concentration of 450 mu L/mL, standing for 4min after all the mixture sample is immersed into the activated carbon extraction plate, and adsorbing at least phospholipid and protein in the mixture sample by using the activated carbon extraction plate;
step 3) elution: and (3) eluting the active carbon extraction plate by adopting 1.7 mL of isooctane or n-hexane for three times, and laminating the active carbon extraction plate and the collection plate which are overlapped up and down by using a positive pressure device after the last elution is finished, so that the collection plate collects the eluent containing 25(OH) D3, wherein the opening pressure of the positive pressure device is 12psi, and the laminating time is 7 s.
Step 4), re-dissolving: nitrogen was blown to the collection plate using a nitrogen instrument until the solvent of the eluate evaporated to leave an extract of 25(OH) D3 remaining in the pores of the collection plate, and then the extract was redissolved using 150 μ L of a methanol/water mixed solution at a volume ratio of 70:30 as a redissolution to dissolve 25(OH) D3, thereby obtaining a redissolution of 25(OH) D3; the reconstituted solution of 25(OH) D3 was vortexed at 3000rpm for 4min and then centrifuged at 2800rpm at 6 ℃ for 2min (optionally without centrifugation, if impurities were visible in the reconstituted solution, centrifugation is required, otherwise centrifugation is not required, and the impurities were almost removed by centrifugation multiple times in step (1)).
Wherein, the active carbon extraction board adopts the perforated plate (if adopt 96 orifice plates), every downthehole active carbon that is filled with of perforated plate to form 96 extraction columns, the collecting plate adopts porous plastic slab (if adopt 96 orifice plates), the collecting plate have with the same quantity of hole of active carbon extraction board, the plastic slab adopts the polyethylene board. Correspondingly, the positive pressure device adopts a CommaVac 96 hole positive pressure extraction device stably combined with the 96 extraction columns. When the extraction liquid is collected, a 96-hole collection plate is placed under a 96-hole activated carbon extraction plate, the 96-hole activated carbon extraction plate is pressed by a positive pressure device, and the extraction liquid containing 25(OH) D3 is collected by the collection plate.
Example 3:
the embodiment 23 of the invention discloses a method for extracting a vitamin D metabolite-25 (OH) D3 in serum, which comprises the following steps:
step 1) sample extraction: adding 200 mu L of serum sample into a centrifuge tube (1.5mL of sterile centrifuge tube), adding 300 mu L of water/isopropanol mixed solution with the volume ratio of 50:50 as a precipitated protein breaking agent into the centrifuge tube to break precipitated protein in the serum, so that 25(OH) D3 is released from the precipitated protein, uniformly mixing the serum sample and the precipitated protein breaking agent by vortex at 2800rpm, and instantly separating at 12000rpm for 50 s;
after the first transient separation is finished, at least 300 mu L of 50:50 water/isopropanol mixed solution is added into the centrifugal tube, the centrifugal tube is vortexed at 3000rpm for 3min, and then the mixture is uniformly mixed and then is instantaneously separated at 12000rpm for 50s, so that a mixture sample containing 25(OH) D3, phospholipid and protein is finally obtained;
step 2), loading: loading the mixture sample obtained in the step 1) onto an activated carbon extraction plate of 500 mu L/mL, standing for 6min after all the mixture sample is immersed into the activated carbon extraction plate, and adsorbing at least phospholipid and protein in the mixture sample by the activated carbon extraction plate;
step 3) elution: and 2.1mL of isooctane or n-hexane is adopted to elute the activated carbon extraction plate, elution is carried out for three times, and after the last elution is finished, the activated carbon extraction plate and the collection plate which are overlapped up and down are pressed by a positive pressure device, so that the collection plate collects the eluent containing 25(OH) D3, wherein the opening pressure of the positive pressure device is 15psi, and the pressing time is 8 s.
Step 4), re-dissolving: nitrogen was blown to the collection plate by a nitrogen analyzer until the solvent of the eluate was evaporated to leave an extract of 25(OH) D3 remaining in the pores of the collection plate, and then the extract was redissolved using 200 μ L of a methanol/water mixed solution at a volume ratio of 70:30 as a redissolution to dissolve 25(OH) D3, thereby obtaining a redissolution of 25(OH) D3; the reconstituted solution of 25(OH) D3 was vortexed at 4000rpm for 5min and then centrifuged at 3000rpm for 3min at a temperature of 8 ℃ (optionally without centrifugation, where centrifugation is required if impurities are visible to the naked eye, otherwise centrifugation is not required, and the impurities are almost removed by centrifugation multiple times in step (1)).
Wherein, the active carbon extraction board adopts the perforated plate (if adopt 96 orifice plates), every downthehole active carbon that is filled with of perforated plate to form 96 extraction columns, the collecting plate adopts porous plastic slab (if adopt 96 orifice plates), the collecting plate have with the same quantity of hole of active carbon extraction board, the plastic slab adopts the polyethylene board. Correspondingly, the positive pressure device adopts a CommaVac 96 hole positive pressure extraction device stably combined with the 96 extraction columns. When the extraction liquid is collected, a 96-hole collection plate is placed under a 96-hole activated carbon extraction plate, the 96-hole activated carbon extraction plate is pressed by a positive pressure device, and the extraction liquid containing 25(OH) D3 is collected by the collection plate.
The embodiment 4 of the invention also discloses a method for detecting a vitamin D metabolite-25 (OH) D3 in serum, which comprises the following steps: adopting a compound solution of 25(OH) D3 obtained by the extraction method of the vitamin D metabolite-25 (OH) D3 in the serum as an object to be detected; and loading the object to be detected to a liquid chromatogram tandem mass spectrometer, and detecting by adopting the liquid chromatogram tandem mass spectrometer, wherein the detection by the liquid chromatogram tandem mass spectrometer is the conventional detection method at present, and the principle is not described in detail herein. The main innovation of the method for detecting the vitamin D metabolite-25 (OH) D3 in the serum provided by embodiment 4 of the invention is that the above solid phase extraction method is used for extracting 25(OH) D3, the process of extracting 25(OH) D3 by the solid phase extraction method is the sample pretreatment, the determination process is the conventional means, and the determination steps of the liquid chromatography tandem mass spectrometry are roughly listed through a chart and characters as follows:
a) liquid chromatography conditions:
a1) a chromatographic column: waters Xbridge C18(2.1 mm. times.50 mm,3.5 μm);
a2) column temperature: 40 ℃;
a3) sample introduction amount: 5 mu L of the solution;
a4) mobile phase: a mobile phase of methanol containing 0.1% formic acid, water containing 0.1% formic acid;
a5) and (3) an elution mode: gradient elution.
Table 1 mobile phase gradient elution conditions:
b) mass spectrum conditions:
electrospray voltage: 3500V sheath gas pressure: 35arb auxiliary air pressure: 2arb ion transfer tube: 350 ℃ auxiliary gas temperature: acquisition mode at 340 ℃: multiple reaction monitoring
Table 2 ion pair parameters for parent and daughter ion detection for each component:
c) results
FIG. 1 is a chromatogram of 25(OH) D3 assay in serum, showing that the species detected is 25(OH) D3 and the peak on the abscissa 1.41 represents 25(OH) D3; FIG. 2 is a graph showing the ion spectrum of 25(OH) D3 in serum and the result of detecting the peak area of 25(OH) D3 by a liquid chromatography tandem mass spectrometer, which is a verification of the above-mentioned solid phase extraction method, i.e., the content of 25(OH) D3 can be determined by performing pre-treatment of a sample and then performing on-machine measurement by using the above-mentioned solid phase extraction method;
table 3 shows the normalized recovery results:
table 4 compares the normalized recovery results for two different extraction methods:
the recovery rate of the solid-phase extraction method can be seen from table 3, and the comparison between the recovery rates of the solid-phase extraction method and the conventional liquid-liquid extraction method can be seen from table 4, and the recovery rate can represent the extraction efficiency, so that it can be clearly seen that the extraction efficiency of the extraction method (solid-phase extraction method) of 25(OH) D3 in serum disclosed in the embodiment of the present invention is higher than that of the conventional liquid-liquid extraction method, and therefore, the extraction method (solid-phase extraction method) of 25(OH) D3 in serum provided in the embodiment of the present invention is more advantageous, and the sensitivity of the subsequent liquid chromatography tandem mass spectrometry detection can be improved.
In addition, the extraction method of 25(OH) D3 in serum provided by the invention is simple to operate, saves time and labor, and shortens the extraction time by more than half compared with the traditional liquid-liquid extraction method; compared with the traditional liquid-liquid extraction method, the extraction method of 25(OH) D3 in serum provided by the invention saves solvent and time, is easy to realize automation, and can realize automatic batch treatment; the amount of sample used for extraction is small (can be as low as 100. mu.L).
Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A method for extracting vitamin D metabolite-25 (OH) D3 in serum is characterized in that: the method comprises the following steps:
step 1) sample extraction: placing a set amount of serum into a centrifuge tube, adding a precipitated protein breaking agent into the centrifuge tube to break precipitated protein in the serum so as to release 25(OH) D3 from the precipitated protein, uniformly mixing the serum sample with the precipitated protein breaking agent, and then performing instant separation at least once to obtain a mixture sample containing 25(OH) D3, phospholipid and protein;
step 2), loading: loading the mixture sample obtained in the step 1) onto an activated carbon extraction plate, standing after all the mixture sample is immersed into the activated carbon extraction plate, and adsorbing at least phospholipid and protein in the mixture sample by the activated carbon extraction plate;
step 3) elution: carrying out elution treatment on the activated carbon extraction plate at least once, after elution is finished, superposing the activated carbon extraction plate and the collection plate integrally from top to bottom, and then pressing the activated carbon extraction plate and the collection plate together so that the collection plate collects an eluent containing 25(OH) D3;
step 4), re-dissolving: the collection plate was blown with gas until the solvent of the eluent evaporated and the 25(OH) D3 extract remained in the wells of the collection plate, and a re-solvent was added to the wells of the collection plate to re-dissolve the extract, thereby obtaining a re-solution of 25(OH) D3.
2. The method for extracting vitamin D metabolite-25 (OH) D3 from blood serum according to claim 1, wherein the method comprises the following steps: in the step 1), 100-; preferably, after the first transient separation is finished, at least 100-300 μ L50:50 of water/isopropanol mixed solution is added into the centrifugal tube, and the centrifugal tube is vortexed at the rotating speed of 2500-3000rpm for at least 1min, and then is instantaneously separated at the rotating speed of 7000-12000rpm for 10-50s after being uniformly mixed.
3. The method for extracting vitamin D metabolite-25 (OH) D3 from blood serum according to claim 2, wherein the method comprises the following steps: in the step 2), the mixture sample obtained in the step 1) is loaded on a 400-fold 500 μ L/mL activated carbon extraction plate, and the mixture sample is left for 3-6min after being immersed in the activated carbon extraction plate.
4. The method for extracting vitamin D metabolite-25 (OH) D3 from blood serum according to claim 3, wherein the method comprises the following steps: in the step 3), the active carbon extraction plate is eluted by isooctane or normal hexane for three times, and after the last elution is finished, the active carbon extraction plate and the collection plate which are overlapped up and down are pressed by a positive pressure device; preferably, the opening pressure of the positive pressure device is 10-15psi, and the pressing time is 5-8 s; preferably, the activated carbon extraction plate is eluted with 1.2-2.1 mL of isooctane or n-hexane.
5. The method for extracting vitamin D metabolite-25 (OH) D3 from blood serum according to claim 4, wherein the method comprises the following steps: in the step 4), a nitrogen blow-drying collecting plate is manufactured by a nitrogen instrument; and/or, 100-; preferably, the 25(OH) D3 redissolution is vortexed at 2000-4000rpm for at least 3min and then centrifuged at 2500-3000rpm for at least 1min at a temperature of 4-8 ℃.
6. The method for extracting vitamin D metabolite-25 (OH) D3 from blood serum according to any one of claims 1-5, wherein the method comprises the following steps: the active carbon extraction plate adopts a porous plate, each hole of the porous plate is filled with active carbon, the collection plate adopts a porous plastic plate, and the collection plate is provided with holes with the same number as the active carbon extraction plate; preferably, the plastic plate is a polyethylene plate.
7. The method for extracting vitamin D metabolite-25 (OH) D3 from blood serum according to any one of claims 1-5, wherein the method comprises the following steps:
the preparation method of the activated carbon comprises the following steps:
carbonizing an organic raw material at least comprising shells, fruit stones, coal and wood at a high temperature of 600-900 ℃ under the condition of air isolation to obtain a carbide; the char is reacted with air such that its surface is eroded to produce a microporous structure.
8. A method for detecting a vitamin D metabolite-25 (OH) D3 in serum, comprising: using a re-solution of 25(OH) D3 obtained by the method of claim 7 for extracting 25(OH) D3 from serum as an analyte; and loading the object to be detected to a liquid chromatogram tandem mass spectrometer, and detecting by adopting the liquid chromatogram tandem mass spectrometer.
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