CN118191124A - Quantitative analysis method for concentration of anti-gout medicine components in health care product - Google Patents
Quantitative analysis method for concentration of anti-gout medicine components in health care product Download PDFInfo
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- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 32
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- 230000036541 health Effects 0.000 title claims abstract description 20
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims abstract description 60
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- DBABZHXKTCFAPX-UHFFFAOYSA-N probenecid Chemical compound CCCN(CCC)S(=O)(=O)C1=CC=C(C(O)=O)C=C1 DBABZHXKTCFAPX-UHFFFAOYSA-N 0.000 claims abstract description 31
- 229960003081 probenecid Drugs 0.000 claims abstract description 31
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- 229960003459 allopurinol Drugs 0.000 claims abstract description 30
- 229960001338 colchicine Drugs 0.000 claims abstract description 30
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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Abstract
The invention discloses a quantitative analysis method for the concentration of anti-gout medicine components in a health care product, which comprises the following steps: sample treatment: grinding the health product into powder, mixing with acetonitrile solution, performing ultrasonic extraction, centrifuging, and collecting supernatant to obtain a sample solution; and (3) quantitatively analyzing the sample solution by adopting a liquid chromatography-mass spectrometry system: the liquid chromatography is used as a separation system to separate and elute the sample solution, and the mass spectrum is used as a detection system to detect the eluted sample solution, so that the concentrations of 5 anti-gout drug components including tribromone, allopurinol, probenecid, febuxostat and colchicine can be obtained simultaneously. The quantitative analysis method has the advantages of simple pretreatment, rapidness, sensitivity, strong specificity, high accuracy, good reproducibility, short analysis time, capacity of obtaining satisfactory peak shape, chromatographic retention time and the like, and is suitable for rapid mass detection of the prescription drugs of tribromone, allopurinol, probenecid, febuxostat and colchicine which are added in a illegal way in the health-care product.
Description
Technical Field
The invention relates to the field of detection application of illegal drug addition, in particular to a quantitative analysis method for the concentration of anti-gout drug components in a health product.
Background
At present, with the continuous improvement of living standard, the patients with gout induced by the rise of uric acid level in blood are obviously increased, and the variety of domestic health care products for resisting gout is also rapidly increased. The industrial scale of anti-gout health foods is continuously increased, but monitoring of prescription drugs in anti-gout health foods is a weak link at present. The drugs applied to acute gout are colchicine, and the common therapeutic drugs for recurrent chronic gout patients are allopurinol, probenecid, febuxostat and benzbromarone. The 5 chemical medicines are prescription medicines, and can produce great toxic and side effects on liver and kidney functions and aggravate cardiovascular and cerebrovascular diseases after long-term administration. At present, main methods for detecting febuxostat, colchicine, probenecid, allopurinol and benzbromarone include liquid chromatography, mass spectrometry, gas chromatography, capillary electrophoresis and the like, and detection objects mainly comprise biological samples such as blood, urine and the like, and the research on gout medicine components in health-care foods is less. Therefore, it is necessary to establish a sensitive, rapid and comprehensive detection method for screening and measuring gout medicine components in health food.
Disclosure of Invention
The invention aims to provide a quantitative analysis method for the concentration of anti-gout medicine components in a health care product, which has the advantages of simple and rapid pretreatment, strong specificity, high accuracy, good reproducibility and short analysis time.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
The invention provides a quantitative analysis method for the concentration of anti-gout medicine components in a health care product, which comprises the following steps:
(1) Sample treatment: grinding the health product into powder, mixing with acetonitrile solution, performing ultrasonic extraction, centrifuging, and collecting supernatant to obtain a sample solution;
(2) And (3) quantitatively analyzing the sample solution by adopting a liquid chromatography-mass spectrometry system: separating and eluting the sample solution by using liquid chromatography as a separation system, and detecting the eluted sample solution by using mass spectrum as a detection system to obtain the concentration of the anti-gout medicine component;
The prescription gout medicine components comprise tribromone, allopurinol, probenecid, febuxostat and colchicine.
Further, in the step (2), the quantitative analysis of the anti-gout drug components is probenecid m/z:284.2 to 140.0, CE 25V; benzbromarone m/z:423.1 to 250.9, CE value 32V; febuxostat m/z:317.05 to 261.00, CE value 27V; allopurinol m/z:136.9 to 109.9, CE value 18V; colchicine m/z:400.1 to 358.1, CE value 30v.
Further, in the step (2), the mass spectrum adopts an ion source as an ESI ion source, and positive ion detection is carried out in a multi-stage reaction monitoring MRM mode; the conditions of the mass spectrum are the dry gas temperature: 350 ℃; drying gas flow rate: 11 L/min; atomizer pressure: 30 A psi; capillary voltage: 4000v (+).
Further, in the mass spectrometry detection in the step (2), probenecid and benzbromarone adopt ESI －, febuxostat, allopurinol and colchicine adopt an ESI ＋ mode; monitoring by MRM mode for 0-3.2 min, and simultaneously scanning ESI ＋ and ESI - for 0.01 min.
Further, in the mass spectrometry detection in the step (2), the retention time of probenecid is 1.7 min; the retention time of benzbromarone is 2.2 min; febuxostat retention time was 1.85 min; the retention time of allopurinol is 0.4 min; colchicine retention time was 0.38 min.
Further, the mobile phase A of the liquid chromatography in the step (2) adopts a mixed solution of acetonitrile-formic acid water containing formic acid; column temperature of the liquid chromatography: 30 ℃; the flow rate of the mobile phase is 0.4 mL/min; the sample loading was 2. Mu.L.
Further, the ratio of the mobile phase A changes with time as follows: 0-0.5 min,90% A;0.5-1 min,90% A-20% A;1-2.5 min,20% A;2.5-3.2 min,20% A-90% A.
Further, the specific steps of the sample treatment in the step (1) are as follows: grinding the health product into powder by using a mortar, precisely weighing 1.0 mg, adding 1mL of acetonitrile solution, mixing for 1min by vortex oscillation, performing ultrasonic extraction for 30min, centrifuging for 10min at 14000 r/min, collecting supernatant, placing in a centrifuge tube, and diluting for 10 times to obtain a sample solution.
Further, the liquid chromatography-mass spectrometry system in the step (2) comprises a triple quadrupole liquid chromatography-mass spectrometer and a liquid chromatograph.
Further, the linear range of the method for quantitatively analyzing probenecid, benzbromarone and febuxostat is 5 mug/kg-200 mug/kg, and the lowest quantitative lower limit is 5 mug/kg; the linear range for allopurinol and colchicine is 10 μg/kg-100 μg/kg, the lowest lower limit of quantification is 10 μg/kg.
In summary, the invention provides a quantitative analysis method for the concentration of anti-gout medicine components in a health care product, which comprises the following steps:
(1) Sample treatment: grinding the health product into powder by adopting a mortar, precisely weighing 1.0 mg, adding 1mL of acetonitrile solution, mixing for 1min by vortex oscillation, performing ultrasonic extraction for 30min, centrifuging for 10min at 14000 r/min, collecting supernatant, placing in a centrifuge tube, diluting for 10 times to obtain a sample solution, and performing liquid analysis;
(2) And (3) quantitatively analyzing the sample solution by adopting a liquid chromatography-mass spectrometry system:
Separating and eluting the sample solution by using liquid chromatography as a separation system: the mobile phase A adopts acetonitrile containing formic acid and mixed solution of formic acid water for gradient elution; the chromatographic column of the liquid chromatograph is Waters BEH cube C 18 column (50 mm ×2.1 mm,1.7 μm), column temperature: 30 ℃; mobile phase flow rate: 0.4 mL/min; sample injection amount: 2. mu L; the ratio of the mobile phase A changes with time as follows: 0-0.5 min,90% A;0.5-1 min,90% A-20% A;1-2.5 min,20% A;2.5-3.2 min,20% A-90% A;
Detecting the eluted sample solution by using mass spectrum as a detection system: adopting an ion source as an ESI ion source, carrying out positive ion detection in a multi-stage reaction monitoring MRM mode, and adopting mass spectrum conditions as dry gas temperature: 350 ℃; drying gas flow rate: 11 L/min; atomizer pressure: 30 A psi; capillary voltage: 4000V (+); the quantitative analysis of the anti-gout medicine components is as follows: probenecid m/z:284.2 to 140.0, CE 25V; benzbromarone m/z:423.1 to 250.9, CE value 32V; febuxostat m/z:317.05 to 261.00, CE value 27V; allopurinol m/z:136.9 to 109.9, CE value 18V; colchicine m/z:400.1 to 358.1, CE value 30v; probenecid and benzbromarone adopt ESI －, febuxostat, allopurinol and colchicine adopt an ESI ＋ mode; monitoring by using an MRM mode, wherein the monitoring time is 0-3.2 min, ESI ＋ and ESI - are scanned simultaneously, and the switching time is 0.01min; probenecid retention time was 1.7min; the retention time of the benzbromarone is 2.2 min; the non-cloth retention time was 1.85 min; allopurinol retention time was 0.4 min; colchicine retention time 0.38 min; and finally, simultaneously obtaining the concentrations of the anti-gout medicine components such as tribromone, allopurinol, probenecid, febuxostat and colchicine.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. the quantitative analysis method has short measurement time, can detect the target compound when the whole chromatographic analysis measurement process is 3.0 min, and is particularly suitable for centralized monitoring of a large number of samples;
2. the invention can monitor 5 anti-gout medicine components at the same time by one sample injection, thereby greatly reducing the workload, reducing the use amount of organic reagents and meeting the requirements of energy conservation and environmental protection;
3. The pretreatment method is simple, convenient and quick, is suitable for routine quantitative analysis and detection, is especially suitable for detection of a large number of samples, and reduces operation steps and operation time;
4. the lowest quantitative lower limit of the quantitative analysis method is probenecid, benzbromarone and febuxostat 5 mug/kg, and allopurinol and colchicine 10 mug/kg are lower than the prior art; and the recovery rate is high, and the precision (relative standard deviation, RSD) is less than 15%.
Drawings
FIG. 1 is a block diagram of an anti-gout drug detected using the quantitative analysis method of the present invention; wherein A is benzbromarone; b is allopurinol; c probenecid; d is febuxostat; e is colchicine.
FIG. 2 is a chromatogram of an anti-gout drug ingredient; wherein A is MRM chromatogram of benzbromarone; b is the MRM chromatogram of allopurinol; c is the MRM chromatogram of probenecid; d is the MRM chromatogram of febuxostat; e is the MRM chromatogram of colchicine.
FIG. 3 is a blank matrix chromatogram and a lowest quantitative lower limit chromatogram; wherein A1, B1, C1, D1 and E1 are chromatograms of a blank matrix, and A2, B2, C2, D2 and E2 are chromatograms of an object to be detected, wherein the minimum quantitative lower limit is added to the blank matrix.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the specific embodiments.
Example 1: quantitative analysis method for concentration of benzbromarone, allopurinol, probenecid, febuxostat and colchicine (structural formula shown in figure 1) in health care product
1. Instrument and reagent
(1) Instrument for measuring and controlling the intensity of light
Agilent 6460 type triple quadrupole liquid chromatograph and 1290 Infinicity II liquid chromatograph.
(2) Reagent(s)
Acetonitrile: damas-beta (4L), HPLC grade purity 99.90%; methanol damas-beta (4L), HPLC grade purity 99.90%; formic acid: 50 mL, thermo, united states.
2. Experimental procedure
(1) Sample treatment:
For the tablet or granule health product, grinding into powder with a mortar, precisely weighing 1.0 mg, adding 1mL acetonitrile solution, mixing for 1min under vortex oscillation, ultrasonic extracting for 30min, centrifuging at 14000 r/min for 10min, collecting supernatant, placing in a centrifuge tube, diluting for 10 times, and performing liquid analysis;
(2) Detection sample determination is carried out by adopting a liquid chromatography-mass spectrometry system:
Separating the sample by using liquid chromatography as a separation system, wherein the mobile phase adopts mixed solution gradient elution of acetonitrile containing formic acid and formic acid water, and the chromatographic column of the liquid chromatography is Waters BEH section C 18 column (50 mm multiplied by 2.1 mm,1.7 μm), and the column temperature is: 30 ℃; the flow rate of the mobile phase is 0.4 mL/min; sample injection amount: 2. mu L. The flow match ratio varies with time as: 0-0.5 min,90% A;0.5-1 min,90% A-20% A;1-2.5 min,20% A;2.5-3.2 min,20% A-90% A.
(3) Detecting the health product sample subjected to gradient elution by taking a mass spectrum as a detection system, adopting an ion source as an ESI ion source, and carrying out positive ion detection in a multi-stage reaction monitoring MRM mode, wherein the mass spectrum is prepared by the following conditions: 350 ℃; drying gas flow rate: 11L/min; atomizer pressure: 30 A psi; capillary voltage: 4000v (+);
The quantitatively analyzed ions were respectively: probenecid m/z 284.2 to 140.0, CE 25V; the m/z of the benzbromarone is 423.1 to 250.9, and the CE value is 32V; febuxostat m/z is 317.05-261.00, and the CE value is 27V; allopurinol m/z is 136.9-109.9, and the CE value is 18V; colchicine m/z 400.1.fwdarw.358.1, CE value 30v. Probenecid and benzbromarone are monitored by adopting ESI － and ESI ＋ modes of the rest compounds and MRM modes for 0-3.2 min, ESI ＋ and ESI - are scanned simultaneously, and switching time is 0.01 min. Probenecid retention time was 1.7 min; the retention time of the benzbromarone is 2.2 min; febuxostat retention time was 1.85 min; allopurinol retention time was 0.4 min; colchicine retention time 0.38 min.
3. Experimental results
The MRM profile of benzbromarone, allopurinol, probenecid, febuxostat and colchicine is shown in figure 2.
Example 2: quantitative analysis method for detecting concentration of benzbromarone, allopurinol, probenecid, febuxostat and colchicine
1. Specialization of
In the quantitative analysis method, the specificity of the object to be detected is evaluated by comparing the lowest concentration point of the standard curve with a blank matrix operated by the same method. The chromatograms of the blank substrates are shown in A1, B1, C1, D1 and E1 of FIG. 3 respectively; the chromatograms of the object to be measured with the lowest quantitative lower limit of the blank matrix are shown as A2, B2, C2, D2 and E2 in FIG. 3 respectively. As can be seen from fig. 3, the endogenous substances in the blank matrix do not interfere with the measurement of the analyte and the internal standard, indicating that the quantitative analysis method of the present invention has specificity for the measurement of colchicine, benzbromarone, febuxostat, allopurinol and probenecid.
2. Matrix effect
The matrix effect in the invention is compared with the mixed standard working solution prepared by using a blank matrix solution and the mixed standard working solution prepared by using a pure acetonitrile solution, and the matrix effect of 5 anti-gout drugs in the health care food is examined (see table 1). As can be seen from Table 1, the matrix effect values of the method of the present invention are between 0.90 and 1.10, and thus the matrix effect is not greatly affected.
Table 1: linear equation, correlation coefficient, linear range, detection limit, quantitative lower limit and matrix effect of 5 objects to be detected
3. Standard curve, linear range and lower limit of quantification
Standard curve dilution solutions of colchicine, benzbromarone, febuxostat, allopurinol and probenecid were prepared from 60% acetonitrile water (v acetonitrile: v water = 6:4): taking a blank matrix extracting solution, adding a solution containing 5 components to be detected, and preparing a series of standard solutions with concentration. 100. Mu.L of each sample was prepared according to the procedure for pretreatment of the sample in example 1, and LC-MS/MS analysis was performed, and regression analysis was performed using a weighted least squares method to obtain a standard curve. In this example, 3 batches of samples were used for the experiment. The obtained linear regression equation is shown in table 1, y represents the ratio of the peak area of the drug to be tested to the peak area of the internal standard, and x represents the concentration of the drug to be tested. The linear range of the quantitative analysis method for probenecid, benzbromarone and febuxostat is 5-200 mug/kg, and the lowest quantitative lower limit is 5 mug/kg; the standard curve range for allopurinol and colchicine is 10-100 μg/kg, and the lowest lower limit of quantification is 10 μg/kg.
4. Recovery rate and precision
Preparing quality control samples with low, medium and high concentrations of an object to be detected according to the steps of a standard curve series solution preparation method, carrying out 6 sample analyses on each concentration, and recording chromatographic peaks; and then preparing a pure sample solution without a matrix of the final measured concentration for analysis to obtain a corresponding peak area, and examining the extraction recovery rate of the sample according to the ratio of the peak area to the peak area. The analysis and measurement results are shown in Table 2, which shows that the quantitative analysis method of the invention has high recovery rate and high precision.
Table 2: extraction recovery rate (n=6) and precision of 5 samples
Example 3 sample measurement
The quantitative analysis method of the invention is adopted to measure 2 batches of tabletting candy health products with uric acid blood reducing effect, test sample solutions are prepared according to the steps in the example 1, the response values of the test sample solutions are respectively measured, and the content of 5 anti-gout drugs, namely colchicine, benzbromarone, febuxostat, allopurinol and probenecid, is calculated.
The results are shown in Table 3, and the febuxostat and benzbromarone with higher concentrations are detected in 2 batches of samples, and other samples are not detected, so that the quantitative analysis method provided by the invention is sensitive in detection and high in accuracy.
Table 3:2 detection results (mg/kg) of anti-gout drug concentration in commercial uric acid reducing tablet candy
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. The quantitative analysis method for the concentration of the anti-gout medicine component in the health care product is characterized by comprising the following steps:
(1) Sample treatment: grinding the health product into powder, mixing with acetonitrile solution, performing ultrasonic extraction, centrifuging, and collecting supernatant to obtain a sample solution;
(2) And (3) quantitatively analyzing the sample solution by adopting a liquid chromatography-mass spectrometry system: separating and eluting the sample solution by using liquid chromatography as a separation system, and detecting the eluted sample solution by using mass spectrum as a detection system to obtain the concentration of the anti-gout medicine component;
The prescription gout medicine components comprise tribromone, allopurinol, probenecid, febuxostat and colchicine.
2. The quantitative analysis method according to claim 1, wherein in the step (2), the quantitative analysis of the anti-gout pharmaceutical components is performed by: probenecid m/z:284.2 to 140.0, CE 25V; benzbromarone m/z:423.1 to 250.9, CE value 32V; febuxostat m/z:317.05 to 261.00, CE value 27V; allopurinol m/z:136.9 to 109.9, CE value 18V; colchicine m/z:400.1 to 358.1, CE value 30v.
3. The quantitative analysis method according to claim 1, wherein in the step (2), the mass spectrum adopts an ion source as an ESI ion source, and positive ion detection is performed in a multistage reaction monitoring MRM mode; the conditions of the mass spectrum are the dry gas temperature: 350 ℃; drying gas flow rate: 11 L/min; atomizer pressure: 30 A psi; capillary voltage: 4000v (+).
4. The method according to claim 3, wherein in the mass spectrometry in the step (2), probenecid and benzbromarone are in ESI －, febuxostat, allopurinol and colchicine are in ESI ＋ mode; monitoring by MRM mode for 0-3.2 min, and simultaneously scanning ESI ＋ and ESI - for 0.01 min.
5. The method of claim 1, wherein the probenecid retention time in the mass spectrometry of step (2) is 1.7 min; the retention time of benzbromarone is 2.2 min; febuxostat retention time was 1.85 min; the retention time of allopurinol is 0.4 min; colchicine retention time was 0.38 min.
6. The quantitative analysis method according to claim 1, wherein the mobile phase a of the liquid chromatography of step (2) is a mixed solution of acetonitrile-formic acid water containing formic acid; the column temperature of the liquid chromatography is 30 ℃; the flow rate of the mobile phase is 0.4 mL/min; the sample loading was 2. Mu.L.
7. The quantitative analysis method according to claim 6, wherein the change of the mobile phase a ratio with time is: 0-0.5 min,90% A;0.5-1 min,90% A-20% A;1-2.5 min,20% A;2.5-3.2 min,20% A-90% A.
8. The quantitative analysis method according to claim 1, wherein the specific steps of the sample treatment in the step (1) are: grinding the health product into powder by using a mortar, precisely weighing 1.0 mg, adding 1mL of acetonitrile solution, mixing for 1min by vortex oscillation, performing ultrasonic extraction for 30min, centrifuging for 10min at 14000 r/min, collecting supernatant, placing in a centrifuge tube, and diluting for 10 times to obtain a sample solution.
9. The quantitative analysis method according to claim 1, wherein the liquid chromatography system in step (2) comprises a triple quadrupole liquid chromatography and a liquid chromatograph.
10. The quantitative analysis method according to claim 1, wherein the linear range of the quantitative analysis of probenecid, benzbromarone and febuxostat by the method is 5 μg/kg-200 μg/kg, and the lowest lower limit of quantification is 5 μg/kg; the linear range for allopurinol and colchicine is 10 μg/kg-100 μg/kg, the lowest lower limit of quantification is 10 μg/kg.
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