CN117761203A - Analysis method for determining salbutamol concentration in K2EDTA human plasma - Google Patents
Analysis method for determining salbutamol concentration in K2EDTA human plasma Download PDFInfo
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- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 229960002052 salbutamol Drugs 0.000 title claims abstract description 31
- 238000004458 analytical method Methods 0.000 title claims abstract description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 39
- 238000001514 detection method Methods 0.000 claims abstract description 34
- 239000000243 solution Substances 0.000 claims abstract description 23
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000002156 mixing Methods 0.000 claims abstract description 14
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 12
- 238000001556 precipitation Methods 0.000 claims abstract description 11
- 239000012224 working solution Substances 0.000 claims abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 8
- 238000000861 blow drying Methods 0.000 claims abstract description 5
- 238000007664 blowing Methods 0.000 claims abstract description 5
- 239000000523 sample Substances 0.000 claims description 68
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- 150000002500 ions Chemical class 0.000 claims description 26
- 238000012360 testing method Methods 0.000 claims description 25
- 239000013062 quality control Sample Substances 0.000 claims description 21
- 238000010828 elution Methods 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 12
- 238000012544 monitoring process Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 3
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 claims description 3
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 3
- 239000012071 phase Substances 0.000 description 26
- 238000004140 cleaning Methods 0.000 description 14
- 210000002381 plasma Anatomy 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 12
- 239000007788 liquid Substances 0.000 description 10
- 238000001819 mass spectrum Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 6
- 235000019253 formic acid Nutrition 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- 238000010926 purge Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000002552 multiple reaction monitoring Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000004743 Polypropylene Substances 0.000 description 3
- 235000019257 ammonium acetate Nutrition 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000000622 liquid--liquid extraction Methods 0.000 description 3
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- -1 polypropylene Polymers 0.000 description 3
- 229920001155 polypropylene Polymers 0.000 description 3
- 230000006920 protein precipitation Effects 0.000 description 3
- 238000000638 solvent extraction Methods 0.000 description 3
- NDAUXUAQIAJITI-GQALSZNTSA-N 4-[2-[[1,1,1,3,3,3-hexadeuterio-2-(trideuteriomethyl)propan-2-yl]amino]-1-hydroxyethyl]-2-(hydroxymethyl)phenol Chemical compound [2H]C([2H])([2H])C(C([2H])([2H])[2H])(C([2H])([2H])[2H])NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-GQALSZNTSA-N 0.000 description 2
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 229960004194 lidocaine Drugs 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- WRMRXPASUROZGT-UHFFFAOYSA-N monoethylglycinexylidide Chemical compound CCNCC(=O)NC1=C(C)C=CC=C1C WRMRXPASUROZGT-UHFFFAOYSA-N 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 229940044601 receptor agonist Drugs 0.000 description 2
- 239000000018 receptor agonist Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 1
- 208000009079 Bronchial Spasm Diseases 0.000 description 1
- 208000014181 Bronchial disease Diseases 0.000 description 1
- 206010006482 Bronchospasm Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 208000031481 Pathologic Constriction Diseases 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000004989 laser desorption mass spectroscopy Methods 0.000 description 1
- 235000020997 lean meat Nutrition 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 230000036262 stenosis Effects 0.000 description 1
- 208000037804 stenosis Diseases 0.000 description 1
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- 238000002560 therapeutic procedure Methods 0.000 description 1
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Classifications
-
- 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
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to the technical field of analysis and detection, in particular to an IPC G01N30, and more particularly relates to an analysis method for determining salbutamol concentration in K2EDTA human plasma. The method comprises the steps of treating samples with ammonia water solution and internal standard working solution, adding precipitation solution into each sample, uniformly mixing, centrifuging, and blow-drying each sample by using a nitrogen blowing instrument; then adding methanol into the blow-dried sample, mixing uniformly, centrifuging, and performing liquid chromatography-tandem mass spectrometry detection to obtain the analysis method for determining the salbutamol concentration in K2EDTA human plasma with low detection limit, wherein the minimum detection limit of the method is 10pg/mL, the accuracy and the precision are high, the percentage CV is less than 10%, and the minimum detection limit in the prior art is at ng/mL level and cannot reach pg/mL level.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to an IPC G01N30, and more particularly relates to an analysis method for determining salbutamol concentration in K2EDTA human plasma.
Background
Salbutamol is beta 2 Receptor agonists are widely used in various industries, and in the aquaculture industry, nutritional ingredients in salbutamol animals are redistributed, so that the metabolism of the animal is regulated and controlled, the lipolysis is enhanced, the protein synthesis is promoted, the carcass lean meat percentage and feed return are remarkably improved, and the receptor agonists can enter a human body through a food chain and accumulate in the human body, so that symptoms such as muscle tremors, arrhythmia and headache are caused. In addition, salbutamol has excellent smooth muscle relaxing effect and can relieve bronchospasm and stenosis caused by asthma. In addition to being used in animal husbandry and in therapy, albuterol is also often used as an stimulant. However, the device is strictly forbidden to be used in the stadium and the outside of the stadium, thereby improving the fairness of the competition. Therefore, the salbutamol concentration in human plasma needs to be detected, so that the accuracy of diagnosis is improved, and the detection rate of the stimulant is improved.
The prior patent CN202310325186.7 discloses a method for simultaneously detecting the concentration of lidocaine and metabolites MEGX and salbutamol thereof in blood plasma, wherein a blood plasma sample to be detected, acetonitrile and an internal standard are mixed for pretreatment to obtain a sample injection to be detected; and (3) carrying out liquid chromatography-tandem mass spectrometry detection on the sample liquid to be detected to obtain the concentrations of lidocaine, MEGX and salbutamol in the plasma sample to be detected, wherein the detection limit of the method is higher, and the salbutamol with low concentration cannot be detected.
Disclosure of Invention
In order to solve the problems in the prior art, the first aspect of the present invention provides an analysis method for determining the concentration of albuterol in K2EDTA human plasma, comprising the steps of: treating the samples with ammonia water solution and internal standard working solution, adding precipitation solution into each sample, uniformly mixing, centrifuging, and blow-drying the samples by using a nitrogen blowing instrument; and then adding methanol into the blow-dried sample, uniformly mixing, centrifuging, and then carrying out liquid chromatography-tandem mass spectrometry detection.
Preferably, the specific analysis method for determining the salbutamol concentration in K2EDTA human plasma comprises the following steps:
s1: respectively adding an ammonia solution and an internal standard working solution into the standard curve sample, the quality control sample and the test sample to obtain a mixed standard curve sample, a mixed quality control sample and a mixed test sample;
s2: respectively adding a precipitation solution into the mixed standard curve sample, the mixed quality control sample and the mixed test sample, uniformly mixing by vortex at room temperature, and centrifuging for 10-20 min after uniform mixing to obtain a centrifuged standard curve sample, a centrifuged quality control sample and a centrifuged test sample;
s3: transferring supernatant in the standard curve sample after centrifugation, the quality control sample after centrifugation and the test sample after centrifugation to an empty EP tube at room temperature, and blow-drying by a nitrogen blower; adding methanol, mixing at room temperature, centrifuging for 2-5 min, and placing into an automatic sampler for liquid chromatography-tandem mass spectrometry detection.
Preferably, the volume ratio of the sample to the ammonia water solution to the internal standard working solution is (8-15): 1: (2-3); further 10:1:2.5.
according to the invention, by adding the ammonia water solution, not only is the extraction recovery rate of the sample improved, but also the signal of the test result of the sample is improved, and the detection limit is improved. The inventors found that when ammonia water was added, there was a greater effect on the improvement of the signal in the liquid chromatograph-tandem mass spectrum, and the response and peak shape of the signal were improved.
Preferably, the concentration of the internal standard working solution is 5.0-15.0 ng/mL; further preferably 10.0ng/mL.
Preferably, the precipitation solution is methanol and acetonitrile, and the volume ratio is (0.5-1.5): 1, a step of; further preferred is 1:1.
preferably, the volume ratio of the sample to the precipitation solution is 1: (6-10); further preferred is 1:8.
preferably, the temperature of the centrifugation is 4-8 ℃, and the rotation speed of the centrifugation is 3000-5000 rpm; further preferably, the temperature is 4℃and the rotational speed of the centrifugation is 4000rpm.
In the invention, more complex liquid-liquid extraction or solid phase extraction is optimized into simple protein precipitation through a specific pretreatment process, so that the time and cost of sample pretreatment are reduced. The inventor finds that in the prior art, the analysis method for determining the salbutamol concentration in human plasma by using LC-MS/MS adopts liquid-liquid extraction and solid phase extraction as pretreatment, and the two pretreatment methods are long in time consumption, high in price and easy to produce cross contamination in the treatment process, and the specific protein precipitation is adopted in the invention, and particularly the volume ratio of methanol to acetonitrile is 1: the pretreatment method of the solution is simple, efficient, short in time consumption, not easy to pollute and capable of improving the stability of the method. Meanwhile, the dosage of methanol and acetonitrile in the invention is small, thus reducing the possibility of reagent pollution.
Preferably, the liquid chromatography conditions in the liquid chromatography-tandem mass spectrometry detection include: the settings of chromatographic column, column temperature, autosampler temperature, mobile phase elution procedure, switching valve procedure, autosampler needle wash and sample volume.
Preferably, the chromatographic column used in the liquid chromatography-tandem mass spectrometry detection is5μm C18(2)ACQUITY/>BEH C18，ACQUITY/>HSS T3, any one of InertSustrin AQ-C18.
Preferably, the InertSustin AQ-C18 has a gauge of 2.1mm by 50mm,3 μm.
In the invention, a specific chromatographic column is selected, so that the response of the peak shape of a chromatographic peak is improved. In the development process, the inventor screens chromatographic columns of different types, including:5μm C18(2)/>ACQUITY/>BEH C18，ACQUITY/>HSS T3, inertSustin AQ-C18, wherein InertSustin AQ-C18 has the best peak shape and response.
Preferably, the column temperature is 30-50 ℃; the temperature of the automatic sampler is 5-10 ℃; mobile phase A is acetic acid water solution with mass fraction of 0.1% and mobile phase B is acetonitrile.
Preferably, the mobile phase elution procedure is a gradient elution procedure: 0.0 to 1.5min, and the volume fraction of the mobile phase B is 4 percent; 1.5 to 1.61min, the volume fraction of the mobile phase B is changed from 4 percent to 50 percent; 1.61-2.0 min, the volume fraction of the mobile phase B is changed from 40% to 4%; flow rate of mobile phase system: 0.6mL/min.
In the present invention, the interference between peak shapes is reduced and the response is improved by a specific mobile phase elution procedure. The inventors found that the response was highest when water, acetonitrile, methanol, water containing 0.1% formic acid, methanol containing 0.1% formic acid, acetonitrile containing 0.1% formic acid, water containing 0.1% acetic acid, methanol containing 0.1% acetic acid, acetonitrile containing 0.1% acetic acid, water containing 0.1% formic acid and 10mM ammonium acetate, methanol containing 0.1% formic acid and 10mM ammonium acetate, acetonitrile containing 0.1% formic acid and 10mM ammonium acetate, and the like were confirmed to be water containing 0.1% acetic acid when mobile phase A was acetonitrile and mobile phase B was acetonitrile.
Preferably, the switching valve program: feeding waste liquid for 0-0.9 min; feeding mass spectrum for 0.9-1.6 min; 1.6 to 2.0, and feeding waste liquid.
Preferably, the automatic injector washes the needle: cleaning solvent (R0): 50% methanol; sample suction rate (Sampling speed): 5.0 μL/sec; volume of displaced cleaning fluid (Measuring Line Purge Volume) in the flow path is measured: 100. Mu.L; use air gap volume: no; needle cleaning Type (risse Type): external only; replacement wash flow rate (ringing Speed): 35 μL/sec; cleaning liquid type (Rinse Port liquid Selection): r0; displacing the Volume of the cleaning fluid (ringing Volume): 500. Mu.L; needle tube cleaning Mode (risse Mode): wash before and after aspiration of sample (Before and after aspiration); needle tube purge Time (Rinse Time): 2sec; needle tube cleaning Method (Rinse Method): a standard purge Port dip syringe (Rinse Port Only); needle cannula immersion purge Time (Rinse Dip Time): 0sec.
Preferably, the sample injection volume is 5-15 mu L.
Preferably, the mass spectrum conditions in the liquid chromatography-tandem mass spectrometry detection comprise ionization mode, ion source parameters, mass spectrum acquisition time and settings for monitoring selective reaction ions.
Preferably, the ionization mode includes electrospray ion source, positive ion mode, multiple Reaction Monitoring (MRM).
Preferably, the ion source parameters include a collision gas (CAD): 7.00psi; curtain gas (CUR):
20.0psi; ion source gas 1 (GS 1): 60.0psi; ion source gas 2 (GS 2): 60.0psi; ion source spray voltage (IS): 5500V; ion source Temperature (TEM): 550.00 ℃; resolution Q1/Q3 (Resolution Q1/Q3): unit/unit; pause time (MR Pause): 5.0070msec.
Preferably, the mass spectrometry acquisition time is 2.0min.
Preferably, the selective reactive ion species monitoring comprises selective reactive ion species monitoring as set forth in table 1.
TABLE 1
| Salbutamol | Salbutamol-d 9 | |
| Monitoring ion pairs | 240.100/148.200 | 249.300/149.100 |
| De-cluster voltage (DP) | 80.00V | 80.00V |
| Inlet voltage (EP) | 10.00V | 10.00V |
| Outlet Voltage (CXP) | 10.00V | 10.00V |
| Impact energy (CE) | 26.00eV | 27.00eV |
| Residence time (Dwell) | 200.00msec | 200.00msec |
Advantageous effects
1. In the invention, more complex liquid-liquid extraction or solid phase extraction is optimized into simple protein precipitation through a specific pretreatment process, so that the time and cost of sample pretreatment are reduced.
2. According to the invention, by adding the ammonia water solution, not only is the extraction recovery rate of the sample improved, but also the signal of the test result of the sample is improved, and the detection limit is improved. In addition, the supernatant after precipitation is dried by nitrogen and then redissolved, and the response is improved in an enrichment mode.
3. In the invention, a specific chromatographic column is selected, so that the response of the peak shape of a chromatographic peak is improved.
4. In the present invention, the interference between peak shapes is reduced and the response is improved by a specific mobile phase elution procedure.
5. According to the invention, ammonia water is added, a specific precipitation solution is used, enrichment is carried out through a nitrogen blowing instrument, a specific chromatographic column is adopted, a mobile phase elution program is set, and mass spectrum conditions are simultaneously optimized, so that an analysis method for measuring the salbutamol concentration in K2EDTA human plasma with a low detection limit is obtained, the minimum detection limit of the method is 10pg/mL, the accuracy and precision are high, the% CV is less than 10%, the minimum detection limit in the prior art is at ng/mL level at present, and the pg/mL level cannot be reached.
Drawings
FIG. 1 is a partial screenshot of the detection chromatographic peak of LLOQ QC 10pg/mL quality control sample in example 1.
FIG. 2 is a partial screenshot of the detection chromatographic peak of the test sample in example 1.
FIG. 3 is a partial screenshot of the detection chromatographic peak of the test sample in comparative example 1.
Detailed Description
Example 1
The first aspect of the present embodiment provides a specific analysis method for determining salbutamol concentration in K2EDTA human plasma, comprising the steps of:
s1: respectively adding 10 mu L of ammonia water solution and 25 mu, 10ng/mL of internal standard working solution into 100 mu L of standard curve sample, 100 mu L of standard quality control sample and 100 mu L of standard test sample placed on a 2.2mL 96-well polypropylene plate at 25 ℃ under the condition of white light to obtain a mixed standard curve sample, a mixed quality control sample and a mixed test sample;
s2: respectively adding 800 mu L of methanol-acetonitrile solution (v/v, 1:1) into the mixed standard curve sample, the mixed quality control sample and the mixed test sample, uniformly mixing for 15min at room temperature by vortex, and centrifuging (4 ℃ at 4000 rpm) for 10min after uniform mixing to obtain a centrifuged standard curve sample, a centrifuged quality control sample and a centrifuged test sample;
s3: transferring 600 mu L of supernatant in the standard curve sample after centrifugation, the quality control sample after centrifugation and the test sample after centrifugation to an empty EP tube at 25 ℃ to be placed in a 96-hole polypropylene plate, and drying by a nitrogen blowing instrument; then 150. Mu.L of 10% aqueous methanol solution is added, the mixture is uniformly mixed for 3min at room temperature, and the mixture is centrifuged (4 ℃ C., 4000 rpm) for 3min and then placed into an automatic sampler for liquid chromatography-tandem mass spectrometry detection.
The concentration of salbutamol in the standard curve samples was 10.0pg/mL,20.0pg/mL,50.0pg/mL,200pg/mL,400pg/mL,800pg/mL,1600pg/mL and 2000pg/mL, respectively.
The quality control samples are LLOQ QC 10pg/mL, LQC 30.0pg/mL, GMQC 150.0pg/mL, MQC 600.0pg/mL and HQC1500.0 pg/mL.
A partial screenshot of the detection result data of the LLOQ QC 10pg/mL quality control sample is shown in FIG. 1.
The test sample is a K2EDTA human plasma containing salbutamol, and is from a clinical test sample with the number of HLJ-FLKYY.
The results of partial detection of the test sample are shown in FIG. 2.
The liquid chromatography conditions in the liquid chromatography-tandem mass spectrometry detection comprise: the settings of chromatographic column, column temperature, autosampler temperature, mobile phase elution procedure, switching valve procedure, autosampler needle wash and sample volume.
The chromatographic column adopted in the liquid chromatography-tandem mass spectrometry detection is InertSustin AQ-C18.
The InertSustin AQ-C18 has a specification of 2.1mm by 50mm and 3 μm.
The column temperature is 30-50 ℃; the temperature of the automatic sampler is 5-10 ℃; mobile phase A is acetic acid water solution with mass fraction of 0.1% and mobile phase B is acetonitrile.
The mobile phase elution procedure is a gradient elution procedure: 0.0 to 1.5min, and the volume fraction of the mobile phase B is 4 percent; 1.5 to 1.61min, the volume fraction of the mobile phase B is changed from 4 percent to 50 percent; 1.61-2.0 min, the volume fraction of the mobile phase B is changed from 40% to 4%; flow rate of mobile phase system: 0.6mL/min.
The switching valve program: feeding waste liquid for 0-0.9 min; feeding mass spectrum for 0.9-1.6 min; 1.6 to 2.0, and feeding waste liquid.
The automatic injector washes the needle: cleaning solvent (R0): 50% methanol; sample suction rate (Sampling speed): 5.0 μL/sec; volume of displaced cleaning fluid (Measuring Line Purge Volume) in the flow path is measured: 100. Mu.L; use air gap volume: no; needle cleaning Type (risse Type): external only; replacement wash flow rate (ringing Speed): 35 μL/sec; cleaning liquid type (Rinse Port liquid Selection): r0; displacing the Volume of the cleaning fluid (ringing Volume): 500. Mu.L; needle tube cleaning Mode (risse Mode): wash before and after aspiration of sample (Before and after aspiration); needle tube purge Time (Rinse Time): 2sec; needle tube cleaning Method (Rinse Method): a standard purge Port dip syringe (Rinse Port Only); needle cannula immersion purge Time (Rinse Dip Time): 0sec.
The sample injection volume was 10. Mu.L.
The mass spectrum conditions in the liquid chromatography-tandem mass spectrometry detection comprise ionization mode, ion source parameters, mass spectrum acquisition time and setting of selective reaction ion monitoring.
The ionization mode includes electrospray ion source, positive ion mode, multiple Reaction Monitoring (MRM).
The ion source parameters include collision gas (CAD): 7.00psi; curtain gas (CUR): 20.0psi; ion source gas 1 (GS 1): 60.0psi; ion source gas 2 (GS 2): 60.0psi; ion source spray voltage (IS): 5500V; ion source Temperature (TEM): 550.00 ℃; resolution Q1/Q3 (Resolution Q1/Q3): unit/unit; pause time (MR Pause): 5.0070msec.
The mass spectrum acquisition time is 2.0min.
The selective reactive ion species monitoring includes selective reactive ion species monitoring as set forth in table 1.
TABLE 1
| Salbutamol | Salbutamol-d 9 | |
| Monitoring ion pairs | 240.100/148.200 | 249.300/149.100 |
| De-cluster voltage (DP) | 80.00V | 80.00V |
| Inlet voltage (EP) | 10.00V | 10.00V |
| Outlet Voltage (CXP) | 10.00V | 10.00V |
| Impact energy (CE) | 26.00eV | 27.00eV |
| Residence time (Dwell) | 200.00msec | 200.00msec |
Decision criteria for results: the collection of chromatograms and integration of chromatographic peaks of analytes and internal standards was done by AB Sciex analysis software (version 1.7.2 and above). The standard curve is obtained by adopting Watson LIMS system (version 7.6.1 and above) regression, the chromatographic response ratio of the analyte and the internal standard is taken as an ordinate, the weighted (W=1/x 2) least square method is used for carrying out linear regression on the concentration (x) of the analyte in the blood plasma and the response ratio (y), the regression equation (y=ax+b) is the standard curve, and the drug concentration of the sample to be detected is calculated by the standard curve equation obtained by fitting.
Comparative example 1
The embodiment of comparative example 1 is the same as example 1, except that the mobile phase elution procedure is a gradient elution procedure: 0.0 to 0.9min, and the volume fraction of the mobile phase B is 7 percent; 0.9-1.31 min, the volume fraction of the mobile phase B is changed from 7% to 90%; 1.31-2.0 min, the volume fraction of the mobile phase B is changed from 90% to 7%; flow rate of mobile phase system: 0.6mL/min.
The partial detection data of the detection sample are shown in FIG. 3.
Comparative example 2
The embodiment of comparative example 2 is the same as example 1, except that S1: and respectively adding 10ng/mL of internal standard working solution into 100 mu L of standard curve sample, 100 mu L of standard quality control sample and 100 mu L of standard test sample placed on a 2.2mL 96-well polypropylene plate at 25 ℃ under the condition of white light to obtain a mixed standard curve sample, a mixed quality control sample and a mixed test sample.
Testing limit, accuracy and precision
1. The concentration of albuterol in the quality control samples of LLOQ QC 10pg/mL, LQC 30.0pg/mL, GMQC 150.0pg/mL, MQC 600.0pg/mL and HQC1500.0 pg/mL (6 replicates for each concentration) in example 1 was tested and the data recorded in Table 2.
Conclusion: the salbutamol measured by each quality control sample accords with the concentration of the quality control sample, and the deviation from the theoretical concentration is small and accurate.
TABLE 2
2. The extraction recovery rates in example 1 and comparative example 2 were measured. See table 1.
TABLE 3 Table 3
| Group of | Recovery rate of extraction |
| Example 1 | 95％ |
| Comparative example 2 | 50％ |
3. In the development process, the liquid phase elution gradient is optimized. Before optimization, the chromatographic peak is shown in comparative example 1, the chromatographic peak is shown in fig. 3, the chromatographic peak is shown in example 1 after development, and the chromatographic peak is shown in fig. 2, so that before optimization, the interference peak beside the target peak is not completely separated. After optimization, the target peak is completely separated from the interference peak.
Claims (10)
1. An analytical method for determining salbutamol concentration in K2EDTA human plasma, comprising the steps of: treating the samples with ammonia water solution and internal standard working solution, adding precipitation solution into each sample, uniformly mixing, centrifuging, and blow-drying the samples by using a nitrogen blowing instrument; and then adding methanol into the blow-dried sample, uniformly mixing, centrifuging, and then carrying out liquid chromatography-tandem mass spectrometry detection.
2. The method for determining the concentration of albuterol in K2EDTA human plasma according to claim 1, characterized in that it comprises the following steps:
s1: respectively adding an ammonia solution and an internal standard working solution into the standard curve sample, the quality control sample and the test sample to obtain a mixed standard curve sample, a mixed quality control sample and a mixed test sample;
s2: respectively adding a precipitation solution into the mixed standard curve sample, the mixed quality control sample and the mixed test sample, uniformly mixing by vortex at room temperature, and centrifuging for 10-20 min after uniform mixing to obtain a centrifuged standard curve sample, a centrifuged quality control sample and a centrifuged test sample;
s3: transferring supernatant in the standard curve sample after centrifugation, the quality control sample after centrifugation and the test sample after centrifugation to an empty EP tube at room temperature, and blow-drying by a nitrogen blower; adding methanol, mixing at room temperature, centrifuging for 2-5 min, and placing into an automatic sampler for liquid chromatography-tandem mass spectrometry detection.
3. The analytical method for determining salbutamol concentration in K2EDTA human plasma according to claim 1, wherein the volume ratio of the sample to the ammonia solution to the internal standard working solution is (8-15): 1: (2-3).
4. The analytical method for determining salbutamol concentration in K2EDTA human plasma according to claim 1, wherein the precipitation solution is methanol and acetonitrile, and the volume ratio is (0.5 to 1.5): 1.
5. the method for determining the concentration of albuterol in K2EDTA human plasma according to claim 1, characterized in that the volume ratio of sample to precipitation solution is 1: (6-10).
6. The method for determining the concentration of albuterol in K2EDTA human plasma according to claim 1, wherein the conditions of liquid chromatography in the liquid chromatography-tandem mass spectrometry detection include: the settings of chromatographic column, column temperature, autosampler temperature, mobile phase elution procedure, switching valve procedure, autosampler needle wash and sample volume.
7. The method according to claim 6, wherein the chromatographic column used in the liquid chromatography-tandem mass spectrometry is5μm C18(2)/>ACQUITY/>BEH C18，ACQUITY/>HSS T3, any one of InertSustrin AQ-C18.
8. The method for determining the concentration of albuterol in K2EDTA human plasma according to claim 6, characterized in that the column temperature is 30-50 ℃; the temperature of the automatic sampler is 5-10 ℃; mobile phase A is acetic acid water solution with mass fraction of 0.1% and mobile phase B is acetonitrile.
9. The method of claim 6, wherein the mobile phase elution procedure is a gradient elution procedure: 0.0 to 1.5min, and the volume fraction of the mobile phase B is 4 percent; 1.5 to 1.61min, the volume fraction of the mobile phase B is changed from 4 percent to 50 percent; 1.61-2.0 min, the volume fraction of the mobile phase B is changed from 40% to 4%; flow rate of mobile phase system: 0.6mL/min.
10. The method according to claim 1, wherein the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry detection include ionization mode, ion source parameters, mass spectrometry acquisition time and settings for selective reactive ion monitoring.
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