CN115856171A - Method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones - Google Patents
Method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones Download PDFInfo
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- CN115856171A CN115856171A CN202211625226.1A CN202211625226A CN115856171A CN 115856171 A CN115856171 A CN 115856171A CN 202211625226 A CN202211625226 A CN 202211625226A CN 115856171 A CN115856171 A CN 115856171A
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Abstract
The invention discloses a method for simultaneously detecting multiple fat-soluble vitamins and steroid hormones, which comprises the following steps: adding an internal standard into the serum sample, uniformly mixing, and then adding methyl tert-butyl ether for extraction; extracting the tested components into an organic solvent, centrifuging, drying the extract liquid by nitrogen, redissolving, and performing sample injection analysis by a liquid chromatography-mass spectrometry system. The detection method can simultaneously detect the contents of five fat-soluble vitamins and seventeen steroid hormones in serum (blood plasma), simultaneously test 22 substances to be detected, is accurate and can carry out quantitative detection. The invention adopts the scheme of changing the conventional prior art that the isotope of the substance to be detected is used as the internal standard substance, and the trenbolone cyclohexylmethyl carbonate is used as the internal standard substance, so that 22 substances to be detected can be simultaneously quantified, and the workload is greatly reduced.
Description
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 multiple steroid hormones.
Background
Fat-soluble vitamins (fat-soluble vitamins) are a group of vitamins containing a ring structure and a long aliphatic hydrocarbon chain, are insoluble in water and soluble in fat and organic solvents, and mainly comprise vitamin A, vitamin D, vitamin E and vitamin K. Vitamin A can maintain normal vision, prevent nyctalopia, promote growth and development, and treat xerophthalmia, and is commonly found in radix Dauci Sativae, animal liver, vegetables, and fruits. Vitamin D regulates the calcium and phosphorus metabolism of human bodies and promotes the growth and development of bones, and is commonly found in animal livers, fish and milk products. Vitamin E is helpful for delaying aging and resisting oxidation, and is commonly found in edible oil, fruits, vegetables and grains. Vitamin K is a substance necessary for maintaining blood coagulation, and if the body lacks vitamin K, the body often has a tendency of bleeding, and the vitamin K is often found in onions, green vegetables, milk products and the like.
Steroid hormones are a large class of hormones, mainly including glucocorticoids, mineralocorticoids, gonadal hormones, etc., which are secreted by different layers of the adrenal cortex. Steroids in the body are numerous and each exerts a different action. For example: glucocorticoids: mainly comprises cortisone, prednisone and the like which are commonly used in clinic, has the functions of resisting inflammation, resisting allergy and increasing blood sugar, and is easy to cause full moon face, buffalo back and central obesity when excessive glucocorticoid is applied; mineralocorticoid: can regulate the water and salt metabolism of the body, and if the mineralocorticoid is excessive, the patient is easy to have the fluctuation of blood pressure; gonadal hormones: the gonadal hormones mainly comprise progestational hormone, estrogen and the like, and testosterone, androsterone and the like, can promote the development of sexual organs and maintain second sexual characteristics, and can easily cause menstrual disorder, infertility and the like if the gonadal hormone level is abnormal; other hormones: 1, 25-dihydroxyvitamin D3 is a hormone which helps calcium absorption, also belongs to steroid hormones, and is mainly used for regulating calcium and phosphorus. If the synthesis of 1, 25-dihydroxyvitamin D3 is insufficient, osteoporosis, rickets and the like are easy to occur. In addition, the steroid hormones in the body are of many kinds, and should be timely visited to the hospital for diagnosis once abnormality occurs. Steroid hormones play an important role in the body, and if the level of the steroid hormone is too low or the cholesterol content in the body is too low, the synthesis and secretion of the steroid hormone are affected, the metabolism of the hormone level in the body is abnormal, and the body can have pathological states.
After retrieval: chinese patent application CN110133135A discloses a content determination method of multiple fat-soluble vitamins, which detects a serum sample by a liquid-phase mass spectrometry, brings in a standard curve to obtain the content of the multiple fat-soluble vitamins in the serum sample, and takes isotopes as internal standards. Compared with the existing detection method, the method of the invention can simultaneously detect a plurality of fat-soluble vitamins.
Chinese patent application CN114689740A discloses a method for simultaneously determining six related steroid hormones CAH. The six related steroid hormones are 17 alpha-hydroxyprogesterone, androstenedione, 11-deoxycorticosterol, 21-deoxycorticosterol, cortisol and corticosterone; the determination method comprises the following steps: step S1: respectively preparing a mixed internal standard substance, a quality control substance and a standard curve product, and adopting an isotope as an internal standard substance; step S2: preparing a sample extracting solution; and step S3: extracting and incubating the sample; and step S4: concentrating and redissolving the sample; step S5: and (3) determining the redissolved solution by using a liquid chromatography tandem mass spectrometer, drawing a calibration curve, and calculating the concentrations of 17 alpha-hydroxyprogesterone, androstenedione, 11-deoxycorticosterol, 21-deoxycorticosterol, cortisol and corticosterone in a sample to be detected. The method can simultaneously detect multiple steroid hormones.
At present, the two substances are respectively measured by a liquid chromatography-mass spectrometry method, the number of fat-soluble vitamins or steroid hormones which are simultaneously measured is not more than six, and the internal standard substances are isotopes of target substances, and a plurality of internal standard substances are prepared for quantitative analysis. The scheme not only needs large sample amount, but also wastes time and labor, and has limitation on the condition of small sample volume and more items.
Disclosure of Invention
In order to make up the defects of the existing method, the invention provides a measuring method which has high selectivity and can simultaneously and accurately quantify the content of multiple steroid hormones and multiple fat-soluble vitamins in serum (blood plasma).
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones comprises the following steps:
adding an internal standard into the serum sample, uniformly mixing, and then adding an extracting agent for extraction; extracting the component to be detected into an organic solvent, centrifuging, transferring the extract into another centrifuge tube, drying by blowing, redissolving, and feeding into a liquid chromatography-mass spectrometry system for sample analysis; preparing a standard solution at the same time;
liquid chromatography conditions:
mobile phase A:0.1 to 0.5 percent of formic acid solution; mobile phase B: methanol solution containing 0.1-0.5% formic acid; the type of the chromatographic column: c18, 2.0X 150mm,3.0 μm; flow rate: 0.3-0.4ml/min; column temperature: 40-45 ℃;
gradient elution was used, the procedure for gradient elution being as follows: 0-0.5min, the volume percentage of the mobile phase B is 45%; the volume percentage of the mobile phase B is gradually reduced to 90 percent within 0.5-3 min; 3-5min, the volume percentage of the mobile phase B is 90%, and the fluidity B is reduced to 45% from 5-5.1 min; 5.1-5.5min, the volume percentage of the mobile phase B is 45%;
mass spectrum conditions: monitoring by adopting multiple reactions in an atmospheric pressure chemical ionization source positive ion mode; ion source voltage was 4kv (APCI +); the temperature of the ion transmission tube is 340-360 ℃; the temperature of the atomized gas is 340-360 ℃, and the flow rate of the sheath gas is 40-50L/min.
Preferably, the concentration of the internal standard substance is trenbolone cyclohexylmethyl carbonate is 0.8-1.2. Mu.g/ml, preferably 1. Mu.g/ml. Internal standards of the invention: trenbolone cyclohexylmethyl carbonate is a chemical substance with a chemical structure similar to that of the object to be tested and has the chemical formula C 26 H 34 O 4 CAS number 23454-33-3, molecular weight 410.55. Density 1.17g/cm 3 Boiling point 607.9 deg.C at 760mmHg, flash point 261.7 deg.C.
Preferably, the extractant is methyl tert-butyl ether, and the ratio of the volume of the extractant to the volume of the sample is (5-10): 1.
Preferably, the extract is blown dry with nitrogen.
Preferably, the redissolved cosolvent is methanol.
Preferably, the liquid chromatography detection conditions are as follows: athena C18-WP, 2.1X 150mm,3 μm; flow rate: 0.4ml/min; column temperature: 45 ℃; mobile phase: a:0.1% formic acid water; b: methanol with 0.1% formic acid.
The percentage content of the invention is mass percentage content.
Compared with the prior art, the invention has the advantages that:
1. the detection method can simultaneously detect the contents of five steroid hormones and seventeen fat-soluble vitamins in serum (blood plasma), simultaneously test 22 substances to be detected, is accurate and can carry out quantitative detection.
2. The invention adopts the scheme of changing the conventional prior art that the isotope of the substance to be detected is used as the internal standard substance, and the trenbolone cyclohexylmethyl carbonate is used as the internal standard substance, so that 23 substances to be detected can be quantified simultaneously, and the workload is greatly reduced.
Drawings
FIG. 1 is a Total ion plot (TIC);
FIG. 2 is an extracted ion map of VitA;
FIG. 3 is an extracted ion graph of VitE;
FIG. 4 is an extracted ion graph of VitK 1;
FIG. 5 is an extracted ion graph of 25-OHVitD 2;
FIG. 6 is an extracted ion graph of 25-OHVitD 3;
FIG. 7 is an extracted ion map of E1;
FIG. 8 is an extracted ion map of E2;
FIG. 9 is an extracted ion map of E3;
FIG. 10 is the extracted ion map of A4;
FIG. 11 is an extracted ion map of P;
FIG. 12 is an extracted ion map of TESTO;
FIG. 13 is an extracted ion map of DHT;
FIG. 14 is an extracted ion map of the PREG;
FIG. 15 is an extracted ion map of HCOR;
FIG. 16 is an extracted ion map of DHEA;
FIG. 17 is an extracted ion map of ALD;
FIG. 18 is an extracted ion map of CORTISONE;
FIG. 19 is an extracted ion map of 11 DOCORs;
FIG. 20 is an extracted ion map of 17-OHP;
FIG. 21 is an extracted ion map of 17-OHPPREG;
FIG. 22 is an extracted ionogram for the 21 DOCOR;
FIG. 23 is an extracted ion map of 21 OHPRG;
FIG. 24 is an extracted ion diagram of TCMHC.
Detailed Description
The detailed structure of the present invention will be further described with reference to the accompanying drawings and the detailed description.
Example 1
1. Reagents (all reagents are commercially available on the general market): testosterone, estrone, progesterone, estradiol, estriol, androstenedione, dihydrotestosterone, pregnenolone, 17-hydroxyprogesterone (17 alpha-hydroxyprogesterone), dehydroepiandrosterone, 17 alpha-hydroxyprogesterone, 21-hydroxyprogesterone, cortisol, corticosterone, aldosterone, desoxycorticosterone, 11-desoxycortisol, 18-hydroxypcortol, 21-desoxycortisol, vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1, and the internal standard is trenbolone cyclohexylmethyl carbonate.
Methanol, acetonitrile, methyl tert-butyl ether, formic acid, artificial serum.
2. Mobile phase A:0.1 to 0.5 percent of formic acid aqueous solution; mobile phase B: methanol (containing 0.1-0.5% formic acid);
preparing standard solution by accurately weighing 10mg of each standard substance in a 10mL volumetric flask, dissolving with methanol, adding to scale to obtain a concentration of 1mg/mL, storing at-80 deg.C, and diluting with methanol to obtain working solution when in use.
3. Preparing a working curve: the standard solution mixture was diluted with artificial serum to the following concentration gradients as shown in table 1:
TABLE 1 gradient chart of dilution concentration of artificial serum
4. The apparatus comprises an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, a high-speed refrigerated centrifuge, a mixer, a nitrogen blowing instrument and an analytical balance.
5. Sample treatment: taking 100 mu L of serum, adding 10 mu L of internal standard (1 mu g/mL trenbolone cyclohexylmethyl carbonate), uniformly mixing, adding 600 mu L of methyl tert-butyl ether, extracting for 5 minutes on a uniformly mixing device, standing for 1 minute, transferring an upper layer organic phase into another tube, drying under nitrogen, redissolving by 100 mu L of methanol, vortex and uniformly mixing for 1 minute, centrifuging for 10 minutes at 14000 r/min, transferring a supernatant into a sample introduction bottle, injecting 5 mu L of sample, and taking the area ratio concentration of a measured object and the internal standard as a regression curve to calculate the content of the measured object.
6. Ultra-high performance liquid chromatography conditions:
mobile phase A:0.1 to 0.5 percent of formic acid solution; mobile phase B: methanol (containing 0.1-0.5% formic acid); the type of the chromatographic column: c18, 2.0X 150mm,3.0 μm;
gradient elution was used, the gradient elution being as in table 2:
TABLE 2 gradient elution procedure
7. Mass spectrum conditions:
multiple Reaction Monitoring (MRM) is adopted in an atmospheric pressure chemical ionization source (APCI) positive ion mode; ion source voltage was 4kv (APCI +); the temperature of the ion transmission tube is 350 ℃; the temperature of the atomizing gas is 350 ℃, and the flow rate of the sheath gas is 45L/min. Mass spectrometry ion pairs and parameters are as follows in table 3:
TABLE 3 Mass Spectroscopy ion Pair and parameters
8. The method is used for measuring the normal human serum sample, the result calculation adopts an internal standard method, and a regression equation is made according to a standard curve: y = ax + b, y-is the ratio of the peak areas of the measured substance and the internal standard, x-is the concentration of the measured substance, a-is the slope, b-is the intercept, each component has a regression equation, the concentration (x) of each component is calculated by substituting the ratio (y) of the measured peak area of each component of the normal person to the peak area of the internal standard into the equation, and the measurement results are shown in table 4:
TABLE 4 measurement of normal human serum samples
9. Methodology validation
a. The linear division is: 22 the linear range of the measured components is shown in Table 1, and the linear correlation coefficient (r) is more than or equal to 0.990.
b. And (3) recovery rate: the tested object is prepared into samples with high, medium and low concentrations by using artificial serum, each concentration is repeated by 5 parts, the ratio of the result measured by the method to the result measured by a pure standard with the corresponding concentration is multiplied by 100 percent to obtain the recovery rate, and the recovery rates of 23 components (including an internal standard) are all between 93 percent and 102 percent.
c. Repeatability and stability: the test object is prepared into samples with high, medium and low concentrations by using artificial serum, each concentration is repeated for 5 parts, the obtained result is measured according to the method, the Relative Standard Deviation (RSD) between days and within days is calculated continuously for 3 days, and the result shows that the RDS within the day of 23 components (including an internal standard) is less than or equal to 6 percent, and the RSD within the day is less than or equal to 10 percent.
The verification shows that the analysis method is accurate.
The above description is for the purpose of describing particular embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and its concept within the scope of the present invention, and the technical solution and its concept should be covered by the scope of the present invention.
Claims (7)
1. A method for simultaneously detecting a plurality of fat-soluble vitamins and a plurality of steroid hormones, which is characterized in that: the method comprises the following steps:
adding an internal standard substance into a serum sample, uniformly mixing the internal standard substance and the trenbolone cyclohexylmethyl carbonate, and adding an extracting agent for extraction; extracting the component to be detected into an organic solvent, centrifuging, transferring the extract into another centrifuge tube, drying by blowing, redissolving, and feeding into a liquid chromatography-mass spectrometry system for sample analysis; preparing a standard solution and drawing a standard curve;
liquid chromatography conditions:
mobile phase A:0.1 to 0.5 percent of formic acid solution; mobile phase B: methanol solution containing 0.1-0.5% formic acid; the type of the chromatographic column: c18, 2.0X 150mm,3.0 μm; flow rate: 0.3-0.4ml/min; column temperature: 40-45 ℃; gradient elution is adopted;
mass spectrum conditions: under the positive ion mode of the atmospheric pressure chemical ionization source, multiple reaction monitoring is adopted; ion source voltage was 4kv (APCI +); the temperature of the ion transmission tube is 340-360 ℃; the temperature of the atomized gas is 340-360 ℃, and the flow rate of the sheath gas is 40-50L/min.
2. The method for simultaneously detecting multiple fat-soluble vitamins and multiple steroid hormones as claimed in claim 1, wherein: the concentration of the internal standard substance is trenbolone cyclohexyl methyl carbonate and is 0.8-1.2 mu g/ml.
3. The method for simultaneous detection of multiple fat-soluble vitamins and multiple steroid hormones as claimed in claim 1 or 2, wherein: the extractant is methyl tert-butyl ether, and the ratio of the volume of the extractant to the volume of the sample is (5-10): 1.
4. The method for simultaneous detection of multiple fat-soluble vitamins and multiple steroid hormones as claimed in claim 1 or 2, wherein: the extract was blown dry with nitrogen.
5. The method for simultaneous detection of multiple fat-soluble vitamins and multiple steroid hormones as claimed in claim 1 or 2, wherein: the redissolved cosolvent is methanol.
6. The method for simultaneous detection of multiple fat-soluble vitamins and multiple steroid hormones as claimed in claim 1 or 2, wherein: the detection conditions of the liquid chromatogram are as follows: athena C18-WP, 2.1X 150mm,3 μm; flow rate: 0.4ml/min; column temperature: 45 ℃; mobile phase: a:0.1% formic acid water; b: methanol with 0.1% formic acid.
7. The method for simultaneous detection of multiple fat-soluble vitamins and multiple steroid hormones as claimed in claim 1 or 2, wherein: the gradient elution procedure was as follows: 0-0.5min, the volume percentage of the mobile phase B is 45%; the volume percentage of the mobile phase B is gradually reduced to 90 percent in 0.5-3 min; 3-5min, the volume percentage of the mobile phase B is 90%, and the fluidity B is reduced to 45% from 5-5.1 min; 5.1-5.5min, and the volume percentage of the mobile phase B is 45%.
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| CN116519850A (en) * | 2023-06-29 | 2023-08-01 | 四川大学华西医院 | Method for rapidly detecting 16 hormone concentrations in serum sample |
| CN116519850B (en) * | 2023-06-29 | 2023-09-19 | 四川大学华西医院 | Method for rapidly detecting 16 hormone concentrations in serum sample |
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