CN112345660A - Kit and method for determining activity of human plasma sample renin - Google Patents

Kit and method for determining activity of human plasma sample renin Download PDF

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CN112345660A
CN112345660A CN202011044849.0A CN202011044849A CN112345660A CN 112345660 A CN112345660 A CN 112345660A CN 202011044849 A CN202011044849 A CN 202011044849A CN 112345660 A CN112345660 A CN 112345660A
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kit
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angiotensin
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刘鹏云
马金飞
曲亮
沈凌晓
刘华芬
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Hangzhou Calibra Diagnostics Co ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The invention relates to a kit and a method for determining activity of human plasma sample renin, wherein the kit comprises (1) a standard curve working solution preparation kit; (2) preparing a kit by using an internal standard working solution; (3) preparing a kit by using a sample pretreatment buffer solution; (4) configuring a kit for a quality control sample; (5) a reaction stop solution kit; (6) liquid chromatography gradient eluent. The kit and the method have the advantages of high efficiency, low cost, simple and convenient operation, high accuracy, high sensitivity and the like.

Description

Kit and method for determining activity of human plasma sample renin
Technical Field
The invention relates to the technical field of medical treatment, and mainly relates to a kit and a method for determining activity of human plasma sample renin.
Background
Plasma Renin Activity (PRA) is an index that measures the efficiency of angiotensin I production in peripheral blood under the catalysis of Renin, and is of great significance in the differential diagnosis of hypertension caused by the Renin Angiotensin Aldosterone System (RAAS), with significantly increased Activity in hypertensive patients caused by renal artery stenosis and decreased Activity in patients with secondary aldosteronism. Renin activity is also an important measure of the effectiveness of alternative hormone therapy in patients with inadequate adrenal function, which is normal in the case of patients with adequate hormone replacement therapy, and elevated in the case of patients with inadequate replacement therapy. Renin activity is also the most important index for screening Primary Aldosteronism (PA). Currently, immunoassay techniques are commonly used to detect Plasma Renin Activity (PRA) by measuring the rate of angiotensin I production or Direct Renin Concentration (DRC) by directly measuring plasma Renin Concentration by radioimmunoassay. Currently, the direct renin concentration detection method is continuously improving, the measurement results obtained by different methods or reagents are very different, and large-scale clinical tests or population researches are required to be carried out, so that the renin activity detection can be replaced by the first-line detection method. The activity of renin is measured by an immunoassay, so that the inherent defects of the immunoassay exist, the specificity of the method is poor, cross-coupling reaction exists, the sensitivity is low, the linear range is narrow, the pretreatment process is complex, and the detection requirement of the PRA of the patient with the actual clinical protoaldosis at a lower level cannot be met. However, the existing high performance liquid chromatography mass spectrometry detection method needs complex pretreatment Solid Phase Extraction (SPE) operation or needs to additionally add or modify a two-dimensional liquid chromatography system, the method is complex, the cost of detection instruments and consumables is high, the technical difficulty is high, the method is not suitable for the requirement of clinical large-scale sample treatment, and the popularization difficulty is high. In addition, the result of the renin activity assay is easily affected by experimental conditions, such as buffer salt concentration, pH, plasma matrix, incubation temperature, etc., and detection deviation between rooms and rooms is often encountered, so that how to control the accuracy and reproducibility of the result and data is also an important difficult problem in the existing renin activity assay. Therefore, an improved high performance liquid chromatography tandem mass spectrometry method for detecting activity of human plasma sample renin and a complete quality control system matched with the method and capable of covering the whole process from pretreatment to instrument detection are urgently needed, so that the method has the advantages of high efficiency, high flux, low cost, simplicity and convenience in operation, high accuracy and high sensitivity, and meanwhile, the accuracy and the result reproducibility of the whole detection process are effectively monitored, the accuracy of actual clinical renin activity detection is ensured, and the method is convenient for clinical popularization and application.
Disclosure of Invention
The invention aims to provide an improved high performance liquid chromatography tandem mass spectrometry kit and an improved high performance liquid chromatography tandem mass spectrometry method for detecting activity of human plasma sample renin aiming at the defects of the prior art. In order to realize the purpose of the invention, the following technical scheme is adopted:
the invention relates to a kit for determining activity of human plasma sample renin, which is a high performance liquid chromatography tandem mass spectrometry kit and comprises the following components:
(1) preparing a kit for a standard curve working solution: it comprises angiotensin I standard substance, 40-60% methanol water solution by volume, 1-8% bovine serum albumin BSA water solution by mass;
(2) preparing an internal standard working solution kit: which comprises angiotensin I isotope internal standard ANG I-13C15N, 40-60 volume percent methanol aqueous solution, 50-100mg/mL zinc sulfate aqueous solution and methanol;
(3) preparing a kit from a sample pretreatment buffer solution: benzyl sulfonyl fluoride methanol solution with the concentration of 50-200mM, 0.5-2mol/L Tris and 0.1-0.5mol/L EDTA buffer solution with the pH value of 5.45-5.50;
(4) preparing a quality control sample preparation kit: it comprises EDTA anticoagulated plasma sample and angiotensin I standard solution;
(5) reaction stop solution kit: formic acid;
(6) liquid chromatography gradient eluent: the volume ratio of the formic acid aqueous solution with the volume ratio of 0.05-0.2% to the methanol solution is 90-5% in the gradient elution procedure: 10 to 95 percent.
In a preferred embodiment of the invention, the concentration of angiotensin I in the standard curve working fluid is between 0.2ng/mL and 30 ng/mL. By controlling the concentration range of angiotensin I in the standard curve working solution, a good linear relationship can be obtained.
In a preferred embodiment of the present invention, the concentration of the aqueous BSA solution in the standard curve working solution preparation kit is 1 to 8% by mass. The present invention facilitates the simulation of biological matrices by controlling BSA within a preferred range.
In a preferred embodiment of the invention, the quality control sample kit is stored at-20 to-80 ℃.
The invention also relates to a human plasma sample pretreatment process for determining renin activity, which comprises the following steps of treating the human plasma sample by using the kit:
(1) preparing a standard stock solution and a standard curve working solution by adopting a standard curve working solution preparation kit;
(2) preparing an internal standard stock solution and an internal standard working solution by adopting an internal standard working solution preparation kit;
(3) preparing a quality control sample by adopting a quality control sample preparation kit, wherein the quality control sample comprises an angiotensin I low-concentration quality control sample and a high-concentration quality control sample, and is obtained by additionally adding angiotensin I standard stock solutions or standard solutions with different concentrations or volumes in the step (1) into an EDTA anticoagulated blood plasma sample; the quality control samples comprise low-activity quality control samples and high-activity quality control samples with different renin activities, wherein the high-activity plasma samples are plasma samples which are collected freshly, and the low-activity plasma samples are plasma samples which are treated by placing for 3-7 days at the temperature of 2-8 ℃;
(4) preparing a sample pretreatment buffer solution by adopting a sample pretreatment buffer solution preparation kit, and placing the sample pretreatment buffer solution in a porous plate;
(5) taking 2 parts of a sample to be tested in parallel, placing the sample into sample holes of 2 multi-hole plates, stopping the incubation of one multi-hole plate for 2-4 hours by adopting a reaction stopping solution formic acid, adding an internal standard working solution prepared by an internal standard working solution preparation kit to obtain an incubated sample, and centrifuging to obtain a supernatant; and after the other porous plate is uniformly mixed, immediately stopping by adopting a reaction stopping solution formic acid, adding an internal standard working solution prepared by an internal standard working solution preparation kit to obtain an unincubated sample, and centrifuging to obtain a supernatant.
The invention also relates to a method for determining the activity of human plasma sample renin, which comprises the human plasma sample pretreatment process and the detection of the incubated sample and the non-incubated sample respectively by adopting a liquid chromatography tandem mass spectrometry method.
In a preferred embodiment of the invention, the liquid chromatography adopts gradient elution, the chromatographic column is a C18 column, the mobile phase A is 0.05-0.2 vol% formic acid water solution, the mobile phase B is 0.05-0.2 vol% formic acid methanol solution, and the volume ratio of the mobile phase A to the mobile phase B is 90-5%: 10 to 95 percent.
In a preferred embodiment of the invention, the gradient elution procedure is as follows:
Figure BDA0002707668740000031
Figure BDA0002707668740000041
in a preferred embodiment of the invention, the mass spectrometer is a triple quadrupole mass spectrometer and mass spectrometric detection is performed using an electrospray ion source and positive ion mode (ESI +) and Multiple Reaction Monitoring (MRM) mode.
In a preferred embodiment of the invention, the renin activity is (post-incubation sample angiotensin I concentration-non-incubated sample angiotensin I concentration)/incubation time.
In a preferred embodiment of the invention, the quality control of the detection error is to evaluate the accuracy of the detection result by comparing the deviation between the detection index value of the quality control sample and the theoretical target value, wherein the detection index comprises the concentration of angiotensin I in the quality control sample after the non-incubation and the renin activity level of the quality control sample.
The invention has the advantages that (1) the pretreatment operation is simple, the protein precipitation method is adopted, the operations such as concentration, blow drying, redissolution and the like are avoided, the requirement on operators is lower, and the manpower and the working time are saved; (2) the principle that the ESI detection mode can be used for detecting multi-charge charged particles is directly utilized, direct detection can be carried out on the multi-charge charged particles, and digestion and hydrolysis of angiotensin I are not needed, so that extra and unnecessary labor is avoided; (3) the pretreatment equipment is conventionally configured in a laboratory, the reagent is low in cost and easy to purchase, and the project is easy to develop in clinic; (4) the operation is simple and convenient, the conventional treatment can be carried out on a 96-well plate, and the high-flux detection and the automation of a detection mode are convenient to realize; (5) the quality control system is provided with two sets of quality control systems which are not incubated and are incubated, so that the accuracy and the repeatability of the detection and pretreatment processes (particularly the incubation process) of the sample instrument are ensured, the problems are conveniently found, and the correction is carried out in time.
Particularly, the kit and the method have the advantages of high efficiency, low cost, simple and convenient operation, high accuracy, high sensitivity and the like.
Drawings
Figure 1 chromatogram of the lower limit of quantitation for angiotensin I detection: (A is AngI, B is an AngI internal standard);
FIG. 2 is a linear regression graph of a standard curve created by the internal standard method.
The specific implementation mode is as follows:
the present invention is further illustrated by the following examples, which are intended to be illustrative of the present invention and are not to be construed as limiting the invention, and any modifications and variations of the present invention are intended to fall within the spirit and scope of the appended claims.
Example 1: analysis method for detecting activity of human plasma sample renin by liquid chromatography tandem mass spectrometry technology
Firstly, preparing a solution
Preparation of stock solution
Angiotensin I primary stock: accurately weighing 1mg of angiotensin I standard substance, dissolving in 10mL of 50% methanol aqueous solution, and preparing into 100 μ g/mL primary stock solution.
Preparation of angiotensin I secondary stock solution: 100 μ L of the primary stock solution of angiotensin I was aspirated and diluted with 900 μ L of 50% aqueous methanol to give a 10 μ g/mL secondary stock solution.
Note: the stock solutions prepared above were all stored in a refrigerator at-20 ℃.
Preparing a standard curve working solution:
(1) draw 100 μ L of the secondary stock of angiotensin I and dilute it with 900 μ L of 50% aqueous methanol to a 1 μ g/mL tertiary stock.
Table 1: preparation of three-stage stock solution
Figure BDA0002707668740000051
(2) The tertiary stock solution was diluted with 1% BSA aqueous solution to form a series of standard curves (SD)1, 2, 3, 4, 5, 6, 7, the specific formulation of which is shown in the table below.
Table 2: standard curve preparing table
Figure BDA0002707668740000052
Figure BDA0002707668740000061
Note: storing the prepared stock solution in a refrigerator at the temperature of-80 ℃; the standard curve working solution is packaged into small parts, the temporarily unused parts are stored in a refrigerator at minus 80 ℃, and the working solution is stored in the refrigerator at 4 ℃.
Preparing an internal standard stock solution:
(1) angiotensin I isotope internal standard primary stock solution: adding 0.1mg of angiotensin I isotope internal standard (ANG I-13C15N) was added to a sample bottle containing 1mL of 50% aqueous methanol to prepare a first-order stock solution of 100. mu.g/mL.
(2) Angiotensin I isotope internal standard secondary stock solution: precisely sucking 10 mu L of primary stock solution of the angiotensin I isotope internal standard with the concentration of 100 mu g/mL, adding 990 mu L of 50% methanol aqueous solution into the primary stock solution, and diluting the primary stock solution into secondary stock solution of the angiotensin I isotope internal standard with the concentration of 1 mu g/mL.
Preparing an internal standard working solution:
precisely sucking 50 mu L of 1 mu g/mL angiotensin I internal standard secondary stock solution and 1mL of 89mg/mL zinc sulfate (ZnSO4.7H2O) aqueous solution, placing the two solutions in a 10mL centrifuge tube, adding 8950 mu L of methanol into the centrifuge tube, and uniformly mixing to obtain 5ng/mL angiotensin I internal standard working solution.
Preparing a quality control sample:
mixing fresh EDTA anticoagulated human plasma sample as background, and subpackaging into 2 parts, wherein the mixed plasma A is stored at-20 deg.C, and the other part of mixed plasma B is stored at-20 deg.C after being placed at 2-8 deg.C for 7 days.
Placing 10mL of mixed plasma A on a frozen ice box or an ice bag, adding 5 mu L of angiotensin I tertiary stock solution, carrying out vortex for 30 seconds, and carrying out quick subpackage with the volume of about 250 mu L to obtain low-concentration quality control (L);
and (3) placing 10mL of mixed plasma B on a frozen ice box or an ice bag, adding 50 mu L of angiotensin I tertiary stock solution, performing vortex for 30 seconds, and quickly subpackaging the mixture to obtain the high-concentration quality control H with the volume of about 250 mu L.
Preparation of other solutions:
ZnSO4preparing a solution: weighing 3.56 g of ZnSO4.7H2Dissolving O in 40mL of water to prepare 89mg/mL ZnSO4.7H2And (4) O aqueous solution. The volume can be proportionally adjusted according to the requirement, and the product is stored at room temperature.
Preparation of phenylmethylsulfonyl fluoride (PMSF) solution: 0.174g of PMSF was added to 10mL of methanol to prepare a 100mM solution of PMSF in methanol under storage conditions of 2 to 8 ℃.
Generating a buffer solution: 12.11g of TRIS (hydroxymethyl) aminomethane (TRIS) and 7.4g of EDTA (ethylenediaminetetraacetic acid) were put in a 100mL volumetric flask, and deionized water was added to 90mL, followed by sonication for 30min until the solution was uniformly dissolved. Adding deionized water to the scale mark, and mixing. Transferring to a storage container made of polypropylene. Adjusting pH to 5.45-5.50 with acetic acid, and storing at-20 deg.C.
Generation of buffer a: on the day of assay, 100. mu.L of 100mM PMSF solution was added to 10mL of generation buffer to prepare generation buffer A (pH 5.4-5.6).
II, specific operation steps:
(1) preparation of reagents: first, 20. mu.L of generation buffer A was added to two clean 1.2mL 96-well collection plates for subsequent pre-treatment of the samples.
(2) Sample unfreezing: the plasma sample to be tested is placed in ice water (0 ℃) to be thawed until being thawed.
(3) Sampling: 2 100. mu.L aliquots of the plasma samples were transferred in parallel to two plates prepared in step (1) and tested for the presence of the non-incubated and incubated samples, respectively, and the remaining samples were immediately frozen at-20 ℃.
(4) Treatment of incubation samples: sealing one batch of samples in the step (3) by using a silica gel pad, briefly vortexing, then placing the samples in a water bath at 37 ℃ for 3 hours, adding 12 mu L of formic acid serving as reaction termination solution after 3 hours, vortexing, and then adding 200 mu L of ANG I-containing internal standard containing 5ng/mL angiotensin I isotope13C15And (3) vortexing and uniformly mixing the internal standard working solution of N, centrifuging at the temperature of 4 ℃ and the rpm of 15000 for 10 minutes, taking 100 mu L of supernate, adding the supernate into a 96-hole sample plate, and analyzing by a liquid chromatography tandem mass spectrometer.
(5) Treatment of the unincubated samples: and (4) sealing the other batch of samples left in the step (3) by using a sealing gasket, and then, uniformly mixing in a vortex manner. Immediately adding 12 mu L of reaction termination liquid formic acid, carrying out vortex, adding 200 mu L of internal standard working solution containing angiotensin I isotope internal standard, carrying out vortex mixing, then centrifuging at 4 ℃ and 15000rpm for 10 minutes, taking 100 mu L of supernatant, adding into a 96-hole sample inlet plate, and carrying out sample injection analysis by a liquid chromatography tandem mass spectrometer.
(6) Calculation of renin activity: renin activity is equal to the rate of angiotensin I production in ng/mL/hr, calculated as: (post-incubation angiotensin I concentration-pre-incubation angiotensin I concentration)/incubation time, the relevant assay results are shown in tables 7-10.
When the liquid chromatogram tandem mass spectrometry is carried out, the liquid chromatogram adopts gradient elution, and the reverse phase chromatography establishes the separation conditions of the substances to be detected as follows: the chromatographic column is Phenomenex Kinetex C18(2.6,100A,50x2.1mm), the flow rate is 0.6mL/min, and the column temperature is 40 ℃; wherein the mobile phase A is a formic acid aqueous solution with the volume ratio of 0.1%, the mobile phase B is a methanol solution with the volume ratio of 0.1%, and the volume ratio of the mobile phase A to the mobile phase B is 90-5%: 10 to 95 percent. Gradient program as shown in table 4 below, the retention times for angiotensin I and its isotopic internal standard are respectively: 1.92 min.
Table 4: gradient elution procedure
Time (min) Flow rate (mL/min) Mobile phase A (%) Mobile phase B (%)
0 0.6 90 10
1 0.6 90 10
2 0.6 5 95
3.2 0.6 5 95
3.25 0.6 90 10
3.8 0.6 90 10
When mass spectrometry is performed, angiotensin I is detected and quantified by a triple quadrupole mass spectrometer with the instrument model of SCIEX 4500MD, mass spectrometry is performed by adopting an electrospray ion source positive ion mode (ESI +) and a multi-reaction monitoring MRM mode, the corresponding mass spectrometry detection method is set as shown in table 5 below, and the mass spectrometry conditions are shown in table 6 below.
Table 5: mass spectrometry detection method
Figure BDA0002707668740000081
Figure BDA0002707668740000091
TABLE 6 Mass Spectrometry conditions
Figure BDA0002707668740000092
A standard curve is established by an internal standard method, the verification record of the linear relation is shown in the following table 7, and the measurement unit is ng/mL.
Table 7: standard curve verification results
Figure BDA0002707668740000101
Table 8: results of standard curve
Figure BDA0002707668740000102
In the concentration range, the linear relationship of detection is good.
Table 9: accuracy and precision within day:
Figure BDA0002707668740000111
the low-concentration quality control and the high-concentration quality control respectively detect the concentration levels of the non-incubated angiotensin I and the incubated angiotensin I, the accuracy and the precision (CV%) of the detection result in a day are both less than 15%, the accuracy and the precision (CV%) of the renin activity obtained by further calculation are both less than 20%, and the result shows that the reproducibility and the accuracy of the detection result of the samples of the same batch meet the detection requirements.
Table 10: day-to-day accuracy and precision;
Figure BDA0002707668740000121
the low-concentration quality control and the high-concentration quality control are used for respectively detecting the levels of the non-incubated angiotensin I and the incubated angiotensin I, the accuracy and the precision (CV%) of the detection results of 5 different batches are both less than 15%, and the daytime accuracy and the precision of the renin activity obtained by further calculation are both less than 20%, which indicates that the reproducibility and the accuracy of the detection results of samples of different batches of the method meet the detection requirements. In addition, this patent is through monitoring not hatching and the quality control sample testing result after hatching, not only can supervise every inspection batch's instrument testing process and pretreatment renin hatching reaction process in daily clinical examination respectively and whether have unusually, can supervise whole detecting system whether normal in addition, can control the experimentation more systematic and comprehensive, be favorable to detecting deviation and detection quality's control in daily clinical examination, in time discover the problem.
Third, optimization of test conditions
(1) Stock solutions were prepared with 50% methanol water as solvent:
the solubility of angiotensin I is better in water and therefore water was chosen as the dissolution solvent, but in practical experiments it was found that angiotensin I has adsorption problems in conventional plastic centrifuge tubes, leading to a decrease in its practical concentration at low concentrations. Actual results also demonstrate that, as shown in the table below, the actual concentration of the standard solution when diluted with 1000ng/mL of water is 64% of the theoretical concentration, and as the concentration decreases, the values deviate more and more, consistent with the assumptions. Further, a 50% methanol solution and a bovine serum albumin BSA solution with a mass ratio of 8% are respectively considered, so that an organic solvent methanol is innovatively added to reduce the adsorption effect, and a good recovery rate is obtained, which is shown in the following table. The concentrations of both the 50% methanol and 8% BSA diluted solutions differed very little from the theoretical concentrations. In comparison, a more theoretical concentration of 50% methanol was used, and in addition the addition of an organic solvent prevented the solution from deteriorating, so the stock solution was selected to be prepared for long-term storage of the standard solution with 50% methanol in water instead of the BSA solution as the solvent.
Table 11: comparison of different solvents
Theoretical concentration Actual concentration (Water dilution) Actual concentration (50% methanol) Actual concentration (8% BSA dilution)
1000ng 639 1067 917
100ng 31 103 89
10ng 0.45 10.4 9.2
1ng 0.02 0.925 0.879
(2) The standard curve diluent is 1% Bovine Serum Albumin (BSA)
Because the angiotensin I is an endogenous substance, the actual biological matrix, serum, plasma and the like all contain the concentrations of the angiotensin I with different concentrations, a simulated biological matrix 1% bovine serum albumin solution is adopted as a substitute matrix, and the matrix does not contain the endogenous angiotensin I, so that standard concentration samples with different levels and accurate concentrations can be obtained by adding the angiotensin I solution with standard concentration, and the standard concentration samples are used for configuring the standard curve concentration samples used for a short time.
(3) Matrix of quality control sample:
the prior art often adopts BSA analog matrix to configure a quality control sample, and the method has the problems that the BSA has larger difference with the actual plasma matrix, does not have the bioactivity of renin and the like, and has the following problems. A. The BSA simulated matrix can not reflect the interference and influence of the actual plasma sample matrix on the pretreatment and detection processes, so that the methodological verification of the simulated matrix BSA proves that the accuracy and precision results are good, and the method does not represent that the method has the same performance in the actual plasma sample detection process. B. The existence of angiotensin I synthetase and metabolic enzyme in plasma leads to over-high or under-low detection results, which are similar to the actual detection of unknown plasma samples, but the quality control of the BSA matrix cannot reflect the influence of the enzymes on the detection in the detection process, which is detailed in Table 12. (3) The BSA matrix does not contain angiotensin I convertase and renin, so the quality control cannot be used to monitor the quality of the incubated sample, and thus the quality of the incubation conversion process in the renin activity detection cannot be monitored, and the result deviation of the incubation process of the current detection batch cannot be found, which is detailed in table 13.
TABLE 12 variation of the stability of the quality control of BSA configuration and of the plasma matrix at Normal temperature
Figure BDA0002707668740000141
Figure BDA0002707668740000151
The BSA matrix is controlled at normal temperature, the concentration of the BSA matrix does not change obviously with time, but the plasma matrix sample is obviously reduced (possibly under the catalytic action of angiotensin-metabolizing enzyme) with time at normal temperature, and the BSA matrix and the plasma matrix sample show consistent rules in the control of low-concentration and high-concentration quality. The results show that the stability of the two matrixes is obviously different, and the state of angiotensin I in an actual sample cannot be completely simulated by using the BSA matrix, so the accuracy verification of the method by using the BSA matrix cannot reflect the accuracy and detection performance of the method in the actual sample detection.
The result shows that the standing time of the plasma sample at room temperature in the pretreatment process must be as short as possible, or the operation process keeps a low-temperature state as possible, otherwise the angiotensin I is converted and degraded, and the result of the renin activity detection is deviated.
TABLE 13 variation of quality control of different substrates in incubation environments
Figure BDA0002707668740000152
The results show that before and after the incubation process, the BSA matrix has no obvious change before and after the incubation, while the quality control of the plasma matrix has obvious multiple-fold increase before and after the incubation, and the results show that the BSA matrix can not realize the simulated incubation process and carry out quality monitoring. Therefore, the plasma matrix is superior to the BSA matrix in quality control.
(4) Temperature condition for storing quality control sample
Since various angiotensin I-metabolizing enzymes exist in blood plasma, the related documents mention that these enzymes have an influence on the result during the incubation, and the actual results show that the temperature has a great influence on the result during the configuration process of quality control at normal temperature, thereby influencing the detection results of angiotensin I before and after incubation, as shown in table 14 below.
Table 14: effect of configuration and storage temperature on quality control samples
Figure BDA0002707668740000161
When the quality control is carried out by using the plasma preparation at normal temperature, the recovery rate is found to be remarkably low, the influence of the temperature in the preparation process on the recovery rate is further considered, the recovery rate is higher at low temperature (2-8 ℃), and the recovery rate is lower after the preparation is placed for a long time (more than 1 hour) at room temperature. This is probably due to the low activity of angiotensin I-metabolizing enzymes in plasma at low temperatures and the higher activity at room temperature. The activity of degradation enzyme is inhibited and the degradation of angiotensin I in quality control is reduced by improving the temperature conditions of configuration and storage, so that higher extraction recovery rate is obtained. At present, no literature research on a quality control configuration method of angiotensin I exists.
(5) Quality process of pretreatment process of incubation sample
The calculation of renin activity requires to measure the angiotensin I levels of the sample which is not incubated and the sample incubated at 37 ℃, and then substitutes into a formula to calculate the renin activity, wherein the detection result after incubation is composed of buffer solution in the incubation process, and the concentration, pH and temperature in the incubation process have large influence, so that strict quality monitoring needs to be carried out on the process, thereby ensuring the accuracy of the detection of angiotensin I and the calculation of renin activity result after incubation.
By adopting the method in the prior art, the BSA analog matrix is used for configuring quality control, and the matrix lacks renin, angiotensinogen and angiotensin converting enzyme, and cannot simulate the generation change of angiotensin I in the incubation process, so that the incubation process cannot be monitored. The scheme of the application innovatively adopts a quality control sample of an actual blood plasma matrix, 2 quality control schemes with different angiotensin I concentrations and different renin activities are designed, the deviation caused by the detection method is comprehensively monitored by inspecting indexes such as the angiotensin I level and the renin activity level, and the phenylmethanesulfonyl fluoride (PMSF) and EDTA are added in the pretreatment process to inhibit the activity of Angiotensin Converting Enzyme (ACE) for converting the angiotensin I into the angiotensin II, so that the accuracy of the incubation process and the renin activity result is ensured.
The quality control system for detecting the activity of the renin based on the real plasma matrix is established by solving a series of technical problems, can effectively supervise the detection and pretreatment processes of the activity of the renin, and can also be used for further research and improvement of a renin activity detection method.
(6) Method for pre-treating precipitated protein
The method for processing the angiotensin I before detection in the prior art comprises an online column switching method and an offline SPE solid phase extraction method, wherein the online column switching method is high in automation degree, but the liquid chromatography equipment is difficult to build and maintain, the technical threshold is high, a first-stage separation column needs to be frequently replaced, and the cost is high. The SPE solid phase extraction method has complex pretreatment and needs operations such as activation, sample loading, leaching and elution, concentration, redissolution and the like. Compared with the methods, the method has the advantages that a simple protein precipitation method is adopted in the sample pretreatment process, the method has the advantages of simplicity and easiness in operation, and the difficulty in operation of personnel is low; B. the detection device has no special requirements on instruments and equipment, a two-dimensional chromatographic system does not need to be installed, a positive pressure and negative pressure pretreatment device is also not needed, a liquid phase mass spectrometer configured in a standard mode can meet the detection requirements, the pretreatment equipment is a vortex instrument and a centrifugal machine configured in a laboratory in a conventional mode, the requirements on personnel are low, only the conventional pipettor and the conventional liquid phase mass spectrum operation need to be carried out, the operation is simple, the operation of proteolysis is not needed, and the simple training can be mastered; C. the reagent has low cost and easy popularization, and is suitable for developing and transforming corresponding commercialized kits; D. and the 96-well operation mode is compatible, so that the high flux and the automation of detection are realized conveniently.
Table 15: comparison of the protein precipitation method with the prior art
Online pole switching method SPE solid phase extraction method The patented method
Cost of equipment High (additive two-dimensional liquid chromatography) Middle (adding nitrogen blowing and positive pressure instrument) Is low in
Difficulty of equipment maintenance Height of Is low in Is low in
Difficulty of equipment operation Height of In Is low in
Difficulty of pretreatment operation Simple and easy Medium and high grade Simple and easy
Cost of pretreatment consumables High (on-line processing column) High (SPE plate) Is low in
Time consuming pretreatment Short length Long and long In
Personnel training requirements Height of Height of Is low in
(7) Composition of protein precipitant
The commonly used protein precipitant includes organic solvent, neutral salt solution, strong acid, etc. By comparison, the protein precipitant selected by the invention is a mixed reagent of methanol and zinc sulfate aqueous solution, and the precipitant contains an isotope internal standard of angiotensin I. The internal standard precipitant is added, so that effective protein precipitation of the plasma sample can be realized while the isotope internal standard is added. The solution of protein precipitant was composed of 90% methanol by volume, 10% aqueous zinc sulfate (89 mg/mL). In the pretreatment of the plasma sample, the protein precipitant may be present in a ratio of 1: 1, the full protein precipitation effect can be realized by adding the sample solution before precipitation, the dilution times of the sample are reduced, and the response of the substance to be detected is improved.
In the literature, there is known a method of pretreating a plasma sample by SPE in a foreign laboratory. The method compares the SPE method and the self-constructed Protein Precipitation (PPT) method in the prior art, and respectively detects the angiotensin I content of three quality control samples and ten actual samples, and the comparison results are shown in the following table.
Table 16: comparison of existing SPE methods and Protein Precipitation (PPT) methods of the present application
Figure BDA0002707668740000181
Figure BDA0002707668740000191
The result shows that the method has no obvious deviation with the SPE solid phase extraction detection method in the literature, the error of the result is within 80-120%, and the detection result is relatively consistent, but the detection cost of the precipitated protein method adopted by the method is relatively low.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims (10)

1. A kit for determining activity of human plasma sample renin is a high performance liquid chromatography tandem mass spectrometry kit, and comprises the following components:
(1) preparing a kit for a standard curve working solution: it comprises angiotensin I standard substance (Ang I), 40-60 vol% methanol water solution, 1-8 mass% Bovine Serum Albumin (BSA) water solution;
(2) preparing an internal standard working solution kit: which comprises angiotensin I isotope internal standard ANGI-13C15N, 40-60 volume percent methanol aqueous solution, 50-100mg/mL zinc sulfate aqueous solution and methanol;
(3) preparing a kit from a sample pretreatment buffer solution: 50-200mM phenylmethylsulfonyl fluoride methanol solution (PMSF), 0.5-2mol/L Tris (hydroxymethyl) aminomethane and 0.1-0.5mol/L EDTA buffer solution with pH between 5.45-5.50;
(4) preparing a quality control sample preparation kit: it comprises EDTA anticoagulated plasma sample and angiotensin I standard solution;
(5) reaction stop solution kit: formic acid;
(6) liquid chromatography gradient eluent: the volume ratio of the formic acid aqueous solution with the volume ratio of 0.05-0.2% to the methanol solution is 90-5% in the gradient elution procedure: 10 to 95 percent.
2. The kit of claim 1, wherein the concentration of angiotensin I in the standard curve working fluid is between 0.2ng/mL and 30 ng/mL.
3. The kit according to claim 1, wherein the concentration of the BSA aqueous solution in the standard curve working solution preparation kit is 1 to 8 mass%.
4. The kit of claim 1, wherein the quality control sample preparation kit is stored at-20 to-80 ℃.
5. A process for pretreating a human plasma sample to be tested for measuring renin activity, which comprises treating the human plasma sample to be tested with the kit of any one of claims 1 to 4, comprising the steps of:
(1) preparing a standard stock solution and a standard curve working solution by adopting a standard curve working solution preparation kit;
(2) preparing an internal standard stock solution and an internal standard working solution by adopting an internal standard working solution preparation kit;
(3) preparing a quality control sample by adopting a quality control sample preparation kit, wherein the quality control sample comprises angiotensin I low-concentration and high-concentration quality control samples, and is obtained by additionally adding angiotensin I standard stock solutions or standard solutions in (1) with different volumes or concentrations into EDTA anticoagulated plasma samples; the quality control samples comprise low-activity and high-activity quality control samples with different renin activities, wherein the high-activity plasma sample is a plasma sample obtained after fresh collection, and the low-activity plasma sample is a plasma sample obtained after fresh collection of plasma and placement for 3-7 days at the temperature of 2-8 ℃.
(4) Preparing a sample pretreatment buffer solution by adopting a sample pretreatment buffer solution preparation kit, and placing the sample pretreatment buffer solution in a porous plate;
(5) parallelly taking 2 parts of a sample to be tested, placing the 2 parts of the sample into sample holes of at least 2 porous plates, stopping the incubation of one porous plate for 2-4 hours by adopting a reaction stopping solution formic acid, adding an internal standard working solution prepared by an internal standard working solution preparation kit to obtain an incubated sample, and centrifuging to obtain a supernatant; and after the other porous plate is uniformly mixed, immediately stopping by adopting a reaction stopping solution formic acid, adding an internal standard working solution prepared by an internal standard working solution preparation kit to obtain an unincubated sample, and centrifuging to obtain a supernatant.
6. A method for determining renin activity of a human plasma sample, which comprises the human plasma sample pretreatment process as claimed in claim 5, and a step of detecting the incubated sample and the non-incubated sample respectively by adopting a liquid chromatography tandem mass spectrometry method and a further detection error quality control step.
7. The method according to claim 6, wherein the liquid chromatography adopts gradient elution, the chromatographic column is a C18 column, the mobile phase A phase is 0.05-0.2 vol% formic acid water solution, the mobile phase B phase is 0.05-0.2 vol% formic acid methanol solution, and the volume ratio of the mobile phase A to the mobile phase B is 90-5%: 10 to 95 percent.
8. The method of claim 7, the gradient elution procedure is as follows:
time (min) Flow rate (mL/min) Mobile phase A (%) Mobile phase B (%) 0 0.6 90 10 1 0.6 90 10 2 0.6 5 95 3.2 0.6 5 95 3.25 0.6 90 10 3.8 0.6 90 10
。
9. The method of claim 6, wherein the mass spectrometer is a triple quadrupole mass spectrometer, and mass spectrometric detection is performed using an electrospray ion source and positive ion mode (ESI +) and Multiple Reaction Monitoring (MRM) modes.
10. The method of claim 6, wherein the quality control of the detection error is performed by comparing the deviation between the detection index value of the quality control sample and the theoretical target value, thereby evaluating the accuracy of the detection result, wherein the detection index comprises the concentration of angiotensin I in the quality control sample after the non-incubation and the incubation, and the renin activity level of the quality control sample.
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