CN113866285A - Biomarker for diabetes diagnosis and application thereof - Google Patents
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- CN113866285A CN113866285A CN202010623870.XA CN202010623870A CN113866285A CN 113866285 A CN113866285 A CN 113866285A CN 202010623870 A CN202010623870 A CN 202010623870A CN 113866285 A CN113866285 A CN 113866285A
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- carnitine
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- choline chloride
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a biomarker for diabetes diagnosis and application thereof. Specifically, a biomarker set is provided, the set comprising one or more of acetyl L-carnitine, trimethylamine oxide, choline chloride. Wherein, the acetyl L-carnitine is firstly applied to the diabetes diagnosis. Proved by verification, when the acetyl L-carnitine is used for diagnosing and distinguishing diabetic patients and healthy people, the AUC reaches over 0.8, and when two indexes of the acetyl L-carnitine and choline chloride are jointly applied, the AUC is closer to 1 than that of a single index, and the diagnosis effect is better; when three indexes of acetyl L-carnitine, trimethylamine oxide and choline chloride are jointly applied, the AUC is closer to 1 than that of the joint application of a single index and two indexes, and the diagnosis and distinguishing effect is best. By detecting the biomarker, the diabetes can be diagnosed accurately, with high specificity and high sensitivity.
Description
Technical Field
The present invention belongs to the field of biochemistry and relates to disease diagnosis markers, in particular to biomarkers (groups) for diabetes diagnosis.
Background
With the change of life style and the improvement of living standard of people and the increase of old people, the incidence rate of global diabetes is continuously increased, and the latest global diabetes map released by the international diabetes union shows that: in 2019, the countries with the largest number of adult diabetics are ranked, China is ranked first continuously, and is expected to continue 2030 years, so that the incidence rate is high, and the low diagnosis rate and the low control rate are the current conditions of diabetes in China. Diabetes mellitus is a group of metabolic syndrome with chronic hyperglycemia as a common clinical characteristic, and causes blood sugar increase due to glucose utilization disorder of the body caused by various factors such as insulin secretion defect, insulin resistance and glycogen metabolism abnormality, and is accompanied by metabolic disorders such as fat, protein, water, electrolyte and the like. Serious patients or patients under stress conditions are easy to be complicated with acute metabolic disorders, such as diabetic ketoacidosis or diabetic nonketotic hyperosmolar syndrome, which directly endangers the life of the patients. Long-term hyperglycemia can cause pathological changes of all tissues and organs of the whole body, and can cause acute and chronic complications, such as pancreatic failure, water loss, electrolyte disorder, nutrient deficiency, immunity reduction, renal function impairment, neuropathy, fundus oculi pathological changes and the like. Therefore, early detection and diagnosis of diabetes and good control of blood glucose are currently the most effective methods for the prevention and treatment of diabetes.
2019 world health organization WHO publishes diabetes diagnosis standards: diabetes is diagnosed based on the patient's symptoms (polydipsia, polyuria, polyphagia, weight loss from the body), fasting and postprandial random blood glucose concentrations, the OGTT test, and hemoglobin A1c (HbA1 c). The diagnosis of diabetes is somewhat unreliable depending on symptoms, blood sugar and the like, because more than 50 percent of diabetics have no obvious symptoms and are passive when the diabetes is found depending on the symptoms, and the conventional diagnosis method has complicated operation steps and limited use, so the diagnosis rate of the diabetics in China is very low.
Metabolomics, which reflects the direct relationship of genes, proteins and metabolic activities themselves by measuring the change in the concentration of cellular, tissue and body metabolites, is another new branch of omics research that has emerged following genomics, transcriptomics, proteomics. Because metabonomics change is the final reflection of the body on the effects of genes, diseases, environment, medicines and the like, and endogenous metabolites of the metabonomics are the key or end-point reactions of a series of life events of the body, the metabonomics can help people to better understand various complex interactions and the essence thereof in the body, can be used for diagnosing human diseases and realizing individualized accurate treatment.
The human intestinal flora is a complex community and the intestinal microbiota plays an important role in immunity and defense, digestion and metabolism, inflammation and cell proliferation. The main nutrients choline, betaine and carnitine from red meat, eggs, dairy products and saltwater fish are involved in biological activities such as energy metabolism in human bodies. After ingestion, fermentation of these nutrients by gut microbes results in the release of Trimethylamine (TMA), which is converted to trimethylamine oxide (TMAO) by the host liver enzymes, flavin-containing monooxygenase 3(FMO 3). There is increasing evidence that: TMAO which is one of small molecules of intestinal microorganism metabolism is not only involved in cholesterol metabolism, promotion of platelet high aggregation, increase of thrombus formation and promotion of vascular inflammatory reaction to cause arterial plaque formation, but also involved in insulin resistance, and is closely related to pathogenesis of diabetes. However, no metabolic markers relevant to diabetes diagnosis exist at present.
Therefore, there is a great need in the art to provide biomarkers for accurate, highly sensitive, highly specific diagnosis of diabetes.
Disclosure of Invention
The first object of the present invention is to provide a biomarker (population) for accurate, highly sensitive, highly specific diagnosis of diabetes.
The second object of the present invention is to provide a detection kit based on the biomarker group, which is used for diagnosing diabetes, improving the convenience of diagnosis and promoting the standardization of a diagnosis method.
It is a second object of the present invention to provide a method capable of simultaneously analyzing and detecting a plurality of said biomarkers.
The present invention provides in a first aspect the use of a biomarker, or an isotopic internal standard thereof, or a combination thereof, for the preparation of a diagnostic reagent or kit for: (A) determining whether an object is diabetic; and/or (B) risk prediction for diabetes;
wherein the biomarker comprises a substance selected from the group consisting of:
(a) acetyl L-carnitine;
(b) acetyl L-carnitine and choline chloride;
(c) acetyl L-carnitine and trimethylamine oxide; and
(d) acetyl L-carnitine, trimethylamine oxide and choline chloride.
In another preferred embodiment, the isotope in the isotope internal standard is selected from the group consisting of:2H、3H、11C、13C、14C、15N、17O、18O、36cl, or a combination thereof, preferably, D: (A), (B), (C) and C)2H)、C13Or15N, or a combination thereof.
In another preferred embodiment, when the isotope is D, the D replaces H (an inactive hydrogen) on the C atom.
In another preferred embodiment, the difference between the molecular weight M1 of the isotopic internal standard and the molecular weight M0 of the marker (in natural abundance) (M1-M0) is ≧ 2, more preferably ≧ 3, ≧ 4, such as 4, 5, 6, 7, 8, 9, 10, 11, 12.
In another preferred embodiment, the isotopic internal standard is selected from the group consisting of: acetyl L-carnitine-D3, choline chloride-D9, trimethylamine oxide-D9, or a combination thereof.
In another preferred embodiment, the biomarker is a plasma biomarker.
In another preferred embodiment, the biomarker further comprises one or more substances selected from the group consisting of: betaine, crotonobetaine, gamma-butylbetaine, and L-carnitine.
In a second aspect of the invention, there is provided a set of biomarkers, wherein the biomarkers comprise a substance selected from the group consisting of:
(a) acetyl L-carnitine;
(b) acetyl L-carnitine and choline chloride;
(c) acetyl L-carnitine and trimethylamine oxide; and
(d) acetyl L-carnitine, trimethylamine oxide and choline chloride.
In another preferred embodiment, the biomarker further comprises one or more substances selected from the group consisting of: betaine, crotonobetaine, gamma-butylbetaine, and L-carnitine.
In a third aspect of the present invention, there is provided a kit comprising:
(1) a biomarker standard comprising a substance selected from the group consisting of:
(a) acetyl L-carnitine;
(b) acetyl L-carnitine and choline chloride;
(c) acetyl L-carnitine and trimethylamine oxide; and
(d) acetyl L-carnitine, trimethylamine oxide, and choline chloride;
and (2) an isotopic internal standard of the biomarker of (1).
In another preferred embodiment, the kit further comprises one or more reagents selected from the group consisting of:
(3) blank plasma, preferably carbon-adsorbed blank human plasma;
(4) an extraction solvent, preferably comprising acetonitrile (v/v, percentages being based on total volume of extraction solvent) containing 1.0-3.0% acetic acid, preferably 1.5-2.5%;
(5) a mobile phase additive a comprising formic acid; and/or
(6) A mobile phase additive B comprising ammonia.
In another preferred embodiment, the kit further comprises a component selected from the group consisting of:
a 96 well reaction plate, label or instructions.
In another preferred embodiment, the label or instructions states:
when the detection result of the biomarker in the blood of the subject satisfies one or more of the following conditions, the subject is suggested to have diabetes:
(1) choline chloride is more than or equal to 18 mu M;
(2) acetyl L-carnitine is more than or equal to 8.29 uM; and/or
(3) Trimethylamine oxide is more than or equal to 4.2 uM.
In another preferred embodiment, the standard is a solution of the biomarker, preferably a mixed standard solution of the biomarker.
In another preferred embodiment, the isotope internal standard is a solution (preferably methanol solution) of the isotope internal standard, preferably a mixed internal standard solution.
In another preferred embodiment, the isotopic internal standard is selected from the group consisting of: acetyl L-carnitine-D3, choline chloride-D9, trimethylamine oxide-D9, betaine-D11, gamma-butylbetaine-D9, L-carnitine-D9, or a combination thereof.
In a fourth aspect of the present invention, there is provided a method for detecting a biomarker in a sample by liquid-mass spectrometry, comprising the steps of:
(a) providing the kit according to claim 2 and a test sample, preferably the test sample is a biological sample, more preferably the test sample is selected from the group consisting of: blood, serum, plasma, or a combination thereof, preferably, peripheral whole blood;
(b) adding the solution of the isotope internal standard into a standard substance solution and the sample to be detected respectively, and extracting with an extraction solvent to obtain a standard sample and a sample to be detected respectively; and
(c) and respectively loading the standard sample and the sample to be detected to an HPLC-MS (high performance liquid chromatography-mass spectrometry) and analyzing, wherein the MS adopts a positive ion MRM (multiple Reaction Monitor) mode.
In another preferred example, step (b) further comprises processing a quality control sample obtained by mixing a standard solution with a known concentration with blank plasma in the same manner, wherein the quality control sample is used for evaluating the stability and/or accuracy of the method in the detection process.
In another preferred embodiment, the biomarker is a diabetes-associated biomarker.
In another preferred example, the method further comprises:
(d) the concentration of the biomarker in the sample was calculated by the internal standard curve method based on the ion abundance (daughter or parent) measured by HPLC.
In another preferred embodiment, the LC-MS is HPLC-MS or UPLC-MS LC-MS, such as Shimadzu LC-MS 8050.
In another preferred example, the mass spectrum is triple quadrupole mass spectrometry or ion trap mass spectrometry.
In another preferred embodiment, the ion source of the mass spectrometer is an electrospray ionization source (ESI) or an atmospheric pressure chemical ionization source (APCI).
In another preferred embodiment, the elution conditions of the HPLC comprise one or more characteristics selected from the group consisting of:
(i) the chromatographic column is a reverse phase chromatographic column (such as C18 chromatographic column);
(ii) mobile phase: phase A: water (v/v, based on the total volume of phase a) containing 0.05 to 0.4% formic acid (preferably 0.05 to 0.2%, more preferably 0.08 to 0.12%) and 0.01 to 0.1% ammonia (preferably 0.03 to 0.1%, more preferably 0.05 to 0.7%); phase B: acetonitrile (v/v, based on the total volume of phase B) containing 0.05-0.4% formic acid (preferably 0.07-0.25%, more preferably 0.09-0.15%); and
(ii i) gradient elution.
In another preferred example, the gradient program includes:
starting from the equilibrium state, phase A changes from 1-20% to 55-65% in 2-10min (preferably, 2-5min), the percentage being based on the total flow rate of the mobile phase (v/v), preferably phase A changes from 2-18% to 58-63%, more preferably phase A changes from 4-16% to 59-62%.
In another preferred example, the gradient program comprises, in percentages, in total flow of mobile phase a and mobile phase B (v/v):
time (min) | Mobile phase A% | Mobile phase B (%) |
0.0 | 1-8 | 92-99 |
1.5 | 10-20 | 80-90 |
2.5 | 55-65 | 35-45 |
3.5 | 1-8 | 92-99 |
5.0 | 1-8 | 92-99。 |
In another preferred embodiment, the chromatographic elution conditions further comprise one or more characteristics selected from the group consisting of:
(a) the column temperature of the chromatographic column is 25-50 ℃, preferably 35-45 ℃;
(b) the temperature of the sample injection chamber is 2-20 ℃, preferably 5-15 ℃;
(c) the flow rate is 0.1-1.5mL/min, preferably 0.2-1.0mL/min, more preferably 0.3-0.8mL/min, most preferably 0.4-0.6 mL/min.
In another preferred example, in the mobile phase a, formic acid: ammonia water: the volume ratio of water is (0.05-0.2%): (0.03-0.1%): 100 percent; preferably, (0.08-0.12%): (0.05-0.07%): 100 percent.
In another preferred embodiment, the chromatographic column is an acquired UPLC BEH HILIC.
In another preferred embodiment, the column has a size of (1.8-2.5) × (40-200) mm, 1.5-2.3. mu.M). Typically, the column is of size (2.1X 100mm, 1.7. mu.M).
Typically, the reverse phase chromatography column is an ACQUITY UPLC BEH HILIC (2.1X 100mm, 1.7. mu.M). Preferably, the chromatographic column is also provided with a protective pre-column, such as a protective pre-column: ACQUITY UPLC BEH HILIC VAnGuard pre-column (1.7. mu.M, 2.1X 5 mm). The protective pre-column can intercept part of impurities in the sample, thereby protecting the chromatographic column.
In still another preferred example, the method includes:
(i) ionizing the HPLC eluate by an electrospray ion source (ESI) to produce precursor ions of the biological metabolite and internal standard; and
(ii) the precursor ions are collided within the mass spectrum, respectively producing fragment ions of the precursor ions.
In another preferred embodiment, the detection conditions of the mass spectrum include one or more of the following characteristics:
the flow rate of the atomized gas is 2.5-3.5L/min, preferably 2.8-3.2L/min, more preferably 2.9-3L/min;
heating gas flow rate is 8-12L/min, preferably 9-11L/min, more preferably 9.5-10L/min;
the flow rate of the drying gas is 8-12L/min, preferably 9-11L/min, more preferably 9.5-10L/min;
the interface temperature is 250-350 ℃; preferably, 270-320 ℃, more preferably, 280-300 ℃;
the DL temperature is 250-350 ℃; preferably, 270-320 ℃, more preferably, 280-300 ℃; and/or
The temperature of the heating block is 350-450 ℃; preferably 370-420 deg.C, more preferably 380-400 deg.C.
In another preferred embodiment, the collision energy of the acetyl L-carnitine is-20 to-25 eV, preferably-21 to-22 eV.
In another preferred embodiment, the collision energy of choline chloride is-25 to-35 eV, preferably-28 to-32 eV.
In another preferred embodiment, the collision energy of the trimethylamine oxide is-20 to-25 eV, preferably-21 to-22 eV.
In another preferred example, the monitoring mass-to-charge ratio of the parent ion/daughter ion of the acetyl L-carnitine is 204.00> 85.10.
In another preferred example, the monitored mass-to-charge ratio of parent ion/daughter ion of choline chloride is 104.15> 58.1.
In another preferred embodiment, the mass-to-charge ratio of parent ion/daughter ion of trimethylamine oxide is 76.15> 58.15.
In a fifth aspect of the present invention, there is provided a method for diagnosing diabetes, comprising the steps of:
(1) providing a test sample, preferably a blood sample (e.g., blood, serum, plasma, or a combination thereof, preferably peripheral whole blood);
(2) detecting the concentration of the biomarker in the sample as C1;
(3) comparing the concentration of the biomarker C1 with a control reference value C0,
wherein the biomarker comprises a substance selected from the group consisting of:
(a) acetyl L-carnitine;
(b) acetyl L-carnitine and choline chloride;
(c) acetyl L-carnitine and trimethylamine oxide; and
(d) acetyl L-carnitine, trimethylamine oxide, and choline chloride;
if the biomarker concentration of the test subject is C1 > C0, the test subject is suggested to have diabetes. And vice versa.
In another preferred embodiment, choline chloride has a C0 of 18. mu.M.
In another preferred embodiment, the C0 of acetyl-l-carnitine is 8.29 uM.
In another preferred embodiment, trimethylamine oxide has a C0 of 4.2 uM.
In another preferred embodiment, the biomarker further comprises one or more substances selected from the group consisting of: betaine, crotonobetaine, gamma-butylbetaine, and L-carnitine.
In a sixth aspect of the present invention, a method for establishing a prediction of diabetes risk, the method comprises: identifying a differentially expressed substance in the blood sample between the patient and a healthy control,
wherein the differentially expressed material comprises biomarkers selected from the group consisting of:
(a) acetyl L-carnitine;
(b) acetyl L-carnitine and choline chloride;
(c) acetyl L-carnitine and trimethylamine oxide; and
(d) acetyl L-carnitine, trimethylamine oxide and choline chloride.
In another preferred embodiment, the differentially expressed material further comprises one or more materials selected from the group consisting of: betaine, crotonobetaine, gamma-butylbetaine, and l-carnitine.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a standard regression curve of the standard acetyl L-carnitine solution obtained in example 1, wherein the compound name is acetyl L-carnitine, m/z:204.00>85.10, standard curve formula:
f (x) 0.412947 x +0.00424439, correlation coefficient (R) 0.9961528, and fitting degree (R2) 0.9923204.
FIG. 2 is a standard regression curve of the betaine standard solution obtained in example 1, wherein the compound name: betaine, m/z 118.10>85.10, standard curve formula f (x) 0.229124 x +0.0305053, correlation coefficient (R) 0.9992594, and fitting degree (R2) 0.9985193.
FIG. 3 is a standard regression curve of the choline chloride standard solution obtained in example 1, wherein the name of the compound is choline chloride, m/z is 104.15>85.10, the formula of the standard curve is f (x) 0.254350 x +0.0209732, the correlation coefficient (R) is 0.9952100, and the degree of fitting (R2) is 0.9904429.
FIG. 4 is a standard regression curve of the crotonobetaine standard solution obtained in example 1, wherein the compound name is crotonobetaine, m/z:144.00>85.10, standard curve formula:
f (x) 0.272751 x-0.00613629, correlation coefficient (R) 0.9961319, and fitting degree (R2) 0.9922787.
FIG. 5 is a standard regression curve of the gamma-butylbetaine standard solution obtained in example 1, wherein the compound name is gamma-butylbetaine, m/z:146.00>87.05, and the standard curve formula:
f (x) 0.198595 x-0.0105567, correlation coefficient (R) 0.9957488, and fitting degree (R2) 0.9915156.
Fig. 6 is a standard regression curve of the standard l-carnitine solution obtained in example 1, wherein the compound name: l-carnitine, m/z:162.10>60.10, standard curve formula f (x) -0.373827 x +0.806322, correlation coefficient (R) -0.9997124, and fitting degree (R ^2) -0.9994249.
Fig. 7 is a standard regression curve of the trimethylamine oxide standard solution obtained in example 1, wherein the compound name: trimethylamine oxide, m/z 76.15>58.15, standard curve formula:
f (x) 0.135069 x +0.00872933, correlation coefficient (R) 0.9981007, and fitting degree (R2) 0.9962049.
FIG. 8 is a schematic diagram of a Receiver Operating Curve (ROC) curve model for joint inspection of two indexes by the ROC method.
Detailed Description
The present inventors have conducted extensive and intensive studies, through extensive screening and testing. Biomarkers (populations) are provided that can be used as accurate diagnoses of diabetes. The invention discovers for the first time that acetyl L-carnitine can be used as a biomarker for diagnosing diabetes, and has high diagnosis sensitivity and specificity. When the acetyl L-carnitine, the choline chloride and/or the trimethylamine oxide are used as biomarker groups for diagnosing the diabetes, whether the patient suffers from the diabetes or not can be diagnosed with high specificity, high sensitivity and high accuracy, and the misdiagnosis rate is reduced. The present invention has been completed based on this finding.
Term(s) for
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
As used herein, the term "liquid-mass spectrometry" is short for high performance liquid-mass spectrometry, i.e., "liquid-mass spectrometry" is used interchangeably with "high performance liquid-mass spectrometry".
As used herein, the term "acetyl-l-carnitine" is abbreviated ALC, i.e. "acetyl-l-carnitine" is used interchangeably with "ALC".
As used herein, the term "trimethylamine oxide" is abbreviated TMAO, i.e., "trimethylamine oxide" and "TMAO" are used interchangeably.
As used herein, the term "ultra performance liquid chromatography" is abbreviated UPLC, i.e., "ultra performance liquid chromatography" is used interchangeably with "UPLC".
In the present invention, it is understood that the mobile phase flow rate is the sum of the flow rates of the mobile phase a and the mobile phase B.
As used herein, the terms "acetyl-l-carnitine-D3" and "ALC-D3" are used interchangeably to refer to the deuteroide formed after the substitution of H in acetyl-l-carnitine with deuterium isotope, which can be used as an internal standard for mass spectrometric detection of acetyl-l-carnitine in the present invention.
As used herein, the term "trimethylamine oxide-D9 methanol" is used interchangeably with "TMAO-D9" and in the present invention is used as an internal standard for the mass spectrometric detection of trimethylamine oxide and trimethyllysine.
As used herein, the terms "fragment ion" and "daughter ion" are used interchangeably and refer to those resulting from a single molecular fragmentation reaction of a molecular ion or larger fragment ion.
As used herein, the terms "mobile phase A", "phase A" or "aqueous mobile phase" are used interchangeably to refer to the mobile phase A obtained after adding 0.05-0.4% (v/v) formic acid and 0.01-0.1% (v/v) ammonia water, respectively, to ultrapure water under liquid phase conditions.
As used herein, the terms "mobile phase B", "B phase" or "organic mobile phase" are used interchangeably to refer to the mobile phase B being acetonitrile comprising 0.05-0.4% (v/v) formic acid in liquid phase conditions.
As used herein, the term "HPLC" or "high performance liquid chromatography" refers to liquid chromatography in which the degree of separation is increased by passing a mobile phase under pressure through a stationary phase, typically a tightly packed column.
As used herein, "mass spectrometry" (MS) refers to analytical techniques for identifying compounds by their mass, the MS technique generally comprising (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass to charge ratio (m/z), the compound being ionized and detected by any suitable means. A "mass spectrometer" generally comprises an ionizer (ion source) and an ion detector.
According to the present invention, the term "biomarker panel" refers to one biomarker, or a combination of two or more biomarkers.
According to the invention, the content of the biomarker is indicated by a mass spectrometry signal area normalization value.
As used herein, the term "biomarker," also referred to as a "biomarker," refers to a measurable indicator of a biological state of an individual. Such biomarkers can be any substance in an individual as long as they are related to a particular biological state (e.g., disease) of the subject, e.g., nucleic acid markers (e.g., DNA), protein markers, cytokine markers, chemokine markers, carbohydrate markers, antigen markers, antibody markers, species markers (species/genus markers) and functional markers (KO/OG markers), and the like. Biomarkers are measured and evaluated, often to examine normal biological processes, pathogenic processes, or therapeutic intervention pharmacological responses, and are useful in many scientific fields.
As used herein, the terms "isotopic internal standard" or "isotopic derivative" are used interchangeably to refer to an isotopic derivative formed by the replacement of one or more atoms in a compound by an isotopic atom thereof. The isotope internal standard has basically the same physicochemical properties with the compound, but has different molecular weight, so the isotope internal standard is very suitable to be used as an internal standard substance in mass spectrum detection. Common isotopes include, but are not limited to:2H、3H、11C、13C、14C、15N、17O、18O、36cl, or a combination thereof, preferably, D: (A), (B), (C) and C)2H)、C13Or15N, or a combination thereof. Typically, the difference in molecular weight of the isotopic internal standard compared to the original compound (M1-M0) is ≥ 2, more preferably ≥ 3, ≥ 4, such as 4, 5, 6, 7, 8, 9, 10, 11, 12.
The term "about" as used herein in reference to a quantitative measurement means that the indicated value is plus or minus 10%.
ROC-AUC
The ROC-AUC is a method for evaluating model accuracy, and is a coordinate graph formed by a Receiver operating characteristic curve (Receiver operating characteristic curve), a False positive probability (False positive rate) as a horizontal axis and a True positive probability (True positive rate) as a vertical axis, and is a comprehensive index reflecting continuous variables of sensitivity and specificity. AUC is the Area under the ROC curve (Area under the curve). The ROC-AUC value is between 1.0 and 0.5, the closer to 1, the better the diagnosis effect is, the lower the accuracy is at 0.5-0.7, the certain accuracy is at 0.7-0.9, and the higher the accuracy is at AUC above 0.9. When AUC is 0.5, the diagnostic method is completely ineffective and is not valuable. AUC <0.5 does not correspond to the real case and occurs rarely in practice.
Biomarkers and uses thereof
In the invention, Acetyl L-Carnitine (Acetyl L-Carnitine) is found to be used as a marker for diagnosing diabetes for the first time. In the ROC curve, the single index AUC > 0.85, suggesting a high correlation between acetyl l-carnitine and diabetes.
Preferably, on the basis of acetyl-L-carnitine, in combination with choline chloride and/or triethylamine oxide (i.e. trimethylamine N-oxide), a biomarker panel for the diagnosis of diabetes is formed. Experiments prove that when the acetylcholine and the choline chloride or the triethylamine oxide are simultaneously used for evaluating the diabetes, the AUC is more than 0.95, which shows that the diagnosis has high accuracy.
More preferably, when three indexes of acetyl L-carnitine, choline chloride and triethylamine oxide are simultaneously used for evaluating diabetes, surprisingly, AUC is more than 0.995, and the diabetes can be accurately diagnosed.
In the present invention, the biomarkers can also be used in combination with other common biomarkers for the diagnosis of diabetes. Such as (but not limited to): betaine, crotonobetaine, gamma-butylbetaine, L-carnitine, etc.
Generally, the determination of whether a subject has diabetes can be made by measuring the concentration of the biomarker in a blood-related sample. Preferably, the sample is selected from the group consisting of: blood, serum, plasma, or a combination thereof, preferably, peripheral whole blood.
In the present invention, the biomarkers are metabolic markers, especially intestinal microbial metabolic markers.
The biomarkers of the invention may also be used for risk prediction of diabetes. Such as when the concentration of the biomarker in the blood of a subject differs from a reference value (healthy control) and a reference value (patient control), to make a risk prediction.
Reagent kit
In the present invention, the kit of the invention comprises a collection according to the second aspect of the invention.
In another preferred embodiment, each biomarker in the collection according to the first aspect of the invention is used as a standard.
In another preferred embodiment, the kit further comprises an isotopic internal standard for the biomarker.
In another preferred embodiment, the kit further comprises an instruction which describes a reference data set describing the levels (e.g. amounts) of the individual biomarkers of the panel according to the first aspect of the invention, derived from diabetic patients and/or healthy controls.
Detection method
The invention also provides a detection method for detecting various biomarkers represented by acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butyl betaine, L-carnitine and trimethylamine oxide in a sample.
Preferably, the method of the invention is a LC-MS assay comprising the steps of:
(i) ionizing said one or more biomarkers and an internal standard by an electrospray ion source (ESI) to produce at least one precursor ion of said one or more biomarkers and said internal standard, respectively;
(ii) generating one or more fragment ions of the precursor ions of the one or more biomarkers and the internal standard, respectively; and
(iii) (iii) comparing the amount of the one or more biomarkers and the one or more ions of the internal standard produced in step (i) or (ii) or both to determine the amount of the one or more biomarkers in the biological sample; wherein, formic acid and ammonia water are in a mobile phase in the chromatographic detection process.
The method is particularly suitable for detecting acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butyl betaine, L-carnitine and trimethylamine oxide.
In a preferred embodiment, the detection method of the present invention comprises the steps of:
(1) the standard substance, the internal standard substance and the extraction solvent are added into a quality control product and a blood sample to be detected, after full reaction, supernatant is taken, and the content of the markers in the blood sample to be detected is determined and analyzed through high performance liquid chromatography-mass spectrometry.
In a preferred embodiment, the chromatographic conditions of the high performance liquid are as follows:
(i) the column is a reverse phase column (such as C18 column).
(ii) Mobile phase: phase A: water (v/v, based on the total volume of phase a) containing 0.05 to 0.4% formic acid (preferably 0.05 to 0.2%, more preferably 0.08 to 0.12%) and 0.01 to 0.1% ammonia (preferably 0.03 to 0.1%, more preferably 0.05 to 0.7%). And adding formic acid and ammonia water into the phase A for adjusting the peak shape of the chromatographic peak. The formic acid and the part of the ammonia water may also be added in the form of ammonium formate or the like.
Phase B: acetonitrile (v/v, based on the total volume of phase B) containing 0.05-0.4% formic acid (preferably 0.07-0.25%, more preferably 0.09-0.15%).
(iii) Gradient elution.
Preferably, the procedure of gradient elution comprises:
in another preferred embodiment, the gradient elution procedure comprises:
in another preferred embodiment, the gradient elution procedure comprises:
in another preferred embodiment, the gradient elution procedure comprises:
in another preferred embodiment, the high performance liquid further has one or more chromatographic conditions selected from the group consisting of:
temperature of the column: 25-50 ℃, preferably 35-45 ℃;
sample chamber temperature: 2-20 ℃, preferably 5-15 ℃;
mobile phase (mobile phase a + mobile phase B) flow rate: 0.1-1.5mL/min, preferably 0.2-1.0mL/min, more preferably 0.3-0.8mL/min, most preferably 0.4-0.6 mL/min.
In another preferred embodiment, the reverse phase chromatographic column is an acquired UPLC BEH HILIC.
In another preferred embodiment, the reverse phase chromatography column has a specification of (1.8-2.5) × (40-200) mm, 1.5-2.3. mu.M). Typically, the reverse phase chromatography column is of size (2.1X 100mm, 1.7. mu.M).
Typically, the reverse phase chromatography column is an ACQUITY UPLC BEH HILIC (2.1X 100mm, 1.7. mu.M). Preferably, the chromatographic column is also provided with a protective pre-column, such as a protective pre-column: ACQUITY UPLC BEH HILIC VAnGuard pre-column (1.7. mu.M, 2.1X 5 mm). The protective pre-column can intercept part of impurities in the sample, thereby protecting the chromatographic column.
Typically, in the mass spectrometry phase, the sample undergoes the following phases:
(i) ionizing the HPLC eluate by an electrospray ion source (ESI) to produce precursor ions of the biological metabolite and internal standard; and
(ii) the precursor ions are collided within the mass spectrum, respectively producing fragment ions of the precursor ions.
The methods of the invention are non-therapeutic and non-diagnostic in vitro.
The main advantages of the invention include:
(a) the invention discovers that acetyl L-carnitine has high correlation (AUC > 0.85) with diabetes for the first time and can be used as a marker for diagnosing the diabetes.
(b) Furthermore, the invention finds that the accuracy, sensitivity and specificity of the diabetes diagnosis can be obviously improved by the joint detection of the acetyl L-carnitine and the choline chloride and/or the TMAO, the ACU is close to 1, the diagnosis distinction is good, and the accurate diagnosis of the diabetes can be realized.
(b) The invention also provides a liquid chromatography-mass spectrometry method for detecting various diabetes-related biomarkers in a complex biological sample (such as blood), the method can realize qualitative and quantitative detection of various biomarkers through single detection, and the detection method has the advantages of simple operation, short analysis time (joint detection only needs 5 minutes), accurate detection result and high result reproduction rate (CV is less than 10 percent), and is very suitable for automatic sample injection detection of large-scale samples.
(c) The kit provided by the invention can be used for diagnosing diabetes by a liquid chromatography-mass spectrometry, improves the diagnosis convenience and promotes the standardization of a diagnosis method.
The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1:
1. origin of specimen
After patient consent, 29 plasma samples of clinically diagnosed diabetic patients (fasting blood glucose > 7.0mmol/L, two hours postprandial blood glucose > 11.1mmol/L) and 29 healthy persons were collected and matched for age and gender with diabetic patients.
2. Main apparatus and equipment:
shimadzu LC-MS 8050; labsolutions instruments software.
3. Reagents and materials:
acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butyl betaine, L-carnitine and trimethylamine oxide standard substances; acetyl L-carnitine-D3, gamma-butyl betaine-D9, betaine-D11, choline chloride-D9, L-carnitine-D9, trimethylamine oxide-D9, methanol, acetonitrile, ethanol, formic acid, ammonia water and carbon adsorbed human plasma are all commercially available reagents.
4. Liquid chromatography and mass spectrometry conditions:
chromatographic conditions are as follows:
Mobile phase A: an aqueous solution of 0.1% (v/v) formic acid (A) + 0.025% (v/v) aqueous ammonia (B); mobile phase B: 0.1% (v/v) formic acid (A) in acetonitrile; based on the total volume of the mobile phase B; flow rate 0.6 mL/min: column temperature 45 ℃: sample chamber temperature: 8 ℃; the sample volume is 1 mu L; needle washing liquid: 50% methanol water.
Mobile phase gradient method:
mass spectrum conditions:
detecting in positive ion MRM mode by using an electrospray ionization source (ESI);
compound (I) | Monitoring ions | Q1 Pre deviation | Energy of collision | Q3 Pre deviation |
Acetyl L-carnitine | 204.00>85.10 | -14V | -22.0eV | -22V |
Betaine | 118.10>58.10 | -12V | -25.0eV | -21V |
Choline chloride | 104.15>58.10 | -11V | -31.0eV | -10V |
Crotonobetaine | 144.00>58.10 | -10V | -25.0eV | -10V |
Gamma-butylbetaine | 146.00>87.05 | -10V | -17.0eV | -16V |
L-carnitine | 162.10>60.10 | -11V | -17.0eV | -10V |
Oxetamine | 76.15>58.15 | -12V | -22.0eV | -10V |
Acetyl L-carnitine-D3 | 207.15>85.05 | -10V | -22.0eV | -14V |
betaine-D11 | 129.10>66.15 | -14V | -30.0eV | -11V |
Gamma-butylbetaine-D9 | 155.20>87.00 | -15V | -20.0eV | -15V |
Choline chloride-D9 | 113.05>66.30 | -22V | -32.0eV | -25V |
L-carnitine-D9 | 171.20>69.20 | -12V | -19.0eV | -12V |
TMAO-D9 | 85.15>66.20 | -10V | -24.0eV | -12V |
The flow rate of the atomizing gas is 3L/min, and the flow rate of the heating gas is 10L/min; the flow rate of the drying gas is 10L/min; interface temperature: 300 ℃; DL temperature: 300 ℃; temperature of the heating block: at 400 ℃.
5. The experimental process comprises the following steps:
5-1 solution preparation
5-1-1 preparation of mixed standard curve solution:
accurately weighing appropriate amounts of acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butyl betaine, L-carnitine and trimethylamine oxide standard substances, and dissolving with 50% methanol aqueous solution to respectively prepare standard substance stock solutions with the concentration of 5 mmol/L. Then, the standard substance stock solution with the concentration of 5mmol/L is sequentially diluted into the following standard curve working solution by 50 percent methanol aqueous solution.
Mixing standard solutions, wherein the concentrations of the standard curves of the acetyl L-carnitine, the crotonobetaine and the gamma-butyl betaine are 0.05, 0.25, 1, 2.5, 5, 10 and 25 mu mol/L; the concentrations of the standard curves of the four substances of the betaine, the choline chloride, the L-carnitine and the trimethylamine oxide are 0.1, 0.5, 2, 5, 10, 20 and 50 mu mol/L; the preparation matrix of the mixed standard solution is 50% methanol aqueous solution.
5-1-2 preparation of mixed internal standard solution:
accurately weighing proper amounts of acetyl L-carnitine-D3, gamma-butyl betaine-D9, betaine-D11, choline chloride-D9, L-carnitine-D9 and trimethylamine oxide-D9, dissolving with methanol to obtain a 1mmol/L mixed internal standard storage solution, and diluting with methanol to obtain a 10 mu mol/L mixed internal standard working solution.
5-1-3 extraction solvent
The components of the extraction solvent are acetonitrile and acetic acid, and the volume ratio of the acetic acid is 2.0%.
5-2, preparation of a quality control product I and a quality control product II:
and (3) taking a proper amount of Seralab carbon to adsorb blank human plasma, adding a small amount of solution with the highest concentration point of the mixed standard substance, and respectively preparing a quality control I and a quality control II.
5-3, pretreatment and sample injection analysis of a plasma sample:
respectively adding 25 mu L of mixed standard substance working solution and 25 mu L of to-be-detected plasma sample into different wells of a 96-well plate, sequentially adding 20 mu L of mixed internal standard into each well, adding 400 mu L of acetic acid acetonitrile solution (the concentration of acetic acid is 2%), performing vortex for 1min, centrifuging at 4000rpm for 5min, taking 120 mu L of supernatant, respectively injecting 1 mu L of supernatant into a liquid chromatograph-mass spectrometer, and determining and analyzing the content (unit is mu M) of each metabolite of the intestinal flora metabolites in the to-be-detected plasma sample. Wherein the standard regression curves of the above compounds are shown in FIGS. 1-7.
As shown in FIGS. 1 to 7, the standard regression curves of the above compounds are very linear, R2Greater than 0.99, and meets the performance requirement.
5-4 results
As shown in table 1 below, the sensitivity and specificity data automatically derived from the ROC curve method, which specifically fixed all indicators had a specificity of 96.6% (i.e., only one positive in healthy samples), and the AUC areas and thresholds obtained from the ROC curve are shown in table 1. In the ROC curve evaluation method, when the area value AUC under the ROC curve is greater than 0.5, the closer to 1, the better the diagnostic effect. AUC has lower accuracy at 0.5-0.7, certain accuracy at 0.7-0.9, and higher accuracy at 0.9 or above. Greater than the threshold is defined as + and less than the threshold is defined as-.
AUC and threshold value of each index in Table 1
Example 2: constructing ROC curve, and comparing the above intestinal flora metabolites singly or in combination to diagnose and differentiate diabetic patients from healthy people
The ability of the test subjects to diagnose diabetes was determined by the expression levels of acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butylbetaine, L-carnitine, and trimethylamine oxide in 29 diabetic samples and 29 non-diabetic normal plasma samples, as verified by the Receiver Operating Curve (ROC) method. Sensitivity and specificity data automatically derived from the ROC curve method, so assuming that the specificity of all indices is 96.6% (i.e. only one of healthy samples is positive), the sensitivity, AUC and threshold values obtained from the ROC curve are shown in table 2.
TABLE 2 ability of Single differential metabolite diagnosis to differentiate diabetic patients from healthy people
Single differential metabolite | Specificity of | Sensitivity of the probe | AUC | Threshold value |
Choline chloride | 96.6% | 82.8% | 0.967 | 18uM |
Acetyl L-carnitine | 96.6% | 75.9% | 0.893 | 8.29uM |
Oxetamine | 96.6% | 65.5% | 0.861 | 4.2uM |
L-carnitine | 96.6% | 17% | 0.413 | 102uM |
Crotonobetaine | 96.6% | 10% | 0.22 | 8uM |
Gamma-butylbetaine | 96.6% | 6.7% | 0.536 | 1.5uM |
Betaine | 96.6% | 6.7% | 0.429 | 116.5uM |
As can be seen from Table 2, the ability of acetyl L-carnitine, choline chloride and TMAO to differentiate diabetic patients from healthy people is strong, and AUC is above 0.85. The invention discovers for the first time that acetyl L-carnitine can be applied to diagnosis and differentiation of diabetic patients and healthy people. Among the three difference indexes, the choline chloride diagnosis has the strongest capacity of distinguishing the diabetic patients from the healthy people, the AUC is 0.967, the sensitivity is 82.8 percent, and the specificity is 96.6 percent. Second, the ability of acetyl L-carnitine diagnosis to distinguish diabetic patients from healthy persons was 0.893, with a sensitivity of 75.9% and a specificity of 96.6%. TMAO diagnosis has the worst ability to distinguish diabetic patients from healthy people, with AUC of 0.861, sensitivity of 65.5% and specificity of 96.6%.
Wherein, the index joint inspection ROC curve model graph refers to fig. 8.
Example 3
In order to further verify whether the two indexes of acetyl L-carnitine, choline chloride and trimethylamine oxide, which are firstly applied to diagnosis and differentiation of diabetic patients and healthy people, have a combined detection effect.
TABLE 3 ability of two differential metabolite combination diagnostics to differentiate diabetic patients from healthy people
As shown in Table 3, when acetyl L-carnitine and choline chloride are used in combination, the ability of diagnosing and distinguishing diabetic patients from healthy people can be obviously improved, the AUC is 0.993, and the diagnosis and distinguishing effect is good; when the specificity is 96.6%, the sensitivity is respectively improved from 82.8% and 75.9% in the single detection to 96.6% in the joint detection. When the acetyl L-carnitine and the TMAO are applied in a combined manner, the diagnosis and the distinguishing capability of the diabetic patients and healthy people are inferior to the combined effect of the acetyl L-carnitine and the choline chloride, and the AUC is 0.96; when the specificity is 96.6%, the sensitivity is respectively improved from 75.9% and 65.5% in the single detection to 82.8% in the joint detection.
TABLE 4 ability of three differential metabolite combination diagnostics to differentiate diabetic patients from healthy people
Three differential metabolite combinations | Specificity of | Sensitivity of the probe | AUC |
Acetyl L-carnitine, choline chloride and TMAO | 93.1% | 100% | 0.996 |
As shown in table 4, when the three indicators of acetyl-l-carnitine, choline chloride and TMAO are used in combination, AUC is closer to 1 than any two indicators in combination, AUC is 0.996, and the diagnosis and differentiation effect is better; surprisingly, the sensitivity is improved from 82.8%, 75.9% and 65.5% in the single test to 100% in the joint test, respectively, and the specificity is still as high as 93.1%.
Example 4
Preparation of detection kit
A detection kit is prepared based on the biomarkers provided by the invention, and the kit comprises the following components: quality control product, isotope internal standard extracting solution, extracting solvent, mobile phase additive A and mobile phase additive B. Preferably, the test kit further comprises a control, a 96-well reaction plate, a 96-well filter plate, instructions, and the like. The kit can be used for detecting the content of metabolites of three intestinal flora, namely acetyl L-carnitine, choline chloride and TMAO.
Specifically, in the kit, a reference substance and/or a quality control substance contains acetyl L-carnitine, choline chloride and TMAO, an isotope internal standard extracting solution contains acetyl L-carnitine-D3, choline chloride-D9 and TMAO-D9, the concentration of the isotope internal standard extracting solution is 5 mu M, an extracting solvent comprises a component (i) acetonitrile and a component (ii) acetic acid, the volume ratio (v/v) is 2.0%, a mobile phase additive A is formic acid, and a mobile phase additive B is ammonia water. The kit is stored at 2-8 ℃.
Of course, when designing a detection kit, it is not necessary to completely contain the above-mentioned three biomarker standards, and only a few of them may be used in combination. The standard products can be packaged separately or made into mixture package. The kit is designed based on the biomarker group provided by the invention, and can be used for diagnosing and distinguishing diabetic patients from healthy people.
In conclusion, the invention discovers for the first time that acetyl L-carnitine can be used as a biomarker for diagnosing diabetes, and has high diagnosis sensitivity. When two or three of acetyl L-carnitine, choline chloride and/or trimethylamine oxide are used as biomarker groups for diagnosing diabetes, whether a patient has the diabetes can be diagnosed with high specificity, high sensitivity and high accuracy, the diabetes can be diagnosed simply, accurately and quickly, the misdiagnosis rate is reduced, and the method has remarkable progress.
The invention also provides a high performance liquid chromatography-mass spectrometry combined analysis method for detecting the biomarkers in the sample, which can complete the separation and detection of the diabetes-related intestinal microbial metabolism markers (especially the biomarkers of the invention) in complex samples in a single experiment, and the method has high accuracy, high sensitivity, high stability and good reproducibility, and is very suitable for being used as a standard analysis method for detecting the intestinal microbial metabolism markers in biological samples.
The detection of the biomarker provides possibility for developing a method for preventing diabetes by pertinently regulating intestinal microorganism metabolic markers, thereby meeting the requirement of clinical early diagnosis of diabetes and being expected to realize accurate treatment of diabetes.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. Use of a biomarker, or an isotopic internal standard thereof, or a combination thereof, for the preparation of a diagnostic reagent or kit for: (A) determining whether an object is diabetic; and/or (B) risk prediction for diabetes;
wherein the biomarker comprises a substance selected from the group consisting of:
(a) acetyl L-carnitine;
(b) acetyl L-carnitine and choline chloride;
(c) acetyl L-carnitine and trimethylamine oxide; and
(d) acetyl L-carnitine, trimethylamine oxide and choline chloride.
2. The use of claim 1, wherein the isotope in the isotope internal standard is selected from the group consisting of:2H、3H、11C、13C、14C、15N、17O、18O、36cl, or a combination thereof, preferably, D: (A), (B), (C) and C)2H)、C13Or15N, or a combination thereof.
3. The use of claim 1, wherein the biomarker further comprises one or more substances selected from the group consisting of: betaine, crotonobetaine, gamma-butylbetaine, and L-carnitine.
4. A set of biomarkers, wherein the biomarkers comprise a substance selected from the group consisting of:
(a) acetyl L-carnitine;
(b) acetyl L-carnitine and choline chloride;
(c) acetyl L-carnitine and trimethylamine oxide; and
(d) acetyl L-carnitine, trimethylamine oxide and choline chloride.
5. A kit, comprising:
(1) a biomarker standard comprising a substance selected from the group consisting of:
(a) acetyl L-carnitine;
(b) acetyl L-carnitine and choline chloride;
(c) acetyl L-carnitine and trimethylamine oxide; and
(d) acetyl L-carnitine, trimethylamine oxide, and choline chloride;
and (2) an isotopic internal standard of the biomarker of (1).
6. The kit of claim 5, wherein the kit further comprises one or more reagents selected from the group consisting of:
(3) blank plasma, preferably carbon-adsorbed blank human plasma;
(4) an extraction solvent, preferably comprising acetonitrile (v/v, percentages being based on total volume of extraction solvent) containing 1.0-3.0% acetic acid, preferably 1.5-2.5%;
(5) a mobile phase additive a comprising formic acid; and/or
(6) A mobile phase additive B comprising ammonia.
7. A method for the combined liquid-mass spectrometric detection of a biomarker in a sample, comprising the steps of:
(a) providing the kit of claim 2 and a test sample, preferably selected from the group consisting of: blood, serum, plasma, or a combination thereof, preferably, peripheral whole blood;
(b) adding the solution of the isotope internal standard into a standard substance solution and the sample to be detected respectively, and extracting with an extraction solvent to obtain a standard sample and a sample to be detected respectively; and
(c) and respectively loading the standard sample and the sample to be detected to an HPLC-MS (high performance liquid chromatography-mass spectrometry) and analyzing, wherein the MS adopts a positive ion MRM (multiple Reaction Monitor) mode.
8. The method of claim 7, wherein the elution conditions of the HPLC comprise one or more characteristics selected from the group consisting of:
(i) the chromatographic column is a reverse phase chromatographic column (such as C18 chromatographic column);
(ii) mobile phase: phase A: water (v/v, based on the total volume of phase a) containing 0.05 to 0.4% formic acid (preferably 0.05 to 0.2%, more preferably 0.08 to 0.12%) and 0.01 to 0.1% ammonia (preferably 0.03 to 0.1%, more preferably 0.05 to 0.7%); phase B: acetonitrile (v/v, based on the total volume of phase B) containing 0.05-0.4% formic acid (preferably 0.07-0.25%, more preferably 0.09-0.15%); and
(iii) gradient elution.
10. the method of claim 7, wherein the method comprises:
(i) ionizing the HPLC eluate by an electrospray ion source (ESI) to produce precursor ions of the biological metabolite and internal standard; and
(ii) the precursor ions are collided within the mass spectrum, respectively producing fragment ions of the precursor ions.
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