CN112858504A - Detection method for measuring cGMP concentration in biological body fluid sample by liquid chromatography-mass spectrometry - Google Patents
Detection method for measuring cGMP concentration in biological body fluid sample by liquid chromatography-mass spectrometry Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention belongs to the technical field of drug analysis, relates to an in-vivo drug analysis and determination method, and particularly relates to a detection method for determining cGMP concentration in a biological body fluid sample by liquid chromatography-mass spectrometry. The invention discloses a high-sensitivity method for measuring cAMP concentration in a biological body fluid sample by liquid chromatography-mass spectrometry, which comprises the following steps: (1) preprocessing a sample to be detected; (2) preparing a standard curve by liquid chromatography-mass spectrometry detection; (3) and (3) detecting the concentration of cGMP in the sample to be detected by liquid chromatography-mass spectrometry. The method is rapid, accurate and high in sensitivity, the sample pretreatment method is simple, the repeatability is good, the precision and the accuracy are high, the sensitivity is high, and the detection limit is low.
Description
Technical Field
The invention belongs to the technical field of drug analysis, relates to an in-vivo drug analysis and determination method, and particularly relates to a detection method for determining cGMP concentration in a biological body fluid sample by liquid chromatography-mass spectrometry.
Background
Cyclic guanosine monophosphate (cGMP) is widely distributed in various tissues, has a content of about 1/10 to 1/100 of cAMP, is produced by catalyzing GTP with guanylate cyclase, and is decomposed by phosphodiesterase. cGMP has the function of acetylcholine, inhibiting myocardial contractility, reducing heart rate, increasing nerve excitability, stimulating the release of hydrolase from leukocyte lysosome, stimulating the division and proliferation of lymphocytes, inhibiting gluconeogenesis and stimulating parasympathetic nerve. Therefore, the cGMP concentration in animal plasma and urine can be detected in an important physiological sense, and the cGMP concentration detection method also has an important role in evaluating the influence of the medicine on the secretion of cyclic adenosine monophosphate by animals.
At present, enzyme-linked immunosorbent assay (ELISA) is the most commonly used method for detecting cAMP in body fluids (plasma, urine, cerebrospinal fluid, etc.). Although the ELISA is widely applied, the method still has some defects, mainly including that the operation process is somewhat complicated, false positive results are easy to occur, and the repeatability is to be improved. Compared with ELISA, the liquid chromatogram tandem mass spectrometry has the advantages of being simpler, quicker and more accurate.
The cynomolgus monkey is similar to human in heredity and physiology, is a common model organism for biomedical research and drug development, and a literature report for determining the concentration of cyclic phosphorus guanosine in the plasma and urine of the cynomolgus monkey is not available at present. The method not only establishes the cGMP liquid chromatography tandem mass spectrometry detection method in the body fluid of the plasma and the urine of the cynomolgus monkey for the first time, but also improves and optimizes the defects of complicated detection process and long detection time of the existing method so as to save the detection time and ensure the accuracy and reliability of the detection, thereby being more favorable for evaluating the high-throughput screening of the drugs and clinical samples.
Disclosure of Invention
The invention aims to provide a high-sensitivity method for measuring cGMP concentration in a biological body fluid sample by liquid chromatography-mass spectrometry.
The invention discloses a high-sensitivity method for measuring cGMP concentration in a biological body fluid sample by liquid chromatography-mass spectrometry, which comprises the following steps:
(1) preprocessing a sample to be detected;
(2) preparing a standard curve by liquid chromatography-mass spectrometry detection;
(3) detecting the cGMP concentration in a sample to be detected by liquid chromatography-mass spectrometry;
the pretreatment of the sample to be detected is to add phosphodiesterase inhibition (PDEs) agent as a stabilizer before the sample to be detected is treated and collected.
In a preferred embodiment, the biological fluid sample is plasma, urine and/or cerebrospinal fluid;
in a preferred embodiment, the phosphodiesterase inhibitor is preferably a PDE1 selective inhibitor or a PDE5 selective inhibitor, more preferably IBMX (3-isobutyl-1-methylxanthine), nimodipine, vinpocetine, IC86340, IC224, EHNA, BAY60-7750, IC933, dipyridamole, cilostazol, cilostamide, milrinone, amrinone, enoximone and, cyquazodan, theophylline, rolipram, pyraclostrobin, roflumilast, cilomilast, BRL-50481, IC242, a quinazoline small molecule compound S14, a thiadiazole small molecule compound VP 1.15; most preferred is IBMX.
In a preferred embodiment, the sample to be detected is preprocessed into plasma or urine, EDTA-K2 and IBMX are used as an anticoagulant and a stabilizer, a deuterated compound of an object to be detected is used as an internal standard, a certain volume of a plasma and urine sample is taken, a certain volume of a deuterated internal standard mixed solution is added, after mixing, liquid-liquid extraction or solid-phase extraction is adopted, and a supernatant is obtained by centrifugation, so that a sample to be detected is obtained;
in a preferred embodiment, the pretreatment of the sample to be detected is that plasma takes EDTA-K2 as an anticoagulant, deuterated compounds of respective substances to be detected are taken as internal standards, 1% of 100mM IBMX is added as a stabilizer before sample treatment and collection, 50.0uL of internal standard mixed solution is added into 50.0uL of plasma sample, after mixing, 300uL of acetonitrile is added, vortex mixing is carried out for 10min, centrifugation is carried out for 10min at 2600g under the condition of 4 ℃, and 200 muL of supernatant is taken to obtain the sample to be detected;
in a preferred embodiment, the liquid chromatography-mass spectrometry detection is as follows:
liquid chromatography determination is carried out under the conditions that: a chromatographic column: chromatography column Ultimate Diol 3 μm2.1 × 100 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: 2mM ammonium formate (10.0mg/L citric acid); mobile phase B: 100% ACN solution; washing liquid: 50% ACN solution and ACN/MeOH (1:1, v: v); the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 2uL and analysis time of 10.0 min;
measuring mass spectrum under the conditions of: the ion source is an electrospray ion source, and the interface temperature is as follows: 300 ℃, DL temperature: 250 ℃, heating air flow: 10.00L/min, heating block temperature: 400 ℃, dryer flow: 10.00L/min; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z346.10 → m/152.05;
compared with the prior art, the method of the invention has the following advantages:
1. the method is rapid, accurate and high in sensitivity, and IBMX is added in the sample pretreatment as a stabilizer, so that the stability of a plasma sample and a urine sample during treatment can be realized;
2. the specificity is strong: elution was carried out in a gradient using an Ultimate Diol 3 μm2.1 x 100mM column, aqueous 2mM ammonium formate (10.0mg/L citric acid) and 100% ACN solution as mobile phase. The retention time of the cyclic phosphorus guanosine is about 4.6 min;
3. the sensitivity is high: the minimum quantitative limits of the cyclophosphate guanosine plasma and urine are as follows: 0.500 ng/mL. .
Drawings
FIG. 1 is a chromatogram of cyclophosphamide guanosine and its deuterated internal standard (cyclophosphamide guanosine is shown in the upper figure and internal standard is shown in the lower figure);
FIG. 2 is a mass spectrum of blank plasma (upper diagram is guanosine cyclophosphate, and the lower diagram is an internal standard);
FIG. 3 is a mass spectrum of blank plasma after a standard is added (the upper graph is cyclophosphate guanosine, and the lower graph is an internal standard);
FIG. 4 is a standard curve (5.00-500 ng/mL).
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Both cyclophosphamide guanosine and its isotopic internal standard are provided by canadian TRC. Acetonitrile, methanol, acetic acid, ammonium acetate, formic acid, etc. were all HPLC grade from Fisher, usa, and other chemicals were analytically pure.
CMS-8060 mass spectrometer (Shimadzu, Japan), Lab Solutions 6.84SP1 data acquisition software (Shimadzu, Japan), Mettler XS 105DU electronic balance (Mettler, Switzerland); eppendorf Centrifuge 5424R high speed low temperature Centrifuge (Eppendorf, Germany).
EXAMPLE 1 preparation of stock solutions and working solutions
A proper amount of guanosine cyclophosphate and a certain amount of DMSO are added to prepare stock solutions with the contents of 1.00 mg/mL. Under the condition of room temperature, 10% acetonitrile solution is used for respectively diluting independent stock solution in sequence to prepare working solution of a standard curve sample and a quality control sample. All working solutions were formulated in clear polypropylene tubes and stored in a-20 ℃ freezer refrigerator.
Example 2 sample pretreatment:
adding 1% of 100mM IBMX as a stabilizer into cynomolgus monkey plasma by taking EDTA-K2 as an anticoagulant before treatment, adding 50.0uL of internal standard solution into a 50uL plasma sample, adding 300uL of acetonitrile after mixing, carrying out vortex mixing for 10min, centrifuging for 10min at 2600g at 4 ℃, and taking 200 uL of supernatant to obtain a sample to be detected; (ii) a
Example 3 detection by liquid chromatography-mass spectrometry:
liquid chromatography determination is carried out under the conditions that: a chromatographic column: chromatography column Ultimate Diol 3 μm2.1 × 100 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: 2mM ammonium formate (10.0mg/L citric acid); mobile phase B: 100% ACN solution; washing liquid: 50% ACN solution and ACN/MeOH (1:1, v: v); the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 2uL and analysis time of 10.0 min;
measuring mass spectrum under the conditions of: the ion source is an electrospray ion source, and the interface temperature is as follows: 300 ℃, DL temperature: 250 ℃, heating air flow: 10.00L/min, heating block temperature: 400 ℃, dryer flow: 10.00L/min; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z346.10 → m/152.05;
example 4, standard curve preparation:
diluting the working solution by 20 times by using a plasma or urine substitute matrix (50% acetonitrile), adding 1% of 100mM IBMX as a stabilizer, taking a 50uL plasma sample, adding 50.0uL of deuterated internal standard mixed solution, mixing, adding 300uL acetonitrile, carrying out vortex mixing for 10min, centrifuging for 10min at 2600g at 4 ℃, and taking 200 mu L of supernatant to obtain a standard curve sample. Measuring standard samples with different concentrations by adopting the chromatographic and mass spectrum conditions, drawing a peak area-concentration standard curve, and obtaining a linear regression equation:
cyclic phosphorus guanosine: (x) 1.12866 x +0.0344745, R2＝0.9975124；
Example 5 determination of cyclic adenosine monophosphate concentration in plasma and urine:
preparing the plasma to be detected according to the method for preparing the sample in the embodiment 1, detecting the sample to be detected according to the embodiment 2 by adopting a liquid chromatography-mass spectrometry detection method, recording a peak area corresponding to the cyclic guanosine monophosphate, substituting the peak area ratio of the cyclic guanosine monophosphate concentration and the internal standard into the established standard curve, and calculating to obtain the cyclic guanosine monophosphate concentration in the plasma to be detected.
The concentration of guanosine cyclophosphate in urine was determined in the same manner as in plasma.
Example 6 and comparative example
Precision and accuracy of the method:
concentration of quality control working solution (ng/mL):
| compound (I) | LLOQ QC | LQC | GMQC | MQC | HQC |
| Guanosine cyclophosphate | 10.0 | 30.0 | 80.0 | 400 | 750 |
EDTA-K2 is used as an anticoagulant in plasma, the urine does not contain the anticoagulant, 1% of 100mM IBMX is added as a stabilizer before sample treatment, and a deuterated compound is used as an internal standard; precisely adding 50uL of sample into a 96-deep-well plate, adding 50.0uL of internal standard mixed solution, mixing, adding into a 300uL acetonitrile 96-deep-well plate, carrying out vortex mixing for 10min, centrifuging for 10min at 2600g at 4 ℃, and taking 200 uL of supernatant to a 96-well plate to obtain the quality control sample. Measuring quality control samples with different concentrations by adopting the chromatographic and mass spectrum conditions to obtain peak areas of the cyclic phosphorus guanosine, substituting the peak areas into a peak area-concentration standard curve to obtain the actual concentration of the quality control samples (n is 3): the endogenous concentration was subtracted from the concentration of the quality control sample to obtain the concentration of the exogenous drug (ng/mL).
From the data in the above table it can be seen that:
the experimental results of the method show that RE% is within 15% of acceptable range, and RSD% is within 15% of acceptable range. Therefore, the method has the advantages of good repeatability, high precision and accuracy, high sensitivity and low detection limit.
Example 7 Effect of IBMX on sample stability
Stability of the analyte in plasma at room temperature, stability will be examined by measuring the analyte concentration after storage for about 20 hours at room temperature for two concentration levels of stability samples (one with 1% of 100mM IBMX as a stabilizer and the other without), 3 replicates for each concentration level. The results are as follows:
from the above data, it can be seen that the degradation of guanosine cyclophosphate in plasma without IBMX is significant and the stability is not good. The cyclic phosphorus guanosine in the blood plasma containing IBMX is more stable.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (8)
1. A detection method for measuring cGMP concentration in a biological body fluid sample by liquid chromatography-mass spectrometry comprises the following steps:
(1) preprocessing a sample to be detected;
(2) preparing a standard curve by liquid chromatography-mass spectrometry detection;
(3) detecting the cGMP concentration in a sample to be detected by liquid chromatography-mass spectrometry;
the pretreatment of the sample to be detected is to add a phosphodiesterase inhibitor as a stabilizer before the sample to be detected is treated and collected.
2. The test method according to claim 1, wherein the sample of biological fluid is plasma, urine and/or cerebrospinal fluid.
3. The assay of claim 1 wherein said phosphodiesterase inhibitor is a PDE1 selective inhibitor or a PDE5 selective inhibitor.
4. The assay of claim 1 wherein the phosphodiesterase inhibitor is one of IBMX (3-isobutyl-1-methylxanthine), nimodipine, vinpocetine, IC86340, IC224, EHNA, BAY60-7750, IC933, dipyridamole, cilostazol, cilostamide, milrinone, amrinone, enoximone and, cyguanzodan, theophylline, rolipram, pyraclostrobin, roflumilast, cilomilast, BRL-50481, IC242, the quinazoline small molecule compound S14, and the thiadiazole small molecule compound VP 1.15.
5. The assay of claim 1 wherein the phosphodiesterase inhibitor is IBMX.
6. The detecting method according to claim 1, wherein the sample to be detected is preprocessed into plasma or urine, the EDTA-K2 and IBMX are used as an anticoagulant and a stabilizer, the deuterated compound of the substance to be detected is used as an internal standard, a certain volume of the plasma and urine sample is taken, a certain volume of the deuterated internal standard mixed solution is added, after mixing, liquid-liquid extraction or solid-phase extraction is adopted, and the supernatant is centrifuged to obtain the sample to be detected.
7. The detection method according to claim 3, wherein the pretreatment of the sample to be detected is that plasma is treated with EDTA-K2 as anticoagulant and deuterated compound of each analyte as internal standard, 1% of 100mM IBMX as stabilizer is added before sample treatment and collection, 50.0uL of internal standard mixed solution is added to 50.0uL of plasma sample, after mixing, 300uL of acetonitrile is added, vortex mixing is carried out for 10min, centrifugation is carried out for 10min at 2600g at 4 ℃, and 200 μ L of supernatant is taken to obtain the sample to be detected.
8. The detection method according to claim 1, wherein the detection by liquid chromatography-mass spectrometry is:
liquid chromatography determination is carried out under the conditions that: a chromatographic column: chromatography column Ultimate Diol 3 μm2.1 × 100 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: 2mM ammonium formate (10.0mg/L citric acid); mobile phase B: 100% ACN solution; washing liquid: 50% ACN solution and ACN/MeOH (1:1, v: v); the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 2uL and analysis time of 10.0 min;
measuring mass spectrum under the conditions of: the ion source is an electrospray ion source, and the interface temperature is as follows: 300 ℃, DL temperature: 250 ℃, heating air flow: 10.00L/min, heating block temperature: 400 ℃, dryer flow: 10.00L/min; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z346.10 → m/152.05.
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