CN111198239A - HPLC-MS/MS detection method for plasma amoxicillin concentration - Google Patents
HPLC-MS/MS detection method for plasma amoxicillin concentration Download PDFInfo
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- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 title claims abstract description 68
- 229960003022 amoxicillin Drugs 0.000 title claims abstract description 67
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 238000001514 detection method Methods 0.000 title claims abstract description 23
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 title claims abstract description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 127
- 239000000523 sample Substances 0.000 claims abstract description 62
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000012224 working solution Substances 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 33
- 238000002156 mixing Methods 0.000 claims abstract description 31
- SGTNSNPWRIOYBX-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile Chemical compound C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 SGTNSNPWRIOYBX-UHFFFAOYSA-N 0.000 claims abstract description 29
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- 239000011550 stock solution Substances 0.000 claims abstract description 26
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 20
- 238000001819 mass spectrum Methods 0.000 claims abstract description 20
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000007864 aqueous solution Substances 0.000 claims abstract description 18
- 235000019253 formic acid Nutrition 0.000 claims abstract description 18
- 239000013062 quality control Sample Substances 0.000 claims abstract description 18
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 14
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 239000012488 sample solution Substances 0.000 claims abstract description 9
- 238000013375 chromatographic separation Methods 0.000 claims abstract description 4
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- 150000002500 ions Chemical class 0.000 claims description 60
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- 210000002381 plasma Anatomy 0.000 abstract description 28
- 238000004458 analytical method Methods 0.000 abstract description 3
- DJQOOSBJCLSSEY-UHFFFAOYSA-N Acipimox Chemical compound CC1=CN=C(C(O)=O)C=[N+]1[O-] DJQOOSBJCLSSEY-UHFFFAOYSA-N 0.000 abstract description 2
- 229960003526 acipimox Drugs 0.000 abstract description 2
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000002904 solvent Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 125000002137 amoxicillanyl group Chemical group N[C@@H](C(=O)N[C@H]1[C@@H]2N([C@H](C(S2)(C)C)C(=O)*)C1=O)C1=CC=C(C=C1)O 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 208000007271 Substance Withdrawal Syndrome Diseases 0.000 description 1
- DOQPXTMNIUCOSY-UHFFFAOYSA-N [4-cyano-4-(3,4-dimethoxyphenyl)-5-methylhexyl]-[2-(3,4-dimethoxyphenyl)ethyl]-methylazanium;chloride Chemical compound [H+].[Cl-].C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 DOQPXTMNIUCOSY-UHFFFAOYSA-N 0.000 description 1
- 229960004920 amoxicillin trihydrate Drugs 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000000945 filler Substances 0.000 description 1
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- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 150000003951 lactams Chemical group 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- XQYZDYMELSJDRZ-UHFFFAOYSA-N papaverine Chemical class C1=C(OC)C(OC)=CC=C1CC1=NC=CC2=CC(OC)=C(OC)C=C12 XQYZDYMELSJDRZ-UHFFFAOYSA-N 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
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Images
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
Abstract
The invention relates to an HPLC-MS/MS detection method for the concentration of amoxicillin in blood plasma. The method comprises the following steps: (A) preparing a stock solution; (B) preparing a working solution; (C) preparing a standard curve sample and a quality control sample; (D) sample pretreatment: taking a plasma sample, adding verapamil stock solution, and uniformly mixing; adding acetonitrile, and mixing uniformly; centrifuging and taking supernatant; adding formic acid aqueous solution, and uniformly mixing to obtain a sample solution to be detected (E) for liquid chromatography separation: carrying out liquid phase chromatographic separation on a sample solution to be detected; (F) performing mass spectrometry: and carrying out mass spectrum detection on the sample subjected to liquid chromatography separation. The method for detecting the concentration of the acipimox in the plasma has the advantages of simple operation, quick analysis, high specificity and high separation degree.
Description
Technical Field
The invention relates to the technical field of monitoring of blood concentration of antibiotic drugs, in particular to an HPLC-MS/MS detection method of amoxicillin concentration in blood plasma.
Background
The adverse reaction incidence rate of clinically applied Amoxicillin is about 5-6%, the drug withdrawal person is about 2% due to reaction, the Amoxicillin has the characteristics of small safety range, large individual difference of in vivo metabolism, strong toxicity and the like, the curative effect and the adverse reaction are closely related to the drug concentration in blood plasma, and the drug administration scheme needs to be adjusted according to the blood drug concentration, so that the Amoxicillin and the penicillin have strong bactericidal action and strong capability of penetrating cell walls and are one of the oral penicillins which are widely applied at present.
The method for determining the amoxicillin content specified in the pharmacopoeia is a high performance liquid chromatography, and the specific experimental method is as follows: taking the contents under the items with different filling amounts, mixing uniformly, precisely measuring (about equivalent to 0.125g of amoxicillin), adding mobile phase for dissolving and diluting into a solution containing about 0.5mg in each 1mL, filtering, taking the subsequent filtrate, precisely measuring 20 mu L, injecting into a liquid chromatograph, and recording the chromatogram; and taking a proper amount of amoxicillin reference substance, and determining by the same method. Calculating C in the sample by peak area according to external standard method16H19N3O5The content of S.
Chromatographic conditions and system applicability test:
octadecylsilane chemically bonded silica is used as a filling agent; using 0.05mol/L potassium dihydrogen phosphate solution (pH value is adjusted to 5.0 by 2mol/L potassium hydroxide solution) -acetonitrile (97.5: 2.5) as a mobile phase; a flow rate of about 1mL per minute; the detection wavelength was 254 nm. The number of theoretical plates is not less than 2000 calculated according to amoxicillin.
The analysis method specified in the pharmacopoeia relates to a large number of chemical pretreatment steps, the operation is relatively complicated, and particularly, the preparation and calibration of a phosphate buffer solution are long in time consumption, so that much inconvenience is brought to actual work.
At present, common methods for measuring amoxicillin blood concentration include: high performance liquid chromatography and liquid chromatography-mass spectrometry are combined. Because amoxicillin has large polarity and poor thermal stability, the amoxicillin can not be purified and concentrated by evaporating and redissolving after liquid-liquid extraction, the fussy ultrafiltration method is adopted for the early detection, and the methanol or acetonitrile or perchloric acid which is 2-3 times the amount of the amoxicillin is often adopted for the pretreatment of samples in the later research to precipitate protein. But the precipitated protein can not completely purify the sample, and the matrix effect generated by LC-MS detection can interfere; the supernatant after the precipitant is added and centrifuged is often not enough in sensitivity because of too large dilution factor, so that the sample injection volume needs to be more than 20 mu L to ensure the quantitative sensitivity, the solvent effect caused by mismatching of the supernatant and the mobile phase often generates great interference on the chromatographic peak shape, and a certain proportion of specific solvent is added to eliminate the solvent effect during sample injection. These problems make detection of amoxicillin problematic, resulting in detection by HPLC still being used by researchers today with the prevalence of LC-MS.
Amoxicillin is slightly soluble in water and almost insoluble in ethanol. Free molecular formula and molecular weight C16H19N3O5S/365.40g/mol, hydrate molecular formula and molecular weight C16H19N3O5S·3H2O/419.45g/mol, and the specific structure is shown in formula (I). The structure is characterized in that: the compound is unstable due to the condensed ring tension, and the lactam ring is easy to open and decompose.
The internal standard selected by the method is Verapamil (Verapamul) which is a papaverine derivative and commonly used hydrochloride thereof; is soluble in water and soluble in methanol, ethanol or chloroform. Free molecular formula and molecular weight C27H38N2O4454.60g/mol, hydrochloride molecular formula and molecular weight C27H38N2O4HCl/491.07g/mol, the specific structure is shown in formula (II).
Disclosure of Invention
Based on the method, the invention provides an HPLC-MS/MS detection method for the amoxicillin concentration in the blood plasma. The method is accurate, rapid, and has strong specificity and high separation degree.
An HPLC-MS/MS detection method for plasma amoxicillin concentration comprises the following steps:
(A) preparation of stock solution:
a1: dissolving amoxicillin in (50 +/-5)% acetonitrile to prepare an amoxicillin stock solution;
a2: dissolving verapamil in acetonitrile to prepare a verapamil stock solution;
(B) preparation of working solution:
b1: diluting the amoxicillin stock solution by (50 +/-5)% acetonitrile to prepare a mixed standard curve sample working solution and a mixed quality control sample working solution;
b2: diluting verapamil stock solution with (50 +/-5)% acetonitrile to prepare mixed standard curve sample working solution and mixed quality control sample working solution;
(C) preparation of standard curve sample and quality control sample:
adding a mixed standard curve working solution into blank plasma, and uniformly mixing to prepare an amoxicillin standard curve sample and an amoxicillin quality control sample;
(D) sample pretreatment: taking a plasma sample, adding verapamil stock solution, and uniformly mixing; adding acetonitrile, and mixing uniformly; centrifuging and taking supernatant; adding a formic acid aqueous solution, and uniformly mixing to obtain a sample solution to be detected;
(E) liquid chromatography separation: carrying out liquid phase chromatographic separation on a sample solution to be detected;
(F) performing mass spectrometry: and carrying out mass spectrum detection on the sample subjected to liquid chromatography separation.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the plasma sample to be measured and adds the internal standard working solution, uses acetonitrile to precipitate protein, centrifugalizes and then takes the supernatant, and then adds the diluent, and then only needs a little sample amount to analyze to meet the quantitative requirement, and the sample pretreatment process is greatly simplified. The influence of solvent effect is avoided in the chromatographic detection process, and the chromatographic peak shape is excellent. The method for detecting the concentration of the acipimox in the plasma has the advantages of simple operation, quick analysis, high specificity and high separation degree.
Drawings
FIG. 1 is a diagram showing the detection results of amoxicillin standard substance and verapamil standard substance.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides an HPLC-MS/MS detection method for the concentration of amoxicillin in blood plasma, which comprises the following steps:
(A) preparation of stock solution:
a1: dissolving amoxicillin in (50 +/-5)% acetonitrile to prepare an amoxicillin stock solution;
a2: dissolving verapamil in acetonitrile to prepare a verapamil stock solution;
(B) preparation of working solution:
b1: diluting the amoxicillin stock solution by (50 +/-5)% acetonitrile to prepare a mixed standard curve sample working solution and a mixed quality control sample working solution;
b2: diluting verapamil stock solution with (50 +/-5)% acetonitrile to prepare mixed standard curve sample working solution and mixed quality control sample working solution;
(C) preparation of standard curve sample and quality control sample:
adding a mixed standard curve working solution into blank plasma, and uniformly mixing to prepare an amoxicillin standard curve sample and an amoxicillin quality control sample;
(D) sample pretreatment: taking a plasma sample, adding verapamil stock solution, and uniformly mixing; adding acetonitrile, and mixing uniformly; centrifuging and taking supernatant; adding a formic acid aqueous solution, and uniformly mixing to obtain a sample solution to be detected;
(E) liquid chromatography separation: carrying out liquid phase chromatographic separation on a sample solution to be detected;
(F) performing mass spectrometry: and carrying out mass spectrum detection on the sample subjected to liquid chromatography separation.
In one embodiment, in step (D), the volume ratio of acetonitrile to plasma sample is: 2-5:1, preferably 4-5: 1; the concentration of the formic acid aqueous solution is (0.1 +/-0.02)%. In the sample pretreatment process, the amount of the precipitant is far larger than the amount of the plasma, the proportion of the sample injection solution system to the initial mobile phase is close, the sample injection amount is very low, and the influence of the solvent effect can be completely ignored, so that the separation effect is excellent.
In one embodiment, in step (D), the temperature of the centrifugation is 2-8 ℃, the rotation speed of the centrifugation is 2500-3500rpm, and the centrifugation time is 5-15 min.
In one embodiment, in step (E), the liquid chromatography separation conditions are:
mobile phase: the mobile phase A is 0.1 percent formic acid aqueous solution; the mobile phase B is acetonitrile; gradient elution is adopted; the gradient elution process is as follows: (50 +/-5)% of mobile phase A-mobile phase B in 0-1.3 min; 1.3-1.5 minutes, (50 +/-5)% of mobile phase A-mobile phase B; 1.5-2.3 minutes, (5 +/-2)% of mobile phase A-mobile phase B; 2.3-2.4 minutes, (5 +/-2)% of mobile phase A-% and% of mobile phase B; 2.4-3 minutes, (50 +/-5)% of mobile phase A-mobile phase B; the flow rate is 0.4mL/min to 0.8 mL/min; the sample injection volume is 2 mu L.00-4.00 mu L; the column temperature is set to be 25-35 ℃; the temperature of the automatic sample injector is 2-6 ℃; needle washing liquid: (50 ± 5)% acetonitrile. Washing speed: 30-40 μ L/sec; volume of needle washing liquid: 400-600 μ L.
In one embodiment, in step (E), the gradient elution process is: 50% of mobile phase A-mobile phase B in 0-1.3 min; 1.3-1.5 minutes, 50% of mobile phase A-mobile phase B; 5% of mobile phase A-mobile phase B in 1.5-2.3 minutes; 5% of mobile phase A-mobile phase B in 2.3-2.4 minutes; 2.4-3 minutes, 50% of mobile phase A-mobile phase B; the flow rate is 0.6 mL/min; the sample injection volume is 3.00 mu L; the column temperature was set at 30 ℃; the temperature of the autosampler is 4 ℃; needle washing liquid: 50% acetonitrile; washing speed: 35 μ L/sec; volume of needle washing liquid: 500 μ L.
In one embodiment, in step (F), the mass spectrometry conditions are:
mass spectrum detection conditions: ionization mode: an electrospray ion source; a positive ion mode; monitoring multiple reactions;
ion source parameters: air curtain air: 25 psi; ion source gas 1: 50 psi; ion source gas 2: 50 psi; ion source spray voltage: 5500V; ion source temperature: 550 ℃; resolution Q1/Q3: Unit/Unit; collision gas: medium; pause time: 20 msec; the mass spectrum acquisition time is 3.00 min;
monitoring ion pair quadrupole rod parameters: amoxicillin: ion pairing: 366.2 → 208.1; de-clustering voltage: 30.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 14.00 eV; residence time: 200.0 msec; verapamil: ion pairing: 455.2 → 165.1; de-clustering voltage: 30.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 36.00 eV; residence time: 200.0 msec.
In one embodiment, in step B1, the concentrations of amoxicillin in the mixed standard curve sample working solution are as follows: 300ng/mL, 600ng/mL, 2000ng/mL, 10000ng/mL, 50000ng/mL, 100000ng/mL, 250000ng/mL, and 300000 ng/mL; and/or
In the step B2, the concentrations of amoxicillin in the mixed quality control sample working solution are as follows: the lower limit of quantification quality control is 300ng/mL, the low concentration quality control is 800ng/mL, the medium concentration quality control in geometric mean is 15000ng/mL, the medium concentration quality control in arithmetic mean is 150000ng/mL, and the high concentration quality control is 225000 ng/mL.
In one embodiment, in step (C), the concentrations of amoxicillin in the standard curve sample are: 15.0ng/mL, 30.0ng/mL, 100ng/mL, 500ng/mL, 2500ng/mL, 5000ng/mL, 12500ng/mL, and 15000 ng/mL; and/or
In the quality control sample, the concentrations of amoxicillin are respectively as follows: the lower limit quality control of the quantification is 15ng/mL, the low concentration quality control is 40.0ng/mL, the medium concentration quality control of the geometric mean value is 750ng/mL, the medium concentration quality control of the arithmetic mean value is 7500ng/mL, and the high concentration quality control is 11250 ng/mL.
In one embodiment, in step (E), the model of the liquid chromatography-mass spectrometer used is Shimadzu LC 30AD liquid chromatography combined with Sciex Qtrap5500 mass spectrometer, and the operating system used is analyst 1.6.3; the adopted chromatographic column is Cosmosil 5C18-PAQ of Nacalai; the specification is 4.6 multiplied by 100mm,5.0 μm; the Sciex Qtrap model 5500 mass spectrometer used in the invention has an order of magnitude higher sensitivity than that of the previous instrument.
In one embodiment, in step (D), the sample preprocessing is: taking 40.0 mu L of plasma sample, adding 40.0 mu L of internal standard working solution, uniformly mixing for 1min in a vortex mode, adding 520 mu L of acetonitrile, uniformly mixing for 10min under shaking at room temperature, and then centrifuging for 10min at 4 ℃ and 3000 rpm; and (3) adding 200 mu L of 0.1% formic acid aqueous solution into 100 mu L of supernatant, and shaking and uniformly mixing for 3min to obtain a sample solution to be detected.
The present invention will be described in further detail with reference to specific examples.
Example 1
(1) Preparing a solution:
preparation of stock solution: taking an amoxicillin standard (amoxicillin trihydrate), dissolving in 50% acetonitrile to prepare an amoxicillin stock solution with the concentration of 1.00 mg/mL;
dissolving a proper amount of verapamil standard (verapamil hydrochloride) in 100% acetonitrile to prepare a verapamil stock solution with the concentration of 1.00 mg/mL;
diluting the amoxicillin stock solution by using 50% acetonitrile to prepare a mixed standard curve sample working solution: amoxicillin solutions at concentrations of 300ng/mL, 600ng/mL, 2000ng/mL, 10000ng/mL, 50000ng/mL, 100000ng/mL, 250000ng/mL, and 300000 ng/mL;
diluting the amoxicillin stock solution by using 50% acetonitrile to prepare a mixed quality control sample working solution: the amoxicillin solutions contained concentrations of 300ng/mL (quantitative lower limit quality control), 800ng/mL (low concentration quality control), 15000ng/mL (geometric mean medium concentration quality control), 150000ng/mL (arithmetic mean medium concentration quality control), and 225000ng/mL (high concentration quality control), respectively.
Diluting the verapamil stock solution with 50% acetonitrile to obtain an internal standard working solution, wherein the concentration of the internal standard working solution is as follows: 50.0 ng/mL.
To 380. mu.L of blank plasma was added 20.0. mu.L of the mixed standard curve working solution and mixed well. Eight standard curve samples with different concentrations were obtained as amoxicillin solutions with concentrations of 15.0ng/mL, 30.0ng/mL, 100ng/mL, 500ng/mL, 2500ng/mL, 5000ng/mL, 12500ng/mL and 15000ng/mL, respectively.
To 380. mu.L of blank plasma was added 20.0. mu.L of the mixed standard curve working solution and mixed well. Five quality control samples with different concentrations are obtained, and the samples are amoxicillin solutions with the concentrations of 15ng/mL (quantitative lower limit quality control), 40.0ng/mL (low concentration quality control), 750ng/mL (geometric mean medium concentration quality control), 7500ng/mL (arithmetic mean medium concentration quality control) and 11250ng/mL (high concentration quality control).
(2) Sample pretreatment
And (3) adding 40.0 mu L of human plasma low-concentration sample into 40.0 mu L of internal standard working solution, uniformly mixing for 1min in a vortex mode, adding 520 mu L of acetonitrile, uniformly mixing for 10min under the condition of shaking at room temperature, and then centrifuging for 10min at 4 ℃ and 3000 rpm. And adding 200 mu L of 0.1% formic acid aqueous solution into 100 mu L of supernatant, shaking and mixing uniformly for 3min, and analyzing by sample injection.
(3) Conditions of liquid chromatography
The instrument equipment comprises: shimadzu LC-30AD liquid chromatography system.
A chromatographic column: cosmosil 5C18-PAQ (4.6X 100mm,5.0 μm), Nacalai, column temperature set to 30 ℃ and autosampler temperature set to 4 ℃.
The mobile phase A is 0.1 percent formic acid aqueous solution; mobile phase B was 100% acetonitrile. The total flow rate was 0.60 mL/min.
The mobile phase elution procedure was: from 0 to 1.3 minutes, 50% parts by volume of mobile phase a and 50% parts by volume of mobile phase B; from greater than 1.3 minutes to 1.5 minutes, 50% parts by volume mobile phase a and 50% parts by volume mobile phase B; from greater than 1.5 minutes to 2.3 minutes, 5% parts by volume mobile phase a and 95% parts by volume mobile phase B; from greater than 2.3 minutes to 2.4 minutes, 5% parts by volume mobile phase a and 95% parts by volume mobile phase B; from greater than 2.4 minutes to 3 minutes, mobile phase a is 50% parts by volume and mobile phase B is 50% parts by volume.
The mass spectrum switching valve program is as follows: 0 to 1 minute, valve B (into waste); 1 to 2 minutes, a valve (into mass spectrum); 2 to 3 minutes, valve B (into waste).
The needle wash was 50% acetonitrile. The needle washing mode is external flushing; the washing speed is 35 mu L/sec; flush port liquids R1; the volume of the flushing fluid is 500 mu L; flushing mode, namely flushing before and after needle insertion; the rinsing and soaking time is 0 second; flushing method, only flushing port; the washing time is 2 seconds;
the injection volume is 3.00 mu L, and the amoxicillin retention time is about 1.29 min; the verapamil retention time was about 1.47 min.
(5) Conditions of Mass Spectrometry
The instrument equipment comprises: sciex Qtrap5500 Mass Spectrometry System.
Ionization mode: electrospray ion source (ESI); positive ion mode (Positive); multiple Reaction Monitoring (MRM).
Ion source parameters: air curtain gas (CUR): 25 psi; ion source Gas 1(Gas 1): 50 psi; ion source Gas 2(Gas 2): 50 psi; ion source spray voltage (IS): 5500V; ion source Temperature (TEM): 550 ℃; resolution Q1/Q3(Resolution Q1/Q3): Unit/Unit; collision gas (CAD): medium; pause time (MR Pause): 20 msec; the mass spectrum acquisition time was 3.00 min.
Monitoring ion pair quadrupole rod parameters:
amoxicillin: ion pairing: 366.2 → 208.1; declustering voltage (DP): 30.0V; inlet voltage (EP): 10.00V; outlet voltage (CXP): 25.00V; collision Energy (CE): 14.00 eV; residence time (Dwell): 200.0 msec.
Verapamil: ion pairing: 455.2 → 165.1; declustering voltage (DP): 30.0V; inlet voltage (EP): 10.00V; outlet voltage (CXP): 25.00V; collision Energy (CE): 36.00 eV; residence time (Dwell): 200.0 msec.
Under the condition, the concentration of the amoxicillin with low concentration in the human plasma is measured to be 15.0 ng/mL. The detection results of the amoxicillin standard and the verapamil standard are shown in fig. 1.
Example 2
(1) The solution was prepared in accordance with example 1.
(2) Sample pretreatment
Taking a sample with the concentration of 40.0 mu LBeagle in the plasma of the dog, adding 40.0 mu L of internal standard working solution, mixing uniformly for 1min in a vortex mode, adding 320 mu L of acetonitrile, shaking and mixing uniformly for 10min at room temperature, and then centrifuging for 10min at 4 ℃ and 3000 rpm. And adding 200 mu L of 0.1% formic acid aqueous solution into 100 mu L of supernatant, shaking and mixing uniformly for 3min, and analyzing by sample injection.
(3) Conditions of liquid chromatography
The instrument equipment comprises: shimadzu LC-30AD liquid chromatography system.
A chromatographic column: cosmosil 5C18-PAQ (4.6X 100mm,5.0 μm), Nacalai, column temperature set to 30 ℃ and autosampler temperature set to 4 ℃.
The mobile phase A is 0.1 percent formic acid aqueous solution; mobile phase B was 100% acetonitrile. The total flow rate was 0.60 mL/min.
The mobile phase elution procedure was: from 0 to 1.3 minutes, 50% parts by volume of mobile phase a and 50% parts by volume of mobile phase B; from greater than 1.3 minutes to 1.5 minutes, 50% parts by volume mobile phase a and 50% parts by volume mobile phase B; from greater than 1.5 minutes to 2.3 minutes, 5% parts by volume mobile phase a and 95% parts by volume mobile phase B; from greater than 2.3 minutes to 2.4 minutes, 5% parts by volume mobile phase a and 95% parts by volume mobile phase B; from greater than 2.4 minutes to 3 minutes, mobile phase a is 50% parts by volume and mobile phase B is 50% parts by volume.
The mass spectrum switching valve program is as follows: 0 to 1 minute, valve B (into waste); 1 to 2 minutes, a valve (into mass spectrum); 2 to 3 minutes, valve B (into waste).
The needle wash was 50% acetonitrile. The needle washing mode is external flushing; the washing speed is 35 mu L/sec; flush port liquids R1; the volume of the flushing fluid is 500 mu L; flushing mode, namely flushing before and after needle insertion; the rinsing and soaking time is 0 second; flushing method, only flushing port; the washing time is 2 seconds;
the injection volume is 3.00 mu L, and the amoxicillin retention time is about 1.29 min; the verapamil retention time was about 1.47 min.
(4) Conditions of Mass Spectrometry
The instrument equipment comprises: sciex Qtrap5500 Mass Spectrometry System.
Ionization mode: electrospray ion source (ESI); positive ion mode (Positive); multiple Reaction Monitoring (MRM).
Ion source parameters: air curtain gas (CUR): 25 psi; ion source Gas 1(Gas 1): 50 psi; ion source Gas 2(Gas 2): 50 psi; ion source spray voltage (IS): 5500V; ion source Temperature (TEM): 550 ℃; resolution Q1/Q3(Resolution Q1/Q3): Unit/Unit; collision gas (CAD): medium; pause time (MR Pause): 20 msec; the mass spectrum acquisition time was 3.00 min.
Monitoring ion pair quadrupole rod parameters:
amoxicillin: ion pairing: 366.2 → 208.1; declustering voltage (DP): 30.0V; inlet voltage (EP): 10.00V; outlet voltage (CXP): 25.00V; collision Energy (CE): 14.00 eV; residence time (Dwell): 200.0 msec.
Verapamil: ion pairing: 455.2 → 165.1; declustering voltage (DP): 30.0V; inlet voltage (EP): 10.00V; outlet voltage (CXP): 25.00V; collision Energy (CE): 36.00 eV; residence time (Dwell): 200.0 msec.
Under these conditions, the concentration of amoxicillin in Beagle dog plasma was found to be 11250 ng/mL.
Example 3
(1) The solution was prepared in accordance with the examples.
(2) Sample pretreatment
And (3) adding 40.0 mu L of human plasma low-concentration sample into 40.0 mu L of internal standard working solution, uniformly mixing for 1min in a vortex mode, adding 520 mu L of acetonitrile, uniformly mixing for 10min under the condition of shaking at room temperature, and then centrifuging for 10min at 4 ℃ and 3000 rpm. And adding 200 mu L of 0.1% formic acid aqueous solution into 100 mu L of supernatant, shaking and mixing uniformly for 3min, and analyzing by sample injection.
(3) Conditions of liquid chromatography
The instrument equipment comprises: shimadzu LC-30AD liquid chromatography system.
A chromatographic column: cosmosil 5C18-PAQ (4.6X 100mm,5.0 μm), Nacalai, column temperature set to 30 ℃ and autosampler temperature set to 4 ℃.
The mobile phase A is 0.1 percent formic acid aqueous solution; mobile phase B was 100% acetonitrile. The total flow rate was 0.60 mL/min.
The mobile phase elution procedure was: from 0 to 1.3 minutes, 50% parts by volume of mobile phase a and 50% parts by volume of mobile phase B; from greater than 1.3 minutes to 1.5 minutes, 50% parts by volume mobile phase a and 50% parts by volume mobile phase B; from greater than 1.5 minutes to 2.3 minutes, 5% parts by volume mobile phase a and 95% parts by volume mobile phase B; from greater than 2.3 minutes to 2.4 minutes, 5% parts by volume mobile phase a and 95% parts by volume mobile phase B; from greater than 2.4 minutes to 3 minutes, mobile phase a is 50% parts by volume and mobile phase B is 50% parts by volume.
The mass spectrum switching valve program is as follows: 0 to 1 minute, valve B (into waste); 1 to 2 minutes, a valve (into mass spectrum); 2 to 3 minutes, valve B (into waste).
The needle washing mode is external flushing; the washing speed is 35 mu L/sec; flush port liquids R1; the volume of the flushing fluid is 500 mu L; flushing mode, namely flushing before and after needle insertion; the rinsing and soaking time is 0 second; flushing method, only flushing port; the washing time is 2 seconds;
the injection volume is 5.00 mu L, and the amoxicillin retention time is about 1.29 min; the verapamil retention time was about 1.47 min.
(4) Conditions of Mass Spectrometry
The instrument equipment comprises: sciex Qtrap5500 Mass Spectrometry System.
Ionization mode: electrospray ion source (ESI); positive ion mode (Positive); multiple Reaction Monitoring (MRM).
Ion source parameters: air curtain gas (CUR): 25 psi; ion source Gas 1(Gas 1): 50 psi; ion source Gas 2(Gas 2): 50 psi; ion source spray voltage (IS): 5500V; ion source Temperature (TEM): 550 ℃; resolution Q1/Q3(Resolution Q1/Q3): Unit/Unit; collision gas (CAD): medium; pause time (MR Pause): 20 msec; the mass spectrum acquisition time was 3.00 min.
Monitoring ion pair quadrupole rod parameters:
amoxicillin: ion pairing: 366.2 → 208.1; declustering voltage (DP): 30.0V; inlet voltage (EP): 10.00V; outlet voltage (CXP): 25.00V; collision Energy (CE): 14.00 eV; residence time (Dwell): 200.0 msec.
Verapamil: ion pairing: 455.2 → 165.1; declustering voltage (DP): 30.0V; inlet voltage (EP): 10.00V; outlet voltage (CXP): 25.00V; collision Energy (CE): 36.00 eV; residence time (Dwell): 200.0 msec.
Under the condition, the concentration of amoxicillin with high concentration in human plasma can be measured to be 25000 ng/mL.
Example 4
(1) The solution was prepared in accordance with the examples.
(2) Sample pretreatment
And (3) adding 40.0 mu L of human plasma sample with each standard curve concentration into 40.0 mu L of internal standard working solution, uniformly mixing for 1min in a vortex mode, adding 520 mu L of acetonitrile, uniformly mixing for 10min by shaking at room temperature, and then centrifuging for 10min at 4 ℃ and 3000 rpm. And adding 200 mu L of 0.1% formic acid aqueous solution into 100 mu L of supernatant, shaking and mixing uniformly for 3min, and analyzing by sample injection.
(3) Conditions of liquid chromatography
The instrument equipment comprises: shimadzu LC-30AD liquid chromatography system.
A chromatographic column: cosmosil 5C18-PAQ (4.6X 100mm,5.0 μm), Nacalai, column temperature set to 30 ℃ and autosampler temperature set to 4 ℃.
The mobile phase A is 0.1 percent formic acid aqueous solution; mobile phase B was 100% acetonitrile. The total flow rate was 0.60 mL/min.
The mobile phase elution procedure was: from 0 to 1.3 minutes, 50% parts by volume of mobile phase a and 50% parts by volume of mobile phase B; from greater than 1.3 minutes to 1.5 minutes, 50% parts by volume mobile phase a and 50% parts by volume mobile phase B; from greater than 1.5 minutes to 2.3 minutes, 5% parts by volume mobile phase a and 95% parts by volume mobile phase B; from greater than 2.3 minutes to 2.4 minutes, 5% parts by volume mobile phase a and 95% parts by volume mobile phase B; from greater than 2.4 minutes to 3 minutes, mobile phase a is 50% parts by volume and mobile phase B is 50% parts by volume.
The mass spectrum switching valve program is as follows: 0 to 1 minute, valve B (into waste); 1 to 2 minutes, a valve (into mass spectrum); 2 to 3 minutes, valve B (into waste).
The needle washing mode is external flushing; the washing speed is 35 mu L/sec; flush port liquids R1; the volume of the flushing fluid is 500 mu L; flushing mode, namely flushing before and after needle insertion; the rinsing and soaking time is 0 second; flushing method, only flushing port; the washing time is 2 seconds;
the injection volume is 3.00 mu L, and the amoxicillin retention time is about 1.29 min; the verapamil retention time was about 1.47 min.
(4) Conditions of Mass Spectrometry
The instrument equipment comprises: sciex Qtrap5500 Mass Spectrometry System.
Ionization mode: electrospray ion source (ESI); positive ion mode (Positive); multiple Reaction Monitoring (MRM).
Ion source parameters: air curtain gas (CUR): 25 psi; ion source Gas 1(Gas 1): 50 psi; ion source Gas 2(Gas 2): 50 psi; ion source spray voltage (IS): 5500V; ion source Temperature (TEM): 550 ℃; resolution Q1/Q3(Resolution Q1/Q3): Unit/Unit; collision gas (CAD): medium; pause time (MR Pause): 20 msec; the mass spectrum acquisition time was 3.00 min.
Monitoring ion pair quadrupole rod parameters:
amoxicillin: ion pairing: 366.2 → 208.1; declustering voltage (DP): 30.0V; inlet voltage (EP): 10.00V; outlet voltage (CXP): 25.00V; collision Energy (CE): 14.00 eV; residence time (Dwell): 200.0 msec.
Verapamil: ion pairing: 455.2 → 165.1; declustering voltage (DP): 30.0V; inlet voltage (EP): 10.00V; outlet voltage (CXP): 25.00V; collision Energy (CE): 36.00 eV; residence time (Dwell): 200.0 msec.
Under the condition, the relationship between the areas of the amoexin peaks and the concentrations in human plasma can be measured as follows: y 0.0002346+0.0006688x, R2The lowest detection limit was 10ng/mL and the lower limit of quantitation was 15ng/mL, at 0.9970.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. An HPLC-MS/MS detection method for the amoxicillin concentration in plasma is characterized by comprising the following steps:
(A) preparation of stock solution:
a1: dissolving amoxicillin in (50 +/-5)% acetonitrile to prepare an amoxicillin stock solution;
a2: dissolving verapamil in acetonitrile to prepare a verapamil stock solution;
(B) preparation of working solution:
b1: diluting the amoxicillin stock solution by (50 +/-5)% acetonitrile to prepare a mixed standard curve sample working solution and a mixed quality control sample working solution;
b2: diluting verapamil stock solution with (50 +/-5)% acetonitrile to prepare mixed standard curve sample working solution and mixed quality control sample working solution;
(C) preparation of standard curve sample and quality control sample:
adding a mixed standard curve working solution into blank plasma, and uniformly mixing to prepare an amoxicillin standard curve sample and an amoxicillin quality control sample;
(D) sample pretreatment: taking a plasma sample, adding verapamil stock solution, and uniformly mixing; adding acetonitrile, and mixing uniformly; centrifuging and taking supernatant; adding a formic acid aqueous solution, and uniformly mixing to obtain a sample solution to be detected;
(E) liquid chromatography separation: carrying out liquid phase chromatographic separation on a sample solution to be detected;
(F) performing mass spectrometry: and carrying out mass spectrum detection on the sample subjected to liquid chromatography separation.
2. The method of claim 1, wherein in step (D), the volume ratio of acetonitrile to plasma sample is: 4-5: 1; the concentration of the formic acid aqueous solution is (0.1 +/-0.02)%.
3. The method according to claim 1, wherein in step (D), the temperature of the centrifugation is 2-8 ℃; the rotation speed of the centrifugation is 2500-; the centrifugation time is 5min-15 min.
4. The method of claim 1, wherein in step (E), the liquid chromatography separation conditions are:
mobile phase: the mobile phase A is (0.1 plus or minus 0.02)% formic acid aqueous solution; the mobile phase B is acetonitrile; gradient elution is adopted;
the gradient elution process is as follows: (50 +/-5)% of mobile phase A-mobile phase B in 0-1.3 min; 1.3-1.5 minutes, (50 +/-5)% of mobile phase A-mobile phase B; 1.5-2.3 minutes, (5 +/-2)% of mobile phase A-mobile phase B; 2.3-2.4 minutes, (5 +/-2)% of mobile phase A-% and% of mobile phase B; 2.4-3 minutes, (50 +/-5)% of mobile phase A-mobile phase B;
the flow rate is 0.4mL/min-0.8 mL/min; the sample injection volume is 2 mu L.00-4.00 mu L; the column temperature is set to be 25-35 ℃; the temperature of the automatic sample injector is 2-6 ℃; needle washing liquid: (50 ± 5)% acetonitrile flakes; washing speed: 30-40 μ L/sec; volume of needle washing liquid: 400-600 μ L.
5. The method according to claim 4, wherein in step (E), the mobile phase A is 0.1% formic acid aqueous solution; the gradient elution process is as follows: 50% of mobile phase A-mobile phase B in 0-1.3 min; 1.3-1.5 minutes, 50% of mobile phase A-mobile phase B; 5% of mobile phase A-mobile phase B in 1.5-2.3 minutes; 2.3-2.4 minutes, 5% of mobile phase A-% and 5% of mobile phase B; 2.4-3 minutes, 50% of mobile phase A-mobile phase B;
the flow rate is 0.6 mL/min; the sample injection volume is 3.00 mu L; the column temperature was set at 30 ℃; the temperature of the autosampler is 4 ℃; needle washing liquid: 50% acetonitrile; washing speed: 35 μ L/sec; volume of needle washing liquid: 500 μ L.
6. The method of claim 1, wherein in step (F), the mass spectrometry conditions are:
mass spectrum detection conditions: ionization mode: an electrospray ion source; a positive ion mode; monitoring multiple reactions;
ion source parameters: air curtain air: 25 psi; ion source gas 1: 50 psi; ion source gas 2: 50 psi; ion source spray voltage: 5500V; ion source temperature: 550 ℃; resolution Q1/Q3: Unit/Unit; collision gas: medium; pause time: 20 msec; the mass spectrum acquisition time is 3.00 min;
monitoring ion pair quadrupole rod parameters: amoxicillin: ion pairing: 366.2 → 208.1; de-clustering voltage: 30.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 14.00 eV; residence time: 200.0 msec; verapamil: ion pairing: 455.2 → 165.1; de-clustering voltage: 30.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 36.00 eV; residence time: 200.0 msec.
7. The method of claim 1, wherein in step B1, the concentrations of amoxicillin in the mixed standard curve sample working solution are as follows: 300ng/mL, 600ng/mL, 2000ng/mL, 10000ng/mL, 50000ng/mL, 100000ng/mL, 250000ng/mL, and 300000 ng/mL; and/or
In the step B2, the concentrations of amoxicillin in the mixed quality control sample working solution are as follows: the lower limit of quantification quality control is 300ng/mL, the low concentration quality control is 800ng/mL, the medium concentration quality control in geometric mean is 15000ng/mL, the medium concentration quality control in arithmetic mean is 150000ng/mL, and the high concentration quality control is 225000 ng/mL.
8. The method of claim 7, wherein in step (C), the concentrations of amoxicillin in the standard curve sample are: 15.0ng/mL, 30.0ng/mL, 100ng/mL, 500ng/mL, 2500ng/mL, 5000ng/mL, 12500ng/mL, and 15000 ng/mL; and/or
In the quality control sample, the concentrations of amoxicillin are respectively as follows: the lower limit quality control of the quantification is 15ng/mL, the low concentration quality control is 40.0ng/mL, the medium concentration quality control of the geometric mean value is 750ng/mL, the medium concentration quality control of the arithmetic mean value is 7500ng/mL, and the high concentration quality control is 11250 ng/mL.
9. The method according to any one of claims 1 to 8, wherein in step (E), the liquid chromatography mass spectrometer used is a Shimadzu LC 30AD liquid chromatograph in combination with a Sciex Qtrap5500 mass spectrometer, and the operating system used is Analyst 1.6.3; the chromatographic columns used were: nacalai's Cosmosil 5C 18-PAQ.
10. The method of any one of claims 1 to 8, wherein the plasma is from a human or animal.
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