CN117420245A

CN117420245A - Detection method of human dry blood spot ceramide and kit for detecting ceramide

Info

Publication number: CN117420245A
Application number: CN202311380837.9A
Authority: CN
Inventors: 张伟; 成晓亮; 李美娟
Original assignee: Nanjing Pinsheng Medical Technology Co ltd; Shanghai Ammonia Biotechnology Co ltd; Nanjing Pinsheng Medical Laboratory Co ltd
Current assignee: Nanjing Pinsheng Medical Technology Co ltd; Shanghai Ammonia Biotechnology Co ltd; Nanjing Pinsheng Medical Laboratory Co ltd
Priority date: 2023-10-24
Filing date: 2023-10-24
Publication date: 2024-01-19

Abstract

The invention provides a detection method of human dry blood spot ceramide and a kit for detecting ceramide, wherein the detection method comprises the following steps: (1) Preprocessing a human dry blood spot sample to prepare a sample to be tested; (2) And carrying out high performance liquid chromatography-tandem mass spectrometry detection on the sample to be detected, and obtaining the content of 12 ceramides in the sample to be detected according to the detection result. The detection method provided by the invention has the advantages of short sample pretreatment time, simple operation, short analysis time, high chromatographic separation degree, high precision and high sensitivity, provides a reliable quantitative analysis method for clinical research on the correlation of broad-spectrum ceramide and cardiovascular diseases, and can be used for diagnosis and health assessment of 12 kinds of ceramide and cardiovascular diseases in clinical human dry blood spots.

Description

Detection method of human dry blood spot ceramide and kit for detecting ceramide

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a detection method of human dry blood spot ceramide and a kit for detecting ceramide.

Background

Ceramides are a class of bioactive lipids that play an important role in cellular functions such as apoptosis and inflammation. New research data shows that ceramide not only has important biochemical significance, but also may have diagnostic uses. Many studies have revealed a close relationship between ceramide and cardiac metabolic status, with ceramide having independent predictive value for cardiovascular adverse events and type 2 diabetes onset. Studies have shown that different circulating levels of ceramide rise in diabetics, and that some specific ceramides can predict the onset of type 2 diabetes even a few years before onset [ Turpin-Nolan S M, bryning J C.The role of ceramides in metabolic disorders: when size and localization matters [ J ]. Nature Reviews Endocrinology,2020,16 (4): 224-233 ]. Ceramide concentrations have been shown to be predictive of cardiovascular atherosclerotic disease, such as coronary artery disease, stroke, and heart failure and atrial fibrillation [ Choi R H, tatum S M, symons J D, et al Ceramides and other sphingolipids as drivers of cardiovascular disease [ J ]. Nature reviews Cardiology,2021,18 (10): 701-711 ]. These associations were initially observed in small case control studies, comparing healthy controls to those of patients with stable coronary heart disease or acute coronary syndromes. These findings facilitate a series of larger scale cohort studies and randomized clinical trials.

The data obtained from these various studies helps to determine the clinical utility of clinical tests of ceramides, but the significance of individual ceramides and their effect on disease pathogenesis is still uncertain. This hampers the development of single ceramide clinical trials. Furthermore, panel tests involving several ceramide concentrations may be difficult to apply to clinical decisions and are not attractive diagnostic reporting options. To address these shortcomings, based on a large number of published data, risk scores using circulating ceramide concentrations have been developed and adapted for routine clinical practice. Currently, circulating ceramides are used clinically as biomarkers for effective risk of primary and secondary prevention of cardiovascular atherosclerotic disease [ Havulina A S, sysi-Aho M, hilvo M, et al, circulating ceramides predict cardiovascular outcomes in the population-based FINRISK 2002cohort [ J ], arteriosclerosis, thrombisis, and vascular biology,2016,36 (12): 2424-2430 ].

In these studies, which are basically carried out on serum or plasma as a matrix, the transport and storage of samples are extremely important in the accurate measurement of compounds in plasma/serum, and especially the transport of serum/plasma samples and the transport conditions thereof are challenges, which increases the cost of developing clinical studies. Over the past few years, dry Blood Spot (DBS) technology has become an advantageous tool for qualitative and quantitative biological analysis. DBS technology is a minimally invasive method for collecting small amounts of whole blood (.ltoreq.50. Mu.L) from the finger or heel and applying it to specially prepared filter papers for drying. DBS is considered to be the most suitable method for sampling biological material, and has particular inherent advantages, including that DBS samples do not require phlebotomy, that the required biological material is small in volume, that it can be stored and transported under ambient conditions, that the stability of the analyte is improved, and that the risk of infection caused by contaminating the sample is reduced. Existing DBS stability studies, although limited, have demonstrated the stability of analytes under various storage conditions [ Meesters RJ W, hooff GP.State-of-the-art dried blood spot analysis: an overview of recent advances and future trends [ J. ] Bioanalysis 2013,5 (17): 2187-2208 ]. DBS has been widely used in clinical practice, basic research and crowd-based research to date. The most common and widely accepted clinical application of DBS is for neonatal screening programs, which mainly involve screening detection of metabolic disorders. In addition, DBS is also used for quantitative analysis of endogenous substances such as human hormone and 25 hydroxy vitamin D, and exogenous substances such as drugs.

Because the volume of DBS sampling is smaller, the requirements on the detection sensitivity and chromatographic separation degree of quantitative analysis are higher, and meanwhile, the requirements on the precision between DBS samples are higher, so that the requirements on the sampling preparation of DBS are also higher, and the reliability and reliability of sample test are ensured. Therefore, DBS endogenous metabolite analysis, such as human hormone, 25 hydroxy vitamin D, is mostly performed by derivatization reaction to increase detection sensitivity, but at the same time, derivatization steps are complicated, and detection cost [ Demirev P A.driven blood spots: analysis and applications [ J ]. Analytical chemistry,2013,85 (2): 779-789 ]. In DBS quantitative analysis, the treatment of DBS samples is extremely critical, and currently reported pretreatment methods of DBS samples mainly comprise oscillation extraction, ultrasonic extraction and the like, such as human hormone and 25 hydroxy vitamin D [ Eyles D, anderson C, ko P, et al A active LC/MS/MS assay of 25OH vitamin D3and 25OH vitamin D2 in dried blood spots[J ]. Clinica chimica acta,2009,403 (1-2): 145-151 ] in DBS, and absolute quantitative analysis of DBS ceramide is freshly reported. In addition, in the quantitative analysis of DBS, the standard curve sample is prepared by replacing the whole blood matrix, so that time and labor are wasted, and the detection cost is increased.

Different analytical techniques have been used to measure ceramide in whole blood, plasma or serum, including gas chromatography, "shotgun" analysis, among which the most prominent is the liquid chromatography tandem mass spectrometry detection method. However, in analytical studies of these ceramides, pretreatment methods are complicated and the analysis time is long, and qualitative or semi-quantitative analysis is used. In a recent study [ Meikopoulos T, begou O, theodordis G, et al Ceramides biomarkers determination in quantitative dried blood spots by UHPLC-MS/MS [ J ]. Analytica Chimica Acta,2023, 1255:34131 ], four ceramides were analyzed using a DBS technique based on micro-quantitative sampling blood cards, including Cer (d18:1/16:0), cer (d18:1/18:0), cer (d18:1/24:0) and Cer (d18:1/24:1). In the detection method, the DBS pretreatment sequentially adopts the methods of ultrasonic treatment, grinding and crushing and nitrogen blowing and re-dissolution, so that the pretreatment steps are complicated, the treatment time is long, the required blood quantity of the dried blood spots is large (2 quantitative dried blood spots of 10 mu L are required for one-time measurement), the analysis sensitivity is low, and the purchase cost of the micro quantitative sampling blood sampling card is increased. In addition, in the detection method, methanol water with the volume fraction of less than 50% is adopted for preparing the standard substance solution of the ceramide and preparing the standard curve sample, and the ceramide is insoluble in water and methanol or acetonitrile, so that the detection accuracy is difficult to ensure. Similarly, in another study for quantitative detection of ceramide in DBS [ Chuang W L, pacheco J, zhang K.A Simple, high-Throughput Method for Analysis of Ceramide, glucopyranosyl amide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS [ J ]. Clinical Applications of Mass Spectrometry in Biomolecular Analysis: methods and Protocols,2016:263-272 ], methanol/acetonitrile/water (80:15:5, V/V/V) was used as an extract of ceramide in DBS in the treatment of DBS samples, and it was difficult to efficiently extract ceramide in DBS by shaking and ultrasonic extraction, resulting in low extraction recovery, resulting in inaccurate concentration detection. Most current studies have focused on four ceramide studies, cer (d18:1/16:0), cer (d18:1/18:0), cer (d18:1/24:0) and Cer (d18:1/24:1), and several lipidomic studies have reported that there may be a close association between other ceramides and disease [ Zheng W, kollmeyer J, symolon H, et al Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamic, signaling and autophagy [ J ]. Biochimica et Biophysica Acta (BBA) -Biomembranes,2006,1758 (12): 1864-1884 ].

In view of the foregoing, a method for determining ceramide in human dry blood spots, which has high specificity, high accuracy, simple and convenient operation, short determination time, comprehensive determination types and low detection cost, is needed to help to improve the quality of clinical detection of ceramide, and can more comprehensively analyze dynamic changes of ceramide, is a focus of research in the field.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a detection method of human dry blood spot ceramide and a kit for detecting ceramide, and the method has the advantages of short sample pretreatment time, simple operation, short analysis time, high chromatographic resolution, high precision and high sensitivity, and provides a reliable quantitative analysis method for clinical research on the correlation of broad-spectrum ceramide and cardiovascular diseases.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for detecting human dry blood patch ceramide, the method comprising the steps of:

(1) Preprocessing a human dry blood spot sample to prepare a sample to be tested;

(2) And carrying out high performance liquid chromatography-tandem mass spectrometry detection on the sample to be detected, and obtaining the content of 12 ceramides in the sample to be detected according to the detection result.

According to the invention, after the human dry blood spot sample is subjected to pretreatment, the high performance liquid chromatography is utilized to separate the target object to be detected from the interference components in the sample matrix, then the mass spectrometer is utilized to detect the response of the target object to be detected and the corresponding isotope internal standard, the isotope internal standard method is used for quantification, and the concentrations of 12 kinds of ceramide in the human dry blood spot are respectively calculated.

Preferably, the pretreatment method of the sample to be tested is as follows: and dissolving the human dry blood spot sample in an internal standard extraction liquid, performing ultrasonic extraction and centrifugation, and taking supernatant to obtain the sample to be tested.

Preferably, the solvent of the internal standard extract comprises isopropanol and dimethylsulfoxide.

Preferably, the solvent of the internal standard extract is a combination of isopropanol and dimethylsulfoxide.

Preferably, the volume ratio of isopropyl alcohol to dimethyl sulfoxide is (3-5): 1, which may be, for example, 3.5:1, 4:1, 4.5:1, etc., preferably 4:1.

Preferably, the internal standard extract contains 4 isotope internal standards, namely Cer (d18:1/16:0) -d7, cer (d18:1/18:0) -d7, cer (d18:1/24:0) -d7 and Cer (d18:1/24:1) -d7.

Preferably, the concentrations of the 4 isotope internal standard substances in the internal standard extract liquid are respectively as follows: 25nmol/LCer (d18:1/16:0) -d7, 12.5nmol/L Cer (d18:1/18:0) -d7, 25nmol/L Cer (d18:1/24:0) -d7, 25nmol/L Cer (d18:1/24:1) -d7.

Preferably, the pretreatment method of the sample to be tested is as follows: punching a dried blood sheet with the diameter of 3mm from a dried blood spot to obtain a dried blood spot sample, dissolving the dried blood spot sample in 400-800 mu L of an internal standard extraction liquid, wherein the solvent of the internal standard extraction liquid is a combination of isopropanol and dimethyl sulfoxide, the volume ratio of the isopropanol to the dimethyl sulfoxide is (3-5): 1, extracting for 10-30min at the temperature of 25-50 ℃ by ultrasonic, centrifuging for 5-20min at the temperature of 14000-16000rpm and the temperature of 10-20 ℃, and taking supernatant to obtain the sample to be tested.

Wherein 400-800. Mu.L may be, for example, 500. Mu.L, 600. Mu.L, 700. Mu.L, etc.; 25-50deg.C, such as 26deg.C, 28deg.C, 30deg.C, 35deg.C, 40deg.C, 45deg.C, etc.; 10-30min, such as 15min, 20min, 25min, etc.; 14000-16000rpm, for example 14500rpm, 15000rpm, 15500rpm, etc.; 10-20deg.C, for example, 13 deg.C, 15 deg.C, 18 deg.C, etc.; 5-20min, for example, 8min, 10min, 15min, 18min, etc.

Preferably, the pretreatment method of the sample to be tested is as follows: punching a dried blood sheet with the diameter of 3mm from a dried blood spot to obtain a dried blood spot sample, dissolving the dried blood spot sample in 600 mu L of an internal standard extraction liquid, wherein the solvent of the internal standard extraction liquid is a combination of isopropanol and dimethyl sulfoxide, the volume ratio of the isopropanol to the dimethyl sulfoxide is 4:1, extracting for 20min at the ultrasonic temperature of 37 ℃, centrifuging for 10min at the temperature of 14800rpm and the temperature of 15 ℃, and taking supernatant to obtain the sample to be tested.

Preferably, the 12 ceramides are Cer (d18:1/16:0), cer (d18:1/18:0), cer (d18:1/24:0), cer (d18:1/24:1), cer (d18:1/14:0), cer (d18:1/20:0), cer (d18:1/22:0), cer (d18:0/16:0), cer (d18:0/18:0), cer (d18:0/24:0), cer (d18:0/24:1), lacCer (d18:1/24:1).

Preferably, the conditions of the high performance liquid chromatography in the high performance liquid chromatography tandem mass spectrometry detection are as follows: the chromatographic mobile phase comprises mobile phase A and mobile phase B, and is performed by gradient elution, wherein the flow rate is 0.4-0.6mL/min, preferably 0.5mL/min, the column temperature is 30-45deg.C, preferably 40deg.C, and the sample injection volume is 0.5-5 μL, preferably 1 μL.

Preferably, the mobile phase A is an aqueous formic acid solution, and the volume fraction of formic acid in the aqueous formic acid solution is 0.05-0.2%, preferably 1%.

Preferably, the mobile phase B is an isopropanol-acetonitrile mixed solution containing formic acid, the volume fraction of the formic acid in the mixed solution is 0.05-0.2%, preferably 1%, and the volume ratio of the isopropanol to the acetonitrile is (2-3): (3-2), preferably 1:1.

Preferably, the conditions of mass spectrum in the high performance liquid chromatography tandem mass spectrometry detection are as follows: the ion source is an electrospray ion source, the scanning mode is a positive ion scanning mode of multi-reaction monitoring, the capillary voltage is 2-4kV, preferably 3kV, and the nozzle voltage is 0V; the temperature of the drying gas is 250-350 ℃, preferably 300 ℃, the temperature of the sheath gas is 300-350 ℃, preferably 320 ℃, the flow rate of the drying gas is 6-8L/min, preferably 7L/min, the flow rate of the sheath gas is 10-12L/min, preferably 11L/min, and the pressure of the atomizing gas is 40-50psi, preferably 45psi.

Preferably, the detection method further comprises the step of preparing a standard solution.

Preferably, the solvent and the blank matrix adopted in the preparation process of the standard substance solution are mixed solutions of isopropanol and dimethyl sulfoxide, and the volume ratio of the isopropanol to the dimethyl sulfoxide is (3-5): 1, preferably 4:1.

Preferably, the preparation of the standard solution comprises the following steps: and dissolving a standard sample in the internal standard extract, oscillating, and sucking the supernatant to obtain a standard solution.

Preferably, the detection method comprises the steps of:

(1) Dissolving a human dry blood spot sample in an internal standard extract, performing ultrasonic extraction and centrifugation, and taking a supernatant to obtain the sample to be tested;

the solvent of the internal standard extract is a combination of isopropanol and dimethyl sulfoxide;

the volume ratio of the isopropanol to the dimethyl sulfoxide is (3-5): 1;

the internal standard extract liquid contains 4 isotope internal standard substances, wherein the isotope internal standard substances are Cer (d18:1/16:0) -d7, cer (d18:1/18:0) -d7, cer (d18:1/24:0) -d7 and Cer (d18:1/24:1) -d7 respectively;

(2) Performing high performance liquid chromatography tandem mass spectrometry detection on the sample to be detected, and obtaining the content of 12 ceramides in the sample to be detected according to a detection result;

The conditions of the high performance liquid chromatography are as follows: the chromatographic mobile phase comprises a mobile phase A and a mobile phase B, which are performed in a gradient elution mode, wherein the flow rate is 0.4-0.6mL/min, the column temperature is 30-45 ℃, and the sample injection volume is 0.5-5 mu L;

the mobile phase A is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.2%;

the mobile phase B is isopropanol-acetonitrile mixed solution containing formic acid, the volume fraction of the formic acid in the mixed solution is 0.05-0.2%, and the volume ratio of the isopropanol to the acetonitrile is (2-3) (3-2);

the conditions of mass spectrometry were: the ion source is an electrospray ion source, the scanning mode is a positive ion scanning mode of multi-reaction monitoring, the capillary voltage is 2-4kV, and the nozzle voltage is 0V; the temperature of the drying gas is 250-350 ℃, the temperature of the sheath gas is 300-350 ℃, the flow rate of the drying gas is 6-8L/min, the flow rate of the sheath gas is 10-12L/min, and the pressure of the atomizing gas is 40-50psi.

Preferably, the detection method comprises the steps of:

the volume ratio of the isopropanol to the dimethyl sulfoxide is 4:1;

the conditions of the high performance liquid chromatography are as follows: the chromatographic mobile phase comprises a mobile phase A and a mobile phase B, which are performed in a gradient elution mode, wherein the flow rate is 0.5mL/min, the column temperature is 40 ℃, and the sample injection volume is 1 mu L;

the mobile phase A is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.1%;

the mobile phase B is isopropanol-acetonitrile mixed solution containing formic acid, the volume fraction of the formic acid in the mixed solution is 0.1%, and the volume ratio of the isopropanol to the acetonitrile is 1:1;

the conditions of mass spectrometry were: the ion source is an electrospray ion source, the scanning mode is a positive ion scanning mode of multi-reaction monitoring, the capillary voltage is 3kV, and the nozzle voltage is 0V; the temperature of the drying gas is 300 ℃, the temperature of the sheath gas is 320 ℃, the flow rate of the drying gas is 7L/min, the flow rate of the sheath gas is 11L/min, and the atomizing pressure is 45psi.

In a second aspect, the invention provides a kit for detecting ceramide, wherein the kit adopts an internal standard extract liquid comprising 4 isotope internal standards, and the isotope internal standards are Cer (d18:1/16:0) -d7, cer (d18:1/18:0) -d7, cer (d18:1/24:0) -d7 and Cer (d18:1/24:1) -d7 respectively.

Preferably, the solvent of the internal standard extract adopted by the kit is a combination of isopropanol and dimethyl sulfoxide, and the volume ratio of the isopropanol to the dimethyl sulfoxide is 4:1.

Preferably, the kit can be used to detect 12 ceramides, which are Cer (d18:1/16:0), cer (d18:1/18:0), cer (d18:1/24:0), cer (d18:1/24:1), cer (d18:1/14:0), cer (d18:1/20:0), cer (d18:1/22:0), cer (d18:0/16:0), cer (d18:0/18:0), cer (d18:0/24:0), cer (d18:0/24:1), lacCer (d18:1/24:1).

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

Compared with the prior art, the invention has the following beneficial effects:

(1) According to the method for measuring 12 kinds of ceramide in the human dry blood spots, on one hand, the common isopropanol/dimethyl sulfoxide mixed solution is used as a matrix prepared by the standard curve sample and an extraction reagent of the ceramide in DBS, so that the solubility of the ceramide is enhanced, the process of standard curve preparation is simplified, the DBS sample is treated by an ultrasonic extraction method, and the ceramide in the DBS can be effectively extracted, so that a higher extraction recovery rate is obtained; on the other hand, only a DBS blood sampling card which is common in the market is needed to prepare a DBS sample, and ceramide substances in 12 human blood can be monitored simultaneously within 7 minutes, so that dynamic changes of ceramide in the human body can be monitored more comprehensively;

(2) The detection method provided by the invention can monitor the ceramide concentration of the venous blood dry blood spots and the fingertip blood dry blood spots simultaneously, so that the sample type applicability of the method is expanded, and the ceramide concentration monitoring of the venous blood dry blood spots and the fingertip blood dry blood spots has good consistency;

(3) The detection method provided by the invention has the advantages of short sample pretreatment time, simple operation, short analysis time, high chromatographic separation degree, high precision and high sensitivity, provides a reliable quantitative analysis method for clinical research on the correlation of broad-spectrum ceramide and cardiovascular diseases, and can be used for diagnosis and health assessment of 12 kinds of ceramide and cardiovascular diseases in clinical human dry blood spots.

Drawings

FIG. 1 is a selective ion flow chromatogram of 12 ceramide compounds of a standard solution;

FIG. 2 is a selective ion flow chromatogram of 12 ceramide compounds of a human dry blood spot sample.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

"optional" or "any" means that the subsequently described event or event may or may not occur, and that the description includes both cases where the event occurs and cases where the event does not.

The indefinite articles "a" and "an" preceding an element or component of the invention are not limited to the requirement (i.e. the number of occurrences) of the element or component. Thus, the use of "a" or "an" should be interpreted as including one or at least one, and the singular reference of an element or component includes the plural reference unless the amount clearly dictates otherwise.

The description of the terms "one embodiment," "some embodiments," "exemplarily," "specific examples," or "some examples," etc., herein described means that a specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this document, the schematic representations of the above terms are not necessarily for the same embodiment or example.

The reagents or instrument sources in the following examples are as follows:

standard substance: cer (d18:1/16:0), cer (d18:1/18:0), cer (d18:1/24:0), cer (d18:1/24:1), cer (d18:1/14:0), cer (d18:1/20:0), cer (d18:1/22:0), cer (d18:0/16:0), cer (d18:0/18:0), cer (d18:0/24:0), cer (d18:0/24:1), lacCer (d18:1/24:1) standards were all purchased from Avanti company;

Isotopic internal standard: cer (d18:1/16:0) -d7, cer (d18:1/18:0) -d7, cer (d18:1/24:0) -d7, cer (d18:1/24:1) -d7 were all purchased from Avanti company;

instrument: qlife Lab 9000plus triple quadrupole mass spectrometer (supplied by pin healthcare);

qlife Lab 9000 high performance liquid chromatography (G7167A autosampler, supplied by pin-life medical);

the system operating software is MS quantitative analysis 10.0.10.0;

SCILOGEX D2012 high speed table centrifuge (usa);

ultrapure water meter (ELGA LabWater, uk);

multitube Vortex mixer (Vortex genie2, usa);

an adjustable pipette (Eppendorf 0.5-10. Mu.L, 10-100. Mu.L, 100-1000. Mu.L);

methanol (MS grade, fisher);

acetonitrile (MS grade, fisher);

isopropanol (MS grade, fisher);

formic acid (MS grade, fisher);

dimethyl sulfoxide (HPLC grade, belgium technology);

column Kinetex XB-C18 (2.6 μm,3.0×50mm, phenomenex);

blood sample collection card for dried blood spot (provided by Ridiner).

The correspondence between 12 kinds of ceramides and the isotope internal standard in the human dry blood spots measured in the invention is shown in table 1:

TABLE 1

Sequence number	Ceramide	Internal standard
			1	Cer(d18:1/16:0)	Cer(d18:1/16:0)-d7
2	Cer(d18:1/18:0)	Cer(d18:1/18:0)-d7
			3	Cer(d18:1/24:0)	Cer(d18:1/24:0)-d7
4	Cer(d18:1/24:1)	Cer(d18:1/24:1)-d7
			5	Cer(d18:1/14:0)	Cer(d18:1/24:0)-d7
6	Cer(d18:1/20:0)	Cer(d18:1/24:1)-d7
			7	Cer(d18:1/22:0)	Cer(d18:1/24:0)-d7
8	Cer(d18:0/16:0)	Cer(d18:1/18:0)-d7
			9	Cer(d18:0/18:0)	Cer(d18:1/18:0)-d7
10	Cer(d18:0/24:0)	Cer(d18:1/18:0)-d7
			11	Cer(d18:0/24:1)	Cer(d18:1/24:1)-d7
12	LacCer(d18:1/24:1)	Cer(d18:1/24:1)-d7

The mass spectrum parameters of 12 ceramides in human dry blood spots measured in the present invention are shown in table 2:

TABLE 2

Preparation example 1

The preparation example provides an internal standard extract, and the preparation method comprises the following steps:

the isotope stock solutions in the following table are prepared from 4 isotope internal standard substances by using isopropanol/dimethyl sulfoxide mixed solution (4:1, V/V), then the isotope stock solutions with various concentrations are prepared into mixed internal standard SI solution by using isopropanol/dimethyl sulfoxide mixed solution (4:1, V/V) (see table 3 for details), 15 mu L of mixed internal standard SI solution is taken, 60mL of isopropanol/dimethyl sulfoxide mixed solution (4:1, V/V) is added, and the mixture is uniformly mixed to obtain the internal standard extract. Wherein the internal standard extract contains 25nmol/L Cer (d18:1/16:0) -d7, 12.5nmol/LCer (d18:1/18:0) -d7, 25nmol/L Cer (d18:1/24:0) -d7 and 25nmol/L Cer (d18:1/24:1) -d7.

TABLE 3 Table 3

Preparation example 2

The preparation example provides a series of standard substance solutions, and the preparation method is as follows:

(1) Weighing 12 standard substances, and respectively adding isopropanol/dimethyl sulfoxide (4:1, V/V) to completely dissolve to prepare standard substances with the concentration of 1mmol/L of Cer (d18:1/16:0), 1mmol/L of Cer (d18:1/18:0), 1mmol/L of Cer (d18:1/24:0), 2mmol/L of Cer (d18:1/24:1), 0.2mmol/L of Cer (d18:1/14:0), 2mmol/L of Cer (d18:1/20:0), 0.2mmol/L of Cer (d18:1/22:0), 0.2mmol/L of Cer (d18:0/16:0), 1mmol/L of Cer (d18:0/24:0), 2mmol/L of Cer (d18:0/24:1), and 1mmol/L of LacCer (d18:1/24:1);

(2) The stock solutions of the above standards were then formulated as mixed solutions containing 40. Mu. Mol/L Cer (d18:1/16:0), 10. Mu. Mol/L Cer (d18:1/18:0), 200. Mu. Mol/L Cer (d18:1/24:0), 100. Mu. Mol/L Cer (d18:1/24:1), 4. Mu. Mol/L Cer (d18:1/14:0), 10. Mu. Mol/L Cer (d18:1/20:0), 40. Mu. Mol/L Cer (d18:1/22:0), 4. Mu. Mol/L Cer (d18:0/16:0), 4. Mu. Mol/L Cer (d18:0/18:0), 20. Mu. Mol/L Cer (d18:0/24:1), 40. Mu. Mol/L Cer (d18:1/24:1) and 40. Mu. Mol/L Cer (d18:1:0) as specified in Table 4S standard solutions;

TABLE 4 Table 4

(3) Taking 16 mu L of mixed standard S0 solution to a 1.5mL centrifuge tube, adding 144 mu L of blank matrix (isopropanol/dimethyl sulfoxide (4:1, V/V) mixed solution) to obtain a first high-value concentration point S8, and gradually diluting to S1, wherein the preparation method of the standard solution is shown in Table 5, the concentration of each standard solution is shown in Table 6, and the concentration unit is mu mol/L.

TABLE 5

TABLE 6

Ceramide	S1	S2	S3	S4	S5	S6	S7	S8
									Cer(d18:1/16:0)	0.02	0.04	0.1	0.2	0.4	1	2	4
Cer(d18:1/18:0)	0.005	0.01	0.025	0.05	0.1	0.25	0.5	1
									Cer(d18:1/24:0)	0.1	0.2	0.5	1	2	5	10	20
Cer(d18:1/24:1)	0.05	0.1	0.25	0.5	1	2.5	5	10
									Cer(d18:1/14:0)	0.002	0.004	0.01	0.02	0.04	0.1	0.2	0.4
Cer(d18:1/20:0)	0.005	0.01	0.025	0.05	0.1	0.25	0.5	1
									Cer(d18:1/22:0)	0.02	0.04	0.1	0.2	0.4	1	2	4
Cer(d18:0/16:0)	0.002	0.004	0.01	0.02	0.04	0.1	0.2	0.4
									Cer(d18:0/18:0)	0.002	0.004	0.01	0.02	0.04	0.1	0.2	0.4
Cer(d18:0/24:0)	0.01	0.02	0.05	0.1	0.2	0.5	1	2
									Cer(d18:0/24:1)	0.005	0.01	0.025	0.05	0.1	0.25	0.5	1
LacCer(d18:1/24:1)	0.02	0.04	0.1	0.2	0.4	1	2	4

(4) And taking 20 mu L of standard substance samples at each concentration point, putting the samples into a 1.5mL centrifuge tube, adding 600 mu L of internal standard extract of isopropanol/dimethyl sulfoxide (4:1, V/V) mixed solution, carrying out vortex oscillation for 5min, and sucking clear solution to obtain a series of standard substance solutions.

Preparation example 3

The preparation example provides a series of quality control sample, wherein the quality control sample is divided into three concentrations of low concentration, medium concentration and high concentration, namely QC-L, QC-M, QC-H, and the specific preparation method is as follows:

QC-L is obtained by diluting the mixed standard S0 solution to 40 times with EDTA anticoagulated venous blood whole blood sample with a predetermined concentration, wherein the volume ratio of the S0 solution to the whole blood is 1:39, uniformly mixing, sucking 50 mu LQC-L whole blood sample each time, spotting the sample to the center of a dry blood spot on a dry blood spot blood sampling card filter paper, and air-drying the sample for 4 hours in a dark place to prepare a QC-L dry blood spot sample, wherein the concentration of various ceramides in the sample is shown in Table 7;

the QC-M is obtained by diluting the mixed standard S0 solution to 100 times by using EDTA anticoagulated venous blood sample with a predetermined concentration, wherein the volume ratio of the S0 solution to the whole blood is 1:99, uniformly mixing, absorbing 50 mu L of QC-L whole blood sample each time, spotting the mixture to the center of a dry blood spot on a dry blood spot blood sampling card filter paper, and air-drying the mixture for 4 hours in a dark place to prepare a QC-M dry blood spot sample, wherein the concentration of various ceramides in the sample is shown in Table 7;

QC-H is obtained by diluting the mixed standard S0 solution to 200 times with EDTA anticoagulated venous blood whole blood sample with a predetermined concentration, wherein the volume ratio of the S0 solution to the whole blood is 1:199, uniformly mixing, sucking 50 mu L QC-L whole blood sample each time, spotting the sample to the center of a dry blood spot on a dry blood spot blood sampling card filter paper, and air-drying the sample for 4 hours in a dark place, wherein the content of various ceramides in the sample is shown in Table 7, and the content unit in Table 7 is mu mol/L.

TABLE 7

Ceramide	QC-L	QC-M	QC-H
				Cer(d18:1/16:0)	0.2	0.4	1
Cer(d18:1/18:0)	0.05	0.1	0.25
				Cer(d18:1/24:0)	1	2	5
Cer(d18:1/24:1)	0.5	1	2.5
				Cer(d18:1/14:0)	0.02	0.04	0.1
Cer(d18:1/20:0)	0.05	0.1	0.25
				Cer(d18:1/22:0)	0.2	0.4	1
Cer(d18:0/16:0)	0.02	0.04	0.1
				Cer(d18:0/18:0)	0.02	0.04	0.1
Cer(d18:0/24:0)	0.1	0.2	0.5
				Cer(d18:0/24:1)	0.05	0.1	0.25
LacCer(d18:1/24:1)	0.2	0.4	1

Example 1

The embodiment provides a method for quantitatively detecting ceramide in human dry blood spots, which comprises the following steps:

1. sample pretreatment:

sample to be measured: sample application of human venous blood whole blood containing EDTA anticoagulant on a blood sampling card to obtain a dried blood spot sample, punching a dried blood sheet with the diameter of 3mm from the dried blood spot sample into a 1.5mL centrifuge tube, adding 600 mu L of the internal standard extraction liquid of preparation example 1, then placing the sample in ultrasonic at 37 ℃ for 20min, finally centrifuging at 15 ℃ and 14800rpm for 10min, sucking 100 mu L of supernatant, and carrying out sample injection;

standard solution: taking 20 mu L of standard sample of each concentration point in preparation example 2 into a 1.5mL centrifuge tube, adding 600 mu L of internal standard extract of preparation example 1, carrying out vortex oscillation for 5min, sucking 100 mu L of supernatant, and carrying out sample injection;

quality control product solution: taking quality control substances QC-L, QC-M and QC-H in preparation example 3 respectively, punching dried blood slices with the diameter of 3mm in a 1.5mL centrifuge tube, adding 600 mu L of the internal standard extract in preparation example 1, then placing the mixture in ultrasonic at 37 ℃ for 20min, and finally centrifuging at 15 ℃ at 14800rpm for 10min, sucking 100 mu L of supernatant respectively, and carrying out sample injection;

2. high performance liquid chromatography tandem mass spectrometry detection:

(1) High performance liquid chromatography conditions:

mobile phase a: an aqueous solution containing 0.1% formic acid (V/V);

Mobile phase B:0.1% formic acid (V/V) in isopropanol/acetonitrile (1:1, V/V);

chromatographic column: kineex XB-C18 (2.6 μm, 3.0x50 mm);

gradient elution is shown in Table 8, the flow rate is 0.5mL/min, the column temperature is 40 ℃, and the sample injection volume is 1 mu L;

TABLE 8

Elution time (min)	Flow rate (mL/min)	A(％)	B(％)
				0	0.5	10	90
0.5	0.5	1	99
				5	0.5	1	99
5.1	0.5	10	90
				7	0.5	10	90

(2) In electrospray ionization mode, positive ion mode scanning is performed by adopting multi-reaction monitoring, and the responses of 12 ceramides and corresponding internal standards are monitored, wherein the ion source parameters are as follows: the capillary voltage is 3.0kV, and the nozzle voltage is 0V; the temperature of the drying gas is 300 ℃, the temperature of the sheath gas is 320 ℃, the flow rate of the drying gas is 7L/min, the flow rate of the sheath gas is 11L/min, the atomization pressure is 45psi, and the mass spectrum parameters of 12 ceramides in the human dry blood spots are shown in Table 2;

3. experimental results:

3 samples of the actual human venous blood dried blood spots were taken, and the concentration of 12 kinds of ceramides in the human dried blood spots was measured according to the method of example 1, as shown in Table 9 below:

TABLE 9

Ceramide	Sample 1 (mu mol/L)	Sample 2 (mu mol/L)	Sample 3 (mu mol/L)
				Cer(d18:1/16:0)	0.839	0.802	1.34
Cer(d18:1/18:0)	0.304	0.23	0.387
				Cer(d18:1/24:0)	3.49	3.15	3.65
Cer(d18:1/24:1)	2.17	2.69	5.86
				Cer(d18:1/14:0)	0.0131	0.0135	0.0108
Cer(d18:1/20:0)	0.180	0.114	0.186
				Cer(d18:1/22:0)	0.721	0.647	0.779
Cer(d18:0/16:0)	0.0887	0.0373	0.0930
				Cer(d18:0/18:0)	0.163	0.0493	0.149
Cer(d18:0/24:0)	0.682	0.394	0.475
				Cer(d18:0/24:1)	0.123	0.0771	0.339
LacCer(d18:1/24:1)	1.83	1.28	3.24

4. Methodological verification

(1) Specialization of

The selective ion flow chromatograms of 12 ceramide compounds in the standard solution are shown in fig. 1, and the selective ion flow chromatograms of 12 ceramide compounds in the human dry blood spot sample are shown in fig. 2, so that the 12 ceramide compounds have consistent chromatographic retention time in the standard solution and the actual sample, no interference is generated near the peaks of each compound, and the specificity is good.

(2) Standard curve

And (3) using an isotope internal calibration method, using MS quantitative analysis 10.0.0 software to set the concentration ratio of the standard substance and the internal standard substance as an X axis and the peak area ratio of the standard substance and the internal standard substance as a Y axis, establishing a calibration curve, and calculating the concentration of the object to be detected. The following linear fitting equation of each concentration range of 12 ceramides has good linearity and a correlation coefficient of more than 0.99, and is shown in Table 10.

Table 10

(3) Inspection of recovery rate by adding marks

The mixed standard S0 solution with known concentration is added into whole blood to prepare a low-medium-high three-concentration whole blood quality control product, then 50 mu L of the whole blood quality control product is sucked and spotted into a blood sampling card filter paper dry blood spot hole to prepare the low-medium-high three-concentration dry blood spot quality control product, the three-concentration dry blood spot quality control product is processed for 6 times in parallel, and the calculated recovery rate is shown in a table 11. The result shows that the standard recovery rate of 12 kinds of ceramide in the human dry blood spot sample is between 85% and 115%, and the standard recovery rate meets the requirements.

TABLE 11

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(4) Precision test

Taking dry blood spot quality control products with various concentrations, repeatedly processing 6 batches in one day for 3 days, quantitatively determining the concentration of 12 kinds of ceramide by an isotope internal standard method, and continuously counting the precision in batches every day for three days; the results of 3 batches within three days and calculating the precision between batches are shown in Table 12. The results show that the results of the in-batch and inter-batch precision tests of 12 ceramides in the dried blood spot sample are all below 15%, which shows that the reproducibility of the test method provided by the invention is good.

Table 12

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Example 2

The difference between the method and the method in the embodiment 1 is that the sample to be detected is different from the internal standard extraction liquid in the pretreatment of the sample, and the volume ratio of isopropanol/dimethyl sulfoxide in the internal standard extraction liquid in the embodiment is 3:1; other materials, amounts and test methods were the same as in example 1.

The measurement results were mainly different from those of example 1 in the peak areas of the respective ceramide compounds, and the peak area comparison is shown in table 13, and in example 2, the peak areas of the respective compounds for quality control of the standard sample solution and the dried blood spots were not greatly different from those of example 1, and the peak areas of the respective compounds were increased or decreased by 10% or less from those of example 1.

TABLE 13

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Example 3

The present embodiment provides a method for quantitatively detecting ceramide in human dry blood spots, which is different from embodiment 1 only in that the ultrasonic time for preparing a sample to be detected and a quality control solution is adjusted to be 10min during sample pretreatment; other materials, amounts and test methods were the same as in example 1.

The results obtained by taking 3 parts of standard solution and dried blood spot sample at each concentration using the measurement methods of example 1 and example 3 are shown in Table 14, and the results show that: the concentration of 12 kinds of ceramide in each dried blood spot among the measurement results of example 3 was slightly lower than that of example 1, but the overall difference was not significant.

TABLE 14

Example 4

The present embodiment provides a method for quantitatively detecting ceramide in human dry blood spots, which is different from embodiment 1 only in that, when a sample is pretreated, the ultrasonic temperature of the sample to be detected and the quality control solution is adjusted to be 50 ℃; other materials, amounts and test methods were the same as in example 1.

The results obtained by taking 3 parts of standard solution and dried blood spot sample at each concentration using the measurement methods of example 1 and example 4 are shown in Table 15, and the results show that: the concentration of 12 kinds of ceramide in each dried blood spot among the measurement results of example 4 was slightly lower than that of example 1, but the overall difference was not significant.

TABLE 15

Comparative example 1

This comparative example provides a method for quantitatively detecting ceramide in human dry blood spots, which differs from example 1 only in that the standard sample is used in the preparation of the standard solution at the time of sample pretreatment, the standard sample used in this comparative example is prepared by adjusting the solvent of the mixed standard S0 solution from isopropanol/dimethylsulfoxide (4:1, V/V) to 30% by volume fraction of aqueous methanol solution, and adjusting the blank substrate from isopropanol/dimethylsulfoxide (4:1, V/V) to 20% by volume fraction of aqueous methanol solution (see conditions used in the documents [ Meikopoulos T, begou O, theodordisis G, et al Ceramides biomarkers determination in quantitative dried blood spots by UHPLC-MS/MS [ J ] Analytica Chimica Acta,2023, 1255:34131 ]); other materials, amounts and test methods were the same as in example 1.

3 samples of the S8 standard were taken, and the results obtained by the measurement methods of example 1 and comparative example 1 are shown in Table 16, and the results show that: the peak areas of 12 kinds of ceramides in the measurement result of comparative example 1 were lower than those of example 1, the difference was large, and the concentration of each of the ceramides was lower by more than 15%.

Table 16

Comparative example 2

This comparative example provides a method for quantitatively detecting ceramide in human dry blood spots, which differs from example 1 only in that, during pretreatment of a sample, an internal standard extraction working solution used in preparation of a sample to be tested is adjusted from a mixed solution of isopropanol/dimethyl sulfoxide (4:1, V/V) to a mixed solution of isopropanol/methanol (1:1, V/V) (see conditions used in documents [ Meikopoulos T, begou O, therodonsis G, et al, ceramides biomarkers determination in quantitative dried blood spots by UHPLC-MS/MS [ J ]. Analytica Chimica Acta,2023, 1255:34131 ]); other materials, amounts and test methods were the same as in example 1.

3 parts of each concentration of the standard solution and each of the dried blood spots were collected, and the concentrations of 12 kinds of ceramides in the dried blood spots were measured under the chromatographic conditions described in comparative example 2. The results of measuring the concentration of 12 kinds of ceramide in each dried blood spot were compared with example 1, and the concentration of each ceramide was generally lower, the lower amplitude was 17% to 24%, and the specific differences are shown in Table 17.

TABLE 17

Comparative example 3

This comparative example provides a method for quantitatively detecting ceramide in human dry blood spots, which differs from example 1 only in that, in the pretreatment of a sample, ultrasonic treatment is carried out for 20 minutes at 37℃and then grinding and crushing extraction is carried out (20 grains of 1.4mm grinding porcelain beads, 30 seconds grinding for 2 cycles) after ultrasonic treatment for 20 minutes at 37℃is carried out (see conditions used in the literature [ Meikopoulos T, begou O, theodoridis G, et al Ceramides biomarkers determination in quantitative dried blood spots by UHPLC-MS/MS [ J ]. Analytica Chimica Acta,2023, 1255:34131 ]); other materials, amounts and test methods were the same as in example 1.

The results obtained by taking 3 parts of standard solution and dried blood spot sample at each concentration using the measurement methods of example 1 and comparative example 3 are shown in table 18, and the results show that: the results of measuring the concentration of 12 kinds of ceramides in each dried blood spot were substantially the same as those in example 1, and the overall difference was not obvious.

TABLE 18

Comparative example 4

This comparative example provides a method for quantitatively detecting ceramide in human dry blood spots, which differs from example 1 only in that the internal standard extract used in the preparation of the sample to be tested differs from that used in the pretreatment of the sample in that isopropanol/dimethyl sulfoxide (4:1, V/V) is adjusted to methanol/acetonitrile/water (80:15:5, V/V) (see conditions used in the references [ Chuang W L, pacheco J, zhang K.A Simple, high-Throughput Method for Analysis of Ceramide, glucosyl cepamide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS [ J ]. Clinical Applications of Mass Spectrometry in Biomolecular Analysis: methods and Protocols,2016:263-272. ]); other materials, amounts and test methods were the same as in example 1.

The results obtained by taking the above S8 standard solutions and quality control QC-L, QC-M, QC-H and measuring the same in example 1 and comparative example 4 are shown in Table 19, and the results show that: the measurement results of the ceramide compound in each quality control product of comparative example 3 are significantly lower in peak area than in example 1, and the overall lower degree ranges from 13 to 30%.

TABLE 19

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Comparative example 5

This comparative example provides a method for quantitatively detecting ceramide in human dry blood spots, which differs from example 1 only in that, when the sample is pre-treated, the sample to be measured is adjusted to the corresponding fingertip dry blood spot of the same individual; other materials, amounts and test methods were the same as in example 1.

The concentration of 12 kinds of ceramide in the fingertip blood dry blood spot was measured by taking 3 parts of each concentration of the standard solution and the fingertip blood dry blood spot sample, respectively, according to the chromatographic conditions described in comparative example 5. The results of measuring the concentration of 12 kinds of ceramide in each dry blood spot are compared with the example 1, the total concentration variation range is within 84% -110%, and the results show that the concentration of each ceramide in the human venous blood dry blood spot and the corresponding fingertip blood dry blood spot has consistency, and the detection results are shown in table 20.

Table 20

The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.

Claims

1. A method for detecting human dry blood spot ceramide, which is characterized by comprising the following steps:

2. The method according to claim 1, wherein the pretreatment method of the sample to be tested is as follows: dissolving a human dry blood spot sample in an internal standard extract, performing ultrasonic extraction and centrifugation, and taking a supernatant to obtain the sample to be tested;

preferably, the solvent of the internal standard extract comprises isopropanol and dimethyl sulfoxide;

preferably, the solvent of the internal standard extract is a combination of isopropanol and dimethyl sulfoxide;

preferably, the volume ratio of isopropanol to dimethyl sulfoxide is (3-5): 1, preferably 4:1;

3. The method according to any one of claims 1 or 2, wherein the 12 ceramides are Cer (d18:1/16:0), cer (d18:1/18:0), cer (d18:1/24:0), cer (d18:1/24:1), cer (d18:1/14:0), cer (d18:1/20:0), cer (d18:1/22:0), cer (d18:0/16:0), cer (d18:0/18:0), cer (d18:0/24:0), cer (d18:0/24:1), lacCer (d18:1/24:1).

4. The method according to any one of claims 1 to 3, wherein the conditions of high performance liquid chromatography in the high performance liquid chromatography tandem mass spectrometry detection are: the chromatographic mobile phase comprises mobile phase A and mobile phase B, and is performed by gradient elution, wherein the flow rate is 0.4-0.6mL/min, preferably 0.5mL/min, the column temperature is 30-45deg.C, preferably 40deg.C, and the sample injection volume is 0.5-5 μL, preferably 1 μL.

5. The method according to any one of claims 1 to 4, wherein the mobile phase a is an aqueous formic acid solution, and the volume fraction of formic acid in the aqueous formic acid solution is 0.05-0.2%, preferably 1%;

6. The method according to any one of claims 1 to 5, wherein the conditions for mass spectrometry in the high performance liquid chromatography tandem mass spectrometry detection are: the ion source is an electrospray ion source, the scanning mode is a positive ion scanning mode of multi-reaction monitoring, the capillary voltage is 2-4kV, preferably 3kV, and the nozzle voltage is 0V; the temperature of the drying gas is 250-350 ℃, preferably 300 ℃, the temperature of the sheath gas is 300-350 ℃, preferably 320 ℃, the flow rate of the drying gas is 6-8L/min, preferably 7L/min, the flow rate of the sheath gas is 10-12L/min, preferably 11L/min, and the pressure of the atomizing gas is 40-50psi, preferably 45psi.

7. The method of any one of claims 1-6, further comprising the step of preparing a standard solution;

8. The method according to any one of claims 1 to 7, wherein the method comprises the steps of:

the volume ratio of the isopropanol to the dimethyl sulfoxide is (3-5): 1;

9. The method according to any one of claims 1 to 8, wherein the method comprises the steps of:

the volume ratio of the isopropanol to the dimethyl sulfoxide is 4:1;

10. A kit for detecting ceramide, characterized in that an internal standard extract adopted by the kit comprises 4 isotope internal standards, wherein the isotope internal standards are Cer (d18:1/16:0) -d7, cer (d18:1/18:0) -d7, cer (d18:1/24:0) -d7 and Cer (d18:1/24:1) -d7 respectively;

Preferably, the solvent of the internal standard extract adopted by the kit is a combination of isopropanol and dimethyl sulfoxide, and the volume ratio of the isopropanol to the dimethyl sulfoxide is 4:1;