CN112964808A

CN112964808A - Biological body fluid total homocysteine detection kit and detection method

Info

Publication number: CN112964808A
Application number: CN201911282478.7A
Authority: CN
Inventors: 张晓哲; 刘欣欣; 刘丹; 程孟春; 赵楠
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2021-06-15

Abstract

The invention discloses a detection kit and detection method for total homocysteine in biological fluids. The kit includes a reducing agent and a protein precipitating agent. The kit of the invention provides convenience for the determination of the total homocysteine content in biological fluids, shortens the detection time, improves the detection efficiency, and improves the detection accuracy. The detection method of the invention has simple pretreatment operation, does not need to derivatize the sample, and improves the repeatability of the detection result; the pretreatment time is short, and the analysis time is only 3 minutes, which meets the requirements of clinical detection on timeliness and economic cost , with clinical application value.

Description

Biological body fluid total homocysteine detection kit and detection method

Technical Field

The invention belongs to the technical field of analytical chemistry and clinical examination, and particularly relates to a biological body fluid total homocysteine detection kit and a detection method.

Background

Homocysteine (Hcy) is a sulfhydryl-containing amino acid and an intermediate metabolite of methionine in the human body. Hcy has been considered by the international medical community as an independent risk factor for the onset of cardiovascular and cerebrovascular diseases such as atherosclerosis and thrombosis, coronary heart disease, etc., and its clinical significance has been considered to exceed that of cholesterol. In addition, elevated Hcy is a sensitive biomarker reflecting human folate and vitamin B12 deficiency.

The Hcy detection is widely used for clinical diagnosis and treatment of cardiovascular and cerebrovascular diseases, and plays an important role in disease prevention and diagnosis and treatment. The conventional detection methods for clinical Hcy include biochemical method, immunological method and liquid chromatography, and usually use imported special instruments or reagents. Due to the fact that the molecular weight of Hcy is small, detection in a biological sample is subject to objective technical bottlenecks such as specificity, the existing Hcy detection method in the market has the problems of low accuracy and poor comparability, and a reliable detection result is difficult to provide for clinical diagnosis.

The liquid chromatography tandem mass spectrometry (LC-MS/MS), which is regarded as a gold standard method for the quantification of small molecular compounds by the clinical medical inspection community, has the advantages of good accuracy, precision and specificity and high flux, and the clinical mass spectrometry detection method of related items has been established in recent years and gradually becomes a powerful supplement of the traditional detection method. For example, Chinese patent CN201910644151.3, liquid chromatography-mass spectrometry detection is carried out after solid-liquid extraction of a sample; performing liquid chromatography-mass spectrometry detection on a sample after derivatization treatment in Chinese patent CN201710447034.9 and Chinese patent CN 201811044060.8; chinese patent CN201810128855.0 and Chinese patent CN201010581678.5, the sample is internally marked by cysteine isotope markers, and then liquid chromatography-mass spectrometry detection is carried out. However, the above method is complicated in process and long in pretreatment time, and is not favorable for rapidly obtaining the detection result; or the system deviation correction still exists in the detection process, and the detection accuracy is not enough.

Disclosure of Invention

In order to solve the technical problems, the invention provides a biological body fluid total homocysteine detection kit and a detection method, which are used for detecting a pretreated biological fluid sample containing an isotope internal standard substance by liquid chromatography-mass spectrometry, comparing the obtained detection data with a standard curve, and calculating to obtain the content of the biological fluid total homocysteine, so that the total homocysteine content can be quickly and accurately obtained.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

on one hand, the invention provides a total homocysteine detection kit for biological body fluid, which comprises a reducing agent and a protein precipitator.

Optionally, the reducing agent comprises at least one of dithiothreitol, mercaptoethanol;

the protein precipitant comprises at least one of trichloroacetic acid, trifluoroacetic acid, acetonitrile and methanol.

Optionally, the reducing agent is 0.01-10M aqueous solution of dithiothreitol;

the protein precipitant is at least one of trichloroacetic acid solution, trifluoroacetic acid solution, acetonitrile and methanol.

Preferably, the trichloroacetic acid solution is 10 to 30 mass percent of trichloroacetic acid aqueous solution; the trifluoroacetic acid solution is 10 to 30 mass percent of trifluoroacetic acid aqueous solution.

Optionally, the kit further comprises an isotope internal standard substance and/or an isotope internal standard substance solution;

the internal isotope standard substance comprises homocystine¹³A C label or a deuterated label;

the solvent of the isotope internal standard substance solution is 0.01-10M acid solution;

the concentration of the isotope internal standard substance solution is 5-150 mu mol/L;

preferably, the isotopic internal standard is d 8-homocystine.

Specifically, the acidic solution may be any one selected from a hydrochloric acid solution, an aqueous formic acid solution, and an aqueous acetic acid solution. The concentration of the acidic solution is 0.01-10M.

The acidic aqueous solution is prepared by hydrochloric acid in the embodiment of the invention, and the preparation of the isotope internal standard working solution specifically comprises the following steps: adding 0.01-10M hydrochloric acid into the isotope internal standard substance to prepare stock solution with a certain concentration, and then diluting the stock solution to the required concentration by using deionized water.

Optionally, the kit further comprises:

at least one of standard substance/standard substance working solution, blank matrix, eluent and quality control substance/quality control substance working solution.

Optionally, the blank matrix is 0.05 wt% -5 wt% of calf serum solution, and the solvent is a buffer salt solution with pH of 7;

the eluent comprises: eluent A and eluent B;

the eluent A is a volatile acid solution with the volume fraction of 0.01-2%; the eluent B is methanol solution containing volatile acid with volume fraction of 0.01-2%; the volatile acid comprises at least one of formic acid, acetic acid and trifluoroacetic acid.

The standard working solution comprises homocysteine standard solutions with at least 4 different concentrations;

the quality control product working solution comprises at least 2 homocysteine solutions with different concentrations;

the minimum concentration of the quality control working solution is higher than that of the standard working solution;

the highest concentration of the quality control working solution is lower than that of the standard working solution.

Specifically, in the embodiment of the invention, the solvent of the calf serum solution is 1-100 mM ammonium acetate aqueous solution, and the pH of the calf serum solution is adjusted to 7 by using ammonia water.

Specifically, the concentration of the working solution of the quality control product is divided into low, medium and high 3 concentrations: QC (L), QC (M), QC (H). The quality control product working solution is used for ensuring the stability and reliability of the detection system. In the embodiment of the invention, the quality control product is a standard product.

The quality control product working solution is the standard product solution with different concentrations, and the obtained quality control product detection data is compared with the standard curve, so that whether the standard curve is accurate or not can be detected, and a basis is provided for a final detection result.

Meanwhile, the quality control product working solution is subjected to liquid phase-mass spectrometry detection at different periods of liquid phase-mass spectrometry detection of the batch organism liquid sample, whether detection data change due to instrument reasons or not in the whole detection process can be detected, and therefore basis is provided for the final detection result.

Optionally, the kit comprises:

1) standard working solution: homocysteine solution with 4 concentrations;

2) quality control product working solution: homocysteine solution with 3 concentrations;

3) internal standard working solution: preparing an isotope internal standard substance into 1mg/mL stock solution by using 0.01-10M hydrochloric acid, and diluting to 5-100 mu mol/L by using water;

4) blank matrix: 0.05 wt% -5 wt% of calf serum solution, wherein the solvent is a buffer salt solution with the pH value of 7;

5) reducing agent: 0.01-10M aqueous solution of dithiothreitol;

6) protein precipitant: at least one of trichloroacetic acid solution, trifluoroacetic acid solution, acetonitrile and methanol;

7) eluent: the eluent A is a volatile acid solution with the volume fraction of 0.01-2%; the eluent B is methanol solution with volatile acid with the volume fraction of 0.01-2%.

In particular to a single organism liquid sample detection kit,

the volume ratio of the internal standard working solution to the quality control working solution is 1: 0.05 to 10;

the volume ratio of the reducing agent to the standard substance working solution to the quality control substance working solution is 3-30: 1

The volume ratio of the protein precipitant to the standard substance working solution and the quality control substance working solution is 3-30: 1;

the volume ratio of the blank substrate to the standard substance working solution to the quality control substance working solution is 3-30: 1: 1.

if the kit is used for detecting a plurality of biological fluid samples, the reducing agent, the protein precipitator and the internal standard working solution are increased according to the number of the biological fluid samples.

On the other hand, the invention provides a method for detecting total homocysteine in biological body fluid, which adopts any one of the above biological body fluid total homocysteine detection kits to detect, and the method at least comprises the following steps:

pretreating a biological fluid sample containing homocysteine to obtain a sample to be detected;

detecting and analyzing a sample to be detected to obtain a detection result of the total homocysteine in the biological body fluid;

wherein the pre-processing comprises: reduction treatment and protein precipitation treatment.

Optionally, the detection method includes:

adding an internal standard working solution and a blank matrix into the standard working solution and the quality control working solution to obtain a standard working solution containing an internal standard substance and a quality control working solution containing an internal standard substance;

adding the internal standard working solution into the biological fluid sample to obtain a biological fluid sample containing an internal standard substance;

sequentially adding a reducing agent and a protein precipitator into a standard substance working solution containing an internal standard substance, a quality control substance working solution containing the internal standard substance and a biological fluid sample containing the internal standard substance for pretreatment to respectively obtain a standard sample to be detected, a sample to be detected and a quality control substance to be detected;

respectively carrying out liquid phase-mass spectrum detection on a standard sample to be detected, a sample to be detected and a quality control product to be detected to obtain standard sample detection data, sample detection data to be detected and quality control product detection data;

preparing a standard curve by adopting standard sample detection data;

and comparing the sample detection data with the standard curve, and calculating to obtain the total homocysteine content of the biological body fluid.

Wherein, adopt standard sample testing data preparation standard curve, specifically do:

taking the concentration of the standard substance as an x axis, taking the peak area ratio of the standard substance obtained by mass spectrometry and the isotope internal standard substance as a y axis, and drawing a standard curve; the linear range of the standard curve is 1-100 mu mol/L.

Optionally, the volume ratio of the biological fluid sample to the internal standard working fluid is 1-15: 1;

the volume ratio of the reducing agent to the biological fluid sample is 1-3: 1;

the volume ratio of the reducing agent to the standard substance working solution to the quality control substance working solution is 3-30: 1;

the volume ratio of the protein precipitant to the biological fluid sample is 1-3: 1;

the volume ratio of the blank substrate to the standard substance working solution to the quality control substance working solution is 3-30: 1.

specifically, the volume ratio of the biological fluid sample to the internal standard working fluid is independently selected from 1: 1. 3: 1. 5: 1. 10: 1. 15: 1.

specifically, the volume ratio of the standard working solution/quality control product working solution to the internal standard working solution is independently selected from 0.05: 1. 0.5: 1. 1: 1. 5: 1. 10: 1.

specifically, the volume ratio of the reducing agent to the volume ratio of the biological fluid sample is independently selected from 1: 1. 1.5: 1. 2: 1. 2.5: 1. 3: 1.

specifically, the volume ratio of the reducing agent to the standard working solution/quality control product working solution is independently selected from 3: 1. 10: 1. 15: 1. 20: 1. 30: 1.

specifically, the volume ratio of the protein precipitant to the biological fluid sample is independently selected from the group consisting of 1: 1. 1.5: 1. 2: 1. 2.5: 1. 3: 1.

specifically, the volume ratio of the protein precipitator to the standard substance working solution/quality control substance working solution is independently selected from 3: 1. 10: 1. 15: 1. 20: 1. 30: 1.

specifically, the volume ratio of the blank matrix to the standard working solution/quality control product working solution is independently selected from 3: 1. 10: 1. 15: 1. 20: 1. 30: 1.

optionally, the biological fluid sample comprises at least one of plasma, serum, urine, and saliva.

The invention has the beneficial effects that:

1. the kit provided by the invention provides convenience for measuring the total homocysteine content in the biological body fluid, shortens the detection time, improves the detection efficiency and improves the detection accuracy.

2. The detection method comprises the steps of firstly preprocessing a biological fluid sample containing an isotope internal standard substance, and carrying out liquid chromatography-mass spectrometry detection to obtain detection data of a sample to be detected; then the detection data of the sample to be detected is compared with the standard curve, the content of the total homocysteine in the biological body fluid is calculated, the sample does not need to be subjected to derivatization reaction or solid-liquid extraction, the pretreatment time is shortened, the repeatability of the detection result is improved, and the detection cost is reduced.

3. The detection method adopts the isotope label of homocystine as the isotope internal label, and homocystine is reduced to cysteine in the pretreatment process, thereby not only correcting the pretreatment process and the deviation of instruments, but also correcting the reduction efficiency of the reducing agent, and further improving the accuracy of the detection result.

4. In the detection method, a buffer system with the pH of 7 is used as a blank matrix solvent in the standard curve preparation, the isotope internal standard working solution prepared by inorganic acid is subjected to buffering capacity, the standard solution is a real condition closer to biological body fluid, and the inaccurate measurement result caused by the influence of acid-base difference on mass spectrum signals can be avoided.

5. The linear range of the standard curve adopted by the detection method covers the range of the reference value, the detection limit and the recovery rate both meet the clinical detection requirement, the pretreatment process is simple, the time consumption is short, the analysis time is only 3 minutes, the flux is high, the cost is low, and the requirement of the clinical homocysteine liquid quality detection can be met.

Drawings

FIG. 1 is an extracted ion flow diagram of homocysteine and its internal isotope label at the lowest concentration point of 5 μmol/L in the standard curve in the example, wherein the left figure is the extracted ion flow diagram of homocysteine, and the right figure is the extracted ion flow diagram of the internal isotope label;

FIG. 2 is an extracted ion flow graph of homocysteine and its isotope internal standard substance of No. 37 actually measured sample in example, wherein the left graph is the extracted ion flow graph of homocysteine, and the right graph is the extracted ion flow graph of isotope internal standard substance;

FIG. 3 is a standard graph of homocysteine obtained in example;

FIG. 4 is a histogram of homocysteine concentration distribution in 84 serum samples of example.

Detailed Description

The invention is further illustrated with reference to the following figures and specific examples.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

Examples

The embodiment of the invention detects the total homocysteine content of 84 serum samples.

1. Sample collection

Samples were collected in the harbor hospital of Dalian city, and all volunteers sampled blood on an empty stomach at 7: 00-8: 00 am. Collecting whole blood with a vacuum blood collection tube without anticoagulant, standing for coagulation, centrifuging at 3000rpm for ten minutes, taking upper layer serum, subpackaging, and storing at-80 ℃ for later use.

2. Kit composition

1) Standard solution: the 4 concentrations were: series of standard solutions of 50 mu mol/L, 150 mu mol/L, 200 mu mol/L and 450 mu mol/L;

2) quality control product working solution: the concentrations of QC (L), QC (M), QC (H) and QC (H) are respectively 100 mu mol/L, 250 mu mol/L and 400 mu mol/L.

3) Internal standard working solution: d 8-homocystine hydrochloride aqueous solution with a concentration of 25. mu. mol/L.

The preparation method comprises the following steps: d 8-homocystine was prepared as a 1mg/mL stock solution with 0.1M hydrochloric acid, and the stock solution was diluted to 25. mu. mol/L with deionized water.

4) Blank matrix: 2.5 wt% calf serum solution in 50mM ammonium acetate in water (pH 7 with ammonia);

5) reducing agent: 0.3M aqueous dithiothreitol solution;

6) protein precipitant: 15 wt% aqueous trichloroacetic acid.

3. Sample pretreatment

1) And (3) standard substance: adding 5 mul of series standard working solution into 45 mul of blank substrate, adding 10 mul of internal standard working solution and 50 mul of reducing agent, whirling for 60 seconds, and standing for 30 minutes at room temperature; then adding 50 mu L of protein precipitator, whirling for 1 minute, centrifuging for 5 minutes at 15000rpm, and taking supernatant to obtain a series of standard samples;

2) quality control product: adding 5 mul of quality control working solution into 45 mul of blank matrix, adding 10 mul of internal standard working solution and 50 mul of reducing agent, whirling for 60 seconds, and standing for 30 minutes at room temperature; then adding 50 mu L of protein precipitator, whirling for 1 minute, centrifuging for 5 minutes at 15000rpm, and taking supernatant to obtain a quality control sample;

3) serum samples: adding 10 mul of internal standard working solution and 50 mul of reducing agent into 50 mul of serum sample, whirling for 60 seconds, and standing for 30 minutes at room temperature; then 50 μ L of protein precipitant is added, vortex for 1 minute, and after centrifugation for 5 minutes at 15000rpm, the supernatant is taken to obtain the sample to be measured.

4. High performance liquid chromatography tandem mass spectrometry

Respectively carrying out high performance liquid chromatography tandem mass spectrometry on the obtained series of standard samples, quality control samples and samples to be detected:

the apparatus used was ABI Q TRAP 5500(AB SCIEX);

high performance liquid chromatography conditions: the mobile phase A is a formic acid solution with the volume fraction of 0.2 percent; the mobile phase B is methanol (containing 0.2% by volume of formic acid); the chromatographic column is as follows: InfinityLabPoroshell120EC-C18,50mm × 2.1mm,2.7 μm; the flow rate is 0.3mL/min, the column temperature is 35 ℃, and the sample injection volume is 5 mu L; elution gradient 10% B by volume;

the mass spectrum conditions are as follows: electrospray ionization source, positive ion mode, scanning mode using Multiple Reaction Monitoring (MRM); air curtain gas (CUR) is 20 kPa; collision gas (CAD) is Medium; the spray voltage (IS) IS 5000V; the heating gas temperature is 500 ℃; quantitative ion pairs, declustering voltage (DP) and Collision Energy (CE) for homocysteine and d 4-homocysteine are shown in Table 1:

TABLE 1

5. Results

FIG. 1 is an extracted ion-flow graph of homocysteine and its internal isotope standard detected by mass spectrometry at the lowest concentration point of the standard curve of 5. mu. mol/L (shown in the left and right panels, respectively). The extracted ion flow graph of the lowest concentration point of the standard curve reflects whether the detection limit (namely the lowest point of the standard curve) meets the requirement, and the signal-to-noise ratio (S/N) requirement of the detection limit is more than 10. As can be seen from fig. 1, the signal-to-noise ratio is much greater than 10.

FIG. 2 is an extracted ion-flow diagram of homocysteine and its internal isotope standard from No. 37 actual measurement samples detected by mass spectrometry (shown in the left and right panels, respectively). By randomly extracting the detection data of the No. 37 actually measured sample, it can be seen that the mass spectrum data of homocysteine and its internal standard in the actual sample correspond to the mass spectrum data of FIG. 1, indicating that the standard curve can be used for calculating the concentration of the actually measured sample.

It can be seen from FIGS. 1 and 2 that the time to peak for homocysteine is only 1.1 min, so the entire assay can be completed in 3 min with high flux; the shortened analysis time greatly reduces the cost required for detection.

Taking the concentration of the standard substance as an x axis, taking the peak area ratio of the standard substance and the isotope internal standard substance as a y axis to draw a standard curve, and calculating the total homocysteine content;

before the detection sequence of the sample to be detected begins and after the detection of all the sample sequences to be detected is completed, a standard curve (a standard curve 1 and a standard curve 2) is obtained by respective detection, and the standard curve is fitted to calculate the concentration of homocysteine in the sample.

The standard curve obtained by fitting is shown in fig. 3: y is 0.0803x +0.0297(r is 0.9998), homocysteine has good linearity in the concentration range of 5-45 mu mol/L, and the recovery rate of each point of the standard curve is shown in tables 2 and 3.

And substituting the quality control detection data into the standard curve, and calculating the recovery rate, wherein the recovery rate is in the range of 80-120%, and the detection system is stable and the result is reliable.

TABLE 2 standard curve 1 normalized recovery (%)

TABLE 3 Standard Curve 2 normalized recovery (%)

As can be seen from the table above, the recovery rates of the sample to be detected before and after the sequence detection are within the error range, which indicates that the detection process is stable.

And respectively comparing the homocysteine data of 84 serum samples detected by mass spectrometry with a standard curve, and calculating to obtain the homocysteine content of the 84 serum samples.

The distribution histogram of homocysteine concentration in 84 serum samples is shown in FIG. 4, and homocysteine concentration in 84 volunteers is basically in accordance with normal distribution, wherein the concentration of homocysteine in 4 volunteers is higher than the reference value range (5-15. mu. mol/L). The result of the homocysteine concentration detected by the detection method is accurate.

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Claims

1. A total homocysteine detection kit for biological body fluid is characterized by comprising a reducing agent and a protein precipitator.

2. The biological fluid total homocysteine detection kit according to claim 1 characterised in that said reducing agent comprises at least one of dithiothreitol, mercaptoethanol;

3. The kit for detecting the total homocysteine in biological body fluid according to claim 1, wherein said reducing agent is 0.01-10M aqueous solution of dithiothreitol;

the protein precipitator is at least one of trichloroacetic acid solution, trifluoroacetic acid solution, acetonitrile and methanol.

4. The kit for detecting the total homocysteine in biological fluid according to claim 1, characterized in that the kit further comprises an isotope internal standard substance and/or an isotope internal standard substance solution;

the isotope internal standard substance comprises homocystine¹³A C label or a deuterated label;

preferably, the isotopic internal standard is d 8-homocystine.

5. The kit for detecting total homocysteine in biological fluids according to claim 1, characterized in that said kit further comprises:

6. The biological fluid total homocysteine detection kit according to claim 5 characterised in that said blank matrix is a 0.05-5 wt% calf serum solution, the solvent is a buffered saline solution with pH 7;

the eluent comprises: eluent A and eluent B;

the eluent A is a volatile acid solution with the volume fraction of 0.01-2%; the eluent B is a methanol solution containing volatile acid with the volume fraction of 0.01-2%; the volatile acid comprises at least one of formic acid, acetic acid and trifluoroacetic acid.

7. The kit for detecting total homocysteine in biological fluids according to claim 1 characterized in that said kit comprises:

1) standard working solution: homocysteine solution with 4 concentrations;

3) internal standard working solution: preparing an isotope internal standard into 1mg/mL stock solution by using 0.01-10M hydrochloric acid, and diluting the stock solution to 5-100 mu mol/L by using water;

5) reducing agent: 0.01-10M aqueous solution of dithiothreitol;

8. A method for detecting total homocysteine in biological fluids, which is characterized in that the method adopts the biological fluid total homocysteine detection kit of any one of claims 1-7 to detect, and the method at least comprises the following steps:

detecting and analyzing the sample to be detected to obtain a detection result of the total homocysteine in the biological body fluid;

9. The method of detecting total homocysteine in biological fluids according to claim 8 wherein said method comprises:

preparing a standard curve by using the detection data of the standard sample;

and comparing the sample detection data with a standard curve, and calculating to obtain the total homocysteine content of the biological body fluid.

10. The method according to claim 9, wherein the total homocysteine in a biological fluid is detected,

the volume ratio of the biological fluid sample to the internal standard working solution is 1-15: 1;

the volume ratio of the internal standard working solution to the quality control product working solution is 1: 0.05 to 10;

the volume ratio of the protein precipitator to the standard substance working solution to the quality control substance working solution is 3-30: 1;