CN112485340A - Method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry - Google Patents
Method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry, which comprises the following steps: (1) and plasma sample pretreatment: adding the internal standard solution B into the plasma sample, oscillating and centrifuging, and taking supernatant to obtain a pretreated plasma sample; (2) qualitative and quantitative detection: separating 1, 5-sorbitan in the pretreated plasma sample by adopting an ultra-high performance liquid chromatography technology, establishing a calibration curve by using a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the 1, 5-sorbitan in the plasma sample to be detected. The method has the advantages of high detection sensitivity, strong specificity, accuracy and simple pretreatment process of the plasma sample, can finish the separation and detection of 1, 5-anhydro-sorbitol within 6min, and meets the requirements of matrix effect and precision.
Description
Technical Field
The invention relates to the technical field of biochemical analysis and detection, in particular to a method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry.
Technical Field
1, 5-sorbitan () in the human body is mainly derived from common foods, and its molecular structure is similar to that of glucose. The closed pyrano ring structure ensures that 1,5-AG is stably present in the blood and is not metabolized by human organs. Normally, 1,5-AG is reabsorbed by the kidney through the glucose reabsorption channel SGLT 4; the blood content is relatively constant because the part of the urine entering the urine is equivalent to the intake amount. In hyperglycemic patients (>180mg/dL), the absorption function of the kidney for glucose is hindered due to changes in osmotic pressure, and the high glucose content in the kidney competitively inhibits reabsorption of 1,5-AG in the renal tubules, resulting in the excretion of large amounts of 1,5-AG with urine. In this case, the 1,5-AG content in blood is decreased and the urine content is increased. Compared with the glycated hemoglobin (HbA1c), 1,5-AG can accurately reflect the change of the blood sugar of a patient within 1-2 weeks and even 24 hours before the blood sugar, namely, the slight fluctuation of the blood sugar can be accurately measured. The method can be used together with HbA1c to accurately and effectively monitor blood sugar. In addition, a study containing 2000 specimens from japan and an 11-year follow-up published in atherosclosis in 2011 showed that 1,5-AG could predict cardiovascular disease (CVD) risk in the male population. Also in 2015, researchers at the university of John Hodgkin, USA reported that patients with low blood levels of 1,5-AG had an increased incidence of coronary heart disease, stroke and heart failure, indicating that 1,5-AG is one of the important biomarkers for diabetes and the cardiovascular complications associated with diabetes.
In conclusion, the increase or decrease of 1,5-AG in human body is closely related to the risk of diabetes and cardiovascular diseases, and the content of 1,5-AG in blood plasma is very important for evaluating the health of human body.
Disclosure of Invention
The invention aims to overcome the defects and provides a method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry, which has the advantages of high detection sensitivity, strong specificity and accuracy, simple pretreatment process of a plasma sample, capability of completing separation and detection of 1, 5-sorbitan within 6min, and matrix effect and precision meeting the requirements.
In order to achieve the aim, the invention provides the following scheme, and the method for detecting the 1, 5-sorbitan in the plasma by the ultra-high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
(1) and plasma sample pretreatment: adding the internal standard solution B into the plasma sample, oscillating and centrifuging, and taking supernatant to obtain a pretreated plasma sample;
(2) qualitative and quantitative detection: separating 1, 5-sorbitan in the pretreated plasma sample by adopting an ultra-high performance liquid chromatography technology, establishing a calibration curve by using a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the 1, 5-sorbitan in the plasma sample to be detected, wherein the specific chromatographic conditions are as follows:
(a) ultra-high performance liquid chromatography conditions:
mobile phase A: ultrapure water containing 0.0028-0.005% formic acid and 0.16-0.25 mmol/L ammonium formate in volume fraction;
mobile phase B: acetonitrile;
flow rate of the column: 0.3-0.5 mL/min;
column temperature of the column: 35-50 ℃;
sample injection volume of chromatographic column: 0.5-1.5 muL;
adopting a gradient elution mode;
(b) mass spectrum conditions:
in electrospray ionization negativeUnder the ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spraying voltage is 2-3 kV; the temperature of the desolvation is 110-130 ℃; the temperature of atomizing gas is 380-420 ℃, the airflow speed of atomizing gas is 780-820L/h, and the airflow speed of taper holes is 130-170L/h; simultaneously monitoring the target 1, 5-anhydrosorbitol (m/z162.90 → 100.88) and the isotope internal standard 1, 5-anhydrosorbitol-13C6(m/z 168.93→104.88)；
The parameters of the multi-reaction monitoring method of the target object are as follows:
de-clustering voltage: 10V;
collision voltage: 13V;
retention Time (RT): 1.63 min.
Preferably, the plasma sample is human or animal plasma.
Preferably, the ultra-high performance liquid chromatography conditions are:
mobile phase A: ultrapure water containing 0.004% by volume of formic acid and 0.2mmol/L ammonium formate;
mobile phase B: acetonitrile;
flow rate of the column: 0.4 mL/min;
column temperature of the column: 40 ℃;
sample injection volume of chromatographic column: 1 mu L of the solution;
gradient elution mode is adopted.
Preferably, the type of the chromatographic column is ACQUITY UPLC BEH Amide, and the specification of the chromatographic column is as follows: the length is 100mm, the inner diameter is 2.1mm, and the particle diameter is 1.7 μm.
Preferably, the gradient elution procedure for the chromatography column is: 0-3 min, wherein the volume fraction of the mobile phase A is 15%, and the volume fraction of the mobile phase B is 85%; 3-3.5 min, wherein the volume fraction of the mobile phase A is 70%, and the volume fraction of the mobile phase B is 30%; 3.5-6 min, wherein the volume fraction of the mobile phase A is 70%, and the volume fraction of the mobile phase B is 30%; 6min, the volume fraction of the mobile phase A is 15%, and the volume fraction of the mobile phase B is 85%; wherein the flow rate is kept constant during the gradient elution procedure.
Preferably, the mass spectrometry conditions are: the spraying voltage is 2.5 kV; the desolvation temperature is 120 ℃; the temperature of the atomizing gas is 400 ℃, the airflow speed of the atomizing gas is 800L/h, and the airflow speed of the taper hole is 150L/h.
Preferably, the pretreated plasma sample is prepared according to the following method: adding 20 mu L of plasma into a centrifuge tube, adding 1200 mu L of internal standard solution B into the centrifuge tube, then oscillating for 10min after swirling for several seconds, centrifuging for 10min at 15000r/min, and taking 70 mu L of supernatant to obtain the pretreated plasma sample.
Preferably, the internal standard solution B is prepared according to the following method: taking 25.00mg of isotope internal standard 1, 5-anhydrosorbitol-13C6, adding 1.471mL of methanol with volume fraction of 50% for complete dissolution to obtain an isotope internal standard solution with the concentration of 100mmol/L, then diluting the isotope internal standard solution with the methanol with the volume fraction of 50% to 1mmol/L to obtain an internal standard solution A, and adding 10 mu L of the internal standard solution A into 20mL of acetonitrile solution to obtain an internal standard solution B.
Compared with the prior art, the invention has the beneficial effects that: the method has the advantages of high sensitivity, strong specificity, accuracy and simpler pretreatment process of the plasma sample, can finish the separation and detection of the 1, 5-sorbitan within 6min, meets the requirements on matrix effect and precision, can be used for the qualitative and quantitative analysis of the 1, 5-sorbitan clinically, and provides a reliable detection method for the clinical health assessment of diabetes risk and cardiovascular disease risk.
Drawings
FIG. 1 is a total ion flow chromatogram of 1, 5-anhydrosorbitol and its internal isotope standard in the standard of the examples.
FIG. 2 is a total ion flow chromatogram of 1, 5-anhydrosorbitol and its internal isotope standard in plasma samples from examples.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Examples
The plasma samples in this example were from plasma samples collected from the 12 month clinic in 2017 of the heart disease hospital, wuhan asia.
The instruments used in this example include a Xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf 0.5-10 muL, 10-100 muL, 100-1000 muL).
The reagent consumable in this embodiment includes: chromatographically pure acetonitrile (Honeywell, usa); MS grade acetonitrile (Fisher, usa); MS grade formic acid, ammonium formate (Merck, usa); bovine Serum Albumin (BSA) (Merck, usa); column Waters BEH Amide,1.7 μm,2.1X 100mm (Waters corporation).
The standard in this example: 1,5-AG from Merck, 1,5-AG-13C6 was purchased from Omicron Biochemicals and had a purity of 98% or more.
Quality control materials in this example: the blank plasma matrix solution containing 1,5-AG molecules has three concentrations of QC (L), QC (M) and QC (H) in low, medium and high, and is shown in Table 1.
TABLE 11, 5-sorbitan quality control concentrations (in. mu.M)
The embodiment provides a method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry, which comprises the following steps:
(1) and plasma sample pretreatment: adding 20 mu L of plasma into a 1.5mL centrifuge tube, adding 1200 mu L of internal standard solution B, vortexing for several seconds, oscillating for 10min, centrifuging for 10min at 15000r/min, and taking 70 mu L of supernatant to obtain a pretreated plasma sample for later use;
the internal standard solution B is prepared by the following method: taking 25.00mg of isotope internal standard 1, 5-anhydrosorbitol-13C6, adding 1.471mL of 50% methanol by volume fraction to dissolve completely, obtaining the isotopologue with the concentration of 100mmol/LAnd (3) diluting the isotope internal standard solution to 1mmol/L by using methanol with the volume fraction of 50% to obtain an internal standard solution A, and adding 10 mu L of the internal standard solution A into 20mL of acetonitrile solution to obtain an internal standard solution B.
(2) Qualitative and quantitative detection: separating 1, 5-dehydrated sorbitol in the pretreated plasma sample by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the conditions of the ultra-high performance liquid chromatography are as follows: the type of the chromatographic column is ACQUITY UPLC BEH Amide, and the specification of the chromatographic column is as follows: the length is 100mm, the inner diameter is 2.1mm, the particle diameter is 1.7 mu m, the flow rate of a chromatographic column is 0.4mL/min, the column temperature of the chromatographic column is 40 ℃, and the sample injection volume of the chromatographic column is 1 mu L; the mobile phase A in the chromatogram mobile phase is ultrapure water containing 0.004 percent of formic acid and 0.2mmol/L of ammonium formate in volume fraction, and the mobile phase B in the chromatogram mobile phase is acetonitrile; the gradient elution procedure for the column was: 0-3 min, wherein the volume fraction of the mobile phase A is 15%, and the volume fraction of the mobile phase B is 85%; 3-3.5 min, wherein the volume fraction of the mobile phase A is 70%, and the volume fraction of the mobile phase B is 30%; 3.5-6 min, wherein the volume fraction of the mobile phase A is 70%, and the volume fraction of the mobile phase B is 30%; 6min, the volume fraction of the mobile phase A is 15%, and the volume fraction of the mobile phase B is 85%; wherein the flow rate is kept constant during the gradient elution procedure.
Then, establishing a calibration curve by using a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the 1, 5-sorbitan in the plasma sample to be detected;
the mass spectrum conditions are as follows: under an electrospray ionization negative ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spraying voltage is 2-3 kV; the temperature of the desolvation is 110-130 ℃; the temperature of atomizing gas is 380-420 ℃, the airflow speed of atomizing gas is 780-820L/h, and the airflow speed of taper holes is 130-170L/h; simultaneously monitoring the target 1, 5-anhydrosorbitol (m/z162.90 → 100.88) and the isotope internal standard 1, 5-anhydrosorbitol-13C6(m/z 168.93 → 104.88). Wherein the declustering voltage and the collision voltage parameters of the target are shown in table 2.
TABLE 21, 5-sorbitan Mass Spectrometry parameters
Preparing a standard substance: accurately weighing 7.101mg of 1, 5-anhydrosorbitol, adding 4.326mL of 50% methanol, and preparing into a standard mother solution with the concentration of 10 mmol/L;
and (3) standard product treatment: adding 10 mu L of the mother solution of the standard substance into 190 mu L of the blank plasma matrix solution to be used as a first high-value concentration point; and (3) diluting 100 mu L of the first high-value concentration point with an equal volume of blank plasma matrix solution to obtain a second high-value concentration point, and then gradually diluting the second high-value concentration point with 1 volume of the blank plasma matrix solution to obtain other six calibration concentration points. And (3) taking 20 mu L of samples at each concentration point, subpackaging, adding 1200 mu L of internal standard solution B, vortexing for several seconds, oscillating for 10min, centrifuging for 10min at 15000r/min, taking 70 mu L of supernatant, transferring the supernatant into a chromatographic bottle, and carrying out LC-MS/MS analysis.
Preparing a quality control product: taking 1 μ L, 5 μ L and 40 μ L of the mother liquor of the standard substance, and respectively diluting to 1000 μ L with blank plasma matrix solution to obtain quality control substance solutions QC (L), QC (M) and QC (H).
And (3) processing quality control products: respectively taking 20 mu L of prepared quality control substance solutions QC (L), QC (M) and QC (H) in 1.5mL centrifuge tubes, adding 1200 mu L of internal standard solution B into the centrifuge tubes, vortexing for several seconds, oscillating for 10min, centrifuging for 10min at 15000r/min, taking 70 mu L of supernatant, and finally transferring the supernatant into a chromatographic bottle for LC-MS/MS analysis.
The blank plasma matrix in this example is 50mg/mL bovine serum albumin aqueous solution.
Adding films on the upper and lower peripheries of a reagent box in an ultra-high performance liquid chromatography-mass spectrometer, performing shockproof insulation, placing mobile phases A and B at the upper left, and placing 4 x 1mL ampoules at the lower left, wherein the standard solution and the quality control product are respectively placed; 10mL of the diluted solution and 100m of the extract were placed on the right side, respectively. The components of the assay kit are shown in Table 3.
TABLE 31 preparation of 5-sorbitan assay kit Components
And (3) testing results:
(1) total ion current chromatogram: the peak shapes of the standard substance of the 1, 5-anhydrosorbitol and the plasma sample are symmetrical, and no interference of a miscellaneous peak exists, which indicates that good detection can be obtained under the condition, the figure 1 is a total ion flow chromatogram of the 1, 5-anhydrosorbitol and an isotope internal standard thereof, and the figure 2 is a total ion flow chromatogram of the 1, 5-anhydrosorbitol and the isotope internal standard thereof in the plasma.
(2) Matrix Effect (ME) and extraction recovery (EE) studies: because the blank plasma matrix in the experiment is 50mg/mL bovine serum albumin aqueous solution (5% BSA), and the quantitative standard curve is prepared by 5% BSA, the matrix effect and the extraction recovery rate of both 5% BSA and plasma are examined, the matrix effect is obtained by comparing the matrix addition standard after extraction with a standard pure solution with the same concentration, the higher the concentration is, the smaller the influence of the matrix effect is, and therefore, only the extraction recovery rate is examined at high concentration (the higher the concentration is, the smaller the extraction recovery rate is possible). And (3) testing the pure solution of the standard sample with the same concentration by adopting ID-UPLC-MS/MS to obtain a peak area-A, testing the solution of the extracted matrix added with the standard by adopting ID-UPLC-MS/MS to obtain a peak area-B, and testing the matrix effect ME (%) ═ B/A multiplied by 100.
The test results are shown in tables 4 and 5. The test result shows that: 1,5-anhydro sorbitol has almost no matrix effect (both low concentration and high concentration are between 85% and 115%) in 5% BSA, and the extraction recovery rate is higher (more than or equal to 80%); similarly, the matrix effect of 1, 5-sorbitan in plasma is between 85% and 115% after internal standard correction; and the extraction recovery rate of the high-concentration sample is more than 80%.
TABLE 41, 5-sorbitan in 5% BSA matrix effect and extraction recovery results
TABLE 51, 5-sorbitan matrix Effect in plasma and extraction recovery results
(3) Standard curve: and (3) establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the plasma by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fitting equation of the 1, 5-anhydro-sorbitol in each concentration range has good linearity, the correlation coefficient is more than 0.998, and the quantitative requirements are met, which is shown in table 6.
TABLE 61, 5-sorbitan Linear regression equation and Linear correlation coefficient
(4) And (3) precision test: taking normal human plasma samples, repeatedly processing 8 batches within one day, and quantitatively determining the concentration of 1, 5-anhydro-sorbitol by an isotope internal standard method, wherein the batch precision is 6.25 percent, and the results are shown in Table 7; the batch was processed in 3 portions during three days, and the calculated inter-batch precision was 6.32%, and the results are shown in Table 8.
TABLE 7 results of precision test in batches (unit. mu.M)
TABLE 8 results of precision test between lots (Unit. mu.M)
The invention discloses an ultra performance liquid chromatography tandem mass spectrometry method for measuring 1, 5-anhydrosorbitol (1,5-anhydroglucitol,1, 5-anhydrosorbitol) in human plasma. Meanwhile, the method detects the peak time and the ion pair of the target object, has high sensitivity, can greatly eliminate matrix interference by adopting an isotope internal standard method for quantification, is not influenced by the conditions of pretreatment process, sample loading volume and flow and the like, and can achieve accurate quantification.
The matrix effect and the extraction recovery rate of the 1, 5-sorbitan in 5% BSA and plasma are inspected, the 1, 5-sorbitan in 5% BSA has almost no matrix effect (the low concentration and the high concentration are both between 85% and 115%), and the extraction recovery rate is higher (more than or equal to 80%); similarly, the matrix effect of 1, 5-sorbitan in plasma is between 85% and 115% after internal standard correction; and the extraction recovery rate of the high-concentration sample is more than 80%. It is demonstrated that 5% BSA can be used as a blank plasma substitute matrix for accurate quantification of 1, 5-sorbitan in plasma.
The reproducibility of the method indicates that the precision of the 1, 5-anhydrosorbitol in day is 6.25 percent, the precision in day is 6.32 percent, and the method has good reproducibility. Experiments to obtain more stable and sensitive target signals, the types and concentrations of different mobile phases and electrolytes were investigated, and baseline separation of compound and matrix interferences was achieved as much as possible. The pre-treatment process of the established plasma sample is very simple, protein precipitation is completed in one step, and the dosage of the plasma is only 20 mu L.
The method has the advantages of high sensitivity, strong specificity, accuracy and simpler pretreatment process, can complete the separation and detection of the compound within 6min, meets the requirements on matrix effect, extraction recovery rate and precision, can be used for the clinical quantitative analysis of the 1, 5-sorbitan plasma, and provides a reliable detection method for the clinical health assessment of diabetes risk and cardiovascular disease risk.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (8)
1. A method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry is characterized by comprising the following steps:
(1) and plasma sample pretreatment: adding the internal standard solution B into the plasma sample, oscillating and centrifuging, and taking supernatant to obtain a pretreated plasma sample; the internal standard liquid B is formed by isotope internal standard 1, 5-anhydrosorbitol-13C6, methanol and acetonitrile solution;
(2) qualitative and quantitative detection: separating 1, 5-sorbitan in the pretreated plasma sample by adopting an ultra-high performance liquid chromatography technology, establishing a calibration curve by using a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the 1, 5-sorbitan in the plasma sample to be detected, wherein the specific chromatographic conditions are as follows:
(a) ultra-high performance liquid chromatography conditions:
mobile phase A: ultrapure water containing 0.0028-0.005% formic acid and 0.16-0.25 mmol/L ammonium formate in volume fraction;
mobile phase B: acetonitrile;
flow rate of the column: 0.3-0.5 mL/min;
column temperature of the column: 35-50 ℃;
sample injection volume of chromatographic column: 0.5-1.5 muL;
adopting a gradient elution mode;
(b) mass spectrum conditions:
under an electrospray ionization negative ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spraying voltage is 2-3 kV; the temperature of the desolvation is 110-130 ℃; the temperature of atomizing gas is 380-420 ℃, the airflow speed of atomizing gas is 780-820L/h, and the airflow speed of taper holes is 130-170L/h; at the same time monitor the eyesStandard 1, 5-anhydrosorbitol (m/z162.90 → 100.88) and isotopic internal standard 1, 5-anhydrosorbitol-13C6(m/z 168.93→104.88)；
The parameters of the multi-reaction monitoring method of the target object are as follows:
de-clustering voltage: 10V;
collision voltage: 13V;
retention Time (RT): 1.63 min.
2. The method for detecting 1, 5-sorbitan in plasma by ultra performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein the plasma sample is human or animal plasma.
3. The method for detecting 1, 5-sorbitan in plasma by ultra performance liquid chromatography tandem mass spectrometry according to claim 1, wherein the conditions of ultra performance liquid chromatography are as follows:
mobile phase A: ultrapure water containing 0.004% by volume of formic acid and 0.2mmol/L ammonium formate;
mobile phase B: acetonitrile;
flow rate of the column: 0.4 mL/min;
column temperature of the column: 40 ℃;
sample injection volume of chromatographic column: 1 mu L of the solution;
gradient elution mode is adopted.
4. The method for detecting 1, 5-anhydro-sorbitol in plasma by ultra performance liquid chromatography tandem mass spectrometry as claimed in claim 3, wherein the type of said chromatographic column is ACQUITY UPLC BEH Amide, and the specifications of said chromatographic column are: the length is 100mm, the inner diameter is 2.1mm, and the particle diameter is 1.7 μm.
5. The method for detecting 1, 5-sorbitan in plasma by ultra performance liquid chromatography-tandem mass spectrometry according to claim 3, wherein the gradient elution procedure of the chromatographic column is as follows: 0-3 min, wherein the volume fraction of the mobile phase A is 15%, and the volume fraction of the mobile phase B is 85%; 3-3.5 min, wherein the volume fraction of the mobile phase A is 70%, and the volume fraction of the mobile phase B is 30%; 3.5-6 min, wherein the volume fraction of the mobile phase A is 70%, and the volume fraction of the mobile phase B is 30%; 6min, the volume fraction of the mobile phase A is 15%, and the volume fraction of the mobile phase B is 85%; wherein the flow rate is kept constant during the gradient elution procedure.
6. The method for detecting 1, 5-sorbitan in plasma by ultra performance liquid chromatography tandem mass spectrometry according to claim 3, wherein the mass spectrometry conditions are as follows: the spraying voltage is 2.5 kV; the desolvation temperature is 120 ℃; the temperature of the atomizing gas is 400 ℃, the airflow speed of the atomizing gas is 800L/h, and the airflow speed of the taper hole is 150L/h.
7. The method for detecting 1, 5-sorbitan in plasma by ultra performance liquid chromatography-tandem mass spectrometry according to any one of claims 1 to 6, wherein the pretreated plasma sample is prepared by the following method: adding 20 mu L of plasma into a centrifuge tube, adding 1200 mu L of internal standard solution B into the centrifuge tube, then oscillating for 10min after swirling for several seconds, centrifuging for 10min at 15000r/min, and taking 70 mu L of supernatant to obtain the pretreated plasma sample.
8. The method for detecting 1, 5-sorbitan in plasma by ultra performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein the internal standard solution B is prepared by the following method: taking 25.00mg of isotope internal standard 1, 5-anhydrosorbitol-13C6, adding 1.471mL of methanol with volume fraction of 50% for complete dissolution to obtain an isotope internal standard solution with the concentration of 100mmol/L, then diluting the isotope internal standard solution with the methanol with the volume fraction of 50% to 1mmol/L to obtain an internal standard solution A, and adding 10 mu L of the internal standard solution A into 20mL of acetonitrile solution to obtain an internal standard solution B.
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