CN116223690A - LC-MS/MS kit for extracting and detecting 6 catecholamines and metabolites thereof from plasma - Google Patents
LC-MS/MS kit for extracting and detecting 6 catecholamines and metabolites thereof from plasma Download PDFInfo
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- CN116223690A CN116223690A CN202310416591.XA CN202310416591A CN116223690A CN 116223690 A CN116223690 A CN 116223690A CN 202310416591 A CN202310416591 A CN 202310416591A CN 116223690 A CN116223690 A CN 116223690A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention provides an LC-MS/MS kit for extracting and detecting 6 catecholamines and metabolites thereof from blood plasma, belonging to the technical field of medical detection. The kit disclosed by the invention can be used for simultaneously detecting 6 catecholamines and metabolites thereof, has the characteristics of high efficiency, strong specificity, strong anti-interference capability, wide detection linear range, high sensitivity and high accuracy, and has a good application prospect in the direction of detecting catecholamines and metabolites thereof.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to an LC-MS/MS kit for extracting and detecting 6 catecholamines and metabolites thereof from blood plasma.
Background
Catecholamines are a class of neurones containing catechol and amine groups, commonly referred to as Norepinephrine (NE), epinephrine (E), and Dopamine (DA), and derivatives thereof. Catecholamines regulate basic physiological functions in vivo, transmit physiological signals, are important signal mediators in normal physiological processes, and also show corresponding changes in the content in pathological processes. Clinically, the kit can be used for auxiliary diagnosis of endocrine-related diseases such as hypertension, hyperthyroidism, pheochromocytoma (PPGLs for short) and the like, and is also important in distinguishing the PPGLs from the most common extracranial solid tumor neuroblastoma of children.
Catecholamine substances have extremely low content in biological samples, extremely poor stability and extremely easy oxidation, and moreover, endogenous chemical interferents of various metabolites with similar structures or same chemical groups exist in the biological samples simultaneously, so that the accurate measurement of catecholamine concentration in the biological samples is extremely difficult, and the accurate measurement of specific catecholamine substances in the biological samples, such as norepinephrine and epinephrine in serum, with high selectivity and high sensitivity is an object which is not yet achieved clinically at present. At present, related researches on content detection and analysis of catecholamine substances at home and abroad are in continuous depth, and a method which is simple and feasible, economical and applicable, accurately quantifies related substances and can be widely applicable is focused on.
The invention discloses a liquid chromatography tandem mass spectrometry detection method of catecholamine and metabolites thereof based on magnetic solid phase extraction, which comprises the following steps of: 1) Preparing a detection sample; 2) Constructing a standard curve of the target object; 3) Detecting by adopting a liquid chromatography tandem mass spectrometry method, and calculating to obtain the content of catecholamine and metabolites thereof in a detection sample by combining the constructed standard curve; wherein catecholamines include dopamine, norepinephrine and epinephrine, and metabolites of catecholamines include 3-methoxytyramine, phenylephrine and norepinephrine. According to the liquid chromatography tandem mass spectrometry detection method based on catecholamine and metabolites thereof through magnetic solid phase extraction, the catecholamine and metabolites thereof detection sample is subjected to pretreatment through a magnetic solid phase extraction technology, and separation and transfer of the to-be-detected object are rapidly realized through application of external magnetic field energy, so that automatic pretreatment is conveniently realized, and meanwhile, the advantages of high selectivity, high purification rate and the like of solid phase extraction are also reserved. However, the magnetic solid phase extraction technique adopted by the method has not been widely used in clinical detection, and the detection may be affected by a high concentration of surfactant during elution. Although the automation is expected to be realized, the method is suitable for large-batch clinical samples, the preparation of the magnetic molecular material is complicated, the single detection cost is high, and the method is not suitable for clinical detection.
Chinese patent application CN202210044388.X filed by Shanghai Rui science and technology Co., ltd.1.14 in 2022, entitled "kit and test method for detecting human plasma catecholamine intermediate metabolite" (publication No. CN 114460188A), discloses a detection method of liquid chromatography tandem mass spectrometry. The invention provides a kit for detecting human plasma catecholamine intermediate metabolites and a test method, wherein the kit comprises the following reagents: (1) calibrator solution: the calibrator is divided into six concentrations, and each concentration of calibrator contains three intermediate metabolites of catecholamines: methoxyepinephrine, methoxynorepinephrine and 3-methoxytyramine, and the calibrator concentration is 10pg/mL-4000pg/mL; (2) quality control product: comprises a quality control product QCL, a quality control product QCM and a quality control product QCH; (3) internal standard solution: a methanol solution mixture comprising 3 intermediate metabolites of catecholamines; (4) buffer I, buffer II; (5) a derivative liquid; (6) a leaching solution; (7) an eluent; (8) 96-well purification plates, 96-well receiving plates; (9) 96 well plate cover film. The sample is treated through the solid phase extraction and the derivatization reaction, so that the detection sensitivity of catecholamine intermediate metabolites in the sample is improved. However, the method adopts solid phase extraction and derivatization in the pretreatment process, the pretreatment process is time-consuming, and the derivatization reagent has extremely high requirement, and side reaction is extremely easy to generate to cause new interferents. In addition, the method only detects three catecholamine compounds, and the linear range is only 10-4000pg/mL.
Chinese patent application 201610907368.5 by the division of the Hangzhou herborist medical test, entitled "method for detecting catecholamines in plasma by liquid chromatography tandem mass spectrometry" (publication No. CN 106442837A). The invention discloses a method for detecting catecholamine in plasma by utilizing liquid chromatography tandem mass spectrometry, which creatively uses a high-flux liquid chromatography tandem triple quadrupole mass spectrometer to detect catecholamine and has the following beneficial effects: acetonitrile is adopted for protein precipitation, dansyl chloride is adopted for derivatization reaction, liquid chromatography is used for tandem triple quadrupole mass spectrometry for detection after extraction, the pretreatment step is simple, plasma matrix interference can be effectively removed, and the specificity is good; after being derivatized by the dansyl chloride, the detection sensitivity can be effectively improved; the high-throughput liquid chromatography tandem mass spectrometer is used for detecting, and simultaneously, the plasma containing 3 catecholamines of dopamine, epinephrine and norepinephrine is qualitatively, accurately and quantitatively detected, so that the detection sensitivity is high, the specificity is good, and the cost is low. However, the method adopts a pretreatment method of adding derivatization to protein precipitation, which takes a long time, is extremely toxic to the dansyl chloride, and only involves detection of 3 catecholamine substances.
Disclosure of Invention
In order to solve the above problems, the present invention provides a pretreatment kit for extracting 6 catecholamines and their metabolites from a plasma sample. The pretreatment method of solid phase extraction is simple and efficient; the LC-MS/MS detection method is adopted, so that the sensitivity is high and the specificity is strong.
The kit is used for measuring catecholamine and metabolites thereof in plasma samples by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method in combination: dopamine (Dopamine, DA), epinephrine (E), norepinephrine (NE), methoxyepinephrine (MN), methoxynorepinephrine (NMN) and trimethoxytyramine (3-MT), and plasma samples were taken from the supernatants after treatment using the pretreatment kit. In the electrospray ionization mode, a multi-reaction monitoring mode (MRM) is adopted for detection, and finally an internal standard method is used for accurate quantitative analysis.
The kit can effectively extract 6 catecholamines and metabolites thereof, has simple pretreatment method, high efficiency and short time consumption, can effectively improve detection flux, and has high specificity and high sensitivity in methodology.
In one aspect, the present invention provides a blood sample release agent combination.
The blood sample release agent combination comprises: 1% -2%V/V formic acid solution, 80% -100% V/V acetonitrile solution and 0.01% -0.1% m/V VC solution.
The concentration of the formic acid solution can be 1% -1.8%, 1% -1.6%, 1% -1.5%, 1% -1.2%, 1.2% -1.8% and 1.5% -2%. The formic acid solution in the blood sample releasing agent combination comprises two different concentrations.
The concentration of the acetonitrile solution can be 80% -82%, 80% -85%, 80% -87%, 80% -89%, 82% -88%, 84% -86%, 84% -85%, 85% -90%, 90% -100%, 95% -100%. The concentration of the VC solution can be 0.01% -0.09%, 0.01% -0.05%, 0.02% -0.08%, 0.04% -0.08%, 0.05% -0.07%, 0.05% -0.06% and 0.06% -0.07%. The acetonitrile solution in the blood sample releasing agent combination comprises two different concentrations.
Preferably, the formic acid solution is 1% and 2% formic acid solution; the acetonitrile solution is 85% and 100% acetonitrile solution; the VC solution is 0.05% VC solution.
The blood sample releasing agent combination can be applied to the pretreatment of plasma samples for measuring catecholamines and metabolites thereof by an LC-MS/MS method.
In another aspect, the present invention provides a method of pretreatment of a blood sample.
The pretreatment method comprises the step of treating with the release agent combination.
Specifically, the pretreatment method may include the steps of:
adding a mixed internal standard working solution into a sample, and uniformly mixing by vortex; centrifuging and transferring the supernatant to a WCX 96-well solution plate which is pretreated by an aqueous solution of 80% -90% V/V acetonitrile containing 1% -2%V/V formic acid and water in sequence; sequentially using ultrapure water and 80% -100% V/V acetonitrile solution to clean the holes; finally, eluting the target from the plate into a 96-hole collecting plate by using an aqueous solution of 80% -90% V/V acetonitrile containing 1% -2%V/V formic acid to obtain an eluent; drying the eluent by a nitrogen blower, adding 0.01-0.1% m/V VC solution, oscillating and centrifuging to obtain supernatant.
Preferably, the pretreatment method may include the steps of:
(1) Adding a mixed internal standard working solution into a sample, and uniformly mixing by vortex; (2) Centrifuging and transferring the supernatant to a WCX 96-well solution plate which is pretreated by an aqueous solution of 80% -90% V/V acetonitrile containing 1% -2%V/V formic acid and water in sequence; (3) Sequentially using ultrapure water and 80% -100% V/V acetonitrile solution to clean the holes; (4) Finally, eluting the target from the plate by using an acetonitrile solution containing 80% -90% of formic acid of 1% -2%V/V to a 96-well collection plate to obtain an eluent; (5) Drying the eluent by a nitrogen blower, adding 0.01-0.1% m/V VC solution, oscillating and centrifuging to obtain supernatant.
Preferably, the concentration of the formic acid solution in the step (2) can be 1% -1.8%, 1% -1.6%, 1% -1.5%, 1% -1.2%, 1.2% -1.8% and 1.5% -2%. Preferably, the concentration of the formic acid solution in the step (4) can be 1% -1.8%, 1% -1.6%, 1% -1.5%, 1% -1.2%, 1.2% -1.8% and 1.5% -2%.
Preferably, the concentration of the formic acid solution in the step (2) and the step (4) is different. Further preferably, the concentration of the formic acid solution in the step (4) is higher than that in the step (2).
The concentration of the acetonitrile solution can be 80% -82%, 80% -85%, 80% -87%, 80% -89%, 82% -88%, 84% -86%, 84% -85%, 85% -90%, 90% -100%, 95% -100%. The concentration of the VC solution can be 0.01% -0.09%, 0.01% -0.05%, 0.02% -0.08%, 0.04% -0.08%, 0.05% -0.07%, 0.05% -0.06% and 0.06% -0.07%.
In some embodiments, the pretreatment method may include the steps of:
(1) Adding a mixed internal standard working solution into a sample, and uniformly mixing by vortex; (2) Centrifuging and transferring the supernatant to a WCX 96-well solution plate which is sequentially pretreated by 85% V/V acetonitrile water solution containing 1%V/V formic acid and water; (3) Sequentially cleaning the holes by using ultrapure water and 100% acetonitrile solution; (4) Finally, eluting the target from the plate into a 96-well collection plate by using an 85% V/V acetonitrile solution containing 2%V/V formic acid to obtain an eluent; (5) Drying the eluent by a nitrogen blower, adding 0.05% m/V VC solution, oscillating and centrifuging to obtain supernatant.
In some embodiments, the centrifugation conditions in step (2) are 14000rpm and centrifugation is performed for 5min.
In some embodiments, the shaking time in step (5) is 5min and the centrifugation time is 3min.
In yet another aspect, the invention provides a kit for extracting 6 catecholamines from blood plasma.
The kit comprises the blood sample release agent combination.
In yet another aspect, the invention provides a kit for detecting 6 catecholamines in blood plasma.
The kit comprises the blood sample release agent combination.
The kit also comprises reagents for chromatographic and mass spectrometry detection. In particular, the reagents for chromatographic detection comprise a mobile phase. Preferably, the reagent for chromatographic detection comprises mobile phase a:0.5% formic acid-0.5% acetic acid aqueous solution; and mobile phase B:0.1% formic acid-methanol.
Preferably, the preparation method of the mobile phase A comprises the following steps: 2.5mL of formic acid and 2.5mL of acetic acid were taken in 500mL of ultrapure water, and sonicated. The preferred duration of the ultrasound is 5min. Preferably, the preparation method of the mobile phase B comprises the following steps: 0.5mL of formic acid was taken in 500mL of methanol and sonicated. The preferred duration of the ultrasound is 5min.
Preferably, the chromatographic column for chromatographic detection is Agilent Pursuit PFP.
Preferably, the chromatographic conditions are: the flow rate is 0.3-0.5mL/min; the sample injection volume is 8-12 mu L; the sample injection temperature is 7-9 ℃; column temperature box 28-32 ℃; the operation time is 5-8min.
In some embodiments, the chromatographic conditions are: the flow rate is 0.4mL/min; sample volume 10. Mu.L; the sample injection temperature is 8 ℃; column temperature box 30 ℃; run time was 6.5min.
The kit also comprises a quality control product, a calibrator and an internal standard solution. The kit also comprises 50% acetonitrile water solution as needle washing liquid 1; and isopropyl alcohol as the needle washing liquid 2.
The invention also provides a use method of the detection kit, which comprises the following steps:
(1) Sample pretreatment; (2) chromatographic detection; (3) mass spectrometry detection; (4) calculating a standard curve.
The kit can be used for detecting patients with clinical pheochromocytoma, paraganglioma or neuroblastoma, and 6 catecholamines and metabolites thereof are extracted and detected from a plasma sample.
Compared with the prior art, the invention has the following advantages:
(1) The pretreatment method is simple and efficient, and the pretreatment reagent can effectively clean interfering substances and enrich target objects; (2) 6 catecholamines and their metabolites can be detected simultaneously; (3) Catecholamine and its metabolite have small molecular weight, so that it is easy to interfere, the elution gradient and the selected ion pair established by the method can effectively raise the detection specificity, and the anti-interference capability is strong; (4) The concentration of the mobile phase additive effectively improves the peak shape of each analyte, and improves the detection sensitivity and accuracy; (5) The linear range is wide, wherein the linear range of 4 compounds DA, MN, NMN, NE can reach 10-10000pg/mL, the linear range of E can reach 21-10000pg/mL, and the linear range of 3-MT can reach 4-1000pg/mL which is higher than the linear range of the prior art.
Drawings
FIG. 1 shows the composition of the kit of example 1. FIG. 2 IS a standard operating curve established with the response ratio of NE and NE-IS as the ordinate. Fig. 3-4 are mass spectrum acquisition plots of NE. FIG. 5 IS a standard operating curve established with NMN and NMN-IS response ratios as ordinate. Figures 6-7 are mass spectrum acquisition plots of NMN. FIG. 8 IS a standard operating curve established with the response ratio of DA and DA-IS as the ordinate. Fig. 9-10 are mass spectrum acquisition plots of DA. FIG. 11 IS a standard operating curve established with response ratios of 3-MT and 3-MT-IS as ordinate. FIGS. 12-13 are mass spectrometry acquisition plots for 3-MT. FIG. 14 IS a standard operating curve established with the response ratio of E and E-IS as the ordinate. Fig. 15-16 are mass spectrum acquisition plots of E. FIG. 17 IS a standard operating curve established with the response ratio of MN and MN-IS as the ordinate. Fig. 18-19 are mass spectrometry acquisition graphs of MN.
The ordinate of the standard curve in the drawing shows the ratio, without units.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
In the following examples, part of the material information is as follows:
(1) Instrument for measuring and controlling the intensity of light
QSight420MD triple quadrupole mass spectrometry detection system (Yikun Biotechnology Co., st.) was used; dynamica Himac CT15RE high-speed refrigerated centrifuge (U.S.); positive pressure device (Biotage, sweden); nitrogen blower (Biotage, sweden); WCX 96-well elision plate (Waters, usa); multitube Vortex mixer (Vortex genie2, usa); an adjustable pipette (Eppendorf 0.5-10. Mu.L, 10-100. Mu.L, 100-1000. Mu.L); glassware, measuring cylinders, etc.
(2) Reagent(s)
MS grade ultrapure water (Fisher, usa); MS grade methanol (Fisher, USA); MS grade formic acid (Fisher, USA); acetonitrile (Fisher, usa); isopropanol (Fisher, usa).
(3) Standard substance
Epinephrine (TRC, canada); epinephrine-d 3 (TRC, canada); norepinephrine hydrochloride (TRC, canada); norepinephrine-d 6 HCL (sigma, usa); dopamine hydrochloride (TRC, canada); dopamine-d 4 (TRC, canada); methoxy Norepinephrine (NMN) (TRC, canada); methoxy norepinephrine-d 3 (TRC, canada); methoxyepinephrine (MN) (TRC, canada); methoxyepinephrine-d 3 (sigma, usa); 3-methoxytyramine (Sigma, USA); 3-methoxytyramine-d 4 (sigma, U.S.).
Example 1
The composition of the kit of this example is shown in FIG. 1, and the kit comprises the following components: calibrator S1-S5, quality control LQC and quality control HQC, internal standard solution, mobile phase additive 1 (formic acid), mobile phase additive 2 (acetic acid) and 96 Kong Guxiang extraction plate.
1. Reagent preparation
(1) Mobile phase a is 0.5% formic acid-0.5% acetic acid aqueous solution, and the specific preparation method comprises: 2.5mL of formic acid and 2.5mL of acetic acid were taken in 500mL of ultrapure water; ultrasound for 5 minutes; the label is CA mobile phase A, and the label is stored at room temperature (the effective period is 7 days).
(2) Mobile phase B is 0.1% formic acid-methanol, specific formulation method: 0.5mL of formic acid was taken in 500mL of methanol; ultrasound for 5 minutes; the label is CA mobile phase B, and the label is stored at room temperature (the effective period is 3 months).
(3) The needle washing liquid 1 is 50% acetonitrile water solution, and the specific preparation method comprises the following steps: 500mL of acetonitrile was added to the reagent bottle; adding 500mL of ultrapure water into the reagent bottle; uniformly mixing and carrying out ultrasonic treatment for 5 minutes; the label is CA needle washing liquid 1, and the needle washing liquid is stored at room temperature (the effective period is 3 months).
(4) The needle washing liquid 2 is isopropanol, and the specific preparation method comprises the following steps: 1000mL of isopropanol was added to the reagent bottle; ultrasound for 5 minutes; the label is CA needle washing liquid 2, and the product is stored at room temperature (the effective period is 3 months).
2. Sample pretreatment
Respectively sucking 400 mu L of standard substance, quality control substance and plasma sample into a 1.5mL EP tube; 200 mu L of mixed internal standard working solution is added respectively, and vortex mixing is carried out for 2min;14000rpm, centrifugation for 5min, transfer supernatant from the EP tube to wCX well plate pretreated with 200. Mu.L of 85% acetonitrile solution containing 1%V/V formic acid (formic acid: 85% acetonitrile=1:99V/V) and 200. Mu.L of water in this order without standing; the wells were washed sequentially with 200. Mu.L of ultrapure water and 200. Mu.L of acetonitrile; finally, 40. Mu.L of 85% acetonitrile solution containing 2%V/V formic acid (formic acid: 85% acetonitrile=2:98V/V) was used to elute the target from the plate into a 96-well collection plate to obtain an eluent; drying the eluent by a nitrogen blower, adding 40 mu L of 0.05% VC solution, oscillating for 5min, centrifuging for 3min, and loading 40 mu L of supernatant into a sample injection bottle containing an inner cannula for detection.
3. Chromatographic conditions
| Mobile phase a | 0.5% formic acid-0.5% acetic acid aqueous solution |
| Mobile phase B | 0.1% formic acid-methanol |
| Flow rate | 0.4mL/min |
| Sample injection volume | 10μL |
| |
8℃ |
| Chromatographic column | Agilent Pursuis PFP (2.0X105 mm,3.0 μm) |
| Column temperature box | 30℃ |
| Run time | 6.5min |
| Needle washing liquid | Acetonitrile/ultrapure water, 1/1 (v/v) |
Liquid phase gradient:
| time (min) | A(％) | B(％) |
| 0.0 | 98 | 2 |
| 2.0 | 95 | 5 |
| 2.5 | 80 | 20 |
| 4.0 | 2 | 98 |
| 5.0 | 2 | 98 |
| 5.01 | 98 | 2 |
| 6.5 | 98 | 2 |
4. Mass spectrometry method
Mass spectrometry source parameters:
| project | Parameters (parameters) |
| Ionization voltage | 5000V(ESI+) |
| Mass spectrometry interface heating (DEG C) | 320℃ |
| Atomizing gas | 200 |
| Source temperature (. Degree. C.) | 300 |
| Blowback drying gas | 100 |
Target compounds and internal standard collection parameter list:
| compounds of formula (I) | Q1 | Q2 | CE(V) | Residence time | CCL2 | EV |
| E | 166.0 | 135.0 | -24 | 30 | -58 | 38 |
| E-d3 | 169.0 | 135.0 | -27 | 30 | -78 | 43 |
| NE | 152.0 | 106.9 | -22 | 30 | -40 | 29 |
| NE-d6 | 158.0 | 113.0 | -23 | 30 | -46 | 25 |
| DA | 154 | 91.0 | -31 | 60 | -52 | 10 |
| DA-d4 | 158.2 | 141.1 | -14 | 60 | -46 | 10 |
| MN | 180.0 | 165.0 | -20 | 150 | -80 | 36 |
| MN-d3 | 183.1 | 168.1 | -23 | 150 | -52 | 38 |
| NMN | 166.0 | 106.1 | -30 | 70 | -50 | 24 |
| NMN-d3 | 169.0 | 137.0 | -20 | 70 | -56 | 23 |
| 3-MT | 151.2 | 91.0 | -25 | 70 | -60 | 37 |
| 3-MT-d4 | 155.0 | 123 | -19 | 70 | -66 | 38 |
Note that: the two ends of the time window can be finely adjusted according to the actual situation.
5. Quality control method
After the standard curve is finished, a group of low-quality control products and a group of high-quality control products are respectively injected into each batch of sample analysis, and after the sample injection analysis of each 40 samples is finished, the accuracy of the quality control products is between 85 and 115 percent, and more than 1/3 of the quality control products of each analysis batch are beyond the range.
6. Standard curve calculation
Regression analysis (weighting factor 1/X2) was used to construct the equation: y=ax+b;
wherein: x = analyte concentration (pg/mL); y = peak area ratio of the test object to the internal standard; a, b=curve factor.
Experimental example 1 Performance parameters of the kit
The performance parameters of the kit of example 1 were determined.
(1) Limit of detection:
preparing a plurality of low-concentration point samples, performing on-machine detection after pretreatment, and selecting the concentration with the average signal-to-noise ratio not less than 3 as a method detection limit, wherein the result is as follows:
(2) Lower limit of quantification
The average value of the detected concentration of the sample and the theoretical value deviation are less than or equal to +/-20 percent (the recovery rate is 80-120 percent), and the minimum concentration of CV is less than or equal to 15 percent is the lower limit of quantification (LLMI) of the kit. LLMI samples were prepared as follows:
| sample numbering | S1-Lot2 | CAs-LLMI1 | CAs-LLMI2 |
| Remove solution (μL) | NA | S1-Lot2 | S1-Lot2 |
| / | / | 2000 | 1500 |
| 0.4％BSA(μL) | / | 5000 | 4500 |
| Final volume (μl) | / | 7000 | 6000 |
| E(pg/mL) | 74.17 | 21.19 | 18.54 |
| NE(pg/mL) | 33.12 | 9.46 | 8.28 |
| MN(pg/mL) | 36.99 | 10.57 | 9.25 |
| NMN(pg/mL) | 32.16 | 9.19 | 8.04 |
| DA(pg/mL) | 37.87 | 10.82 | 9.47 |
| 3-MT(pg/mL) | 15.30 | 4.37 | 3.83 |
The above LLMI samples were tested with the kit and each sample was repeated 10 times with the following results:
(3) Intra-batch/inter-batch precision
Precision in batch: within the same analytical batch (same day), samples at both the high and low concentration levels were independently tested 10 times each:
| NE | target value (pgmL) | NMN | Target value (pgmL) |
| plasma-QC1 | 1367.42975 | plasma-QC1 | 697.82075 |
| plasma-QC2 | 2853.78575 | plasma-QC2 | 2885.02275 |
| E | Target value (pgmL) | MN | Target value (pg/mL) |
| plasma-QC1 | 1458.2335 | plasma-QC1 | 717.20375 |
| plasma-QC2 | 3702.60275 | plasma-QC2 | 2990.0935 |
| DA | Target value (pg/mL) | 3-MT | Target value (pg/mL) |
| plasma-QC1 | 1177.0615 | plasma-QC1 | 182.63175 |
| plasma-QC2 | 2509.31725 | plasma-QC2 | 300.555 |
Precision between batches: three separate batches were tested for the high and low two concentration level samples at three different analytical batches:
(4) Accuracy: the concentration of the target analyte in the reference is determined. The accuracy of the project was assessed by measuring accuracy references with 3 batches of kits, each sample being measured 6 times independently, calculating recovery.
The results were as follows:
experimental example 2 6 Standard working curves of catecholamines and their metabolites and extraction ion flow diagrams
(1) The 5 concentration points of the NE standard working solution are 30, 189.4, 827, 3218 and 8000pg/mL respectively, a standard working curve IS established by taking the response ratio of NE and NE-IS (NE-d 6) as the ordinate, and a linear judgment coefficient R IS established 2 0.99976 as in fig. 2; the NE retention time was 1.304min and the mass spectrum acquisition time range was selected to be 1.1-1.65 minutes as shown in FIGS. 3-4.
(2) The 5 concentration points of the NMN standard working solution are respectively 30, 189.4, 827, 3218 and 8000pg/mL, a standard working curve IS established by taking the response ratio of NMN and NMN-IS (NMN-d 3) as the ordinate, and the linear judgment coefficient R IS established 2 0.99943 as shown in fig. 5; NMN retention time was 1.803min and mass spectrum acquisition time range was chosen to be 1.55-2.5 min, as shown in FIGS. 6-7.
(3) The 5 concentration points of the DA standard working solution are 30, 189.4, 827, 3218 and 8000pg/mL respectively, a standard working curve IS established by taking the response ratio of DA and DA-IS (DA-d 4) as the ordinate, and the linear judgment coefficient R IS established 2 0.999964 as shown in fig. 8; DA retention time was 2.017min and mass spectrum acquisition time range was chosen to be 1.75-2.5 min, as shown in FIGS. 9-10.
(4) The 5 concentration points of the 3-MT standard working solution are respectively 15, 30.7, 93.5, 329 and 800pg/mL, and the response ratio of the 3-MT and the 3-MT-IS (3-MT-d 4) IS taken as the ordinate to establish the standard working curveLine, linear determination coefficient R 2 0.99977, as shown in fig. 11; the 3-MT retention time was 3.543min and the mass spectrum acquisition time range was selected to be 3.2-3.8 min, as shown in FIGS. 12-13.
(5) The 5 concentration points of the E standard working solution are 30, 189.4, 827, 3218 and 8000pg/mL respectively, a standard working curve IS established by taking the response ratio of E and E-IS (E-d 3) as the ordinate, and the linear judgment coefficient R 2 0.99973 as shown in fig. 14; e retention time was 1.562min and mass spectrum acquisition time range was chosen to be 1.3-1.9 min, as shown in FIGS. 15-16.
(6) The 5 concentration points of the MN standard working solution are respectively 30, 189.4, 827, 3218 and 8000pg/mL, a standard working curve IS established by taking the response ratio of MN and MN-IS (MN-d 3) as the ordinate, and a linear judgment coefficient R IS established 2 0.99989 as shown in fig. 17; the MN retention time was 2.569min and the mass spectrum acquisition time range was chosen to be 2.3-2.95 min as shown in FIGS. 18-19.
Claims (10)
1. A blood sample release agent combination comprising: 1% -2%V/V formic acid solution, 80% -100% V/V acetonitrile solution and 0.01% -0.1% m/V VC solution.
2. The blood sample release agent combination of claim 1, wherein the formic acid solution comprises two different concentrations; the acetonitrile solution comprises two different concentrations.
3. The blood sample release agent combination of claim 2, wherein the concentration of the formic acid solution is 1% and 2%; the concentration of the acetonitrile solution is 85% and 100%.
4. The blood sample release agent combination according to claim 1, characterized by being used for the pretreatment of plasma samples for LC-MS/MS assay of catecholamines and their metabolites.
5. A method of pretreatment of a blood sample, comprising treatment with a combination of release agents according to any one of claims 1-4.
6. The method according to claim 5, comprising the steps of:
adding a mixed internal standard working solution into a sample, and uniformly mixing by vortex;
centrifuging and transferring the supernatant to a WCX 96-well solution plate which is pretreated by an aqueous solution of 80% -90% V/V acetonitrile containing 1% -2%V/V formic acid and water in sequence;
sequentially using ultrapure water and 80% -100% V/V acetonitrile solution to clean the holes;
finally, eluting the target from the plate into a 96-hole collecting plate by using an aqueous solution of 80% -90% V/V acetonitrile containing 1% -2%V/V formic acid to obtain an eluent;
drying the eluent by a nitrogen blower, adding 0.01-0.1% m/V VC solution, oscillating and centrifuging to obtain supernatant.
7. A kit for the extraction or detection of 6 catecholamines in blood plasma, comprising a blood sample releasing agent combination according to any of the claims 1-4.
8. The kit of claim 7, further comprising reagents for chromatographic and mass spectrometric detection.
9. The kit of claim 8, wherein the chromatographic conditions are: the flow rate is 0.3-0.5mL/min; the sample injection volume is 8-12 mu L; the sample injection temperature is 7-9 ℃; column temperature box 28-32 ℃; the operation time is 5-8min.
10. The kit of claim 9, wherein the chromatographic conditions are: the flow rate is 0.4mL/min; sample volume 10. Mu.L; the sample injection temperature is 8 ℃; column temperature box 30 ℃; run time was 6.5min.
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