CN114942292B - Method for detecting content of plant sterol substances in blood based on liquid chromatography-tandem mass spectrometry technology - Google Patents
Method for detecting content of plant sterol substances in blood based on liquid chromatography-tandem mass spectrometry technology Download PDFInfo
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Abstract
The invention provides a method for detecting the content of plant sterol substances in blood based on a liquid chromatography-tandem mass spectrometry technology, wherein the plant sterol substances comprise 7-alkene cholanol, and the method comprises the following steps: (1) Preprocessing a blood sample to obtain a sample to be tested containing an internal standard substance; (2) Preparing a standard curve containing an internal standard substance, performing on-machine detection on the standard curve working solution and the sample to be detected by adopting a liquid chromatography tandem mass spectrometry analyzer, and calculating the content of the plant sterol substances by adopting an internal standard method according to a fitted standard curve equation and the peak area of the sample to be detected. The detection method can effectively separate chromatographic peaks of 7-alkene cholanol and cholesterol, avoid the influence of high concentration cholesterol in a sample on quantification, expand the linear range of plant sterols to be detected and shorten the detection time.
Description
Technical Field
The invention relates to the technical field of plant sterol detection, in particular to a method for detecting the content of plant sterols in blood based on a liquid chromatography-tandem mass spectrometry technology.
Background
Sterols are important components of animal and plant cell membranes, including cholesterol and plant sterols, which are structurally similar. Cholesterol is known to be of great importance in the diagnosis and treatment of clinical diseases, while the strong association of plant sterols with cardiovascular disease is poorly understood.
Plant sterols, which may also be referred to as phytosterols, mainly include sitosterol, stigmasterol, campesterol, cholestanol, dehydrocholesterol, 7-enecholestanol. In 1974, it was first reported that a pair of twin sisters contain high concentrations of sitosterol and campesterol in blood, which cause early onset of atherosclerosis, joint xanthoma and the like, and this disease is caused by mutation of ABCG5 and/or ABCG8 genes, which cause resistance of flowback after absorption of plant sterols in vivo, and increase in the concentration of plant sterols in blood, while increase in the level of plant sterols can permeate cell membranes to lower membrane fluidity, permeability and cause lipid metabolism disorder; in addition, plant sterols are more readily oxidized than cholesterol, and their oxidation products severely destroy endothelial cells and penetrate the arterial wall, stimulating monocytes to differentiate into macrophages into tissues, and eventually evolve into foam cells to initiate atherosclerosis. Lipoproteins containing sitosterol can accelerate macrophage death and plaque necrosis, and are important causes of coronary plaque rupture and thrombosis. Because plant sterols are somewhat competitive in inhibiting cholesterol absorption, atherosclerosis due to general cholesterol elevation suggests lowering cholesterol synthesis and administering a certain amount of foods with high plant sterol content, which may be counterproductive to patients with elevated plant sterols.
Plant solid and cholesterol are similar in structure, and cyclopentane polyhydrophenanthrene is taken as a basic structure and contains hydroxyl. The existing conventional biochemical enzyme method for detecting cholesterol mainly comprises the steps of recognizing double chains and 3 hydroxyl groups on C5 and C6, however, partial structures exist in plant sterols, so that the plant sterols and cholesterol cannot be detected separately. Under normal conditions, the proportion of the plant sterol in the whole detection value is very low, and the effect is not great, but for patients with the plant sterol, the content of the plant sterol in blood can be tens times of that of the normal patients with the plant sterol, the conventional detection method can lead to misjudgment, and for vascular damage caused by excessive plant sterol, the misjudgment can be caused, and bad prognosis can be caused by mistreatment. In addition, the total cholesterol of the patients with the phytosteremia is normal or slightly increased, i.e. coronary heart disease, abnormal blood system and the like are easy to occur early, and huge burden is caused to the patients and families, so that the patients with the phytosteremia can be possibly prompted to have stronger atherosclerosis effect than cholesterol, and the concern on the phytosteremia is not limited to the phytosteremia, but the atherosclerosis group.
The intestinal absorption and secretion mechanisms of plant sterols and cholesterol are similar, and plant sterols can be used as surrogate markers for assessing intestinal cholesterol absorption and synthesis, such as dehydrocholesterol, 7-enecholestanol representing synthesis, sitosterol, stigmasterol, campesterol, cholestanol representing absorption. The cholesterol steady state disturbance can be related to hypercholesterolemia, atherosclerosis, obesity, metabolic syndrome and diabetes, and an accurate plant sterol component detection technology can help evaluate the absorption synthesis balance of cholesterol in the body, and better guide clinical lipid-lowering treatment.
With the deep understanding of plant sterols, partial research attempts are made to develop related detection methods, and currently, various methods are reported, including gas chromatography tandem mass spectrometry, gas chromatography tandem flame ionization detector method, liquid chromatography-ultraviolet detector method and liquid chromatography-fluorescence detector method, which are only used in scientific research departments, have complicated operation, require derivatization steps, have no registration certificate of clinical medical instruments and limit popularization and use.
In recent years, high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) technology is rapidly developed in clinical inspection departments, and research and development are also carried out on a method for detecting plant sterol based on the LC-MS/MS technology, but due to the fact that detection difficulty is high, related reports are less, and a plurality of methods still have certain limitations. In particular, the chemical structure and retention time of 7-alkene cholanol are very similar to those of cholesterol, the existing detection method cannot achieve better baseline separation, and the substance is a key marker representing cholesterol synthesis and has important value in accurate detection.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for detecting the content of plant sterols in blood, which can effectively separate chromatographic peaks of 7-alkene cholanol and cholesterol, avoid the influence of high concentration cholesterol in a sample on quantification, and optimize the detection time and the minimum sample size.
In order to achieve the above object, the present invention provides a method for detecting the content of plant sterols including 7-enecholanol in blood based on liquid chromatography tandem mass spectrometry, comprising the steps of:
(1) Preprocessing a blood sample to obtain a sample to be tested containing an internal standard substance;
(2) Preparing a standard curve working solution containing an internal standard substance;
(3) Performing on-machine detection on the standard curve working solution and the sample to be detected by adopting a liquid chromatography tandem mass spectrometry analyzer, fitting according to the detection result of the standard curve working solution to obtain a standard curve equation between the standard substance concentration and the ratio of the standard substance peak area to the internal standard substance peak area, and calculating according to the standard curve equation and the peak area of the object to be detected by an internal standard method to obtain the content of the plant sterol substance;
isocratic elution is carried out on the chromatographic column by using a mobile phase A and a mobile phase B, wherein the mobile phase A consists of acetonitrile, water and formic acid with the mass ratio of 82:18:0.1, and the mobile phase B is absolute ethyl alcohol;
the chromatographic time is noted as t, and the elution procedure is as follows:
when 0min is less than or equal to 14min, introducing a mobile phase A, wherein the flow rate is 0.7mL/min;
when 14min < t is less than or equal to 15.80min, introducing a flow velocity phase B, wherein the flow velocity is 0.35mL/min;
when 15.80min < t is less than or equal to 16.81min, introducing a mobile phase A, wherein the flow rate is 0.35mL/min;
when the flow rate is less than or equal to 17.5min, the mobile phase A is introduced, and the flow rate is 0.7. 0.7 mL/min.
7-alkene cholanol (shown as formula I) and cholesterol (shown as formula II) are isomers, have the same molecular weight and the same mass-to-charge ratio (369.3) during mass spectrometry, so that the separation of chromatographic peaks of the two substances during liquid chromatography tandem mass spectrometry is very difficult, and the accurate quantitative detection of 7-alkene cholanol is difficult.
In addition, most of the prior art separation techniques have the problem that the baseline is gradually raised with multiple sample injections, which results in a decrease in the degree of separation of 7-enecholanol and cholesterol, which has become a detection pain point for 7-enecholanol. As shown in FIG. 1, the chromatographic peaks of 7-enecholanol and cholesterol in the separation profile obtained by the conventional technique are difficult to completely separate.
I is a kind of
II (II)
According to the invention, the steps of powerful cleaning (14 min < t is less than or equal to 15.80 min) and pre-balancing (15.80 min < t is less than or equal to 17.5 min) are added in the chromatographic time interval, so that no residue exists in each sample in the chromatographic column, the baseline is kept clean and is not gradually increased after long-term continuous sample injection, the effective separation of chromatographic peaks of 7-alkene cholanol and cholesterol is realized, the peak outlet time of plant sterols is shortened as a whole, and the detection efficiency is accelerated.
According to a specific embodiment of the present invention, preferably, the chromatography column is a C18 chromatography column having a column temperature of 27 ℃.
According to a specific embodiment of the present invention, preferably, the mass spectrometry conditions comprise:
air curtain gas: 35psi; collision gas: 4 units;
GAS1：40psi；GAS2：60psi；
ion source: an atmospheric pressure chemical ionization source; ion source temperature: 450 ℃;
ionization mode: cation pattern.
The invention optimizes the mass spectrum conditions, can simultaneously realize high responsiveness and high specificity of each plant steroid substance, ensures the sensitivity and specificity of the detection method, and is not easy to be interfered by impurities.
According to a specific embodiment of the present invention, preferably, the plant sterols further include at least one of dehydrocholesterol, campesterol, stigmasterol, beta-sitosterol, cholestanol.
According to a specific embodiment of the present invention, preferably, cholesterol is also contained in the blood sample.
According to a specific embodiment of the invention, preferably, the concentration ratio of cholesterol to the total amount of plant sterols is at least 100:1.
the cholesterol content in normal human blood is about 3-6mmol/l, and the cholesterol content is only about 0.01mmol/l, which is hundreds times higher than that of the plant, and the quantification of low concentration plant sterols is extremely easily affected by high concentration cholesterol if the chromatographic peak separation of the plant sterols from cholesterol is poor. Wherein, the 7-alkene cholane alcohol chromatographic peak is closest to cholesterol, and the high concentration of cholesterol further increases the separation difficulty of the 7-alkene cholane alcohol chromatographic peak and the cholesterol chromatographic peak, and the accurate quantification of the 7-alkene cholane alcohol can be seriously influenced by incomplete separation of the two chromatographic peaks.
According to a specific embodiment of the present invention, preferably, the internal standard is an isotopic internal standard.
According to a specific embodiment of the present invention, the pretreatment of the blood sample preferably comprises the steps of:
centrifuging the blood sample, uniformly mixing the obtained serum, an internal standard substance and absolute ethyl alcohol to obtain a mixture, and then carrying out secondary extraction to obtain the sample to be detected;
the secondary extraction includes: adding water and n-hexane into the mixture, mixing the mixture evenly, taking supernatant, drying the supernatant, and redissolving the supernatant in a mixed solution of isopropanol and acetonitrile.
According to a specific embodiment of the present invention, the pretreatment of the blood sample preferably further comprises: saponification of the mixture prior to the second extraction;
the saponification treatment includes: 10mol/L sodium hydroxide solution is added into the mixture and saponification reaction is carried out under the assistance of ultrasound, the reaction temperature is 68 ℃, and the reaction time is 20-60min.
In the pretreatment of a blood sample, the saponification treatment can cause the bonded phytosterol to be cleaved into the free phytosterol. When the prior treatment does not comprise saponification, the method of the invention can be directly used for detecting the content of free plant sterols; when the pretreatment comprises saponification, the method of the present invention is used to detect the total amount of free and bound plant sterols.
The invention greatly simplifies the pretreatment scheme of the blood sample and can detect the free type and the combined type plant sterol substances at the same time. In the prior art, the pretreatment process still needs a Solid Phase Extraction (SPE) step, which increases cost and time consumption, is unfavorable for large-scale clinical development, and is more difficult to cause personnel difference due to a plurality of steps, thereby being unfavorable for standardization.
According to a specific embodiment of the present invention, the blood sample is preferably used in an amount of 100 μl. The invention can achieve higher sensitivity with smaller sample size based on the common model of clinical laboratory.
According to a specific embodiment of the present invention, the liquid chromatography tandem mass spectrometer is preferably model AB SCIEX 4500MD. The equipment adopted in the prior art is non-clinical conventional equipment, and a method is required to be transferred, verified and adjusted; the model used by the invention is SCIEX 4500MD, has medical instrument registration certificate, is the most popular model for clinical examination at present, and is convenient for large-scale popularization.
By adopting the detection method of the invention, the peak-to-peak sequence of each plant solid matter to be detected is as follows from fast to slow in turn: dehydrocholesterol, 7-cholestanol, stigmasterol, campesterol, cholestanol, sitosterol, and the latest peak time is about 13-14min, and the detection time is shortened.
The technical scheme provided by the invention has the following beneficial effects:
(1) According to the method, the impurity cleaning process and the pre-balancing process for the chromatographic column are added in the chromatographic time interval, so that no residue exists in each sample in the chromatographic column, the baseline is kept clean and is not gradually increased after long-term continuous sample injection, the effective separation of the chromatographic peak of 7-cholesteryl alkanol and cholesterol is realized, the peak outlet time of plant sterol substances is shortened integrally, and the detection efficiency is accelerated;
(2) The method has higher sensitivity and wider linear range, the lowest detection limit is low, and the linear range meets the concentration detection of clinical normal people and phytosteremia people;
(3) The invention simplifies the pretreatment scheme of blood samples and can detect free type and combined type plant sterol substances at the same time.
Drawings
FIG. 1 is a diagram showing the separation of 7-enecholanol and cholesterol in the prior art;
FIG. 2 is a chromatogram (peak time) of each plant steroid measured in example 1 of the present invention;
FIG. 3A is a chart showing the separation of 7-alkylen-cholestanol from an external standard of cholesterol in example 1 of the present invention;
FIG. 3B is a graph showing the separation of 7-alkylen-cholestanol from cholesterol according to example 1 of the present invention;
FIG. 4A is a total ion flow (TIC) chromatogram of the same batch of free plant sterol in example 2 injected into needle 1 of the present invention;
FIG. 4B is a total ion flow chromatogram of the 35 th needle of the same batch of free plant sterols in example 2;
fig. 5 is a total ion flow chromatogram of comparative example 1 without a cleaning step.
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention.
The model of the high performance liquid chromatography tandem mass spectrometry adopted by the embodiment of the invention is SCIEX 4500MD.
Example 1
The present embodiment provides a method for detecting the content of bound and free plant sterols in blood based on liquid chromatography tandem mass spectrometry, wherein the plant sterols in blood include 7-cholestanol (lathosterol), dehydrocholesterol (desmosterol), beta-sitosterol (sitosterol), stigmasterol, campesterol (campestterol) and cholestanol (cholestanol), 6 substances in total, and cholesterol with a concentration of at least 3 mmol/l. The detection method specifically comprises the following steps:
(1) Preparing an internal standard mixed solution:
for the 6 plant sterol substances to be tested, preparing an internal standard mixed solution by adopting corresponding isotope internal standard substances, wherein the solvent is isopropanol, and the concentration of each isotope internal standard substance is shown in table 1:
table 1 internal standard and concentration of each internal standard in internal standard mixture
(2) Pretreatment of blood samples:
centrifuging the blood sample at 3000rpm for 15min, taking 100 mu L of serum+10 mu L of internal standard mixed solution+200 mu L of absolute ethyl alcohol in an EP tube, and uniformly mixing by vortex; adding 20 mu L of 10mol/L sodium hydroxide solution into the mixture and carrying out saponification reaction for 20min at 68 ℃ under the assistance of ultrasound; adding 100 mu L of pure water and 1mL of n-hexane into the saponification reaction solution, swirling for 30s, centrifuging for 5min at 13000rpm, taking 800 mu L of supernatant, drying with nitrogen at room temperature, redissolving the product into 100 mu L of mixed solution of isopropanol and acetonitrile (the mass ratio of the isopropanol to the acetonitrile is 1:1), vibrating for 30s to obtain a sample to be detected, and performing on-machine detection with the sample injection amount of 3 mu L;
(3) Preparing standard curve working solution:
preparing Std 1-Std 7 linear concentration solutions by adopting the 6 standard substances of plant sterols to be detected, wherein the solvent is isopropanol, respectively taking 100 mu L of Std 1-Std 7 linear concentration solutions, adding 10 mu L of internal standard mixed solution, uniformly mixing, and filtering to obtain a standard curve working solution. The concentrations of the standards Std1 to Std7 are shown in Table 2.
TABLE 2 concentration of standards in Std1-Std 7 Linear concentration solutions
(4) And (5) detecting and analyzing on the machine:
performing on-machine detection on standard curve working solutions Std 1-Std 7 and the sample to be detected by adopting a liquid chromatograph tandem mass spectrometer, fitting to obtain a standard curve equation between standard substance concentration and the ratio of standard substance peak area to internal standard substance peak area, and calculating the content of plant sterol substances by adopting an internal standard method by combining the peak area of the sample to be detected;
wherein the chromatographic conditions include: the chromatographic column is a C18 chromatographic column, the column temperature is 27 ℃, the chromatographic column is subjected to isocratic elution by using a mobile phase A and a mobile phase B, the mobile phase A consists of acetonitrile, water and formic acid in a mass ratio of 82:18:0.1, and the mobile phase B is absolute ethyl alcohol;
the chromatographic time is noted as t, and the elution procedure is as follows:
when 0min < t is less than or equal to 14min, introducing a mobile phase A (volume ratio is 100%), wherein the flow rate is 0.7mL/min;
when 14min < t is less than or equal to 15.80min, introducing a flow velocity phase B (volume ratio is 100%), wherein the flow velocity is 0.35mL/min;
when 15.80min < t is less than or equal to 16.81min, introducing a mobile phase A (volume ratio is 100%), wherein the flow rate is 0.35mL/min;
when the flow rate is less than or equal to 17.5min, the mobile phase A (volume ratio is 100%) is introduced, and the flow rate is 0.7 mL/min.
The mass spectrometry conditions included:
air curtain gas: 35psi; collision gas: 4 units; GAS1:40psi; GAS2:60psi; ion source: an atmospheric pressure chemical ionization source; ion source temperature: 450 ℃; ionization mode: cation pattern.
Fig. 2 shows a chromatogram of a plant sterol, and the peak time (retention time) of 6 plant sterols measured by the detection method of this embodiment is as follows in order of fast and slow:
dehydrocholesterol: 4.98min, 7-enecholanol: 7.99min, stigmasterol: 10.64min, campesterol: 10.74min, cholestanol: 11.13min, sitosterol: 13.37min.
As can be seen from figure 2, the cholestanol has the latest peak, the latest peak time is within 14min, and the overall peak time is short. In addition, it can be seen that a higher interference peak appears within the chromatographic time of 8.8-9.0min, and the interference peak is the chromatographic peak of cholesterol in blood after detection.
Fig. 3A and 3B show an external and an internal standard separation spectrum of 7-enecholane and cholesterol, respectively, wherein the low peak is 7-enecholane and the peak is cholesterol, and it can be seen that the detection method of the present invention achieves effective separation of 7-enecholane and cholesterol chromatographic peaks, and the separation degree of 7-enecholane and cholesterol chromatographic peaks is calculated to be > 2 (usually, the separation degree reaches 1.5, which means complete separation).
The linear range of detection of each plant steroid substance achieved by the detection method of this embodiment is as follows:
dehydrocholesterol: 0.05-20mg/L; 7-enecholanol: 0.1-40mg/L; bean sterol: 0.1-40mg/L; campesterol: 0.04-16mg/L; cereal sterol: 0.4-160mg/L; cholestanol: 0.2-80mg/L.
The detection method has the advantages of low minimum detection limit, high sensitivity and wide linear range, and can cover the low concentration value of the plant sterols of normal people and the abnormally high value of patients suffering from the phytosteremia.
The minimum detection limit of the detection method of this example was evaluated, 3 batches were tested in total, intra-batch variation and inter-batch variation were observed, and the intra-batch variation coefficient and the inter-batch variation coefficient were required to be not more than ±20%, and the results shown in table 3 prove that the minimum detection limit of the present invention satisfies the requirements.
Table 3 results of evaluation of lowest limit of detection
Example 2
The present example provides a method for detecting the content of free phytosterols in blood, which is the same as example 1, except that the pretreatment process of the blood sample does not include saponification.
The pretreatment process of the blood sample specifically comprises the following steps:
centrifuging the blood sample at 3000rpm for 15min, taking 100 mu L of serum+10 mu L of internal standard mixed solution+200 mu L of absolute ethyl alcohol in an EP tube, and uniformly mixing by vortex; to the mixture, 100. Mu.L of pure water, 1mL of n-hexane and vortexing for 30s, centrifuging at 13000rpm for 5min, collecting 800. Mu.L of supernatant, blow-drying with nitrogen at room temperature, and redissolving the product in 100. Mu.L of isopropanol: and (3) performing on-machine detection on acetonitrile with shaking for 30s and 3 mu L of sample injection amount.
The other steps are the same as in example 1.
As shown in fig. 4A and 4B, after repeated sampling for 35 times by adopting the method of this embodiment, the baseline can still be stabilized at a certain level, and the baseline does not gradually rise with multiple sampling.
Comparative example 1
This comparative example provides a method for detecting the content of bound and free phytosterols in blood based on liquid chromatography tandem mass spectrometry techniques, which differs from example 1 only in that the column elution procedure does not involve washing with mobile phase B.
The elution procedure was as follows:
when 0min < t is less than or equal to 14min, introducing a mobile phase A (volume ratio is 100%), wherein the flow rate is 0.7mL/min;
when 14min < t is less than or equal to 16.81min, introducing a mobile phase A (volume ratio is 100%), wherein the flow rate is 0.35mL/min;
when the flow rate is less than or equal to 17.5min, the mobile phase A (volume ratio is 100%) is introduced, and the flow rate is 0.7 mL/min.
After 10 sample injections, the baseline is gradually raised as shown in fig. 5 by adopting the detection method of the comparative example, and the quantitative detection of the low-concentration phytostanols is interfered. If only free type plant sterols in blood are detected, the content of plant sterols in the sample is lower and the influence of high concentration cholesterol is larger.
Claims (2)
1. A method for detecting the content of plant sterols in blood based on liquid chromatography tandem mass spectrometry, wherein the plant sterols are 7-enecholestanol, dehydrocholesterol, campesterol, stigmasterol, beta-sitosterol and cholestanol, and the method comprises the following steps:
(1) Preprocessing a blood sample to obtain a sample to be tested containing an internal standard substance;
(2) Preparing a standard curve working solution containing an internal standard substance;
(3) Performing on-machine detection on the standard curve working solution and the sample to be detected by adopting a liquid chromatography tandem mass spectrometry analyzer, fitting according to the detection result of the standard curve working solution to obtain a standard curve equation between the standard substance concentration and the ratio of the standard substance peak area to the internal standard substance peak area, and calculating according to the standard curve equation and the peak area of the object to be detected by an internal standard method to obtain the content of the plant sterol substance;
isocratic elution is carried out on the chromatographic column by using a mobile phase A and a mobile phase B, wherein the mobile phase A consists of acetonitrile, water and formic acid with the mass ratio of 82:18:0.1, and the mobile phase B is absolute ethyl alcohol;
the chromatographic time is noted as t, and the elution procedure is as follows:
when 0min is less than or equal to 14min, introducing a mobile phase A, wherein the flow rate is 0.7mL/min;
when 14min < t is less than or equal to 15.80min, introducing a flow velocity phase B, wherein the flow velocity is 0.35mL/min;
when 15.80min < t is less than or equal to 16.81min, introducing a mobile phase A, wherein the flow rate is 0.35mL/min;
when the flow rate is less than or equal to 16.81min and less than or equal to 17.5min, the mobile phase A is introduced, and the flow rate is 0.7mL/min;
the chromatographic column is a C18 chromatographic column, and the column temperature is 27 ℃;
the mass spectrometry conditions included:
air curtain gas: 35psi; collision gas: 4 units;
GAS1：40psi；GAS2：60psi；
ion source: an atmospheric pressure chemical ionization source; ion source temperature: 450 ℃;
ionization mode: a cationic mode;
the blood sample also contains at least 3mmol/l cholesterol;
the model of the liquid chromatograph tandem mass spectrometer is AB SCIEX 4500MD;
the concentration ratio of cholesterol to total plant sterol is at least 100:1, a step of;
the internal standard is an isotope internal standard, the internal standard of 7-cholestanol is 7-cholestanol-d 7, the internal standard of dehydrocholesterol is dehydrocholesterol-d 6, the internal standard of campesterol is campesterol-d 3, the internal standard of stigmasterol is stigmasterol-d 7, the internal standard of beta-sitosterol is beta-sitosterol-d 7, and the internal standard of cholestanol is cholestanol-d 7;
the pretreatment of the blood sample consists of the following steps:
centrifuging the blood sample, uniformly mixing the obtained serum, the internal standard substance and absolute ethyl alcohol to obtain a mixture, then adding water and n-hexane, uniformly mixing, taking supernatant, drying, and redissolving in a mixed solution of isopropanol and acetonitrile to obtain the sample to be tested.
2. The method of claim 1, wherein the pretreatment of the blood sample consists of:
centrifuging the blood sample, and uniformly mixing the obtained serum, the internal standard substance and absolute ethyl alcohol to obtain a mixture;
adding 10mol/L sodium hydroxide solution into the mixture, and carrying out saponification reaction under the assistance of ultrasound, wherein the reaction temperature is 68 ℃, and the reaction time is 20-60min;
and adding water and n-hexane into the saponification reaction product, uniformly mixing, taking supernatant, drying, and redissolving in the mixed solution of isopropanol and acetonitrile to obtain the sample to be tested.
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| CN106053863A (en) * | 2016-06-02 | 2016-10-26 | 广东佛山国盛医学科技有限公司 | Method for detecting equilibrium state of cholesterol |
| CN206192957U (en) * | 2016-10-21 | 2017-05-24 | 杭州量康科技有限公司 | Test system and preceding processing apparatus of sample of synthetic absorption marker thing of cholesterol |
| WO2018174876A1 (en) * | 2017-03-22 | 2018-09-27 | Mprobe Inc. | Methods and compositions for providing a preeclampsia assessment with metabolites |
| CN111505132A (en) * | 2020-01-10 | 2020-08-07 | 中国医学科学院阜外医院 | Method for detecting novel cardiovascular disease risk marker by liquid chromatography-tandem mass spectrometry |
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| CN106053863A (en) * | 2016-06-02 | 2016-10-26 | 广东佛山国盛医学科技有限公司 | Method for detecting equilibrium state of cholesterol |
| CN206192957U (en) * | 2016-10-21 | 2017-05-24 | 杭州量康科技有限公司 | Test system and preceding processing apparatus of sample of synthetic absorption marker thing of cholesterol |
| WO2018174876A1 (en) * | 2017-03-22 | 2018-09-27 | Mprobe Inc. | Methods and compositions for providing a preeclampsia assessment with metabolites |
| CN111505132A (en) * | 2020-01-10 | 2020-08-07 | 中国医学科学院阜外医院 | Method for detecting novel cardiovascular disease risk marker by liquid chromatography-tandem mass spectrometry |
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