CN111896646A - Kit for detecting 3 lipids in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology - Google Patents

Abstract

A reagent kit for detecting 3 kinds of lipid in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology is disclosed, wherein the 3 kinds of lipid are respectively as follows: α -HB, OA, and LGPC; the kit comprises the following reagents: eluent: eluent A and eluent B, mixed standard substance stock solution, mixed internal standard solution, protein precipitator and quality control product; the invention relates to a kit for detecting 3 lipids in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology, which mixes a plasma sample with mixed internal standard solutions of all objects to be detected, does not need derivatization treatment on the sample, has simple pretreatment, only needs one-step protein precipitation treatment, has small sample dosage, high sensitivity and strong specificity, can simultaneously detect 3 lipids within 5.0 minutes, and can be used for clinical diagnosis and health assessment of plasma lipids.

Description

Kit for detecting 3 lipids in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a kit for detecting 3 lipids in plasma by using an ultra-high performance liquid chromatography tandem mass spectrometry technology.

Background

Diabetes mellitus is one of the most common endocrine metabolic diseases, has genetic susceptibility and is developed under the starting point of environmental factors. With the development of social economy, the change of life style of people and the aging of population, the type 2 diabetes accounts for more than 95 percent of the total population of the diabetes, has high morbidity, hidden onset and unobvious early symptoms, has the annual increasing trend of the incidence rate of the type 2 diabetes in the global range, and particularly has a faster increasing speed and a popular trend in developing countries. Diabetes has now become a third non-infectious disease that threatens human life and health, following cardiovascular ice and tumors.

Epidemiology of type 2 diabetes mainly investigates its prevalence. However, by changing lifestyle, appropriate nutritional management and/or therapeutic intervention, the progression from pre-diabetes to overt type 2 diabetes can be delayed or prevented. Therefore, early detection of pre-diabetes is critical in controlling the prevalence of diabetes. The current definition of pre-diabetes is the use of one or more blood glucose based test criteria, including fasting glucose (FPG), hemoglobin A1c, and 2 hour blood glucose. However, the different pathophysiological states that lead to type 2 diabetes in these detection criteria may only detect pre-diabetes in a limited population of subjects. For more accurate diagnosis of pre-diabetic conditions such as Insulin Resistance (IR) and Impaired Glucose Tolerance (IGT), it is necessary to identify and quantify disease-specific biomarkers.

At present, research shows that three lipids, including alpha-hydroxybutyric acid (alpha-HB), Oleic Acid (OA) and linoleoyl glycerophosphocholine (1-linoleoyl phosphocholine, L-GPC), and insulin measurement are used as biomarkers of insulin antibodies (IR), but the research on how to detect them together is less, so that it is necessary to research and develop a simple, efficient and reliable method for detecting alpha-HB, OA and LGPC in blood plasma to meet the requirement of clinical application.

Disclosure of Invention

The invention aims to solve the technical problem of providing a kit for detecting 3 lipids in plasma by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the 3 lipids are respectively as follows: alpha-hydroxybutyric acid (alpha-HB), Oleic Acid (OA) and Linoleoyl Glycerophosphorylcholine (LGPC);

the isotope internal standard substances corresponding to the 3 lipids are respectively as follows: alpha-hydroxybutyric acid-d 3 (alpha-HB-d 3), oleic acid-13C 18(OA-13C18) and linoleoyl glycerophosphorylcholine-d 9(LGPC-d 9);

the kit comprises the following reagents:

eluent: eluent A: 0.01 to 0.5% formic acid aqueous solution; eluent B: acetonitrile;

mixing standard stock solution: a methanol-chloroform mixed solution containing α -HB, OA and LGPC;

mixing internal standard working solution: a methanol-chloroform mixed solution containing α -HB-d3, OA-13C18 and LGPC-d 9;

protein precipitant: a mixed solution of methanol and acetonitrile;

quality control product: blank plasma matrix solution containing 3 lipids, divided into low, medium and high concentrations, qc (l), qc (m) and qc (h), respectively, wherein:

qc (l) is a 1000-fold dilution of the mixed standard stock in blank plasma matrix;

qc (m) diluted 200-fold with blank plasma matrix for the mixed standard stock;

qc (h) was 20-fold dilution of the mixed standard stock in blank plasma matrix.

Wherein the plasma is human or animal plasma.

Wherein the eluent A is formic acid aqueous solution with the volume concentration of 0.1%.

Wherein the protein precipitator is a mixed solution of methanol and acetonitrile in a volume ratio of 1: 1.

Wherein the blank plasma matrix is 1-10% Bovine Serum Albumin (BSA) aqueous solution.

Wherein the mixed standard stock solution is a methanol-chloroform mixed solution containing 4mM of alpha-HB, 20mM of OA and 80mM of LGPC.

Wherein the mixed internal standard working solution is a methanol-chloroform mixed solution containing alpha-HB-d 320 mu M, OA-13C18100 mu M, LGPC-d9400 mu M.

The preparation method of the kit for detecting 3 lipids in plasma by the ultra-high performance liquid chromatography tandem mass spectrometry technology;

(1) eluent A: preparing 0.01-0.5% formic acid aqueous solution;

eluent B: acetonitrile;

(2) mixing standard stock solution: weighing each standard substance to be detected, adding methanol into alpha-HB and OA for dissolution, adding chloroform into LGPC for complete dissolution, preparing standard substance mother liquor with the concentration of alpha-HB 20mM, OA 100mM and LGPC 200mM in sequence, then respectively transferring alpha-HB 200 mu L, OA200 mu L, LGPC 400 mu L, adding 200 mu L methanol solution, fully and uniformly mixing to obtain 1mL mixed standard substance stock solution, wherein the preparation process is shown in Table 1;

table 1 preparation of stock solutions for mixed standards

(3) Mixing internal standard working solution: weighing each isotope internal standard substance, adding methanol into alpha-HB-d 3 and OA-13C18 for dissolution, adding chloroform into LGPC-d9 for dissolution, and preparing isotope internal standard mother liquor with the concentration of 50mM, 100mM and 200mM in sequence; then respectively transferring alpha-HB-d 34 mu L, OA-13C1810 mu L, LGPC-d920 mu L, adding 966 mu L of methanol, fully and uniformly mixing to obtain 1mL of mixed internal standard solution, wherein the preparation method is shown in Table 2; taking 100 mu L of mixed internal standard solution, adding 900 mu L of methanol, and uniformly mixing to obtain mixed internal standard working solution;

TABLE 2 preparation of mixed internal standard solutions

(4) Protein precipitant: a mixed solution of methanol and acetonitrile in a volume ratio of 1: 1;

(5) quality control product: preparing the mixed standard substance stock solution into three QC (L), QC (M) and QC (H) with different concentrations by using 1-10% BSA-water solution, wherein:

QC (L) includes: α -HB 4 μ M, OA20 μ M, LGPC 80 μ M;

QC (M) comprises: alpha-HB 20 μ M, OA 100 μ M, LGPC 400 μ M;

QC (H) includes: alpha-HB 80 μ M, OA400 μ M, LGPC 1600 μ M.

The application of the kit in detecting 3 lipids in serum by using the ultra performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.

The specific detection method comprises the following steps:

detecting lipid in pretreated plasma by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly separating a target object to be detected from interfering components in a plasma matrix by utilizing the ultra-high performance liquid chromatography, then detecting the mass-to-charge ratio (m/z) of the target object and a corresponding isotope internal standard thereof by utilizing the mass spectrometry, quantifying by using the isotope internal standard method, and respectively calculating the content of 3 types of lipid, wherein the specific chromatographic conditions are as follows:

chromatographic conditions are as follows:

mobile phase A: water containing 0.01 to 0.5% formic acid;

mobile phase B: acetonitrile;

a chromatographic column: ACQUITY UPLC BEH C18 (2.1X 50mm,1.7 μm);

gradient elution is carried out by adopting a mobile phase A and a mobile phase B as a mixed mobile phase, and the gradient elution is shown in a table 3;

the flow rate is 0.3-0.6 mL/min, the column temperature is 40-60 ℃, and the sample injection volume is 0.5-5 muL;

TABLE 3 mobile phase gradient elution parameters

Mass spectrum conditions: mass spectrometry scan mode for Multiple Reaction Monitoring (MRM) in electrospray ionization detection mode; the spraying voltage is 3.0kV (ESI +) and 2.5kV (ESI-); source temperature: 120 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard product and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 4;

TABLE 4 Mass spectrometric parameters

Wherein the pretreated blood plasma is prepared according to the following method: and (3) putting 20 mu L of plasma into a 1.5mL centrifuge tube, adding 20 mu L of mixed internal standard working solution into the centrifuge tube, adding 760 mu L of protein precipitator after vortex, centrifuging for 4-10 min at 12000-15000 r/min and 10-20 ℃, and filling 70 mu L of supernatant into a sample injection bottle with an inner cannula for detection.

The preparation method comprises the following steps of preparing standard solutions with seven different concentration points by using a blank plasma matrix and mixing a mixed standard stock solution by adopting a gradient dilution method, wherein the specific method comprises the following steps:

preparing the mixed standard substance stock solution into calibration substance solutions with seven different concentration points by using a blank plasma matrix, putting 10 mu L of the mixed standard substance stock solution into a 1.5mL centrifuge tube, adding 190 mu L of the blank plasma matrix to obtain a first high-value concentration point (S7), and taking the first high-value concentration point (S7) and diluting the first high-value concentration point by using an equal volume of the blank plasma matrix to obtain a second high-value concentration point (S6); diluting the first high-value concentration point (S7) with blank plasma matrix of 4 times volume to obtain a third high-value concentration point (S5); diluting the second high concentration point (S6) with blank plasma matrix of 4 times volume to obtain a fourth high concentration point (S4); diluting the third high concentration point (S5) with blank plasma matrix of 4 times volume to obtain a fifth high concentration point (S3); diluting the fourth high concentration point (S4) with blank plasma matrix of 4 times volume to obtain a sixth high concentration point (S2); diluting the fifth high-value concentration point (S3) with blank plasma matrix of 4 times volume to obtain a seventh high-value concentration point (S1); the preparation method is shown in Table 5;

TABLE 5 Standard Curve preparation and concentration (unit: μ M)

Has the advantages that: the kit is adopted to detect 3 lipids in serum, including alpha-hydroxybutyric acid, oleic acid and linoleoyl glycerophosphorylcholine, a plasma sample is mixed with a mixed internal standard solution of all substances to be detected, the sample does not need derivatization treatment, the pretreatment is simple, only one-step protein precipitation treatment is needed, the sample dosage is small, the sensitivity is high, the specificity is strong, 3 lipids can be simultaneously detected within 5.0 minutes, and the kit can be used for clinical diagnosis and health assessment of plasma lipids.

Drawings

FIG. 1 is a selective ion flow chromatogram of 3 lipid standards;

FIG. 2 is a selective ion flow chromatogram of 3 lipids in a plasma sample.

Detailed Description

For the purpose of enhancing the understanding of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.

Example 1

1. Material

(1) The instrument comprises the following steps: 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); glassware, graduated cylinders, and the like.

(2) Reagent consumables: MS grade acetonitrile (Fisher, usa); MS grade formic acid (Sigma, usa); HPLC grade methanol (Honeywell, usa); HPLC grade acetonitrile (Honeywell, usa); bovine serum albumin (Sigma, USA) with the column type number ACQUITY UPLC BEH C18 (2.1X 50mm,1.7 μm) (Waters, USA).

(3) And (3) standard substance: alpha-HB was purchased from TRC, alpha-HB-d 3 was purchased from CDN Isotopes, OA-13C18 was purchased from Merck, LGPC was purchased from Echelon Biosciences, LGPC-d9 was purchased from Avanti.

(4) Quality control product: blank plasma matrix solution containing 3 lipids, which are divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H), wherein,

QC (L) includes: α -HB 4 μ M, OA20 μ M, LGPC 80 μ M;

QC (M) comprises: alpha-HB 20 μ M, OA 100 μ M, LGPC 400 μ M;

QC (H) includes: alpha-HB 80 μ M, OA400 μ M, LGPC 1600 μ M.

The upper and lower peripheries of the kit are coated with films, the kit is shockproof and heat-insulated, mobile phases A and B are placed on the upper left, and 11 ampoules are respectively placed on the lower left, wherein the standard solution and the quality control product are respectively; 100mL of protein precipitant was placed on the right side.

2. Method of producing a composite material

(1) Chromatographic conditions are as follows: mobile phase A: water containing 0.01 to 0.5% formic acid; mobile phase B: acetonitrile; a chromatographic column: ACQUITYUPLC BEH C18 (2.1X 50mm,1.7 μm); gradient elution is carried out by adopting a mobile phase A and a mobile phase B as a mixed mobile phase, and the gradient elution is shown in a table 3; the flow rate is 0.3-0.6 mL/min, the column temperature is 40-60 ℃, and the sample injection volume is 0.5-5 muL;

(2) mass spectrum conditions: mass spectrometry scan mode for Multiple Reaction Monitoring (MRM) in electrospray ionization detection mode; the spraying voltage is 3.0kV (ESI +) and 2.5kV (ESI-); source temperature: 120 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard product and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 4;

(3) preparation of mixed standard stock solution

Weighing each standard substance to be detected, adding methanol into alpha-HB and OA for dissolution, adding chloroform into LGPC for complete dissolution, preparing standard substance mother liquor with the concentration of alpha-HB 20mM, OA 100mM and LGPC 200mM in sequence, then respectively transferring alpha-HB 200 mu L, OA200 mu L, LGPC 400 mu L, adding 200 mu L methanol solution, and fully mixing uniformly to obtain 1mL mixed standard substance stock solution, wherein the preparation method is shown in Table 1, and concretely, the mixed standard substance stock solution contains alpha-HB 4mM, OA20 mM and LGPC 80 mM;

(4) preparation of mixed internal standard working solution

Weighing each isotope internal standard substance, adding methanol into alpha-HB-d 3 and OA-13C18 for dissolution, adding chloroform into LGPC-d9 for dissolution, and preparing isotope internal standard mother liquor with the concentration of 50mM, 100mM and 200mM in sequence; then respectively transferring alpha-HB-d 34 mu L, OA-13C1810 mu L, LGPC-d920 mu L, adding 966 mu L of methanol, fully and uniformly mixing to obtain 1mL of mixed internal standard solution, wherein the preparation method is shown in Table 2; specifically, 100 μ L of mixed internal standard solution is taken, 900 μ L of methanol is added, and the mixed internal standard working solution is obtained after uniform mixing.

(5) Preparing a quality control product: preparing the mixed standard substance stock solution into three QC (L), QC (M) and QC (H) with different concentrations by using 1-10% BSA-water solution, wherein:

QC (L) is the above-mentioned mixed standard stock solution diluted to 1000 times with blank plasma matrix;

QC (M) is the above-mentioned mixed standard stock solution diluted to 200 times with blank plasma matrix;

qc (h) was a 50-fold dilution of the above mixed standard stock in blank plasma matrix.

(6) Sample processing

1) Standard curve configuration: preparing a standard curve by adopting a gradient dilution method, and preparing the mixed standard substance stock solution into calibration substance solutions with seven different concentration points by using a blank plasma matrix, wherein the preparation process comprises the following steps: taking 10 mu L of mixed standard substance stock solution to a 1.5mL centrifuge tube, adding 190 mu L of blank plasma matrix to obtain a first high-value concentration point (S7), taking the first high-value concentration point (S7), and diluting the first high-value concentration point with the same volume of the blank plasma matrix to obtain a second high-value concentration point (S6); diluting the first high-value concentration point (S7) with blank plasma matrix of 4 times volume to obtain a third high-value concentration point (S5); diluting the second high concentration point (S6) with blank plasma matrix of 4 times volume to obtain a fourth high concentration point (S4); diluting the third high concentration point (S5) with blank plasma matrix of 4 times volume to obtain a fifth high concentration point (S3); diluting the fourth high concentration point (S4) with blank plasma matrix of 4 times volume to obtain a sixth high concentration point (S2); diluting the fifth high-value concentration point (S3) with blank plasma matrix of 4 times volume to obtain a seventh high-value concentration point (S1); the preparation method is shown in Table 5;

2) pretreatment of standard substance

Taking 20 mu L of each concentration point sample, putting the sample into a 1.5mL centrifuge tube, adding 20 mu L of mixed internal standard for working, and then whirling for 5 s; 760 μ L of methanol was added: mixing acetonitrile (V: V ═ 1:1) solution, and shaking at high speed for 5 min; centrifuging at 15 deg.C for 5min at 15000 r/min; take 70 μ L of supernatant for injection.

3) Preparation of pretreated plasma

mu.L of plasma was taken in a 1.5mL centrifuge tube, 20. mu.L of the mixed internal standard working solution was added thereto, and 760. mu.L of methanol: and (3) centrifuging the mixed solution of acetonitrile (V: V ═ 1:1) at 12000-15000 r/min at 10-20 ℃ for 4-10 min, and taking 70 mu L of supernatant for sample injection.

4) Pretreatment of quality control product

20 μ L of each of the quality control solutions QC (L), QC (M), QC (H) were collected into 1.5mL centrifuge tubes, and then the steps were consistent with the plasma pretreatment steps, which are not repeated here.

The components of the assay kit are shown in Table 6.

TABLE 6 lipid assay kit Components (100 parts)

3. Method verification

1) Selection of ion flow spectrum

The lipid standard and the plasma sample have symmetrical peak shapes and no interference of a hybrid peak, which indicates that the lipid standard and the plasma sample can be well detected under the condition.

2) Calibration 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 lipid 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 3 lipids in each concentration range is good in linearity, and the correlation coefficient is above 0.99, which is detailed in table 7.

TABLE 73 Retention time and Linear Range of lipids

3) Minimum limit of quantitation

The lowest limit of quantitation (LLOQ), which is the lowest point of the standard curvilinear range, also reflects the sensitivity of the method. The lipid content in human body is low, the requirements on the sensitivity and specificity of the method are high, the method is optimized and studied, the current minimum quantitative limit (LLOQ) basically meets the sensitivity requirement of simultaneous detection of 3 lipids, and the concentration of the LLOQ is specifically shown in Table 8.

TABLE 8 quantitative lower limit data table

4) And (4) inspecting the standard recovery rate: randomly selecting one human plasma sample, adding no standard substance to 1 of the human plasma samples, adding low, medium and high 3 concentrations of mixed standard substance stock solution to the other 3 human plasma samples, repeatedly processing and measuring for 5 times by the same step, and calculating the recovery rate result, wherein the result is shown in a table 9; the results show that the results of the standard recovery rate of 3 lipids in the plasma are between 85.27% and 107.35%, and the RSD of 5 times of repeated tests is in the range of 1.20% to 7.01%, and all the results meet the requirements.

TABLE 9 results of recovery of 3 lipids in plasma on a standard basis (. mu.M)

5) And (3) precision test: plasma quality control samples are taken and repeatedly processed for 6 batches in one day and processed for 3 days, 3 kinds of lipid are quantitatively measured by an isotope internal standard method, the calculated batch precision is 1.26% -9.04%, the plasma quality control samples are processed by 3 batches in three days, the calculated batch precision is 3.58% -8.52%, and the results of the batch precision are shown in a table 10.

TABLE 10 results of precision test within and between batches (Unit: μ M)

4. Discussion of the related Art

The invention adopts UPLC-MS/MS method to determine 3 kinds of lipid in human plasma, and provides a kit for determining 3 kinds of lipid in plasma; the detection is carried out according to the peak-out time and the ion pair of the target object, the sensitivity is high, and the specificity is strong. Meanwhile, the isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the influence of the conditions such as a pretreatment process, a sample loading volume and flow is avoided, and accurate quantification can be achieved.

The standard recovery rate of 3 lipids in plasma is inspected, and the standard recovery rate is 85.27-107.35%, and meets the requirements; the reproducibility result of the method shows that the internal precision of 3 lipids in the plasma is 1.26-9.04%, the batch-to-batch precision is 3.58-8.52%, and the reproducibility of the method is good.

The kit provided by the method has higher sensitivity, simple pretreatment, only one-step protein precipitation treatment, small sample dosage, capability of simultaneously detecting 3 lipids within 5.0 minutes, and capability of being used for clinical diagnosis and health assessment of lipids in plasma.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (9)

1. A kit for detecting 3 lipids in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology is characterized in that the 3 lipids are respectively: α -HB, OA, and LGPC;

the kit comprises the following reagents:

eluent: eluent A: 0.01 to 0.5% formic acid aqueous solution; eluent B: acetonitrile;

mixing standard stock solution: a methanol-chloroform mixed solution containing α -HB, OA and LGPC;

mixing internal standard working solution: a methanol-chloroform mixed solution containing α -HB-d3, OA-13C18 and LGPC-d 9;

protein precipitant: a mixed solution of methanol and acetonitrile;

quality control product: blank plasma matrix solution containing 3 lipids, divided into low, medium and high concentrations, qc (l), qc (m) and qc (h), respectively, wherein:

qc (l) is a 1000-fold dilution of the mixed standard stock in blank plasma matrix;

qc (m) diluted 200-fold with blank plasma matrix for the mixed standard stock;

qc (h) was 20-fold dilution of the mixed standard stock in blank plasma matrix.

2. The kit for detecting 3 lipids in plasma according to claim 1, wherein the plasma is human or animal plasma.

3. The kit for detecting 3 lipids in plasma according to claim 1, wherein the eluent A is 0.1% formic acid solution by volume.

4. The kit for detecting 3 lipids in plasma according to claim 1, wherein the protein precipitant is a mixed solution of methanol and acetonitrile at a volume ratio of 1: 1.

5. The kit for detecting 3 lipids in plasma according to claim 1, wherein the blank plasma matrix is 1-10% Bovine Serum Albumin (BSA) aqueous solution.

6. The kit for detecting 3 lipids in plasma according to claim 1, wherein the stock solution of mixed standard is methanol-chloroform mixed solution containing 4mM of α -HB, 20mM of OA and 80mM of LGPC.

7. The kit for detecting 3 lipids in plasma according to claim 1, wherein the mixed internal standard working solution is a methanol-chloroform mixed solution containing α -HB-d320 μ M, OA-13C18100 μ M, LGPC-d9400 μ M.

8. A method for producing the kit according to any one of claims 1 to 7,

(1) eluent A: preparing 0.01-0.5% formic acid aqueous solution;

eluent B: acetonitrile;

(2) mixing standard stock solution: weighing each standard substance to be detected, adding methanol into alpha-HB and OA for dissolution, adding chloroform into LGPC for complete dissolution, preparing standard substance mother liquor with the concentration of alpha-HB 20mM, OA 100mM and LGPC 200mM in sequence, then respectively transferring alpha-HB 200 mu L, OA200 mu L, LGPC 400 mu L, adding 200 mu L methanol solution, and fully mixing to obtain 1mL mixed standard substance stock solution;

(3) mixing internal standard working solution: weighing each isotope internal standard substance, adding methanol into alpha-HB-d 3 and OA-13C18 for dissolution, adding chloroform into LGPC-d9 for dissolution, and preparing isotope internal standard mother liquor with the concentration of 50mM, 100mM and 200mM in sequence; then respectively transferring alpha-HB-d 34 mu L, OA-13C1810 mu L, LGPC-d920 mu L, adding 966 mu L of methanol, and fully and uniformly mixing to obtain 1mL of mixed internal standard solution; taking 100 mu L of mixed internal standard solution, adding 900 mu L of methanol, and uniformly mixing to obtain mixed internal standard working solution;

(4) protein precipitant: a mixed solution of methanol and acetonitrile in a volume ratio of 1: 1;

(5) preparing the mixed standard substance stock solution into three QC (L), QC (M) and QC (H) with different concentrations by using 1-10% BSA-water solution, wherein:

QC (L) includes: α -HB 4 μ M, OA20 μ M, LGPC 80 μ M;

QC (M) comprises: alpha-HB 20 μ M, OA 100 μ M, LGPC 400 μ M;

QC (H) includes: alpha-HB 80 μ M, OA400 μ M, LGPC 1600 μ M.

9. Use of the kit of any one of claims 1 to 8 for detecting lipids in plasma using ultra high performance liquid chromatography tandem mass spectrometry.

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