CN117825592A - Quality control product, kit and method for detecting concentration of antiepileptic drugs in human blood matrix - Google Patents
Quality control product, kit and method for detecting concentration of antiepileptic drugs in human blood matrix Download PDFInfo
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- CN117825592A CN117825592A CN202410250699.0A CN202410250699A CN117825592A CN 117825592 A CN117825592 A CN 117825592A CN 202410250699 A CN202410250699 A CN 202410250699A CN 117825592 A CN117825592 A CN 117825592A
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- quality control
- concentration
- calibrator
- kit
- control product
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Links
- 238000003908 quality control method Methods 0.000 title claims abstract description 88
- 239000001961 anticonvulsive agent Substances 0.000 title claims abstract description 59
- 210000004369 blood Anatomy 0.000 title claims abstract description 58
- 239000008280 blood Substances 0.000 title claims abstract description 58
- 239000011159 matrix material Substances 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 14
- NIJJYAXOARWZEE-UHFFFAOYSA-N Valproic acid Chemical compound CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 claims abstract description 53
- 238000001514 detection method Methods 0.000 claims abstract description 39
- 229960003965 antiepileptics Drugs 0.000 claims abstract description 35
- 241001465754 Metazoa Species 0.000 claims abstract description 33
- CTRLABGOLIVAIY-UHFFFAOYSA-N oxcarbazepine Chemical compound C1C(=O)C2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 CTRLABGOLIVAIY-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229960001816 oxcarbazepine Drugs 0.000 claims abstract description 30
- 229960000604 valproic acid Drugs 0.000 claims abstract description 26
- KJADKKWYZYXHBB-XBWDGYHZSA-N Topiramic acid Chemical compound C1O[C@@]2(COS(N)(=O)=O)OC(C)(C)O[C@H]2[C@@H]2OC(C)(C)O[C@@H]21 KJADKKWYZYXHBB-XBWDGYHZSA-N 0.000 claims abstract description 25
- 229960004394 topiramate Drugs 0.000 claims abstract description 25
- PYZRQGJRPPTADH-UHFFFAOYSA-N lamotrigine Chemical compound NC1=NC(N)=NN=C1C1=CC=CC(Cl)=C1Cl PYZRQGJRPPTADH-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229960001848 lamotrigine Drugs 0.000 claims abstract description 23
- 229960004002 levetiracetam Drugs 0.000 claims abstract description 23
- HPHUVLMMVZITSG-ZCFIWIBFSA-N levetiracetam Chemical compound CC[C@H](C(N)=O)N1CCCC1=O HPHUVLMMVZITSG-ZCFIWIBFSA-N 0.000 claims abstract description 23
- DDBREPKUVSBGFI-UHFFFAOYSA-N phenobarbital Chemical compound C=1C=CC=CC=1C1(CC)C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229960002695 phenobarbital Drugs 0.000 claims abstract description 22
- ZAFYATHCZYHLPB-UHFFFAOYSA-N zolpidem Chemical compound N1=C2C=CC(C)=CN2C(CC(=O)N(C)C)=C1C1=CC=C(C)C=C1 ZAFYATHCZYHLPB-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229960001475 zolpidem Drugs 0.000 claims abstract description 22
- 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 claims abstract description 21
- 229960002036 phenytoin Drugs 0.000 claims abstract description 21
- DGBIGWXXNGSACT-UHFFFAOYSA-N clonazepam Chemical compound C12=CC([N+](=O)[O-])=CC=C2NC(=O)CN=C1C1=CC=CC=C1Cl DGBIGWXXNGSACT-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229960003120 clonazepam Drugs 0.000 claims abstract description 20
- ZRWWEEVEIOGMMT-UHFFFAOYSA-N carbamazepine-10,11-epoxide Chemical compound NC(=O)N1C2=CC=CC=C2C2OC2C2=CC=CC=C12 ZRWWEEVEIOGMMT-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000002360 preparation method Methods 0.000 claims abstract description 17
- BMPDWHIDQYTSHX-UHFFFAOYSA-N licarbazepine Chemical compound C1C(O)C2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 BMPDWHIDQYTSHX-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 10
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 10
- 239000012535 impurity Substances 0.000 claims abstract description 10
- 239000000546 pharmaceutical excipient Substances 0.000 claims abstract description 10
- 239000003755 preservative agent Substances 0.000 claims abstract description 10
- 230000002335 preservative effect Effects 0.000 claims abstract description 10
- 239000003223 protective agent Substances 0.000 claims abstract description 10
- 239000000047 product Substances 0.000 claims description 65
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- 239000000523 sample Substances 0.000 claims description 27
- 238000001179 sorption measurement Methods 0.000 claims description 22
- 239000006228 supernatant Substances 0.000 claims description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 15
- 239000011347 resin Substances 0.000 claims description 14
- 229920005989 resin Polymers 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 10
- FFGPTBGBLSHEPO-LHNTUAQVSA-N 1,2,3,4,5,6,7,8,9,10-decadeuteriobenzo[b][1]benzazepine-11-carboxamide Chemical compound [2H]C1=C([2H])C2=C([2H])C([2H])=C([2H])C([2H])=C2N(C(N)=O)C2=C([2H])C([2H])=C([2H])C([2H])=C21 FFGPTBGBLSHEPO-LHNTUAQVSA-N 0.000 claims description 9
- 235000006708 antioxidants Nutrition 0.000 claims description 9
- 239000011265 semifinished product Substances 0.000 claims description 9
- CXOFVDLJLONNDW-LHNTUAQVSA-N 5,5-bis(2,3,4,5,6-pentadeuteriophenyl)imidazolidine-2,4-dione Chemical compound [2H]C1=C([2H])C([2H])=C([2H])C([2H])=C1C1(C=2C(=C([2H])C([2H])=C([2H])C=2[2H])[2H])C(=O)NC(=O)N1 CXOFVDLJLONNDW-LHNTUAQVSA-N 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- CTRLABGOLIVAIY-DNZPNURCSA-N 1,2,3,4-tetradeuterio-6-oxo-5h-benzo[b][1]benzazepine-11-carboxamide Chemical compound O=C1CC2=C([2H])C([2H])=C([2H])C([2H])=C2N(C(N)=O)C2=CC=CC=C21 CTRLABGOLIVAIY-DNZPNURCSA-N 0.000 claims description 7
- NIJJYAXOARWZEE-WFGJKAKNSA-N 5,5,5-trideuterio-2-(3,3,3-trideuteriopropyl)pentanoic acid Chemical compound [2H]C([2H])([2H])CCC(C(O)=O)CCC([2H])([2H])[2H] NIJJYAXOARWZEE-WFGJKAKNSA-N 0.000 claims description 7
- KJADKKWYZYXHBB-RFYKXYJISA-N [(3as,5ar,8ar,8bs)-2,2,7,7-tetrakis(trideuteriomethyl)-5,5a,8a,8b-tetrahydrodi[1,3]dioxolo[4,5-a:5',4'-d]pyran-3a-yl]methyl sulfamate Chemical compound C1O[C@@]2(COS(N)(=O)=O)OC(C([2H])([2H])[2H])(C([2H])([2H])[2H])O[C@H]2[C@@H]2OC(C([2H])([2H])[2H])(C([2H])([2H])[2H])O[C@@H]21 KJADKKWYZYXHBB-RFYKXYJISA-N 0.000 claims description 7
- 230000010355 oscillation Effects 0.000 claims description 7
- PYZRQGJRPPTADH-ULEDQSHZSA-N 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine Chemical compound N[13C]1=N[13C](N)=NN=[13C]1C1=CC=CC(Cl)=C1Cl PYZRQGJRPPTADH-ULEDQSHZSA-N 0.000 claims description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 229940050561 matrix product Drugs 0.000 claims description 6
- HPHUVLMMVZITSG-QXMLZOKMSA-N (2s)-2-(2,2,3,3,4,4-hexadeuterio-5-oxopyrrolidin-1-yl)butanamide Chemical compound [2H]C1([2H])N([C@@H](CC)C(N)=O)C(=O)C([2H])([2H])C1([2H])[2H] HPHUVLMMVZITSG-QXMLZOKMSA-N 0.000 claims description 5
- DGBIGWXXNGSACT-RHQRLBAQSA-N 5-(2-chloro-3,4,5,6-tetradeuteriophenyl)-7-nitro-1,3-dihydro-1,4-benzodiazepin-2-one Chemical compound ClC1=C([2H])C([2H])=C([2H])C([2H])=C1C1=NCC(=O)NC2=CC=C([N+]([O-])=O)C=C12 DGBIGWXXNGSACT-RHQRLBAQSA-N 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- -1 zolpidem-d 7 Chemical compound 0.000 claims description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- DDBREPKUVSBGFI-ZBJDZAJPSA-N Phenobarbital-d5 Chemical compound C=1C=CC=CC=1C1(C([2H])([2H])C([2H])([2H])[2H])C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-ZBJDZAJPSA-N 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 101100382321 Caenorhabditis elegans cal-1 gene Proteins 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 230000002829 reductive effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- 229930195725 Mannitol Natural products 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 2
- 235000010323 ascorbic acid Nutrition 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 239000011668 ascorbic acid Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000000594 mannitol Substances 0.000 claims description 2
- 235000010355 mannitol Nutrition 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 4
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- FFGPTBGBLSHEPO-UHFFFAOYSA-N carbamazepine Chemical compound C1=CC2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 FFGPTBGBLSHEPO-UHFFFAOYSA-N 0.000 description 9
- 229960000623 carbamazepine Drugs 0.000 description 9
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 8
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
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- 210000002381 plasma Anatomy 0.000 description 5
- 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 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- 206010010904 Convulsion Diseases 0.000 description 3
- 230000001133 acceleration Effects 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- UGJMXCAKCUNAIE-UHFFFAOYSA-N Gabapentin Chemical compound OC(=O)CC1(CN)CCCCC1 UGJMXCAKCUNAIE-UHFFFAOYSA-N 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 206010015037 epilepsy Diseases 0.000 description 2
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 229960002790 phenytoin sodium Drugs 0.000 description 2
- 239000012716 precipitator Substances 0.000 description 2
- FJPYVLNWWICYDW-UHFFFAOYSA-M sodium;5,5-diphenylimidazolidin-1-ide-2,4-dione Chemical compound [Na+].O=C1[N-]C(=O)NC1(C=1C=CC=CC=1)C1=CC=CC=C1 FJPYVLNWWICYDW-UHFFFAOYSA-M 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- MTJGVAJYTOXFJH-UHFFFAOYSA-N 3-aminonaphthalene-1,5-disulfonic acid Chemical compound C1=CC=C(S(O)(=O)=O)C2=CC(N)=CC(S(O)(=O)=O)=C21 MTJGVAJYTOXFJH-UHFFFAOYSA-N 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
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- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229960002870 gabapentin Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229960002623 lacosamide Drugs 0.000 description 1
- VPPJLAIAVCUEMN-GFCCVEGCSA-N lacosamide Chemical compound COC[C@@H](NC(C)=O)C(=O)NCC1=CC=CC=C1 VPPJLAIAVCUEMN-GFCCVEGCSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 229960001233 pregabalin Drugs 0.000 description 1
- AYXYPKUFHZROOJ-ZETCQYMHSA-N pregabalin Chemical compound CC(C)C[C@H](CN)CC(O)=O AYXYPKUFHZROOJ-ZETCQYMHSA-N 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- AEQFSUDEHCCHBT-UHFFFAOYSA-M sodium valproate Chemical compound [Na+].CCCC(C([O-])=O)CCC AEQFSUDEHCCHBT-UHFFFAOYSA-M 0.000 description 1
- 229940084026 sodium valproate Drugs 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000003643 water by type Substances 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
- 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
-
- 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
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/86—Signal analysis
- G01N30/8665—Signal analysis for calibrating the measuring apparatus
-
- 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Landscapes
- 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)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a quality control product, a kit and a method for detecting the concentration of an antiepileptic drug in human blood matrix, and belongs to the technical field of drug detection. The kit comprises a calibrator, a quality control product, an internal standard and a precipitant, wherein the calibrator and the quality control product are freeze-dried preparations; the calibrator and the quality control product are prepared by an animal blood matrix which is filtered and adsorbed to remove interference impurities, and a protective agent, an excipient, a preservative and an antioxidant are added into the animal blood matrix. The calibrator and quality control product of the kit disclosed by the invention are high in stability, and the detection method disclosed by the invention can be used for simultaneously detecting valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam, and is good in accuracy and precision, strong in specificity and high in stability, and is suitable for antiepileptic drug monitoring.
Description
Technical Field
The invention belongs to the technical field of drug detection, and relates to a quality control product, a kit and a method for detecting the concentration of an antiepileptic drug in human blood matrix, in particular to a quality control product, a kit and a method for detecting the concentration of the antiepileptic drug in human blood matrix by adopting liquid chromatography-tandem mass spectrometry.
Background
Epilepsy is a common, complex, chronic neurological disorder with a prevalence of 0.5-1% and is characterized by recurrent attacks. The clinical manifestations of seizures are partial or systemic tics and disturbance of consciousness, and the frequency and rhythm of seizures are largely unpredictable. Accordingly, seizures are not only a considerable psychological and physiological burden for the patient, but can also lead to serious and even life-threatening injuries (e.g., craniocerebral injury upon a fall). Clinical epilepsy is usually treated by single administration or combined administration, and traditional antiepileptic drugs such as phenytoin sodium, carbamazepine and sodium valproate have narrow treatment range, strong toxicity and easy occurrence of adverse reaction, and need to monitor blood concentration periodically. With the improvement of the medical level, a plurality of novel antiepileptic drugs such as lamotrigine, oxcarbazepine, topiramate, levetiracetam and the like are appeared, and compared with the traditional antiepileptic drugs, the antiepileptic drugs have better treatment effect and low toxic and side effects, but need to be taken for a long time. After patients take antiepileptic drugs, there is a large individual difference in pharmacokinetics, and even if the same dose of drugs is used by the same route, different therapeutic responses occur. Therefore, in order to ensure the safety and effectiveness of patient administration, the therapeutic drug dosage of an individual patient must be determined, then timely and accurate therapeutic drug monitoring needs to be performed in clinical treatment, the drug concentration in the patient is periodically detected, and an optimal personalized dosing regimen is established.
The concentration of the antiepileptic drugs in the human blood matrix is detected, and the method has important significance for evaluating the antiepileptic curative effect and avoiding adverse reactions. The clinical detection method of the antiepileptic drug mainly comprises chromatography, and common chromatography comprises High Performance Liquid Chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS) and the like. The Chinese application with publication number of CN112946101A discloses a method for simultaneously measuring the contents of various antiepileptic drugs in blood and application thereof, wherein a protein precipitation method is adopted to combine with HPLC-MS/MS to measure the contents of 9 antiepileptic drugs of carbamazepine, 10, 11-dihydro-10-hydroxycarbazepine, oxcarbazepine, levetiracetam, topiramate, valproic acid, phenytoin, phenobarbital and lamotrigine in blood plasma.
The Chinese application publication No. CN116500158A discloses a blood sample antiepileptic drug detection method and a kit, wherein the antiepileptic drug detected by the detection method comprises carbamazepine, epoxy carbamazepine, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin sodium, lamotrigine, levetiracetam, pregabalin, gabapentin, topiramate, valproic acid, phenobarbital and lacosamide. The detection method comprises the steps of detecting a sample to be detected after pretreatment by using high performance liquid chromatography-tandem mass spectrometry, bringing a detection value into a standard curve, and calculating to obtain the content of antiepileptic drugs in a blood sample; the pretreatment comprises the step of treating a sample to be detected by adopting a precipitator, wherein the precipitator contains an internal standard which is an isotope of an antiepileptic drug.
In recent years, the clinical examination industry is increasingly focusing on the control of quality of detection. The most effective quality control means is to establish a perfect indoor quality control program and participate in the indoor interstitial assessment activity. Since 1982, china began the national inter-clinical laboratory quality evaluation activities, and the quality of the test results was improved to a certain extent. However, the indoor quality control link depends more on the quality control product applied by the hospital, and a reasonable quality control program is established to monitor the daily detection quality. Meanwhile, reasonable and effective indoor quality control is also a necessary guarantee for obtaining good results in indoor interstitial assessment.
The quality control product is used as a carrier for indoor quality control, and has a critical influence on the establishment and effect of an indoor quality control program. Development of quality control products matched with a detection system and anti-epileptic drug substance control products for monitoring system precision and accuracy become the current problems to be solved urgently.
The existing antiepileptic drug concentration detection kit has the defects that the substrate of a calibrator and a quality control product is generally human whole blood, serum or plasma, the source is limited, and the antiepileptic drug concentration detection kit cannot be purchased and sold commercially, so that the whole blood, serum or plasma of animals is required to be used as a substitute substrate. However, interfering substances may be present in the alternative matrix of animal origin, affecting the stability of the kit. The existing antiepileptic drug detection method cannot realize simultaneous detection of valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbamopine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam; in particular, oxcarbazepine is the same as carbamazepine-10, 11-epoxide structural analogues and ion pairs, and the existing detection method can not realize simultaneous detection. The development of a new kit and a detection method are needed to solve the technical problems and better meet the clinical requirements of detecting the concentration of the antiepileptic drugs in human blood matrix.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a kit and a method for detecting the concentration of an antiepileptic drug in human blood matrix, and the stability of the kit is improved by treating animal whole blood, serum or plasma, reducing matrix effect and removing impurity interference; through improving test parameters, oxcarbazepine and carbamazepine 10, 11-epoxide are separated, 10 antiepileptic drugs and 2 active metabolites have good linear relation in respective linear ranges, good accuracy and precision, strong specificity and high stability, meet the analysis requirements of biological samples, meet the detection requirements of common antiepileptic drugs in human blood matrixes, and are suitable for antiepileptic drug monitoring. The aim of the invention is achieved by the following specific technical scheme.
The primary aspect of the invention is to provide a quality control for detecting the concentration of antiepileptic drugs in human blood matrix, including valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam; the quality control product is a freeze-dried preparation, and is prepared by an animal blood matrix which is subjected to filtration and adsorption treatment to remove interference impurities, and the antiepileptic drug, the protective agent, the excipient, the preservative and the antioxidant are added into the animal blood matrix.
Further, the quality control products are Qc-1, qc-2 and Qc-3; the working concentrations of the antiepileptic drugs in each quality control product are shown in table 1.
TABLE 1 working concentration of each substance in each quality control product
Further, the animal blood matrix is rabbit serum or plasma.
Further, the protective agent is one or two of sucrose and trehalose, and the concentration is 0.5-1.5wt% and 1.0-3.0wt% respectively; the excipient is one or two of mannitol and glycine, and the concentration is 1.0-3.0wt% and 1.0-3.0wt% respectively; the preservative is Proclin300, and the concentration is 0.05-0.3wt%; the antioxidant is ascorbic acid with concentration of 0.25-0.75wt%.
The invention also provides a kit for detecting the concentration of an antiepileptic drug in human blood matrix, which comprises a calibrator, the quality control product, an internal standard and a precipitant, wherein the calibrator is a freeze-dried preparation; the calibrator is prepared from an animal blood matrix which is subjected to filtration and adsorption treatment to remove interference impurities, and the antiepileptic drug, the protective agent, the excipient, the preservative and the antioxidant are added into the animal blood matrix. The calibrator and quality control product in the kit can be stably stored for 30 days at 37 ℃ under the heat acceleration stability; and after the calibrator and quality control product prepared by using the matrix without removing impurities are stored for 30 days at 37 ℃ in a heat acceleration stability mode, valproic acid, oxcarbazepine, topiramate and zolpidem have trend changes.
Further, the internal standard is isotope-labeled antiepileptic drugs, which are respectively mixed solutions of carbamazepine-d 10, carbamazepine-10, 11-epoxide-d 10, oxcarbazepine-d 4, 10, 11-dihydro-10-hydroxycarbazepine-d 4, lamotrigine-13C 3, levetiracetam-d 6, zolpidem-d 7, clonazepam-d 4, valproic acid-d 6, phenytoin-d 10, topiramate-d 12 and phenbarbital-d 5, wherein the concentration of the carbamazepine-d 10 is 1.0 mug/mL, the concentration of the 11-epoxide-d 10 is 1.0 mug/mL, the concentration of the oxcarbazepine-d 4 is 1.0 mug/mL, the concentration of the 10, the concentration of the 11-dihydro-10-hydroxycarbazepine-d 4 is 1.0 mug/mL, the concentration of the lamotrigine-13C 3 is 1.0 mug/mL, the concentration of the valproic acid-d 6, the concentration of the topiramate-d 12 and the concentration of the phenytoin-d 5, the concentration of the carbamazepine-d 10 is 1.0 mug/mL, the concentration of the 11-epoxide-d 10 is 1.0 mug/mL, the concentration of the oxepin-d 4 is 1.0 mug/mL, the concentration of the other than the two.
Further, the precipitating agent is methanol.
Further, the calibrator is Cal-1, cal-2, cal-3, cal-4, cal-5 and Cal-6; the working concentrations of antiepileptic drugs in each calibrator are shown in table 1.
Table 2 working concentrations of antiepileptic drugs in each calibrator
In another aspect, the present invention provides a method for preparing the kit, which comprises the following steps:
s1, preparing an animal blood matrix, which specifically comprises the following steps:
s1-1, taking animal blood matrix raw materials, and filtering by using a microporous filter membrane;
s1-2, adding active carbon into the filtered animal blood matrix, wherein the addition amount of the active carbon is 10-50wt% of the blood matrix, adsorbing, oscillating, centrifuging, and taking supernatant;
s1-3, adding an adsorption resin into the supernatant fluid after the adsorption of the activated carbon, wherein the addition amount of the adsorption resin is 10-50wt% of the supernatant fluid after the adsorption of the activated carbon, centrifuging after the adsorption and oscillation, and taking the supernatant fluid; the adsorption resin can be selected from one of XAD-4 macroporous resin, crosslinking resin, 110 resin and other adsorption resins;
s1-4 repeating the steps S1-2 and S1-3 until the impurity content in the animal blood matrix is reduced to below 100 ppm;
s1-5, adding a protective agent, an excipient, a preservative and an antioxidant to obtain an animal blood matrix product;
s2, preparing a calibrator and a quality control product semi-finished product: adding valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam into the animal blood matrix product obtained in the step S1 according to the concentration requirements of different levels of the calibrator and quality control product, uniformly stirring, and checking the concentrations of valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam to obtain a calibrator and quality control product semi-finished product after the calibration is qualified;
s3, preparing a calibrator and a quality control product finished product: subpackaging the calibrator and the quality control semi-finished product obtained in the step S2 according to packaging requirements, and freeze-drying to obtain a calibrator and a quality control finished product;
s4, preparing an internal standard: preparing a mixed solution of carbamazepine-d 10, carbamazepine-10, 11-epoxide-d 10, oxcarbazepine-d 4, 10, 11-dihydro-10-hydroxycarbazepine-d 4, lamotrigine-13C 3, levetiracetam-d 6, zolpidem-d 7, clonazepam-d 4, valproic acid-d 6, phenytoin-d 10, topiramate-d 12, phenobarbital-d 5 according to the internal standard concentration requirement;
s5, assembling a kit: and (3) assembling the calibrator and quality control product obtained in the step (S3), the internal standard prepared in the step (S4) and the precipitant to obtain a kit product.
In another aspect, the present invention provides a method for detecting the concentration of an antiepileptic drug in human blood matrix by using the kit, comprising the following steps:
s1 preparation of solution before detection:
a) Calibrator and quality control product: adding distilled water into a freeze-dried powder bottle containing a calibrator and a quality control product according to requirements at room temperature, and incubating for 20 to 30 minutes at room temperature; rotating the freeze-dried powder bottle to dissolve the content until the content is uniform;
b) Internal standard: taking out the internal standard in the kit, recovering to room temperature, and vortex oscillating before use;
c) Taking out the sample extract, standing at room temperature, and fully balancing for use;
s2, preparation of a detection sample:
a) Sample preparation: taking out the serum sample, recovering to room temperature for standby, and vortex oscillating before use;
b) Sample preparation: transferring the sample to a centrifuge tube by using a pipette, adding an internal standard and methanol, and uniformly mixing for 3-5 minutes by vortex oscillation; placing the centrifuge tube in a centrifuge, and separating supernatant after centrifugation;
s3, sample detection:
transferring the supernatant to a U-shaped plate, placing the plate in a sample injector, and detecting by using a liquid chromatograph-tandem mass spectrometer, wherein the detection parameters are as follows:
chromatographic conditions:
chromatographic column: c18 (100×2.1 mm,3.0 μm, 175 a), or other performance equivalents;
mobile phase A1: taking 998 mL pure water, 1.0mL formic acid and 1.0mL 2M ammonium acetate from 0.1% formic acid-2 mM ammonium acetate aqueous solution, shaking uniformly, and carrying out ultrasonic treatment for 5min;
mobile phase B1: taking 999 mL methanol plus 1.0mL formic acid from 0.1% formic acid methanol solution, shaking uniformly, and carrying out ultrasonic treatment for 5min;
mobile phase A2:2mM ammonium acetate aqueous solution, taking 999 mL pure water plus 1.0mL 2M ammonium acetate, shaking uniformly, and carrying out ultrasonic treatment for 5min;
mobile phase B2: methanol solution, 1000 mL methanol;
column temperature: 30-40 ℃;
flow rate: 0.3-0.4 mL/min;
gradient elution conditions are shown in tables 3 and 4:
TABLE 3 Positive ion mode elution conditions
TABLE 4 anion Pattern elution conditions
Mass spectrometry conditions are shown in tables 5 and 6:
TABLE 5 Source parameters
Table 6 mass spectral parameters
The oxcarbazepine and carbamazepine-10, 11-epoxide can be completely separated through the gradient elution under the chromatographic conditions, and the detection requirements of common antiepileptic drugs in human blood matrixes can be met through the mass spectrum conditions, so that the detection requirements of the common antiepileptic drugs in human blood matrixes can be met, and the method is suitable for antiepileptic drug monitoring.
Compared with the prior art, the invention has the following beneficial technical effects: the calibrator and quality control product in the kit provided by the invention can be stably stored for 30 days at 37 ℃ under the condition of heat acceleration, the uniformity is less than 10%, and the kit can be stored for a long time for use; the detection method can realize the complete separation of oxcarbazepine and carbamazepine-10, 11-epoxide, can realize the simultaneous detection of valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam, can meet the detection requirement of common antiepileptic drugs in human blood matrix, and is suitable for antiepileptic drug monitoring; the antiepileptic drug concentration quality control product assigned by the multi-detection system can be used for monitoring the precision and accuracy of the system.
Detailed Description
The technical scheme of the invention is clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
Example 1
Preparing a kit for detecting the concentration of an antiepileptic drug in human blood matrix, which comprises the following steps:
s1, preparing an animal blood matrix, which specifically comprises the following steps:
s1-1, taking rabbit serum raw material, and filtering by using a 0.2 mu m microporous filter membrane;
s1-2, adding active carbon into the filtered rabbit serum, wherein the addition amount of the active carbon is 20% of that of the rabbit serum, and centrifuging after adsorption and oscillation, and taking supernatant;
s1-3, adding an adsorption resin (XAD-4 macroporous resin, cross-linked resin or 110 resin) into the supernatant after the adsorption of the activated carbon, wherein the addition amount of the adsorption resin is 20% of that of the supernatant, and centrifuging after the adsorption and oscillation, and taking the supernatant;
s1-4 repeating the steps S1-2 and S1-3 until the impurity content in the animal blood matrix is reduced to below 100 ppm;
s1-5, adding a protective agent, an excipient, a preservative and an antioxidant to obtain an animal blood matrix product;
s2, preparing a calibrator and a quality control product semi-finished product: adding an object to be tested (taking 1L as an example) into the animal blood matrix product obtained in the step S1 according to the concentration requirements of the calibrator and the quality control product with different levels:
cal-6 was added sequentially with valproic acid at a concentration of 30mg/mL, 10mg/mL carbamazepine 10, 11-epoxide, 10mg/mL oxcarbazepine, 10mg/mL10, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL clonazepam standard stock 5000. Mu.L, 3000. Mu.L, 1200. Mu.L, 4500. Mu.L, 3000. Mu.L, 4500. Mu.L, 450. Mu.L, 120. Mu.L;
cal-5 is added with valproic acid with the concentration of 30mg/mL, carbamazepine with the concentration of 10mg/mL, 11-epoxide with the concentration of 10mg/mL, oxcarbazepine with the concentration of 10mg/mL and 10mg/mL, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL clonazepam standard stock 3333. Mu.L, 2000. Mu.L, 800. Mu.L, 3000. Mu.L, 2000. Mu.L, 3000. Mu.L, 300. Mu.L, 80. Mu.L;
cal-4 is added with valproic acid with the concentration of 30mg/mL, carbamazepine with the concentration of 10mg/mL, 11-epoxide with the concentration of 10mg/mL, oxcarbazepine with the concentration of 10mg/mL and 10mg/mL, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL clonazepam standard stock 1666. Mu.L, 1000. Mu.L, 400. Mu.L, 1500. Mu.L, 1000. Mu.L, 1500. Mu.L, 150. Mu.L, 40. Mu.L;
cal-3 was added sequentially with valproic acid at a concentration of 30mg/mL, 10mg/mL carbamazepine 10, 11-epoxide, 10mg/mL oxcarbazepine, 10mg/mL10, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL clonazepam standard stock 833. Mu.L, 500. Mu.L, 300. Mu.L, 750. Mu.L, 500. Mu.L, 750. Mu.L, 75. Mu.L, 20. Mu.L;
cal-2 was supplemented with valproic acid at a concentration of 30mg/mL, 10mg/mL carbamazepine 10, 11-epoxide, 10mg/mL oxcarbazepine, 10mg/mL10, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL clonazepam standard stock 333. Mu.L, 200. Mu.L, 80. Mu.L, 300. Mu.L, 200. Mu.L, 300. Mu.L, 30. Mu.L, 8. Mu.L;
cal-1 was supplemented with valproic acid at a concentration of 30mg/mL, 10mg/mL carbamazepine 10, 11-epoxide, 10mg/mL oxcarbazepine, 10mg/mL10, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL clonazepam standard stock 167. Mu.L, 100. Mu.L, 40. Mu.L, 150. Mu.L, 100. Mu.L, 150. Mu.L, 15. Mu.L, 4. Mu.L, sequentially;
qc-3 was added sequentially with valproic acid at a concentration of 30mg/mL, carbamazepine at 10mg/mL, 11-epoxide, oxcarbazepine at 10mg/mL, 10mg/mL10, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL clonazepam standard stock 4000. Mu.L, 2400. Mu.L, 960. Mu.L, 3600. Mu.L, 360. Mu.L, 96. Mu.L;
qc-2 was added sequentially with valproic acid at a concentration of 30mg/mL, carbamazepine at 10mg/mL, 11-epoxide, oxcarbazepine at 10mg/mL, 10mg/mL10, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL clonazepam standard stock 2000. Mu.L, 1200. Mu.L, 480. Mu.L, 1800. Mu.L, 1200. Mu.L, 1800. Mu.L, 180. Mu.L, 48. Mu.L;
qc-1 was added sequentially with valproic acid at a concentration of 30mg/mL, carbamazepine at 10mg/mL, 11-epoxide, oxcarbazepine at 10mg/mL, 10mg/mL10, 11-dihydro-10-hydroxycarbazepine, 10mg/mL phenytoin, 10mg/mL lamotrigine, 10mg/mL levetiracetam, 10mg/mL topiramate, 10mg/mL phenobarbital, 1mg/mL zolpidem, 1mg/mL standard stock solution of clonazepam 500. Mu.L, 300. Mu.L, 120. Mu.L, 450. Mu.L, 300. Mu.L, 450. Mu.L, 45. Mu.L, 12. Mu.L;
uniformly stirring, detecting the concentration of valproic acid, carbamazepine 10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam in the calibrator and the quality control product, and obtaining a calibrator and a semi-finished product of the quality control product after the calibration is qualified;
s3, preparing a calibrator and a quality control product finished product: subpackaging the calibrator and the quality control semi-finished product obtained in the step S2 according to packaging requirements, and freeze-drying to obtain a calibrator and a quality control finished product;
s4, preparing an internal standard: the internal standard is sequentially added with 1mg/mL of valproic acid-d 6, 1mg/mL of carbamazepine-d 10, 1mg/mL of carbamazepine-10, 11-epoxide-d 10, 1mg/mL of oxcarbazepine-d 4 and 1mg/mL of 10, 11-dihydro-10-hydroxycarbamoyl-d 4, 1mg/mL phenytoin-d 10, 1mg/mL lamotrigine-13C 3, 1mg/mL levetiracetam-d 6, 1mg/mL topiramate-d 12, 1mg/mL phenobarbital-d 5 100 mu g/mL zolpidem-d 7, 100 mu g/mL clonazepam-d 4 standard stock solution 10mL, 20mL, 10mL, methanol is used for constant volume to 1L;
and (3) a precipitant: directly taking 1L of methanol;
s5, assembling a kit: and (3) assembling the calibrator and quality control product obtained in the step (S3), the internal standard prepared in the step (S4) and the precipitant to obtain a kit product.
Comparative example 1
Kits were prepared in a similar manner to example 1 except that there were no filtration and adsorption steps for S1-1, S1-2 and S1-3.
Comparative example 2
Kits were prepared in a similar manner to example 1 except that the serum was horse serum.
Comparative example 3
A kit was prepared in a similar manner to example 1, except that the serum was bovine serum.
Example 2
The quality control products of the invention example 1 and the comparative examples 1-3 are stored under the constant temperature and humidity condition of 37 ℃ and sampled for 0, 4, 8, 15, 30 and 37 days respectively, so that the marked volume of distilled water is accurately redissolved and detected, and the slope trend significance is detected according to a t-test table. For a 95% confidence level, when 1 ∣<t 0.05,n-2 ×s(b 1 ) The stability result of the quality control product is not obvious in change trend; otherwise, the change trend of the stability result is obvious, and the detection result is shown in tables 7-10.
TABLE 7 quality control stability test results for example 1
Table 8 results of stability test of quality control product of comparative example 1
Table 9 quality control stability test results of comparative example 2
Table 10 quality control stability test results of comparative example 3
As can be seen from the comparison results of tables 7 to 10, the antiepileptic drug concentration detection kit prepared in example 1 can be stably stored for 30 days under the condition of constant temperature and humidity at 37 ℃, and valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam have no significant trend of change; comparative example 1 was evaluated under the same conditions to show a trending change in valproic acid, oxcarbazepine, topiramate, zolpidem, and comparative example 2 was evaluated under the same conditions to show a trending change in valproic acid; evaluation of comparative example 3 under the same conditions showed a trend change in valproic acid and oxcarbazepine. The kit provided by the invention can be used for detecting the concentration of the antiepileptic drug by treating animal blood matrix, and the animal blood matrix is preferably rabbit serum.
Example 3
The anti-epileptic drug concentration was measured using the kit prepared in example 1, comprising the steps of:
1. preparation of solution before detection:
a) Calibrator and quality control product: the screw cap and rubber stopper were removed from the vials at room temperature, 1.0ml distilled water was added accurately to each vial, the stopper was replaced, and incubation was performed at room temperature for 20 to 30 minutes. The vial was rotated to dissolve the contents until uniform;
b) Internal standard: taking out the internal standard in the kit, recovering to room temperature, and vortex oscillating before use;
c) Taking out the sample extract, standing at room temperature, and fully balancing for use;
2. on-machine sample preparation
a) Sample preparation: taking out the serum sample, recovering to room temperature for standby, and vortex oscillating for 30 seconds before use;
b) Sample preparation: 1) Transferring a 100 uL sample to a 1.5mL centrifuge tube by using a pipette, adding 10uL of isotope internal standard and 300 uL of methanol solution, and uniformly mixing by vortex oscillation for 3-5 minutes; 2) Centrifuging: the 1.5mL centrifuge tube was placed in a centrifuge, centrifuged at 13000rpm at 4℃for 5-10 minutes, and the supernatant was separated.
3. Sample detection
Transfer 100-200 μl of supernatant to 96-well U-plate, place in injector, and detect with liquid chromatograph tandem mass spectrometer.
Chromatographic conditions:
chromatographic column: c18 (100×2.1 mm,3.0 μm, 175 a);
mobile phase A1: taking 998 mL pure water, 1.0mL formic acid and 1.0mL 2M ammonium acetate from 0.1% formic acid-2 mM ammonium acetate aqueous solution, shaking uniformly, and carrying out ultrasonic treatment for 5min;
mobile phase B1: taking 999 mL methanol plus 1.0mL formic acid from 0.1% formic acid methanol solution, shaking uniformly, and carrying out ultrasonic treatment for 5min;
mobile phase A2:2mM ammonium acetate aqueous solution, taking 999 mL pure water plus 1.0mL 2M ammonium acetate, shaking uniformly, and carrying out ultrasonic treatment for 5min;
mobile phase B2: methanol solution, 1000 mL methanol;
column temperature: 35 ℃;
flow rate: 0.35 mL/min.
The gradient elution conditions are shown in tables 3 and 4.
4. The mass spectrum conditions are shown in tables 5 and 6.
Example 4
The quality control product prepared in example 1 was assigned with different measurement systems and tested for precision and accuracy errors.
1. Constant value measuring system
Constant value measurement system 1: the kit prepared in the embodiment 1 and a matched calibrator, quality control and Thermo TSQ Quantiva liquid chromatography tandem mass spectrometer.
Constant value measurement system 2: the kit prepared in the embodiment 1, matched calibrator and quality control product, and YS EXACT 9900MD liquid chromatography tandem mass spectrometer.
Constant value measurement system 3: the kit prepared in the example 1 and matched calibrator and quality control materials, and a Waters ACQUITY UPLC I-Class/Xevo TQ-S IVD liquid chromatograph tandem mass spectrometer.
Constant value measurement system 4: the kit prepared in the embodiment 1 and matched calibrators and quality control products comprise a SCIEX Triple Quad ™ 4500 liquid chromatograph tandem mass spectrometer.
2. Operation validity verification of constant value measurement system
The quality control product prepared in example 1 was measured by a constant value measurement system 1-4, each level of quality control product was repeatedly measured 3 times, and each measured value was within the allowable range of quality control, and the detection system was considered to satisfy the requirements.
3. Quality control product constant value
3.1 measurement: and detecting the extracted concentration monitoring quality control product of the antiepileptic drug to be assigned by using a verified constant value measuring system, measuring 2 bottles of each system, measuring 3 times for each bottle, and recording data.
3.2, calculating: summarizing the data, wherein the total CV value of each horizontal quality control measured value is not more than 10%, if the condition is met, taking the average value of all measured values as the final fixed value result of each horizontal quality control, calculating the deviation among different detection systems, and requiring not more than 10%, wherein the result is shown in Table 11.
Table 11 quality control assignment result comparison
As can be seen from the comparison results of Table 11, the quality control product prepared in example 1 has the advantages that the precision and accuracy errors are smaller than 10% through assignment by different detection systems, the result is good, the quality control product can be used as an antiepileptic drug quality control product for monitoring the precision and accuracy of the system, and the problem of the collapse of the third-party antiepileptic drug quality control product can be effectively solved.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention. The protection scope of the present invention is defined by the claims and the equivalents thereof.
Claims (10)
1. A quality control for detecting the concentration of an antiepileptic drug in a human blood matrix, wherein the antiepileptic drug comprises valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam; the quality control product is a freeze-dried preparation, and is prepared by an animal blood matrix which is subjected to filtration and adsorption treatment to remove interference impurities, and the antiepileptic drug, the protective agent, the excipient, the preservative and the antioxidant are added into the animal blood matrix.
2. The quality control of claim 1, wherein the quality control is Qc-1, qc-2, and Qc-3; the working concentrations of the antiepileptic drugs in each quality control product are shown in the following table:
。
3. the quality control of claim 1 wherein the animal blood substrate is rabbit serum or plasma.
4. The quality control product according to claim 1, wherein the protective agent is one or both of sucrose and trehalose, and the concentration is 0.5-1.5wt% and 1.0-3.0wt%, respectively; the excipient is one or two of mannitol and glycine, and the concentration is 1.0-3.0wt% and 1.0-3.0wt% respectively; the preservative is Proclin300, and the concentration is 0.05-0.3wt%; the antioxidant is ascorbic acid with concentration of 0.25-0.75wt%.
5. A kit for detecting the concentration of an antiepileptic drug in a human blood matrix, which is characterized by comprising a calibrator, a quality control product according to any one of claims 1-4, an internal standard and a precipitant, wherein the calibrator is a freeze-dried preparation; the calibrator is prepared from an animal blood matrix which is subjected to filtration and adsorption treatment to remove interference impurities, and the antiepileptic drug, the protective agent, the excipient, the preservative and the antioxidant are added into the animal blood matrix.
6. The kit of claim 5, wherein the internal standard is an isotopically labeled antiepileptic drug, and the internal standard is a mixed solution of carbamazepine-d 10, carbamazepine-10, 11-epoxide-d 10, oxcarbazepine-d 4, 10, 11-dihydro-10-hydroxycarbazepine-d 4, lamotrigine-13C 3, levetiracetam-d 6, zolpidem-d 7, clonazepam-d 4, valproic acid-d 6, phenytoin-d 10, topiramate-d 12, phenobarbital-d 5, the carbamazepine-d 10 concentration is 1.0 μg/mL, the carbamazepine-d 10, 11-epoxide-d 10 concentration is 1.0 μg/mL, oxcarbazepine-d 4 concentration is 1.0 μg/mL, 10, 11-dihydro-10-hydroxycarbazepine-d 4 concentration is 1.0 μg/mL, zol-C3C 4, valproic acid-d 6, phenytoin-d 10, topiramate-d 12, phenytoin-d 10, and phenytoin-d 10 concentration is 1.0 μg/mL, the 11-epoxide-d 10 concentration is 1.0 μg/mL, the oxepizepine-d 4 concentration is 1.0 μg/mL, the combined solution of 11-dihydro-hydroxycarbazepine-d 4, the combined solution is 1.0 μg/mL.
7. The kit of claim 5, wherein the precipitating agent is methanol.
8. The kit of claim 5, wherein the calibrator is Cal-1, cal-2, cal-3, cal-4, cal-5, and Cal-6, and the working concentrations of each substance in each calibrator are shown in the following table:
。
9. the method of preparing a kit according to any one of claims 5 to 8, comprising the steps of:
s1, preparing an animal blood matrix, which specifically comprises the following steps:
s1-1, taking animal blood matrix raw materials, and filtering by using a microporous filter membrane;
s1-2, adding active carbon into the filtered animal blood matrix, wherein the addition amount of the active carbon is 10-50wt% of the blood matrix, adsorbing, oscillating, centrifuging, and taking supernatant;
s1-3, adding an adsorption resin into the supernatant fluid after the adsorption of the activated carbon, wherein the addition amount of the adsorption resin is 10-50wt% of the supernatant fluid after the adsorption of the activated carbon, centrifuging after the adsorption and oscillation, and taking the supernatant fluid;
s1-4 repeating the steps S1-2 and S1-3 until the impurity content in the animal blood matrix is reduced to below 100 ppm;
s1-5, adding a protective agent, an excipient, a preservative and an antioxidant to obtain an animal blood matrix product;
s2, preparing a calibrator and a quality control product semi-finished product: adding valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam into the animal blood matrix product obtained in the step S1 according to the concentration requirements of different levels of the calibrator and quality control product, uniformly stirring, and checking the concentrations of valproic acid, carbamazepine-10, 11-epoxide, oxcarbazepine, 10, 11-dihydro-10-hydroxycarbazepine, phenytoin, lamotrigine, levetiracetam, topiramate, phenobarbital, zolpidem and clonazepam to obtain a calibrator and quality control product semi-finished product after the calibration is qualified;
s3, preparing a calibrator and a quality control product finished product: subpackaging the calibrator and the quality control semi-finished product obtained in the step S2 according to packaging requirements, and freeze-drying to obtain a calibrator and a quality control finished product;
s4, preparing an internal standard: preparing a mixed solution of carbamazepine-d 10, carbamazepine-10, 11-epoxide-d 10, oxcarbazepine-d 4, 10, 11-dihydro-10-hydroxycarbazepine-d 4, lamotrigine-13C 3, levetiracetam-d 6, zolpidem-d 7, clonazepam-d 4, valproic acid-d 6, phenytoin-d 10, topiramate-d 12, phenobarbital-d 5 according to the internal standard concentration requirement;
s5, assembling a kit: and (3) assembling the calibrator and quality control product obtained in the step (S3), the internal standard prepared in the step (S4) and the precipitant to obtain a kit product.
10. A method for detecting the concentration of an antiepileptic drug in a human blood matrix using a kit according to any one of claims 5-8, comprising the steps of:
s1 preparation of solution before detection:
a) Calibrator and quality control product: adding distilled water into a freeze-dried powder bottle containing a calibrator and a quality control product according to requirements at room temperature, and incubating for 20 to 30 minutes at room temperature; rotating the freeze-dried powder bottle to dissolve the content until the content is uniform;
b) Internal standard: taking out the internal standard in the kit, recovering to room temperature, and vortex oscillating before use;
c) Taking out the sample extract, standing at room temperature, and fully balancing for use;
s2, preparation of a detection sample:
a) Sample preparation: taking out the serum sample, recovering to room temperature for standby, and vortex oscillating before use;
b) Sample preparation: transferring the sample to a centrifuge tube by using a pipette, adding an internal standard and methanol, and uniformly mixing for 3-5 minutes by vortex oscillation; placing the centrifuge tube in a centrifuge, and separating supernatant after centrifugation;
s3, sample detection:
transferring the supernatant to a U-shaped plate, placing the plate in a sample injector, and detecting by using a liquid chromatograph-tandem mass spectrometer.
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