CN117129611A - Method for detecting 7 antihypertensive drugs and metabolites by liquid chromatography-tandem mass spectrometry - Google Patents
Method for detecting 7 antihypertensive drugs and metabolites by liquid chromatography-tandem mass spectrometry Download PDFInfo
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- 239000002207 metabolite Substances 0.000 title claims abstract description 32
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 9
- 239000002220 antihypertensive agent Substances 0.000 title claims description 24
- 229940127088 antihypertensive drug Drugs 0.000 title claims description 21
- 238000001514 detection method Methods 0.000 claims abstract description 35
- XSDQTOBWRPYKKA-UHFFFAOYSA-N amiloride Chemical compound NC(=N)NC(=O)C1=NC(Cl)=C(N)N=C1N XSDQTOBWRPYKKA-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229960002576 amiloride Drugs 0.000 claims abstract description 28
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Classifications
-
- 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
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to the technical field of medicine detection, in particular to a method for detecting 7 antihypertensive medicines and metabolites by using a liquid chromatography-tandem mass spectrometry, wherein the 7 antihypertensive medicines and metabolites comprise: hydrochlorothiazide, chlorthalidone, spironolactone, canrenone, amiloride, triamterene, furosemide, and indapamide, the method specifically comprises: and preparing a standard curve equation, and processing a sample to be detected by adopting a protein precipitation method to obtain a sample injection sample for on-machine detection. The detection method provided by the invention can be used for simultaneously carrying out quantitative detection on 8 substances, and has the advantages of short detection time, short analysis time, good reproducibility, high sample recovery rate, wide linear range and remarkable improvement on the sensitivity and accuracy of detection.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for detecting 7 antihypertensive medicines and metabolites by using a liquid chromatography-tandem mass spectrometry method.
Background
Hypertension is a chronic disease, and most patients need long-term drug treatment, so that the blood pressure can be effectively controlled to a target level. Hypotensive drugs exert a hypotensive effect mainly by affecting the systems of the sympathetic nervous system, the renin-angiotensin-aldosterone system, the endothelin system, and the like, which play an important role in physiological regulation of blood pressure.
Wherein the diuretic antihypertensive drug comprises:
1. thiazines: for example, hydrochlorothiazide (hydrochlorothiazide), chlorothianone (chlororalidone). Hydrochlorothiazide is an oral diuretic and antihypertensive agent widely applied clinically, and is suitable for treating edema and hypertension. The chlorthalidone is mainly used for treating congestive heart failure, ascites due to cirrhosis, nephrotic syndrome, acute and chronic nephritic edema, early stage of chronic renal failure, and sodium retention caused by adrenocortical hormone and estrogen treatment, and can be used alone or in combination with other antihypertensive drugs, and is mainly used for treating primary hypertension.
2. Aldosterone antagonists: for example, spironolactone (Spironolactone), canrenone (Canrenone). Spironolactone is commonly used clinically to treat essential hypertension and oedema with increased aldosterone. The aldosterone antagonist canrenone is commonly used to treat heart failure edema and ascites due to cirrhosis.
3. Potassium retention diuretic: such as Amiloride (Amiloride), triamterene (Triamterene). Amiloride is a potassium-retaining diuretic, has stronger sodium-expelling potassium-retaining diuretic effect for treating liver cirrhosis, edema, primary aldosteronism and the like, and is mainly used for treating mild-moderate hypertension in clinic at present. The triamterene is a potassium-retaining diuretic, acts on the epithelial cells of the distal tubular of the kidney, inhibits reabsorption of sodium ions and chloride ions to produce diuretic effect, and has retention effect on potassium ions.
4. Loop diuretics: such as Furosemide (Furosemide), is commonly used to treat cardiac edema, renal edema, ascites due to cirrhosis, dysfunction or peri-edema caused by vascular disorders.
5. Indapamide (indapamide) is a sulfonamide diuretic, which has the function of reducing blood pressure and promoting urination by inhibiting the reabsorption of the distal tubular cortical dilution, and is widely used for primary hypertension patients clinically.
Because of various types of antihypertensive drugs, the blood concentration of the antihypertensive drugs is monitored, and the method has important significance for reasonable and safe medication of patients. The method commonly adopted for measuring the hypertension medicine at present is a high performance liquid chromatography mass spectrometry method. In the existing method, the defects of less detection substances, more complex pretreatment operation, large sample size requirement, long analysis time and insufficient detection sensitivity of one needle at the same time exist in the detection of the diuretic antihypertensive drug.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting 7 antihypertensive drugs and 1 metabolite by using a liquid chromatography-tandem mass spectrometry, wherein the 7 antihypertensive drugs and the metabolite comprise: hydrochlorothiazide, chlorthalidone, spironolactone, canrenone, amiloride, triamterene, furosemide, and indapamide, comprising at least the steps of:
s1, preparing a standard curve equation, which comprises the following steps: preparing an internal standard working solution and a standard working solution, preparing a sample solution for standard yeast, and detecting the sample solution for standard yeast by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation for calculating the content of 7 antihypertensive drugs and metabolites;
s2, preprocessing a sample to be tested, including: uniformly mixing an internal standard working solution, a sample to be tested and a protein precipitator, centrifuging to obtain a supernatant, and uniformly mixing the supernatant with water I to obtain a sample injection sample; the protein precipitant is methanol;
s3, detecting a sample injection sample, which comprises the following steps: and detecting the sample by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result of the sample into a standard curve equation to obtain the contents of 7 antihypertensive drugs and metabolites in the sample to be detected.
Optionally, the sample to be tested is plasma added with formic acid, and the adding amount of the formic acid is as follows: 2-4 mu L of formic acid is added into each 1-mL plasma.
Optionally, S1 includes: s11, respectively preparing an internal standard working solution and a standard working solution with gradient concentration; s12, respectively taking an internal standard working solution and a standard working solution, adding water II and a protein precipitant, uniformly mixing to obtain a mixed solution, adding water III into the mixed solution, and uniformly mixing to obtain a sample for standard yeast; the protein precipitant is methanol; s13, detecting the sample loading solution for the standard curve by using a high performance liquid chromatography mass spectrometer to obtain a standard curve equation.
Optionally, in S12, the volume ratio of the standard working solution to the internal standard working solution is 1:2.
optionally, in S12, the volume ratio of the standard working solution to the water II is 1:9.
optionally, in S12, the total volume ratio of the standard working solution to the water II and the volume ratio of the protein precipitant are 1: 4-6;
optionally, in S12, the volume ratio of the mixed solution to the water III is 1:0.8 to 1.2.
Optionally, in S2, the volume ratio of the sample to be tested and the protein precipitant is 1: 4-6.
Optionally, in S2, the volume ratio of the sample to be tested to the internal standard working solution is 5:1.
optionally, in S2, the volume ratio of the supernatant to the water I is 1:0.8 to 1.2.
Optionally, in S2: the mixing condition of the internal standard working solution, the sample to be tested and the protein precipitant is 1500-2500 r/min, and the mixing is carried out for 3-6 min.
Optionally, in S2, the conditions for mixing after adding water I are: vortex mixing is carried out for 0.5-1.5 min at 1500-2500 r/min.
Optionally, in S2, the centrifugation condition is 12000-15000 r/min for 8-12 min.
Optionally, in S12, mixing conditions are 1500-2500 r/min vortex mixing for 1-2 min.
Alternatively, the chromatographic column is a C18 chromatographic column; mobile phase: the phase A is an aqueous solution containing 0.0.01% -0.2% formic acid by volume percentage, and the phase B is a methanol solution; flow rate: 0.4-0.5 mL/min; column temperature: 35-45 ℃; sample injection amount: 1-10 mu L; analysis time: 5.5 And (5) min.
The gradient elution conditions were: 0-1.50 minutes, A:65% -20%, B:35% -80%; 1.5 to 2.50 minutes, A:20% -0, B:80% -100%; 2.50-3.50 minutes, A:0, b:100%; 3.51-5.50 minutes, A:65%, B:35%.
Optionally, an electrospray ion source is adopted, positive ions and negative ions are collected simultaneously, and a segmented collection mode is adopted as a collection mode:
in the positive ion mode, the ion pairs expressed as parent/daughter ions are: chlorthalidone: 339.1/322.0; spirolactone: 417.2/341.2; canrenone: 341.3/107.1; amiloride: 230.2/116.1; triamterene: 254.1/237.0; indapamide: 365.9/132.1;
in negative ion mode, the ion pairs expressed as parent/daughter ions are: hydrochlorothiazide: 296.0/268.8; furosemide: 329.1/285.1.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
the detection method provided by the invention can be used for simultaneously detecting the contents of 7 antihypertensive drugs and 1 metabolite by one needle, and has the advantages of short pretreatment time, short detection time and detection result within 5.5 min after the detection method is started. The instrument can analyze 10 groups of samples within 1 hour, and the detection efficiency is remarkably improved.
The pretreatment operation is simple, the time is short, and the sample pretreatment step can be completed within 20-22 min. And the protein precipitant adopts methanol, so that the price is economical and the reagent and personnel cost is reduced.
The detection method has the advantages of good precision, good repeatability, high sample recovery rate, wide linear range and remarkable improvement of the sensitivity and accuracy of the detection result.
Drawings
FIG. 1 is a chromatogram of 7 drugs and metabolites in standard solution in example 1;
FIG. 2 is a chromatogram of 7 drugs and metabolites in a plasma sample of example 1;
FIG. 3 is a chromatogram of canrenone in a plasma sample of example 1;
FIG. 4 is a chromatogram of spirolactone in a plasma sample of example 1;
FIG. 5 is a graph of amiloride chromatograms under D3 conditions in experimental example 3;
FIG. 6 is a chromatogram of amiloride under the test conditions of example 1;
FIG. 7 is a chromatogram under the conditions of experiment 4-1 in Experimental example 4;
FIG. 8 is a chromatogram of the condition D5-1 in Experimental example 5;
FIG. 9 is a chromatogram of the condition D5-2 in Experimental example 5;
FIG. 10 is a chromatogram of the condition D5-3 in Experimental example 5;
FIG. 11 is a chromatogram of the condition D5-4 in Experimental example 5.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention. The embodiment of the invention provides a method for detecting 7 antihypertensive drugs and 1 metabolite by using a liquid chromatography-tandem mass spectrometry, wherein the 7 antihypertensive drugs and 1 metabolite comprise: hydrochlorothiazide, chlorthalidone, spironolactone, amiloride, triamterene, furosemide, and indapamide, with 1 metabolite canrenone (the metabolite of spironolactone), the method comprising at least the steps of:
s1, preparing a standard curve equation, which comprises the following steps:
preparing an internal standard working solution and a standard working solution, preparing a sample solution for standard yeast, and detecting the sample solution for standard yeast by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation for calculating the contents of 7 antihypertensive drugs and 1 metabolite;
s2, preprocessing a sample to be tested, including:
uniformly mixing an internal standard working solution, a sample to be tested and a protein precipitator, centrifuging to obtain a supernatant, and uniformly mixing the supernatant with water I to obtain a sample injection sample; the protein precipitant is methanol;
s3, detecting a sample injection sample, which comprises the following steps:
and detecting the sample by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result of the sample into a standard curve equation to obtain the contents of 7 antihypertensive drugs and 1 metabolite in the sample to be detected.
The pretreatment step of the embodiment of the invention only adopts a protein precipitation method, and has the technical advantages of simple operation and short time. The protein precipitant adopts methanol, has economic price, and can reduce the cost of reagents and personnel.
In the pretreatment step, the solvent effect can be effectively avoided by adding water I into the supernatant.
The invention discovers that the stability of canrenone and spirolactone in blood plasma is poor under the room temperature condition, and the stability can only be 2.5 hours, so that the accuracy of a detection result can not be ensured. Therefore, the invention adopts a mode of adding the stabilizing agent into the sample to be detected so as to ensure the accuracy of the detection result under the normal temperature condition. Screening experiments show that the overall effect is optimal when the stabilizer is formic acid. Further optimization experiments show that the addition amount of formic acid is as follows: 2-4 mu L of formic acid (purity 99.6%) is added to 1-mL plasma, more preferably 2 mu L of formic acid is added to 1-mL plasma, the spironolactone in the plasma can be stabilized at room temperature for 48 hours, and the canrenone can be stabilized at room temperature for 72 hours.
As an improvement of the embodiment of the invention, the sample loading solution for the standard curve is treated in the same way as the sample is in front of, and S1 specifically comprises:
s11, respectively preparing an internal standard working solution and a standard working solution with gradient concentration;
s12, respectively taking an internal standard working solution and a standard working solution, adding water II and a protein precipitant, uniformly mixing, adding water III into the mixed solution, and uniformly mixing to obtain a sample for standard yeast; the protein precipitant is methanol;
s13, detecting the sample loading solution for the standard curve by using a high performance liquid chromatography mass spectrometer to obtain a standard curve equation.
The embodiment of the invention examines the condition that water is used for replacing blood under different proportions of different people, has qualified matrix effect, and proves that the standard curve can be prepared by using water, and the pretreatment of replacing the post-standard curve is simple.
As an improvement of the embodiment of the present invention, in S12: the volume ratio of the standard working solution to the internal standard working solution is 1:2; the volume ratio of the standard working solution to the water II is 1:9, a step of performing the process; the total volume of the standard working solution and the water II and the volume ratio of the protein precipitant are 1: 4-6, and preferably 1:4, a step of; the volume ratio of the mixed solution to the water III is 1:0.8 to 1.2, and preferably 1:1.
as an improvement of the embodiment of the present invention, in S2: the volume ratio of the sample to be tested to the protein precipitant is 1: 4-6, preferably 1:4, a step of; the volume ratio of the sample to be tested to the internal standard working solution is 5:1, a step of; the volume ratio of the supernatant to the water I is 1:0.8 to 1.2, and preferably 1:1.
as an improvement of the embodiment of the invention, the sample to be measured is a plasma sample, and the volume is 50-100 mu L, preferably 50 mu L.
As an improvement of the embodiment of the invention, the specific conditions in the pretreatment process are as follows: the mixing condition of the internal standard working solution, the sample to be tested and the protein precipitant is 1500-2500 r/min vortex mixing for 3-6 min, preferably 2000-r/min vortex mixing for 5 min, and the mixing condition after adding water I is as follows: vortex mixing at 1500-2500 r/min for 0.5-1.5 min, preferably 2000-r/min for 1 min; the centrifugation conditions are 12000-15000 r/min for 8-12 min, preferably 14000 r/min for 10 min.
As a specific implementation manner of the embodiment of the present invention, the sample preprocessing steps are as follows: accurately sucking 10 mu L of internal standard working solution, then transferring 50 mu L of plasma sample (0.1 mu L of formic acid is added), adding 200 mu L of methanol, uniformly mixing for 5 min by a vortex of 2000 r/min, and centrifuging for 10 min at a high speed of 14000 r/min; 100 mu L of distilled water is added into 100 mu L of supernatant, vortex mixing is carried out for 1 min at 2000 r/min, and a sample is obtained.
As an improvement of the embodiment of the present invention, in S12: the mixing condition is 1500-2500 r/min vortex mixing for 0.5-1.5 min, preferably 2000-r/min vortex mixing for 1 min.
As a specific implementation mode of the embodiment of the invention, the preparation method of the sample solution for the standard curve adopts the following steps: taking 5 mu L of working solution of each concentration standard product, 10 mu L of internal standard working solution, 45 mu L of water and 200 mu L of methanol, vortex mixing for 1 min at 2000 r/min, transferring 100 mu L of mixed solution, adding 100 mu L of distilled water, vortex mixing for 1 min at 2000 r/min, and obtaining standard curve sampling solutions of various concentrations.
As an improvement of the embodiment of the present invention, in S1: the internal standard working solution contains isotope internal standards of 7 antihypertensive drugs and metabolites:
the isotopic internal standard of hydrochlorothiazide is: hydrochlorothiazide- [13C6];
the isotopic internal standard of chlorthalidone is: chlorthalidone- [13C6];
the internal isotope of spirolactone is: spirolactone-d 3;
the isotopic internal standard of canrenone is: canrenone-d 4;
the isotopic internal standard of amiloride is: amiloride- [15N3];
the isotopic internal standard of the triamterene is: triamterene-d 5;
the isotope internal standard of furosemide is: furosemide-d 5;
the isotopic internal standard of indapamide is: indapamide-d 3.
The technical effect of adopting the isotope internal standard is as follows: the influence of matrix effect on the result is effectively avoided, and the accuracy of the quantitative result is ensured.
As an improvement of the embodiment of the invention, the standard working solution and the internal standard working solution are diluted by adopting 65-75% of methanol aqueous solution with the volume percentage of preferably 70%; the prepared working solution can be repeatedly taken out for 10 times at the temperature of minus 80 ℃ without affecting the quantitative result.
As an improvement of the embodiment of the present invention, in S12, the preparation method of the internal standard working solution is as follows:
s121, respectively preparing internal standard stock solutions containing isotope internal standards, wherein the triamterene-d 5 internal standard stock solution adopts DMSO and methanol 1:1, preparing the rest internal standard stock solution by adopting pure methanol;
s122, respectively diluting the internal standard stock solution into an internal standard intermediate solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%, preferably 70%;
s123, mixing the internal standard intermediate solutions, and preparing an internal standard working solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%, preferably 70%.
As an improvement of the embodiment of the invention, in S12, the standard working solution is obtained by diluting 65% -75%, preferably 70% of methanol aqueous solution; the prepared standard yeast working solution can be repeatedly taken out for 10 times at the temperature of minus 80 ℃ without affecting the quantitative result.
Specifically, the preparation mode of the standard working solution with gradient concentration is as follows:
s121', preparing standard stock solutions containing standard substances respectively, wherein the standard stock solution of triamterene adopts DMSO and methanol 1:1, preparing the rest standard stock solution by adopting pure methanol;
s122', diluting the amiloride stock solution into amiloride intermediate solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%, preferably 70%; diluting the triamterene stock solution into a triamterene intermediate solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%, preferably 70%;
s123', taking amiloride intermediate liquid, triamcinolone acetonide intermediate liquid and the rest 6 standard stock solutions, and preparing a highest concentration standard working solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%, preferably 70%;
s124', sequentially diluting the standard working solution with the highest concentration with 65-75% by volume, preferably 70% by volume, to obtain the standard working solution with gradient concentration.
As an improvement of the embodiment of the invention, the conditions of the high performance liquid phase when the high performance liquid chromatography mass spectrometer is used for detection are as follows:
the chromatographic column is a C18 chromatographic column, preferably a column having a particle size of 2.5 to 3.0. Mu.m, and more preferably a column having a particle size of 2.7. Mu.m. Still more preferably, the chromatographic column is of the type: poroshell 120 EC-C18 (3 mm X100 mm,2.7 μm);
mobile phase: the phase A is an aqueous solution containing 0.01-0.2% formic acid by volume percent, and the phase B is a methanol solution; further preferably, the phase A is an aqueous solution containing 0.05% formic acid by volume and the phase B is a methanol solution;
flow rate: 0.4 to 0.5 mL/min, preferably 0.4 mL/min,
column temperature: 35-45 ℃, preferably 40 ℃;
sample injection amount: 1-10 mu L, preferably 4 mu L;
analysis time: 5.5 And (5) min.
Gradient elution conditions are shown in table 1:
TABLE 1
As an improvement of the embodiment of the invention, the mass spectrum conditions when the high performance liquid chromatography-mass spectrometer is used for detection are as follows: the mass spectrum detector can adopt an SCEIX AB4500 detector, an electrospray ion source (ESI) is adopted, positive and negative ions are collected simultaneously, and a collection mode adopts a segmented collection (Scheduled MRM) mode:
in the positive ion mode, the ion pairs expressed as parent/daughter ions are:
chlorthalidone: 339.1/322.0; spirolactone: 417.2/341.2; canrenone: 341.3/107.1; amiloride: 230.2/116.1; triamterene: 254.1/237.0; indapamide: 365.9/132.1;
in negative ion mode, the ion pairs expressed as parent/daughter ions are:
hydrochlorothiazide: 296.0/268.8; furosemide: 329.1/285.1.
The positive ion parameters are shown in tables 2 and 3:
table 2: ion source parameters
Table 3: ion pair information
The negative ion parameters are shown in tables 4 and 5:
table 4: ion source parameters
Table 5: ion pair information
The detection method provided by the embodiment of the invention can be used for simultaneously measuring the contents of 8 substances, is short in detection time, can output results within 5.5 min, can analyze 10 groups of samples within 1 hour, and is short in analysis time and high in accuracy. The embodiment of the invention adopts a mode of simultaneously collecting positive and negative ion signals, and the mobile phase adopted by the invention is simple, thereby meeting the response and peak shape of all substances in positive and negative channels. In addition, in the research process, the spirolactone structure is provided with an acetyl sulfhydryl group, the ion source is unstable, a positive ion peak with the mass-to-charge ratio of 341.3 is generated after one molecule of acetyl sulfhydryl group is easily removed, and the positive ion peak is the same as canrenone parent ion, so that ion crosstalk is formed, and the canrenone result is influenced. Therefore, the embodiment of the invention carries out chromatographic separation on spirolactone and canrenone in a short time by improving the liquid chromatography condition, thereby ensuring the accuracy of the detection results of the two substances.
The chemical structural formula of spirolactone:；
canrenone has a chemical structural formula:。
example 1
The embodiment provides an analysis method for detecting 7 antihypertensive drugs and metabolites in blood by liquid chromatography-tandem mass spectrometry, which comprises the following steps:
preparing a standard solution:
1) Standard stock solutions containing each standard substance were prepared separately, and the specific preparation modes are shown in table 6:
TABLE 6
2) Preparing part of standard intermediate liquid:
amiloride intermediate: 50 μl of amiloride mother liquor was taken and 950 μl of methanol was accurately aspirated: water = 7:3 obtaining intermediate solution with the concentration of 100 mg/L, storing at-80 ℃ and the effective period of 3 months, and effectively expecting investigation for a longer time;
intermediate solution of triamterene: 50 μl of triamterene mother liquor was taken, and 950 μl of methanol was accurately aspirated: water = 7:3 obtaining intermediate solution with the concentration of 100 mg/L, storing at-80 ℃ and the effective period of 3 months, and effectively expecting investigation for a longer time;
3) Preparing standard working solution:
amiloride stock solution, triamcinolone acetonide internal standard intermediate solution and the rest 6 standard stock solutions are taken to prepare standard curve L8 point, and the specific preparation method is shown in table 7:
TABLE 7
The standard curve dilution procedure is shown in table 8:
TABLE 8
The concentration values of the pipes are calculated according to the dilution ratio, and the concentration of each level of substances in the standard working solution is shown in table 9:
table 9: unit (ng/mL)
4) Preparation of internal standard stock solution
Internal standard stock solutions containing isotope internal standards were prepared separately, as shown in table 10:
table 10
5) Preparation of internal standard intermediate liquid
The internal standard stock solution is further diluted by adopting 70% methanol aqueous solution by volume percent to obtain an internal standard intermediate solution, and the internal standard intermediate solution is specifically shown in table 11:
TABLE 11
6) Preparation of internal standard working solution
The internal standard intermediate solution is further diluted by adopting 70% methanol aqueous solution by volume percent to obtain an internal standard working solution, and the internal standard working solution is specifically shown in table 12:
table 12
7) Preparation of standard solution
Taking 5 mu L of working solution of each concentration standard product, 10 mu L of internal standard working solution, 45 mu L of water and 200 mu L of methanol into a 1.5 mL centrifuge tube by using a pipettor, carrying out vortex mixing for 1 min at 2000 r/min, transferring 100 mu L of mixed solution into a 1.5 mL centrifuge tube, adding 100 mu L of distilled water, and carrying out vortex mixing for 1 min at 2000 r/min to obtain eight standard solutions with different concentrations.
(II) preparation of a Standard Curve
And detecting the solution by using a high performance liquid chromatography mass spectrometer to obtain eight standard solution chromatograms with different concentrations, respectively obtaining peak areas of a target object and an internal standard object from the standard solution chromatograms, respectively taking the ratio of the peak area of the standard solution of each substance target object to the peak area of the internal standard object as the ordinate y of a standard curve equation, taking the ratio of the concentration in the standard working solution of each substance target object to the concentration of the internal standard working solution as the abscissa x of the standard curve equation, carrying out linear regression on the detected data, fitting to obtain the standard curve equation as y=a x+b, and obtaining the weight coefficients a and b.
(III) pretreatment of sample to be tested
Accurately sucking 10 mu L of internal standard working solution, then transferring 50 mu L of plasma sample (0.1 mu L of formic acid is added), adding 200 mu L of methanol, uniformly mixing for 5 min by a vortex of 2000 r/min, and centrifuging for 10 min at a high speed of 14000 r/min; and transferring 100 mu L of supernatant to a 96-well plate, adding 100 mu L of distilled water into the 96-well plate, and uniformly mixing for 1 min by vortex at 2000 r/min for on-machine detection.
(IV) detection of the sample to be tested
Detecting an upper sample by using a high performance liquid chromatography mass spectrometer to obtain a chromatogram of 8 substances and an internal standard in total of 7 drugs and 1 metabolites of the sample to be detected, obtaining peak areas of the 7 drugs and 1 metabolites and the internal standard peak areas from the chromatogram, substituting the ratio y of the peak areas of the substances and the internal standard into a standard curve equation y=a x+b of the step (two), and obtaining the relative concentration x of 8 substances in total of 7 drugs and 1 metabolites in the blood sample to be detected by calculation, wherein the concentration of an internal standard working solution is known, thereby obtaining the concentration of 8 substances in total of 7 drugs and 1 metabolites in the blood to be detected in the sample by calculation.
The liquid phase conditions were as follows:
chromatographic column: poroshell 120 EC-C18 (2.7 μm,3 mm X100 mm); PN 695975-302;
mobile phase: phase A is aqueous solution containing 0.05% formic acid, and phase B is methanol solution;
column temperature: 40 ℃;
injector temperature: 15 ℃;
sample injection amount: 4. mu L;
analysis time: 5.5 min;
the gradient elution conditions are shown in Table 1.
Needle washing liquid: methanol/water=7/3 (0.2% fa);
the needle washing mode is as follows: washing for 5 seconds before and after sample injection.
The mass spectrum detector is an SCEIX AB4500 detector, an electrospray ion source (ESI) is adopted, positive and negative are collected simultaneously, and a collection mode adopts a segmented collection mode and a Scheduled MRM mode.
The positive ion parameters are shown in tables 2 and 3, and the negative ion parameters are shown in tables 4 and 5.
The chromatograms of 7 drugs and metabolites in the standard solution are shown in figure 1, the chromatograms of 7 drugs and metabolites in the plasma sample are shown in figure 2, the chromatograms of canrenone are shown in figure 3, and the chromatograms of spirolactone are shown in figure 4.
Example 2
The technical process in example 1 is demonstrated as follows:
1. linear and quantitative limits of the method
1. Linear range
Taking 5 mu L of working solution of each concentration standard product by using a pipettor, 10 mu L of internal standard working solution, 45 mu L of water, 200 mu L of protein precipitant (methanol) into a 1.5 mL centrifuge tube, performing vortex mixing for 1 min at 2000 r/min, transferring 100 mu L of mixed solution into a 1.5 mL centrifuge tube, adding 100 mu L of distilled water, performing vortex mixing for 1 min at 2000 r/min, and performing sample injection for 4 mu L to LC-MS/MS analysis. Each substance has good linearity, and the correlation coefficient R2 is greater than 0.99, and the linear range is specifically as follows:
hydrochlorothiazide: 8 ng/mL-4000 ng/mL;
chlorthalidone: 10 ng/mL-5000 ng/mL;
spirolactone: 5 ng/mL-2500 ng/mL;
canrenone: 5 ng/mL-2500 ng/mL;
amiloride: 0.6 ng/mL-300 ng/mL
Furosemide: 100 ng/mL-50000 ng/mL;
triamterene: 1 ng-500 ng/mL;
indapamide: 4 ng/mL-2000 ng/mL;
2. detection limit and quantification limit:
standard solutions of different concentrations were added to the blank plasma and assayed as in example 1 to give the experimental results shown in table 13:
TABLE 13
2. The recovery rate and precision of the method
Standard working solutions of 7 drugs and their metabolites were prepared to 3 concentrations of high, medium and low for sample addition recovery and precision experiments, which were performed by the method of example 1, and repeated analysis was performed for 4 batches, the recovery and precision of which were shown in table 14 below.
TABLE 14
The average recovery rate of 7 drugs and metabolites thereof in the range of 3 addition levels of low, medium and high is 91.43% -104.26%, and the relative standard deviation is 2.01% -5.89%.
3. Matrix effect of the method
Samples with different matrix proportions (90% matrix, 80% matrix, 50% matrix, 20% matrix, 0 matrix) were added to the same standard solution with high concentration (concentration greater than or equal to 10 x the concentration in the matrix samples), each matrix sample being examined for 5 different ratio conditions. The experimental results are shown in table 15.
TABLE 15-1
TABLE 15-2
TABLE 15-3
TABLE 15-4
As can be seen from Table 15, in the detection method of example 1, the RSD of the target area/internal standard area of the same matrix ratio of different matrixes is less than or equal to 10%; target area/internal standard area ratio bias= (X matrix-0 matrix)/0 matrix of different matrix ratios of the same matrix are all less than 10%, all meeting the requirements.
In combination with the verification test, the recovery rate, the precision and other technical indexes of the embodiment 1 meet the requirements, the repeatability is good, the sample adding recovery rate is high, and the accuracy of the detection result is improved.
Experimental example 1
The experimental example is used for explaining the technical effect of adding the stabilizer into the sample to be tested:
the following labeled plasma samples (samples 1-6) were tested according to the method of example 1, with spironolactone and canrenone added in amounts of 1250 ng/mL, with the difference that the stabilizer was added in the pretreatment step as follows:
sample 1: no stabilizer is added to the plasma sample;
sample 2: DTT is added into the plasma sample, and 10 mu L of 30mmol/L DDT is added into each 1 mL plasma;
sample 3: formic acid was added to the plasma samples, 1 μl of formic acid was added per 1 mL plasma;
sample 4: formic acid was added to the plasma samples, 2 μl of formic acid was added per 1 mL plasma;
sample 5: formic acid was added to the plasma samples, 3 μl of formic acid was added per 1 mL plasma;
sample 6: formic acid was added to the plasma samples, with 4 μl of formic acid per 1 mL plasma.
The mixture was left at room temperature for 48 hours and 72 hours.
The results of the spironolactone and canrenone average recovery experiments are shown in table 16:
table 16
As can be seen from Table 16, the room temperature of spirolactone in plasma was stable for 48 hours and the room temperature of canrenone was stable for 72 hours under the detection conditions of the present invention.
Experimental example 2
The experimental example is used for explaining the technical effect of adding different protein precipitants:
preparing a high-concentration sample, and preprocessing the sample to be detected (experiments 1-5) according to the method of the embodiment 1, wherein the difference is that protein precipitants in the preprocessing steps are different:
experiment D2-1: sample pretreatment was performed by the method of example 1, except that acetonitrile was used as the protein precipitant:
experiment D2-2: sample pretreatment was performed by the method of example 1, except that ethanol was used as the protein precipitant:
experiment D2-3: sample pretreatment is carried out by adopting the method of the embodiment 1, wherein the difference is that methanol is selected as a protein precipitant, but the addition amount of the methanol is 100 mu L:
experiment D2-4: sample pretreatment is carried out by adopting the method of the embodiment 1, wherein the difference is that methanol is selected as the protein precipitant, but the addition amount of the methanol is 300 mu L:
experiment D2-5: sample pretreatment was performed by the method of example 1.
The average recovery for the different protein precipitants is shown in Table 17.
Table 17: recovery under different protein precipitants
According to the data shown in Table 17, the recovery rate of example 1 was 92.04% to 104.87% as compared with the recovery rate of example 1, and the pretreatment effect was the best.
Experimental example 3
The experimental example is used for explaining the technical effect of adding water to dilute the supernatant in the pretreatment step:
experiment D3: the test was performed as in example 1, except that: the pretreatment of the sample to be tested in the third step comprises the following steps: mixing the internal standard working solution, the plasma sample and the protein precipitant, centrifuging to obtain supernatant, and directly introducing the supernatant to obtain a chromatogram of amiloride, which is shown in figure 5.
The chromatogram obtained for amiloride according to the detection method of example 1 is shown in FIG. 6.
As can be seen from FIG. 5, amiloride peak formation was poor without the addition of diluent. As can be seen from fig. 6, the peak shape of amiloride is effectively improved after dilution with water, thus demonstrating that the solvent effect can be effectively avoided by dilution with water.
Experimental example 4
The experimental example is used for explaining the technical effect of mobile phase in chromatographic conditions:
the standard solution sample L1 was tested as in example 1, except that the mobile phase was conditioned as follows:
experiment 4-1: the mobile phase is adopted: detecting a standard solution sample L1 by using an aqueous solution as the phase A and methanol as the phase B; experiment 4-1 gave a chromatogram as shown in FIG. 7;
experiment 4-2: the mobile phase is adopted: detecting a standard solution sample L1 by using an aqueous solution containing 0.01% formic acid as the phase A and a methanol solution as the phase B;
experiment 4-3: the mobile phase is adopted: detecting a standard solution sample L1 by using an aqueous solution containing 0.1% formic acid as the phase A and a methanol solution as the phase B;
experiment 4-4: the mobile phase is adopted: detecting a standard solution sample L1 by using an aqueous solution containing 0.2% formic acid as the phase A and a methanol solution as the phase B;
experiment 4-5 (example 1 conditions): the mobile phase is adopted: the phase A is aqueous solution containing 0.05% formic acid, and the phase B is methanol solution for detecting a standard solution sample L1;
the above mobile phase was used for detection and the response of each substance is shown in table 18:
TABLE 18
As shown in fig. 7, the retention time of furosemide is 4.08 min, and the peak type is bad; the triamterene response was lower. As can be seen from Table 18, each material response satisfies the requirements using the mobile phase conditions of the present invention.
Experimental example 5
The experimental example is used for explaining the technical effect of elution conditions in chromatographic conditions:
the standard solution sample L1 was tested as in example 1, except that the elution conditions were as follows:
the elution conditions for experiment D5-1 are shown in Table 19, and the resulting chromatograms are shown in FIG. 8.
TABLE 19
The elution conditions for experiment D5-2 are shown in Table 20, and the resulting chromatograms are shown in FIG. 9.
Table 20
The elution conditions for experiment D5-3 are shown in Table 21, and the resulting chromatograms are shown in FIG. 10:
table 21
Experiment D5-4 elution conditions are shown in Table 22, and the resulting chromatograms are shown in FIG. 11:
table 22
According to the gradient conditions of example 1, as shown in fig. 8-11, each substance has a good peak shape, and spironolactone and canrenone can be separated in a liquid phase, so that the accuracy of the result is ensured.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A method for detecting 7 antihypertensive drugs and metabolites by liquid chromatography tandem mass spectrometry, which is characterized in that the 7 antihypertensive drugs and metabolites comprise: hydrochlorothiazide, chlorthalidone, spironolactone, canrenone, amiloride, triamterene, furosemide, and indapamide, the method comprising at least the steps of:
s1, preparing a standard curve equation, which comprises the following steps:
preparing an internal standard working solution and a standard working solution, preparing a sample solution for standard yeast, and detecting the sample solution for standard yeast by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation for calculating the content of 7 antihypertensive drugs and metabolites;
s2, preprocessing a sample to be tested, including:
uniformly mixing an internal standard working solution, a sample to be tested and a protein precipitant, and centrifuging to obtain a supernatant, wherein water I is added into the supernatant to be uniformly mixed to serve as a sample injection sample; the protein precipitant is methanol;
the sample to be tested is plasma added with formic acid, and the addition amount of the formic acid is as follows: adding 2-4 mu L of formic acid into each 1-mL plasma;
s3, detecting the sample injection sample, which comprises the following steps:
and detecting the sample by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result of the sample into the standard curve equation to obtain the contents of 7 antihypertensive drugs and metabolites in the sample to be detected.
2. The method of claim 1, wherein S1 comprises:
s11, respectively preparing an internal standard working solution and a standard working solution with gradient concentration;
s12, respectively taking the internal standard working solution and the standard working solution, adding water II and a protein precipitant, and uniformly mixing to obtain a mixed solution, wherein water III is added into the mixed solution and uniformly mixed to serve as a sample injection sample for standard curve; the protein precipitant is methanol;
and S13, detecting the standard curve sample solution by using a high performance liquid chromatography mass spectrometer to obtain a standard curve equation.
3. The method according to claim 2, characterized in that in S12:
the volume ratio of the standard working solution to the internal standard working solution is 1:2; and/or the number of the groups of groups,
the volume ratio of the standard working solution to the water II is 1:9, a step of performing the process; and/or the number of the groups of groups,
the total volume ratio of the standard working solution to the water II and the volume ratio of the protein precipitant are 1: 4-6; and/or the number of the groups of groups,
the volume ratio of the mixed solution to the water III is 1:0.8 to 1.2.
4. The method according to claim 1, wherein in S2:
the volume ratio of the sample to be tested to the protein precipitant is 1: 4-6; and/or the number of the groups of groups,
the volume ratio of the sample to be tested to the internal standard working solution is 5:1, a step of; and/or the number of the groups of groups,
the volume ratio of the supernatant to the water I is 1:0.8 to 1.2.
5. The method according to claim 1, wherein in S2:
the conditions for uniformly mixing the internal standard working solution, the sample to be tested and the protein precipitant are 1500-2500 r/min, and vortex mixing is performed for 3-6 min, and/or,
the conditions for mixing after adding the water I are as follows: vortex mixing for 0.5-1.5 min at 1500-2500 r/min; and/or the number of the groups of groups,
the centrifugation conditions are 12000-15000 r/min for 8-12 min.
6. The method according to claim 2, characterized in that in S12: the mixing condition is 1500-2500 r/min vortex mixing for 0.5-1.5 min.
7. The method according to claim 1, wherein the internal standard working fluid contains isotope internal standards of the 7 antihypertensive drugs and metabolites:
the isotopic internal standard of hydrochlorothiazide is: hydrochlorothiazide- [13C6];
the isotopic internal standard of chlorthalidone is: chlorthalidone- [13C6];
the internal isotope of spirolactone is: spirolactone-d 3;
the isotopic internal standard of canrenone is: canrenone-d 4;
the isotopic internal standard of amiloride is: amiloride- [15N3];
the isotopic internal standard of the triamterene is: triamterene-d 5;
the isotope internal standard of furosemide is: furosemide-d 5;
the isotopic internal standard of indapamide is: indapamide-d 3;
and/or the preparation mode of the internal standard working solution is as follows:
s121, respectively preparing internal standard stock solutions containing isotope internal standards, wherein the triamterene-d 5 internal standard stock solution adopts DMSO and methanol 1:1, preparing the rest internal standard stock solution by adopting pure methanol;
s122, respectively diluting the internal standard stock solution into an internal standard intermediate solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%;
s123, mixing the internal standard intermediate solutions, and preparing an internal standard working solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%.
8. The method according to claim 2, wherein in S11, the standard working solution is obtained by diluting 65% -75% by volume of aqueous methanol solution;
and/or the preparation mode of the standard working solution with gradient concentration is as follows:
s121', preparing standard stock solutions containing standard substances respectively, wherein the standard stock solution of triamterene adopts DMSO and methanol 1:1, preparing the rest standard stock solution by adopting pure methanol;
s122', diluting the amiloride stock solution into an amiloride intermediate solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%; diluting the triamterene stock solution into a triamterene intermediate solution by adopting a methanol aqueous solution with the volume percentage of 65% -75%;
s123', taking the amiloride intermediate liquid, the triamcinolone acetonide intermediate liquid and the rest 6 standard stock solutions, and preparing a standard working solution with the highest concentration by adopting a methanol aqueous solution with the volume percentage of 65% -75%;
s124', sequentially diluting the standard working solution with the highest concentration with 65-75% of methanol aqueous solution by volume percent to obtain the standard working solution with gradient concentration.
9. The method of claim 1, wherein the high performance liquid phase conditions when detected using a high performance liquid chromatography mass spectrometer are:
the chromatographic column adopts a C18 chromatographic column;
mobile phase: the phase A is an aqueous solution containing 0.01-0.2% formic acid by volume percent, and the phase B is a methanol solution;
flow rate: 0.4-0.5 mL/min;
column temperature: 35-45 ℃;
sample injection amount: 1-10 mu L;
analysis time: 5.5 min;
the gradient elution conditions were:
0-1.50 minutes, A:65% -20%, B:35% -80%;
1.5 to 2.50 minutes, A:20% -0, B:80% -100%;
2.50-3.50 minutes, A:0, b:100%;
3.51-5.50 minutes, A:65%, B:35%.
10. The method of claim 1, wherein the mass spectrometry conditions when detected using a high performance liquid chromatography mass spectrometer are:
adopting an electrospray ion source, and simultaneously collecting positive and negative ions, wherein a sectional collecting mode is adopted as a collecting mode:
in the positive ion mode, the ion pairs expressed as parent/daughter ions are:
chlorthalidone: 339.1/322.0; spirolactone: 417.2/341.2; canrenone: 341.3/107.1; amiloride: 230.2/116.1; triamterene: 254.1/237.0; indapamide: 365.9/132.1;
in negative ion mode, the ion pairs expressed as parent/daughter ions are:
hydrochlorothiazide: 296.0/268.8; furosemide: 329.1/285.1.
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