CN116754671A - Detection method for detecting concentration of five drugs in blood plasma and application thereof - Google Patents
Detection method for detecting concentration of five drugs in blood plasma and application thereof Download PDFInfo
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- CN116754671A CN116754671A CN202310649809.6A CN202310649809A CN116754671A CN 116754671 A CN116754671 A CN 116754671A CN 202310649809 A CN202310649809 A CN 202310649809A CN 116754671 A CN116754671 A CN 116754671A
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- 210000002381 plasma Anatomy 0.000 title claims abstract description 41
- 239000003814 drug Substances 0.000 title claims abstract description 33
- 229940079593 drug Drugs 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 title abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 47
- 239000012224 working solution Substances 0.000 claims abstract description 30
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims abstract description 24
- 229960004372 aripiprazole Drugs 0.000 claims abstract description 17
- CEUORZQYGODEFX-UHFFFAOYSA-N Aripirazole Chemical compound ClC1=CC=CC(N2CCN(CCCCOC=3C=C4NC(=O)CCC4=CC=3)CC2)=C1Cl CEUORZQYGODEFX-UHFFFAOYSA-N 0.000 claims abstract description 15
- -1 5-diphenyl hydantoin Chemical compound 0.000 claims abstract description 10
- CDONPRYEWWPREK-UHFFFAOYSA-N 7-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butoxy]-1h-quinolin-2-one Chemical compound ClC1=CC=CC(N2CCN(CCCCOC=3C=C4NC(=O)C=CC4=CC=3)CC2)=C1Cl CDONPRYEWWPREK-UHFFFAOYSA-N 0.000 claims abstract description 10
- CTRLABGOLIVAIY-UHFFFAOYSA-N oxcarbazepine Chemical class C1C(=O)C2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 CTRLABGOLIVAIY-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000002347 injection Methods 0.000 claims abstract description 8
- 239000007924 injection Substances 0.000 claims abstract description 8
- PYZRQGJRPPTADH-UHFFFAOYSA-N lamotrigine Chemical compound NC1=NC(N)=NN=C1C1=CC=CC(Cl)=C1Cl PYZRQGJRPPTADH-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229960002036 phenytoin Drugs 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 8
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- 238000000622 liquid--liquid extraction Methods 0.000 claims description 6
- 238000000638 solvent extraction Methods 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 4
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 150000001733 carboxylic acid esters Chemical class 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000005499 Clomazone Substances 0.000 claims description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 2
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 claims description 2
- 150000003868 ammonium compounds Chemical class 0.000 claims description 2
- 150000004945 aromatic hydrocarbons Chemical class 0.000 claims description 2
- KIEDNEWSYUYDSN-UHFFFAOYSA-N clomazone Chemical compound O=C1C(C)(C)CON1CC1=CC=CC=C1Cl KIEDNEWSYUYDSN-UHFFFAOYSA-N 0.000 claims description 2
- 239000003208 petroleum Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 18
- 238000011084 recovery Methods 0.000 abstract description 13
- 230000006920 protein precipitation Effects 0.000 abstract description 11
- HOLCERFFZOJVPE-UHFFFAOYSA-N 4-(chloromethyl)-3H-1,3-oxazol-2-one Chemical compound ClCC=1N=C(OC1)O HOLCERFFZOJVPE-UHFFFAOYSA-N 0.000 abstract description 5
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 238000004140 cleaning Methods 0.000 abstract 1
- 229940126585 therapeutic drug Drugs 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 33
- 239000012071 phase Substances 0.000 description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 24
- 238000002156 mixing Methods 0.000 description 18
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 13
- 239000003085 diluting agent Substances 0.000 description 13
- 239000002904 solvent Substances 0.000 description 11
- 239000011550 stock solution Substances 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 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 8
- 235000019253 formic acid Nutrition 0.000 description 8
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
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- 230000000694 effects Effects 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- CXOFVDLJLONNDW-UHFFFAOYSA-N Phenytoin Chemical compound N1C(=O)NC(=O)C1(C=1C=CC=CC=1)C1=CC=CC=C1 CXOFVDLJLONNDW-UHFFFAOYSA-N 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000000861 blow drying Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229960000623 carbamazepine Drugs 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- CEUORZQYGODEFX-FUEQIQQISA-N 7-[4-[2,2,3,3,5,5,6,6-octadeuterio-4-(2,3-dichlorophenyl)piperazin-1-yl]butoxy]-3,4-dihydro-1h-quinolin-2-one Chemical compound [2H]C1([2H])C([2H])([2H])N(CCCCOC=2C=C3NC(=O)CCC3=CC=2)C([2H])([2H])C([2H])([2H])N1C1=CC=CC(Cl)=C1Cl CEUORZQYGODEFX-FUEQIQQISA-N 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000002514 liquid chromatography mass spectrum Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000012454 non-polar solvent Substances 0.000 description 1
- 229960001816 oxcarbazepine Drugs 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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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 application discloses a detection method for detecting the concentration of five drugs in blood plasma and application thereof, wherein the five drugs are 6- (2, 3-dichlorophenyl) -1,2, 4-triazine-3, 5-diamine, chloromethyl oxazolone, oxcarbazepine metabolite, 5-diphenyl hydantoin, aripiprazole and dehydroaripiprazole respectively, and the method comprises the following steps: 1. preparing a working solution; 2. sample pretreatment; 3. analyzing by high performance liquid chromatography-mass spectrometry sample injection; the detection method solves the problems of environmental protection and difficult impurity removal and cleaning caused by the protein precipitation method in the prior art, greatly improves the recovery rate, and can be applied to the detection of therapeutic drugs for drugs in clinical work.
Description
Technical Field
The application relates to the field of drug detection, and specifically relates to a detection method for detecting five drug concentrations in blood plasma and application thereof, wherein the classification number of the detection method is G01N 30/02.
Background
The measurement of blood drug concentration is the basis of research pharmacokinetics, and animal and human pharmacokinetic studies are important components of preclinical drug studies and primary clinical safety evaluation, and are important pathways for early screening of diseases and fetuses. The plasma contains complex components such as plasma protein, salt ions, water, a drug to be detected and the like, the concentration of the drug is relatively low, and a liquid chromatography-mass spectrometry (LC-MS) technology is integrated with separation and detection technologies, so that the drug can be separated from most of impurities in the body for detection, and the method becomes a main method for detecting the concentration of the drug in the plasma at present.
However, in the existing liquid chromatography-mass spectrometry, the mobile phase of the liquid chromatography generally uses toxic solvents such as acetonitrile and methanol, in addition, for detecting various medicines simultaneously, the problems that various targets are difficult to completely separate by chromatography and flow out simultaneously exist in the liquid chromatography-mass spectrometry, so that the detection sensitivity is greatly reduced, the detection accuracy is influenced, and secondly, the pretreatment of a sample has an important influence on the accuracy of the liquid chromatography-mass spectrometry, but the existing protein precipitation method is generally used, the speed is high, the operation is simple, but the impurity removal degree is very low, a large amount of polar protein impurities contained in plasma cannot be completely removed, so that the precision and the recovery rate are influenced, and the protein precipitation method generally adopts toxic solvents such as acetonitrile and methanol, so that the human body and the environment are greatly influenced.
Chinese patent CN114755348B discloses a method for simultaneously detecting the contents of 20 drugs and metabolites thereof, which uses a protein precipitation method, wherein the protein precipitation method has a high speed, but has a low impurity removal degree, and the protein precipitation method adopts toxic solvents such as acetonitrile, methanol and the like, so that the protein precipitation method can have great influence on human bodies and the environment.
Chinese patent CN110208450a discloses a method for detecting aripiprazole and dehydroaripiprazole in blood, wherein the detection limit of aripiprazole is up to 1.080ng/mL, the linear range is 20ng/mL to 2560ng/mL, the detection limit of aripiprazole is higher, and only two drugs of aripiprazole and dehydroaripiprazole are detected, so that the detection efficiency is lower.
Disclosure of Invention
In order to solve the above-mentioned problems, a first aspect of the present application provides an assay for detecting the concentration of five drugs in blood plasma, 6- (2, 3-dichlorophenyl) -1,2, 4-triazine-3, 5-diamine, chloromethyl oxazolone, oxcarbazepine metabolite, 5-diphenylhydantoin, aripiprazole and dehydroaripiprazole, respectively, comprising the steps of: 1. preparing a working solution; 2. sample pretreatment; 3. and (5) analyzing by high performance liquid chromatography-mass spectrometry sample injection.
Further preferably, the working solution formulation includes: standard stock solution, primary intermediate solution, standard mixed secondary intermediate solution, internal standard stock solution, internal standard intermediate solution, standard working solution, internal standard working solution and standard working solution.
Further preferably, the standard working solution pretreatment is completed and then the standard curve is drawn.
Preferably, the sample pretreatment is a liquid-liquid extraction method, and the extractant in the liquid-liquid extraction method comprises at least one of methyl tertiary butyl ether, n-hexane, carboxylic ester, ethanol, n-butanol, diethyl ether, petroleum ether, isopropanol, chloroform, propylene glycol, aromatic hydrocarbon, benzene, ethane and cyclohexane.
Further preferably, the extractant in the liquid-liquid extraction method is carboxylate,
further preferably, the carboxylic acid ester is ethyl acetate.
Preferably, the extractantIs C n H 2n O 2 Wherein n is not less than 2.
Further preferably, the extractant is C 4 H 8 O 2 。
Preferably, the sample pretreatment includes plasma, and the volume ratio of the plasma to the extractant is 1: (8-12).
Further preferably, the sample pretreatment includes plasma, and the volume ratio of the plasma to the extractant is 1: (9-11).
Further preferably, the sample pretreatment includes plasma, and the volume ratio of the plasma to the extractant is 1:10.
preferably, the bonding group of the chromatographic column in the high performance liquid chromatography-mass spectrum is C18, the particle size is 1.8-5 mu m, and the aperture is 80-100A.
Further preferably, the binding group of the chromatographic column in the high performance liquid chromatography-mass spectrometry is C18, the particle size is 1.8 μm,2.7 μm and 5 μm respectively, and the pore diameter is 90A.
Further preferably, the chromatographic column in the high performance liquid chromatography-mass spectrometry is Shimadzu Shim-pack Velox C18.1 x 100mm,2.7 μm.
Preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry are gradient elution program for 0-5min, and the mobile phase A: carboxylic acid compound and ammonium group compound aqueous solution, mobile phase B: carboxylic acid compound and ammonium compound in methanol.
Further preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry are gradient elution procedure for 0-4min, mobile phase A is water containing 0.1% formic acid and 5mM ammonium formate by volume fraction, mobile phase B is methanol containing 0.1% formic acid and 5mM ammonium formate by volume fraction.
Further preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry are gradient elution procedure for 0-3min.
Preferably, the proportion of the mobile phase A is 65-75% in 2.51-3.00min, and the proportion of the mobile phase B is 25-35% in 2.51-3.00 min.
It is further preferred that the proportion of mobile phase A is 65-70% in 2.51-3.00min and the proportion of mobile phase B is 30-35% in 2.51-3.00 min.
It is further preferred that the proportion of mobile phase A is 65% in time 2.51-3.00min and the proportion of mobile phase B is 35% in time 2.51-3.00 min.
Further preferably, the gradient elution condition schedule is as follows:
| time (min) | Phase A ratio (%) | Comparative example B (%) |
| 0.00 | 65 | 35 |
| 0.50 | 65 | 35 |
| 0.51 | 35 | 65 |
| 2.50 | 35 | 65 |
| 2.51 | 65 | 35 |
| 3.00 | 65 | 35 |
Preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry further comprise a column temperature of 45-55 ℃.
Further preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry further comprise a column temperature of 48-52 ℃.
Further preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry further comprise a column temperature of 50 ℃.
Preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry further comprise a sample injection amount of 1-3 mu L and a flow rate of 0.3-0.4mL/min.
Further preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry further comprise a sample injection amount of 1.2-2.5 mu L and a flow rate of 0.33-0.36mL/min.
Further preferably, the chromatographic conditions in the high performance liquid chromatography-mass spectrometry further comprise a sample injection amount of 2 mu L and a flow rate of 0.35mL/min.
The second aspect of the application provides a method for detecting the concentration of five drugs in blood plasma, and the method is applied to clinical medicine.
In the prior art, a protein precipitation method is generally adopted as a sample pretreatment step, acetonitrile and methanol used in the protein precipitation method have high toxicity and are not environment-friendly, and the extraction speed is high, but the protein precipitation method contains more impurity proteins, so that on one hand, the recovery rate, the precision and the accuracy can be negatively influenced, and on the other hand, in order to improve the separation degree in the subsequent high performance liquid chromatography, the analysis time can be correspondingly prolonged, while in the application, ethyl acetate is adopted as an extractant, the toxicity is low and the environment-friendly, but when the dosage of the ethyl acetate is increased, the polarity of the extractant is large, and a plurality of impurities in blood plasma are polar substances, and the impurities are extracted at the same time of extracting the medicine, so that the volume ratio of the blood plasma to the ethyl acetate is further limited to be 1: and (8-12), at the moment, the impurity peak is obviously reduced, most of impurity interference is removed, and the recovery rate is greatly improved.
Although the interference of most impurities in the plasma is removed by a liquid-liquid extraction method, the method still has the advantage that the tiny part of protein impurities can not be separated, so the gradient elution time is limited to be 2.51-3.00min, the proportion of mobile phases A and B is respectively 65-75% and 25-35%, and the retention time among different components is improved by changing the polarity of the mobile phases in the time period, so that the influence of the tiny part of protein impurities is minimized, and the accuracy and precision are greatly improved. Generally, the faster the flow speed is, the shorter the analysis time is, the poorer the chromatographic separation effect is, but because the application adopts ethyl acetate as an extractant, the purity of a sample is greatly improved, the application adopts chromatographic conditions of 0.33-0.36mL/min, the gradient elution program is 0-3min, the column temperature is 45-55 ℃, on one hand, the better peak shape can be obtained, the separation degree is greatly improved, the separation with matrix impurities is facilitated, the matrix effect is reduced, the sensitivity and the reproducibility can be improved, on the other hand, the flow speed is matched with the flow speed born by mass spectrum, the ionization efficiency is increased, the detection precision is improved, and the chromatographic separation effect is still better under the conditions of shorter analysis time and faster flow speed.
The beneficial effects are that: the application establishes a method for detecting 6- (2, 3-dichlorophenyl) -1,2, 4-triazine-3, 5-diamine, chloromethyl oxazolone, oxcarbazepine metabolite, 5-diphenyl hydantoin and aripiprazole by optimizing sample pretreatment method and high performance liquid chromatography-mass spectrometry condition, wherein the pretreatment method adopts ethyl acetate for liquid phase extraction, solves the problems of environmental protection and most of impurities retention caused by using a protein precipitation method in the prior art, and can effectively remove the impurities, reduce matrix effect and improve recovery rate while protecting environment.
2. The application adopts high performance liquid chromatography-mass spectrometry detection, can accurately and objectively detect the concentration of the medicine, and can be applied to the detection of therapeutic medicines for the medicine in clinical work.
3. The detection method of the application ensures the specificity and accuracy of the detection substances and greatly reduces the influence of the interfering substances.
Examples
Example 1
A method for detecting the concentration of five drugs, 6- (2, 3-dichlorophenyl) -1,2, 4-triazine-3, 5-diamine, clomazone, oxcarbazepine metabolite, 5-diphenylhydantoin, aripiprazole, and dehydroaripiprazole, in plasma, respectively, comprising the steps of: 1. preparing a working solution; 2. sample pretreatment; 3. and (5) analyzing by high performance liquid chromatography-mass spectrometry sample injection.
The preparation of the working solution comprises the following steps: standard stock solution, primary intermediate solution, standard mixed secondary intermediate solution, internal standard stock solution, internal standard intermediate solution, standard working solution, internal standard working solution and standard working solution.
The standard stock solution was chlormethazolone (solvent methanol) at a concentration of 2493 μg/mL, oxcarbazepine metabolite (solvent acetonitrile) at 9000 μg/mL, aripiprazole (solvent acetonitrile containing 0.1% formic acid) at 1745 μg/mL, 5-diphenylhydantoin (solvent methanol: water=1:1), 6- (2, 3-dichlorophenyl) -1,2, 4-triazine-3, 5-diamine 4404 μg/mL (solvent methanol: water=7:3), and dehydroaripiprazole (solvent methanol: water=7:3) at 1000 μg/mL.
The primary intermediate solution was 800. Mu.g/mL of aripiprazole and 200. Mu.g/mL of dehydroaripiprazole. The standard mixed secondary intermediate solution is 8 mug/mL of aripiprazole, 400 mug/mL of chloromethyl oxazolone, 400 mug/mL of oxcarbazepine metabolite, 200 mug/mL of 5, 5-diphenylhydantoin (the solvents are methanol and water with the mass ratio of 7:3), 160 mug/mL of 6- (2, 3-dichlorophenyl) -1,2, 4-triazine-3, 5-diamine (the solvents are methanol and water with the mass ratio of 7:3), and 8 mug/mL of dehydroaripiprazole.
The preparation method of the internal standard stock solution comprises the following steps:
d8-aripiprazole: the standard stock solution was 100. Mu.g/mL and transferred to a 2mL freeze tube immediately after opening the bottle.
D8-dehydroaripiprazole: standard 1.00mg was added directly to the flask and dissolved in 1mL of acetonitrile (0.1% formic acid) to give an internal standard stock solution at a concentration of 1000 μg/mL.
D4-oxcarbazepine metabolite: standard 1.00mg, 1mL acetonitrile was directly added to the flask, dissolved by shaking, and transferred to a 2mL freezing tube to obtain an internal standard stock solution with a concentration of 1000 μg/mL.
D10-5, 5-diphenylhydantoin: the standard is 1.00mg, 1mL of diluent is directly added into a bottle, the diluent is dissolved by shaking, and the solution is transferred into a 2mL freezing tube, so that an internal standard stock solution with the concentration of 1000 mug/mL is obtained.
D3-carbamazepine: the standard is 1.00mg, 1mL of diluent is directly added into a bottle, the diluent is dissolved by shaking, and the solution is transferred into a 2mL freezing tube, so that an internal standard stock solution with the concentration of 1000 mug/mL is obtained.
The preparation method of the internal standard intermediate liquid of the aripiprazole 5, 5-diphenyl hydantoin comprises the following steps:
d8-aripiprazole: mother liquor (100. Mu.g/mL) 100. Mu.L was taken in a 1.5mL centrifuge tube and 900. Mu.L methanol was added: water=7:3 gives an intermediate with a concentration of 10 μg/mL.
D8-dehydroaripiprazole: 10. Mu.L of the mother liquor (1000. Mu.g/mL) was taken in a 1.5mL centrifuge tube and 990. Mu.L of methanol was added: water=7:3 gives an intermediate with a concentration of 10 μg/mL.
D4-oxcarbazepine metabolite: 500. Mu.L of mother liquor (1000. Mu.g/mL) was taken in a 1.5mL centrifuge tube and 500. Mu.L of methanol was added: water=7:3 gives an intermediate with a concentration of 500 μg/mL.
D3-carbamazepine: 200. Mu.L of the mother liquor (1000. Mu.g/mL) was taken in a 1.5mL centrifuge tube and 800. Mu.L of methanol was added: water=7:3 gives an intermediate with a concentration of 200 μg/mL. Aripiprazole 5, 5-diphenylhydantoin
The standard working fluid is prepared as shown in table 1 below: accurately sucking a certain volume of the standard mixed secondary intermediate liquid, and respectively preparing 8 gradient standard working liquids by using a methanol aqueous solution with a volume ratio of 7:3 as a diluent.
Table 1:8 gradients of standard working fluid.
The diluent is methanol and water with a mass ratio of 7:3.
| Mark curved point | Standard curve point dilution process | Total volume of mu L |
| L8 | 150. Mu.L of standard mixed intermediate+850. Mu.L of diluent | 1000 |
| L7 | 150 mu L L8+75 mu L of diluent | 225 |
| L6 | 100 mu L L8+200 mu L of dilution | 300 |
| L5 | 100 mu L L6+100 mu L of dilution | 200 |
| L4 | 100 mu L L5+100 mu L of dilution | 200 |
| L3 | 100 mu L L4+150 mu L of dilution | 250 |
| L2 | 100 mu LL3+100 mu L dilution | 200 |
| L1 | 100ulL2+100 dilution | 200 |
Wherein the concentration of each substance in the standard working solution is shown in table 2:
TABLE 2
The preparation method of the internal standard working solution comprises the following steps: a volume of the internal standard intermediate was added and then diluted to 1mL with a diluent, wherein the concentration of aripiprazole-D8 was 200ng/mL, the concentration of oxcarbazepine metabolite-D4 was 10000ng/mL, the concentration of 5, 5-diphenylhydantoin-D10 was 5000ng/mL, and the concentration of carbamazepine-D3 was 4000ng/mL. The concentration of dehydroepizine-D8 was 200ng/mL.
The standard working solution pretreatment comprises the following steps: mixing 10 mu L of internal standard working solution and 20 mu L of standard working solution, adding the mixture into a centrifuge tube, respectively numbering the mixture into L1-L8, adding 100 mu L of methanol into a blank centrifuge tube, adding 130 mu L of methanol into the blank centrifuge tube, uniformly mixing by vortex, centrifuging, preserving, taking 9 1.5mL centrifuge tubes respectively numbering L8-L1 and blank, sucking 50 mu L of supernatant fluid after centrifuging into the corresponding centrifuge tube, respectively adding 150 mu L of water into the centrifuge tube, uniformly mixing by vortex, respectively sucking 150 mu L of samples of the centrifuge tubes respectively, transferring the samples into the corresponding sample feeding plate, and carrying out instrument analysis, wherein the sample feeding amount is 2 mu L, thus obtaining standard curves of five medicines.
The five drug concentrations for the recovery test are as follows:
wherein QC-H plasma samples are prepared by: after 480 μl of the mixed secondary medium and 3250 μl of blank plasma were mixed, the volume was set to 3730 μl, and QC-M plasma samples were prepared: after mixing 400. Mu.L QC-H plasma sample and 3600. Mu.L blank plasma, the volume was adjusted to 4000. Mu.L, and QC-L plasma sample was prepared: 1250. Mu.L QC-M plasma samples and 2750. Mu.L blank plasma were mixed and the volume was set to 4000. Mu.L.
Preparing QC-working solution: 900. Mu.L of the mixed secondary intermediate solution and 600. Mu.L of the diluent (methanol: water=7:3) were mixed to obtain a QC-H working solution, 150. Mu.L of the QC-H working solution and 1350. Mu.L of the diluent were mixed to obtain a QC-M working solution, and 450. Mu.L of the QC-M working solution and 990. Mu.L of the diluent were mixed to obtain a QC-L working solution.
The sample pretreatment steps are as follows: transferring 10 mu L of internal standard working solution into a 1.5mL centrifuge tube by using a pipette, adding 20 mu L of plasma, adding 200 mu L of ethyl acetate extractant, uniformly mixing by vortex at 2500r/min for 5min, centrifuging at 14000r/min for 10min at a high speed, transferring 150 mu L of supernatant into a 1.5mL plastic centrifuge tube, drying by nitrogen flow, adding 100 mu L of initial mobile phase, uniformly mixing by vortex at 2500r/min for 5min, and taking 90 mu L of sample.
The chromatographic conditions in the high performance liquid chromatography-mass spectrometry are as follows: the column was SHIMADZU Shim-pack Velox C18 (2.1 x 100mm,2.7 μm), column temperature 50 ℃, gradient elution procedure 3min, mobile phase a: water containing 0.1% formic acid and 5mM ammonium formate by volume fraction, mobile phase B was methanol containing 0.1% formic acid and 5mM ammonium formate by volume fraction, flow rate was 0.35mL/min, sample volume: 2. Mu.L.
The needle washing liquid is a methanol water solution with the volume fraction of 50 percent.
Table 3: gradient elution condition schedule.
| Time (min) | Phase A ratio (%) | Comparative example B (%) |
| 0.00 | 65 | 35 |
| 0.50 | 65 | 35 |
| 0.51 | 35 | 65 |
| 2.50 | 35 | 65 |
| 2.51 | 65 | 35 |
| 3.00 | 65 | 35 |
The mass spectrometry conditions are ion sources: an electrospray ion source;
scanning mode: monitoring positive ion multi-reaction; the mass spectrum parameters are: the ionization temperature is 500 ℃, the ionization voltage is 5500V, the air curtain gas is 35L/min, the collision gas is 7L/min, the spray gas is 50L/min, and the auxiliary heating gas is 50L/min.
The specific scan parameters for each ion pair are shown in table 4 below:
TABLE 4 Table 4
Note that: 1. because of the large variety of substances, the collection mode is segmented collection. The 6- (2, 3-dichlorophenyl) -1,2, 4-triazine-3, 5-diamine is internally marked by CBZ-D3, the chloromethyl oxazolone is internally marked by CBZ-D3, the oxcarbazepine metabolite is internally marked by MHD-D4, the 5, 5-diphenylhydantoin is internally marked by PhT-D10, the aripiprazole is internally marked by ARPZ-D8, and the dehydroaripiprazole is internally marked by DHARPZ-D8.
Comparative example 1
The sample pretreatment steps are as follows: transferring 10 mu L of internal standard working solution into a 1.5mL centrifuge tube by using a pipette, adding 30 mu L of plasma, adding 400 mu L of ethyl acetate extractant, uniformly mixing by vortex at 2500r/min for 5min, centrifuging at 14000r/min for 10min at a high speed, transferring 150 mu L of supernatant into a 1.5mL plastic centrifuge tube, drying by nitrogen flow, adding 150 mu L of initial mobile phase, uniformly mixing by vortex at 2500r/min for 5min, and taking 90 mu L of sample. The procedure is as in example 1.
Comparative example 2
The chromatographic conditions in the high performance liquid chromatography-mass spectrometry are as follows: the column was SHIMADZU Shim-pack Velox C18 (2.1 x 100mm,2.7 μm), column temperature 50 ℃, gradient elution procedure 3min, mobile phase a: water containing 0.1% formic acid and 5mM ammonium formate by volume fraction, mobile phase B was methanol containing 0.1% formic acid and 5mM ammonium formate by volume fraction, flow rate was 0.30mL/min, sample volume: 2. Mu.L, analysis time 3.5min, the rest of example 1.
Comparative example 3
The sample pretreatment steps are as follows: transfer 10 μl of internal standard working solution into 2.0mL centrifuge tube with a pipette, then add 20 μl of plasma, and add 200 μl of the solution with a mass ratio of 1:1 and n-hexane extractant, mixing uniformly by vortex at 2000r/min for 5min, centrifuging at 14000r/min for 10min at high speed, transferring 150 mu L of supernatant to a 1.5mL plastic centrifuge tube, blow-drying under nitrogen flow, adding 150 mu L of initial mobile phase, mixing uniformly by vortex at 2500r/min for 5min, and taking 90 mu L of sample. The procedure is as in example 1.
Comparative example 4
The procedure of example 1 was repeated except that the ratio of mobile phase A was changed to 10% in the period of 2.51-3.00min, and the ratio of mobile phase B was changed to 90% in the period of 2.51-3.00 min.
Comparative example 5
The procedure of example 1 was repeated except that the flow rate in the HPLC-MS conditions was changed to 0.50 mL/min.
Evaluation of Performance
1. The experimental data obtained in example 1 are as follows:
(1) Precision (CV), normal level: sample repeatability precision, laboratory precision.
| Name of the name | Sample repeatability precision | Precision in laboratory |
| LMSQ | 2.24 | 2.28 |
| LMZT | 2.10 | 3.27 |
| MHD metabolites | 1.77 | 2.21 |
| PHT | 2.06 | 2.54 |
| ARPZ | 3.51 | 2.98 |
| DHARPZ | 3.72 | 3.45 |
(2) Linear range:
(3) The limit of detection and the limit of quantification of plasma samples are shown in the following table.
(4) And (3) recovery rate detection: (1) Transferring 10 μl of internal standard working solution, 4 μl of QC working solution and 16 μl of blank plasma into 1.5mL centrifuge tube with a pipette, adding 200 μl of ethyl acetate extractant, and adding 2500% of the solution
Mixing r/min by vortex for 5min, centrifuging for 10min under 12000r/min, transferring 150 μl of supernatant to a 1.5mL plastic centrifuge tube, blow-drying with N2, adding 100 μl of mobile phase for dissolution, mixing 2500r/min by vortex for 5min, taking 90 μl of sample to obtain peak area A; (2) First, 10. Mu.L of an internal standard working solution and 4. Mu.L of QC standard solution were mixed in a 1.5mL centrifuge tube at 2500
Mixing with r/min vortex for 2min, blow drying with nitrogen to obtain first solution, mixing 20 μl blank plasma and 10 μl diluent in 1.5mL centrifuge tube, mixing with 2500r/min vortex for 2min, adding 200 μl ethyl acetate extractant, mixing with 2500r/min vortex for 5min, centrifuging at 12000r/min for 10min, collecting 150 μl supernatant in the first solution, mixing with 2500r/min vortex for 2min
Drying with nitrogen, adding 100 μl of mobile phase, dissolving again, and mixing at 2500r/min under vortex for 5 times
After min, 90 μl was sampled to obtain peak area B.
The calculation formula of the recovery rate is as follows: recovery (%) =a/b×100%.
The recovery rate test results are shown in the following table:
2. recovery rate detection and precision test were performed on comparative examples 1 to 5, and the test results are shown in the following table.
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Compared with example 1, the ethyl acetate in comparative example 1 has a larger amount, so that the polarity is stronger, the impurities are easily extracted, the recovery rate is greatly affected, the flow rate in comparative example 2 is slower, the analysis time is longer, the matrix effect is increased, the precision is greatly affected, the type of extractant is changed in comparative example 3, the polarity of the extractant is reduced because n-hexane is a nonpolar solvent, a large amount of impurities remain, the recovery rate is reduced, the change of the fluid phase in a specific time period is changed in comparative example 4, the separation degree is reduced because of the polarity change, the precision is greatly affected, the flow rate in comparative example 5 is faster, the separation effect is reduced, the precision is affected, and the loss of the chromatographic column is also accelerated.
Claims (10)
1. A method for detecting the concentration of five drugs in plasma, wherein the five drugs are 6- (2, 3-dichlorophenyl) -1,2, 4-triazine-3, 5-diamine, clomazone, oxcarbazepine metabolite, 5-diphenylhydantoin, aripiprazole, and dehydroaripiprazole, respectively, the method comprising the steps of: 1. preparing a working solution; 2. sample pretreatment; 3. and (5) analyzing by high performance liquid chromatography-mass spectrometry sample injection.
2. The method according to claim 1, wherein the pretreatment of the sample is a liquid-liquid extraction method, and the extractant in the liquid-liquid extraction method comprises at least one of methyl tert-butyl ether, n-hexane, carboxylic ester, ethanol, n-butanol, diethyl ether, petroleum ether, isopropanol, chloroform, propylene glycol, aromatic hydrocarbon, benzene, ethane, and cyclohexane.
3. The method for detecting the concentration of five drugs in blood plasma according to claim 1, wherein the extractant is C n H 2n O 2 Wherein n is not less than 2.
4. The method for detecting the concentration of five drugs in the blood plasma according to claim 2, wherein the pretreatment of the sample comprises the blood plasma, and the volume ratio of the blood plasma to the extractant is 1: (8-12).
5. The method for detecting the concentration of five drugs in plasma according to claim 1, wherein the binding group of the chromatographic column in the high performance liquid chromatography-mass spectrometry is C18, the particle size is 1.8-5 μm, and the pore diameter is 80-100A.
6. The method for detecting the concentrations of five drugs in blood plasma according to claim 1, wherein the chromatographic conditions in the high performance liquid chromatography-mass spectrometry are gradient elution procedures for 0-5min,
mobile phase a: carboxylic acid compound and ammonium group compound aqueous solution, mobile phase B: carboxylic acid compound and ammonium compound in methanol.
7. The method according to claim 6, wherein the ratio of mobile phase A is 65-75% in 2.51-3.00min and the ratio of mobile phase B is 25-35% in 2.51-3.00 min.
8. The method according to claim 6, wherein the chromatographic conditions in the high performance liquid chromatography-mass spectrometry further comprise a column temperature of 45-55 ℃.
9. The method according to claim 6, wherein the chromatographic conditions in the high performance liquid chromatography-mass spectrometry further comprise a sample injection amount of 1-3 μl and a flow rate of 0.3-0.4mL/min.
10. Use of a method according to any one of claims 1-9 for detecting the concentration of five drugs in plasma in the clinical medical treatment of the claims.
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