CN115236216B - Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof - Google Patents
Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof Download PDFInfo
- Publication number
- CN115236216B CN115236216B CN202210638740.2A CN202210638740A CN115236216B CN 115236216 B CN115236216 B CN 115236216B CN 202210638740 A CN202210638740 A CN 202210638740A CN 115236216 B CN115236216 B CN 115236216B
- Authority
- CN
- China
- Prior art keywords
- immunosuppressant
- calibrator
- mobile phase
- sample
- rapamycin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229960003444 immunosuppressant agent Drugs 0.000 title claims abstract description 72
- 239000003018 immunosuppressive agent Substances 0.000 title claims abstract description 72
- 230000001861 immunosuppressant effect Effects 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 239000008280 blood Substances 0.000 title claims abstract description 46
- 210000004369 blood Anatomy 0.000 title claims abstract description 45
- 238000004128 high performance liquid chromatography Methods 0.000 title claims description 13
- 238000004885 tandem mass spectrometry Methods 0.000 title claims description 7
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 50
- 238000003908 quality control method Methods 0.000 claims abstract description 47
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 claims abstract description 46
- 108010036949 Cyclosporine Proteins 0.000 claims abstract description 46
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 claims abstract description 44
- 229960002930 sirolimus Drugs 0.000 claims abstract description 44
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 claims abstract description 44
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 claims abstract description 43
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 claims abstract description 43
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 claims abstract description 42
- 229960000951 mycophenolic acid Drugs 0.000 claims abstract description 42
- 229960001967 tacrolimus Drugs 0.000 claims abstract description 42
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 claims abstract description 42
- 239000000546 pharmaceutical excipient Substances 0.000 claims abstract description 36
- 229930105110 Cyclosporin A Natural products 0.000 claims abstract description 35
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 30
- 239000011550 stock solution Substances 0.000 claims abstract description 29
- 238000007710 freezing Methods 0.000 claims abstract description 12
- 230000008014 freezing Effects 0.000 claims abstract description 12
- 239000000523 sample Substances 0.000 claims description 64
- 239000000047 product Substances 0.000 claims description 55
- 238000000034 method Methods 0.000 claims description 43
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 27
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 26
- 229960001265 ciclosporin Drugs 0.000 claims description 21
- 238000004108 freeze drying Methods 0.000 claims description 21
- 239000000126 substance Substances 0.000 claims description 21
- 229930182912 cyclosporin Natural products 0.000 claims description 20
- 239000012488 sample solution Substances 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 17
- 229940124531 pharmaceutical excipient Drugs 0.000 claims description 16
- 150000002500 ions Chemical class 0.000 claims description 14
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 13
- 235000019253 formic acid Nutrition 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 12
- 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 11
- 229930195725 Mannitol Natural products 0.000 claims description 11
- 239000000594 mannitol Substances 0.000 claims description 11
- 235000010355 mannitol Nutrition 0.000 claims description 11
- 239000000843 powder Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 10
- 239000008363 phosphate buffer Substances 0.000 claims description 10
- 150000003839 salts Chemical class 0.000 claims description 9
- 238000004806 packaging method and process Methods 0.000 claims description 8
- HPNSFSBZBAHARI-HZIIEIAOSA-N (e)-6-[4-hydroxy-7-methyl-3-oxo-6-(trideuteriomethoxy)-1h-2-benzofuran-5-yl]-4-methylhex-4-enoic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC([2H])([2H])[2H])=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-HZIIEIAOSA-N 0.000 claims description 7
- ZDQSOHOQTUFQEM-XCXYXIJFSA-N ascomycin Natural products CC[C@H]1C=C(C)C[C@@H](C)C[C@@H](OC)[C@H]2O[C@@](O)([C@@H](C)C[C@H]2OC)C(=O)C(=O)N3CCCC[C@@H]3C(=O)O[C@H]([C@H](C)[C@@H](O)CC1=O)C(=C[C@@H]4CC[C@@H](O)[C@H](C4)OC)C ZDQSOHOQTUFQEM-XCXYXIJFSA-N 0.000 claims description 7
- ZDQSOHOQTUFQEM-PKUCKEGBSA-N ascomycin Chemical group C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)O[C@@H]([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C\C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CC)=C\[C@@H]1CC[C@@H](O)[C@H](OC)C1 ZDQSOHOQTUFQEM-PKUCKEGBSA-N 0.000 claims description 7
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 6
- 239000005695 Ammonium acetate Substances 0.000 claims description 6
- 241001494479 Pecora Species 0.000 claims description 6
- 235000019257 ammonium acetate Nutrition 0.000 claims description 6
- 229940043376 ammonium acetate Drugs 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 238000007781 pre-processing Methods 0.000 claims description 4
- 239000012716 precipitator Substances 0.000 claims description 4
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 4
- 229960001763 zinc sulfate Drugs 0.000 claims description 4
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 230000003014 reinforcing effect Effects 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 2
- 238000013441 quality evaluation Methods 0.000 claims 1
- 108010071390 Serum Albumin Proteins 0.000 abstract description 15
- 102000007562 Serum Albumin Human genes 0.000 abstract description 15
- 238000004321 preservation Methods 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 5
- 230000015556 catabolic process Effects 0.000 abstract description 3
- 238000006731 degradation reaction Methods 0.000 abstract description 3
- 238000004090 dissolution Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000006872 improvement Effects 0.000 description 16
- 230000008569 process Effects 0.000 description 14
- 239000011265 semifinished product Substances 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 9
- 238000011835 investigation Methods 0.000 description 8
- 238000011084 recovery Methods 0.000 description 7
- 239000012086 standard solution Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- NPEONIHYDZZZGH-UHFFFAOYSA-N Cyclosporin T Natural products CCC1NC(=O)C(C(O)C(C)CC=CC)N(C)C(=O)C(C(C)C)N(C)C(=O)C(CC(C)C)NC(=O)C(CC(C)C)N(C)C(=O)C(C)NC(=O)C(C)NC(=O)C(CC(C)C)N(C)C(=O)C(C(C)C)NC(=O)C(CC(C)C)N(C)C(=O)CN(C)C1=O NPEONIHYDZZZGH-UHFFFAOYSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 238000007689 inspection Methods 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 4
- 239000000337 buffer salt Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000008176 lyophilized powder Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 4
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 4
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000009210 therapy by ultrasound Methods 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 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 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 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 description 3
- 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 description 3
- 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 description 3
- 229940079593 drug Drugs 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Substances CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 230000000781 anti-lymphocytic effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000000859 sublimation Methods 0.000 description 2
- 230000008022 sublimation Effects 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- HKVAMNSJSFKALM-GKUWKFKPSA-N Everolimus Chemical compound C1C[C@@H](OCCO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 HKVAMNSJSFKALM-GKUWKFKPSA-N 0.000 description 1
- HZQDCMWJEBCWBR-UUOKFMHZSA-N Mizoribine Chemical compound OC1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 HZQDCMWJEBCWBR-UUOKFMHZSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 229960005167 everolimus Drugs 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 229960000556 fingolimod Drugs 0.000 description 1
- KKGQTZUTZRNORY-UHFFFAOYSA-N fingolimod Chemical compound CCCCCCCCC1=CC=C(CCC(N)(CO)CO)C=C1 KKGQTZUTZRNORY-UHFFFAOYSA-N 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000005541 medical transmission Effects 0.000 description 1
- 229960001810 meprednisone Drugs 0.000 description 1
- PIDANAQULIKBQS-RNUIGHNZSA-N meprednisone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)CC2=O PIDANAQULIKBQS-RNUIGHNZSA-N 0.000 description 1
- 229950000844 mizoribine Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003865 nucleic acid synthesis inhibitor Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940046781 other immunosuppressants in atc Drugs 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- OAVGBZOFDPFGPJ-UHFFFAOYSA-N sotrastaurin Chemical compound C1CN(C)CCN1C1=NC(C=2C(NC(=O)C=2C=2C3=CC=CC=C3NC=2)=O)=C(C=CC=C2)C2=N1 OAVGBZOFDPFGPJ-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002054 transplantation 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
-
- 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
- G01N30/04—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)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of medicine detection, in particular to a kit for detecting immunosuppressant in whole blood, a preparation method and a detection method thereof. The kit comprises a calibrator, a quality control product, an internal standard product, a mobile phase stock solution and a protein precipitant; the calibrator and quality control product contain immunosuppressant and pharmaceutical adjuvants with serial concentrations; immunosuppressants include tacrolimus, cyclosporine a, rapamycin and mycophenolic acid; the pharmaceutical adjuvant contains at least one of whole blood, red blood cells and serum albumin. The kit disclosed by the invention has the advantages of no need of low-temperature freezing preservation, difficulty in degradation and convenience in long-distance transportation, and is simple to operate, convenient to use and instant in use and dissolution. The kit of the invention ensures the stability of the immunosuppressant, thereby ensuring the accuracy of the detection method result, and simultaneously has the technical advantages of strong specificity and high sensitivity. The detection method has less pretreatment steps and can adapt to manual treatment and automatic treatment schemes.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a kit for detecting immunosuppressant in whole blood, a preparation method and a detection method thereof.
Background
Immunosuppressants are chemical or biological substances which can reduce tissue injury by inhibiting cellular and humoral immune responses, can inhibit abnormal immune responses of organisms, and are mainly applied to treatment of rejection reaction and autoimmune diseases of organ transplantation. Immunosuppressants currently in clinical use can be largely divided into five major classes: the first class is cytokine inhibitors such as cyclosporine a, tacrolimus, sirolimus, everolimus, and the like; the second category is DNA synthesis inhibitors such as azathioprine, mycophenolic acid, mizoribine, and the like; the third class is glucocorticoids, such as prednisone, methylprednisone, etc.; the fourth class is anti-lymphocyte antibodies, such as anti-lymphocyte globulin, anti-thymus globulin, etc.; the fifth class is other immunosuppressants such as AEB071, FTY720, etc. Immunosuppressants can inhibit the immune function of an organism by affecting the immune response and the immune pathological response of the organism, and different immunosuppressants act on different periods of lymphocyte activation to play a synergistic role, and the whole effectiveness and the safety of a treatment scheme are improved by adopting a combined medication method clinically at present. Due to the narrow therapeutic window of immunosuppressants, individual differences in pharmacokinetics are large. When the amount thereof is too large, serious toxic reaction may be caused; in insufficient doses, patients will develop immune rejection reactions, so that therapeutic drug monitoring (therapeutic drug monitoring, TDM) is necessary to obtain more complete therapeutic information and adjust individual dosing regimens.
Liquid chromatography tandem mass spectrometry (LC-MS/MS) is currently the dominant quantitative analysis method for immunosuppressants. However, due to the instability of the immunosuppressant itself, the related diagnostic reagents for detecting the concentration of the immunosuppressant in whole blood by the liquid chromatography tandem mass spectrometry at present generally need low-temperature freezing preservation, ultralow-temperature preservation and even nitrogen-filled preservation; not only has poor stability, but also consumes energy and electricity. In addition, in the low-temperature preservation kit, the immunosuppressant in the standard product and the quality control product is easy to degrade after repeated freezing and thawing for a long time, and the quality stability of the detection product is difficult to ensure.
Disclosure of Invention
In order to solve the technical problems, the invention provides a kit for detecting immunosuppressant in whole blood, a preparation method and a detection method thereof, and the stability of the immunosuppressant is ensured, so that the accuracy of a detection result is ensured.
The invention provides a kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, which comprises a calibrator, an internal standard, mobile phase stock solution and a protein precipitant; the calibrator contains immunosuppressant and pharmaceutical adjuvants with serial concentrations; immunosuppressants include tacrolimus, cyclosporine a, rapamycin and mycophenolic acid; the pharmaceutical adjuvant contains at least one of whole blood, red blood cells and serum albumin, and the source of the whole blood, the red blood cells and the serum albumin is human or mammal, preferably cow, sheep or pig.
Optionally, the kit further comprises a quality control.
Optionally, the calibrator and the quality control product are freeze-dried powder; the freeze-dried powder contains immunosuppressant with serial concentration and pharmaceutical auxiliary material, and the pharmaceutical auxiliary material also contains excipient and buffer salt; the excipient is at least one of serum albumin, trehalose, mannitol, PVP and sucrose, and the buffer salt is phosphate buffer salt with pH of 7.0-7.4.
Optionally, the mobile phase comprises a mobile phase A stock solution and a mobile phase B stock solution, wherein the mobile phase A stock solution is an aqueous solution containing 2-8% formic acid by volume percentage and 0.5-5 mM ammonium acetate by volume percentage; the stock solution of the mobile phase B is a methanol solution containing 2-8% formic acid by volume percent; the inner standard is acetonitrile solution containing the inner standard, and the inner standard is ascomycin, cyclosporin A-d4, rapamycin-d 3 and mycophenolic acid-d 3; the protein precipitant comprises a protein precipitant I and a protein precipitant II, wherein the protein precipitant I is an aqueous solution of zinc sulfate with the concentration of 0.1-0.3 mol/L; the protein precipitant II is acetonitrile solution containing 0-5% of methanol by volume percent.
The invention provides a preparation method of the kit, which comprises the steps of preparing standard substances and quality control substances, and at least comprises the following steps:
S1, taking an immunosuppressant and pharmaceutical excipients according to a proportion, and preparing to obtain a solution;
s2, subpackaging the solution;
s3, freeze drying, wherein the freeze drying at least comprises the following steps:
s31, precooling: precooling the equipment for 1-3 hours at the temperature of minus 20 ℃ to minus 50 ℃;
s32, pre-freezing: pre-freezing for 2-5 hours at the temperature of minus 30 ℃ to minus 50 ℃;
s33, sublimating: sublimating for 8-12 hours at the temperature of minus 20 ℃ to minus 35 ℃;
s34, analysis and drying: the time is 3-5 hours, and the temperature is 0-20 ℃;
s35, reinforcing drying: the time is 3-10 hours, and the temperature is 15-25 ℃;
s36, packaging.
The invention provides a method for detecting immunosuppressant in whole blood by the kit, which at least comprises the following steps:
s1, preparing a solution: comprising the following steps:
dissolving the calibrator and the quality control product with water respectively for standby; diluting the mobile phase stock solution A with water to obtain a mobile phase A; diluting the mobile phase stock solution B with methanol to obtain a mobile phase B for later use;
s2, preparing a standard curve equation, which comprises the following steps: pretreating a calibrator, and detecting by adopting a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation;
s3, obtaining a sample to be tested, including: taking a whole blood sample to be tested or a quality control product, and obtaining the sample to be tested after pretreatment;
S4, detecting a sample to be detected, including: detecting a sample to be detected by using a high performance liquid chromatography mass spectrometer, and substituting a detection result into a standard curve equation to obtain the concentration of the immunosuppressant in the sample to be detected;
in S2 and S3, the preprocessing includes: taking a sample solution, adding an internal standard substance, wherein the sample solution comprises a whole blood sample to be tested, a calibrator and a quality control substance; adding a protein precipitator I, uniformly mixing, adding a protein precipitator II, uniformly mixing, centrifuging, taking supernatant, centrifuging, taking the supernatant as a sample to be detected for chromatographic analysis; the method also comprises the step of evaluating the precision of the detection method by adopting quality control.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
the kit provided by the invention has strong universality, is suitable for all high performance liquid chromatography-tandem mass spectrometry detection systems, and provides materials and methods for clinically and rapidly detecting tacrolimus, cyclosporine A, rapamycin and mycophenolic acid in human whole blood.
In the preferred technical scheme, the calibrator or quality control product adopts freeze-dried powder, solves the problems of easy degradation, deterioration and the like of tacrolimus, cyclosporine A, rapamycin and mycophenolic acid in the placing process, and provides possibility for long-term stable storage of the kit. Has the advantages of no need of freezing preservation, difficult degradation, convenient long-distance transportation, simple operation, convenient use and instant dissolution. The stability of the immunosuppressant is ensured, thereby ensuring the accuracy of the detection method result, and simultaneously having the technical advantages of strong specificity and high sensitivity.
In the preferred technical scheme, the kit adopts the substituted pharmaceutic adjuvant to substitute the human whole blood, so that the possibility of disease transmission is reduced, and the biological safety is high.
The detection method has less pretreatment steps and can adapt to manual treatment and automatic treatment schemes.
Drawings
FIG. 1 is a standard curve equation obtained in an embodiment of the present invention;
FIG. 2 is a chromatogram of a standard immunosuppressant and an isotopically-internal standard in an embodiment of the invention;
FIG. 3 IS an enlarged view of FK-506 and FK-506-IS of FIG. 2;
FIG. 4 IS an enlarged view of the CSA and CSA-IS of FIG. 2;
FIG. 5 IS an enlarged view of MPA and MPA-IS of FIG. 2;
FIG. 6 IS an enlarged view of the RPM and RPM-IS of FIG. 2;
FIG. 7 is a chromatogram of a whole blood sample immunosuppressant and its isotopic internal standard in an embodiment of the invention;
FIG. 8 IS an enlarged view of FK-506 and FK-506-IS of FIG. 7;
FIG. 9 IS an enlarged view of the CSA and CSA-IS of FIG. 7;
FIG. 10 IS an enlarged view of the MPA and MPA-IS of FIG. 7;
FIG. 11 IS an enlarged view of the RPM and RPM-IS of FIG. 7.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.
The embodiment of the invention provides a kit for detecting immunosuppressants in whole blood by high performance liquid chromatography-tandem mass spectrometry, which is suitable for high performance liquid chromatography-tandem mass spectrometry, can detect the concentration of various immunosuppressant drugs in human whole blood at the same time, and is used for in-vitro diagnosis. The embodiment of the invention is complete in matching and comprises a calibrator, an internal standard, mobile phase stock solution and a protein precipitant. The calibrator contains immunosuppressant with serial concentration, and can be provided with kit containing different immunosuppressant types according to requirement, specifically, immunosuppressant can include tacrolimus (FK-506), cyclosporine A (CSA), rapamycin (RPM) and mycophenolic acid (MPA). The pharmaceutical adjuvant contains at least one of whole blood, red blood cells and serum albumin, and the sources of the whole blood, the red blood cells and the serum albumin are human or mammals.
The kit is used for treating the whole blood sample, and clean treatment fluid can be obtained by a simple protein precipitation-centrifugal extraction pretreatment method without complex purification steps, so that the required sample size is small; different consumables can be matched for use, and manual and automatic processing schemes of whole blood can be flexibly matched; the kit calibrator is prepared by using pharmaceutical excipients which replace whole blood, and the pharmaceutical excipients are evaluated to be consistent with clinical fresh samples. Four different immunosuppressants can be detected simultaneously, and the simultaneous detection of multiple indexes can be realized by a single sample, so that the detection specificity is good, the sensitivity is high, the matrix effect is small, the detection time is short, the flux is high, and the cost is low.
As an improvement of the technical scheme of the embodiment of the invention, the kit also comprises a quality control product, wherein the quality control product contains immunosuppressant with a series of concentration and pharmaceutical auxiliary materials, and the pharmaceutical auxiliary materials contain at least one of whole blood, red blood cells and serum albumin, and the sources of the whole blood, the red blood cells and the serum albumin are human or mammals. The quality control product can more effectively monitor the daily and daytime precision in the test process, and ensure the stability and accuracy in the test sample process.
The calibrator and the quality control product in the embodiment of the invention can be liquid preparations. However, because tacrolimus, cyclosporine A, rapamycin and mycophenolic acid are unstable, the solution is easy to oxidize and deteriorate, the solution needs to be stored at a low temperature, the storage is inconvenient, and the accuracy of the subsequent detection result is affected by repeated freeze thawing. In order to further improve the stability of the kit, the components containing the immunosuppressant in the kit are innovatively prepared into the freeze-dried powder, and the pharmaceutical excipients for ensuring the stability of the immunosuppressant are added into the freeze-dried powder, so that the components do not need to be stored in a freezing way, only can be stored at a low temperature of 2-8 ℃, and the stability is good. Compared with the common solution state, the kit is a freeze-dried product, has less water content (less than 3 percent), stable product quality and is convenient for transportation.
The examples of the present invention find that the sources of whole blood, red blood cells and serum albumin are bovine, ovine or porcine, and further preferably ovine red blood cells and bovine serum albumin.
As an improvement of the technical scheme of the embodiment of the invention, the red blood cells are diluted into the volume percentage concentration of 20-80%, and more preferably 50-80% by adopting phosphate buffer salt with the pH value of 7.4. Through the screening experiment of the concentration of the red blood cells, the stability of 50 to 80 percent of the red blood cells can meet the requirement, but in the experimental operation process, 80 percent of the red blood cells are easy to generate a flocculation state, so that 50 to 70 percent of the red blood cells with the volume percentage concentration are more preferable.
As an improvement of the technical scheme of the embodiment of the invention, the freeze-dried powder contains immunosuppressant and pharmaceutical excipients with a series of concentrations, the pharmaceutical excipients are further added with excipient and buffer salt, the excipient is at least one of serum albumin, trehalose, mannitol, PVP (polyvinylpyrrolidone) and sucrose, and the buffer salt is phosphate buffer salt with pH of 7.0-7.4.
As an improvement of the technical scheme of the embodiment of the invention, the pharmaceutical excipients comprise:
7-12 parts of excipient;
100-800 parts by volume of whole blood or red blood cells;
100-700 parts by volume of phosphate buffer salt;
the excipient is selected from serum albumin and mannitol; wherein, the weight ratio of serum albumin to mannitol is 0.1:1 to 10:1, a step of; when the unit of parts by weight is g, the unit of parts by volume is mL. Preferably:
10 parts of excipient;
250-500 parts by volume of red blood cells;
250-500 parts by volume of phosphate buffer salt;
the excipient is selected from serum albumin and mannitol; wherein, the weight ratio of serum albumin to mannitol is 0.5:1 to 5:1, a step of; when the unit of parts by weight is g, the unit of parts by volume is mL.
As an improvement of the technical scheme of the embodiment of the invention, the specific concentration of the auxiliary materials for the medicines of the calibrator and the quality control product is as follows: bovine serum albumin 0.01g/mL, mannitol 0.01g/mL, red blood cells 0.5mL, phosphate buffer pH7.4 0.5mL.
As an improvement of the technical scheme of the embodiment of the invention, the immunosuppressant with a series of concentrations in the calibrator comprises 6 concentrations of immunosuppressants, and the 6 concentrations of immunosuppressants are as follows:
calibrator C1: 0.9 to 1.1ng/mL of tacrolimus, 9 to 11ng/mL of rapamycin, 0.9 to 1.1ng/mL of cyclosporine A and 90 to 110ng/mL of mycophenolic acid;
Calibrator C2: 4.5 to 5.5ng/mL of tacrolimus, 45 to 55ng/mL of rapamycin, 4.5 to 5.5ng/mL of cyclosporine A and 450 to 550ng/mL of mycophenolic acid;
calibrator C3: tacrolimus 9-11 ng/mL, rapamycin 90-110 ng/mL, cyclosporin A9-11 ng/mL, mycophenolic acid 900-1100 ng/mL;
calibrator C4: 18-22 ng/mL of tacrolimus, 180-220 ng/mL of rapamycin, 18-22 ng/mL of cyclosporine A and 1800-2200 ng/mL of mycophenolic acid;
calibrator C5: tacrolimus 45-55 ng/mL, rapamycin 450-550 ng/mL, cyclosporin A45-55 ng/mL, mycophenolic acid 4500-5500 ng/mL;
calibrator C6: 90-110 ng/mL of tacrolimus, 900-1100 ng/mL of rapamycin, 90-110 ng/mL of cyclosporine A and 9000-11000 ng/mL of mycophenolic acid.
The series of concentrations of immunosuppressant in the quality control product comprises 3 concentrations of immunosuppressant, including:
low value quality control Q7: 1.8 to 2.2ng/mL of tacrolimus, 18 to 22ng/mL of rapamycin, 1.8 to 2.2ng/mL of cyclosporine A and 180 to 220ng/mL of mycophenolic acid;
median quality control Q8: tacrolimus 9-11 ng/mL, rapamycin 180-220 ng/mL, cyclosporin A9-11 ng/mL, mycophenolic acid 900-1100 ng/mL;
high value quality control Q9: tacrolimus 72-88 ng/mL, rapamycin 720-880 ng/mL, cyclosporine A72-88 ng/mL, mycophenolic acid 7200-8800 ng/mL.
As an improvement of the technical scheme of the embodiment of the invention, the kit also comprises an internal standard substance, a mobile phase stock solution and a protein precipitant;
the mobile phase stock solution comprises a mobile phase A stock solution and a mobile phase B stock solution: the stock solution of the mobile phase A is a methanol solution containing formic acid and ammonium acetate, the volume percentage concentration of the formic acid is 2-8%, preferably 4%, and the concentration of the ammonium acetate is 0.5-5 mM, preferably 0.1mM; the stock solution of the mobile phase B is methanol solution containing formic acid, and the concentration of the formic acid in the stock solution of the mobile phase B is 2-8%, preferably 6%.
The internal standard is acetonitrile solution containing the internal standard; the internal standard substances are ascomycin, cyclosporin A-d4, rapamycin-d 3 and mycophenolic acid-d 3; wherein, the internal standard of tacrolimus is ascomycin, the internal standard of cyclosporine A is cyclosporine A-d4, the internal standard of rapamycin is rapamycin-d 3 and the internal standard of mycophenolic acid is mycophenolic acid-d 3. Preferably, the concentration of ascomycin in the internal standard substance is 0.009-0.011 mug/mL, the concentration of cyclosporin A-d4 is 0.4-0.6 mug/mL, the concentration of rapamycin-d 3 is 0.01-0.03 mug/mL, and the concentration of mycophenolic acid-d 3 is 0.8-1.2 mug/mL; more preferably, the concentration of ascomycin in the internal standard is 0.01 μg/mL, the concentration of cyclosporin A-d4 is 0.5 μg/mL, the concentration of rapamycin-d 3 is 0.02 μg/mL, and the concentration of mycophenolic acid-d 3 is 1 μg/mL.
The protein precipitant comprises a protein precipitant I and a protein precipitant II; the protein precipitant I is zinc sulfate aqueous solution with the concentration of 0.1-0.3 mol/L, and the concentration of zinc sulfate is preferably 0.2mol/L; the protein precipitant II is acetonitrile solution containing 0-100% methanol by volume, preferably 100% acetonitrile.
The kit provided by the embodiment of the invention adopts two protein precipitants, has the technical advantages of simple pretreatment, small sample consumption and short detection time, and can effectively remove matrix interference.
As an improvement of the technical scheme of the embodiment of the invention, the kit also comprises a use instruction, a qualification certificate, a lining and a packaging box, and can also contain used consumables such as EP (European patent application) tubes, 96-well plates, heat-sealing films and other products related to the use, storage and transportation processes of the kit.
The embodiment of the invention also relates to a preparation method of the kit, which comprises the steps of preparing calibrator lyophilized powder or quality control product lyophilized powder, and at least comprises the following steps:
s1, taking an immunosuppressant and pharmaceutical excipients according to a proportion, and preparing to obtain a solution;
s2, subpackaging the solution;
s3, freeze drying, at least comprising the following steps:
s31, precooling: precooling the equipment for 1-3 hours at-20 ℃ to-50 ℃, preferably at-20 ℃ for 2 hours;
S32, pre-freezing: prefreezing for 2-5 hours at-30 to-50 ℃, preferably at-35 ℃ for 4 hours;
s33, sublimating: sublimating for 8-12 hours at-20 to-35 ℃, preferably at-25 ℃ for 10 hours;
s34, analysis and drying: the time is 3-5 hours, the temperature is 0-20 ℃, and the drying is carried out for 3 hours at 15 ℃ preferably;
s35, reinforcing drying: the time is 3 to 10 hours, the temperature is 15 to 25 ℃, and the drying is carried out for 4 hours at the temperature of 20 ℃ preferably;
s36, packaging.
The freeze-drying process provided by the embodiment of the invention has the technical advantages of simple process, strong operability, low production cost, safety and reliability. The prepared freeze-dried product has good dissolution and re-solubility, and can be rapidly dissolved into dark red liquid after being dissolved in water and slightly vibrated.
The embodiment of the invention also relates to a method for detecting the immunosuppressant in whole blood by adopting the kit, which uses a high performance liquid chromatography-tandem mass spectrometer to record the chromatogram of a sample to be detected, the peak areas of tacrolimus, cyclosporine A, rapamycin and mycophenolic acid and the peak areas of corresponding internal standards. And (3) preparing a calibration curve by the ratio of the peak area of the TICF (total ion current intensity) peak of FK-506 and CsA, RPM, MPA in the calibrator to the corresponding internal standard peak area and the calibration concentration, so as to calculate the content of FK-506 and CsA, RPM, MPA in the sample to be measured. The embodiment of the invention adopts the method of isotope dilution high performance liquid tandem mass spectrometry to detect, furthest reduces analysis errors caused by loss in the process of sample extraction and detection, and improves the accuracy of a measurement result and the matrix interference resistance of the method.
At least comprises the following steps:
s1, preparing a solution: comprising the following steps:
dissolving the quality control product and the calibrator in water; diluting the mobile phase stock solution A with water; diluting the mobile phase stock solution B with methanol;
s2, preparing a standard curve equation, preprocessing a calibrator, detecting by adopting a high performance liquid chromatography-mass spectrometer, and obtaining the standard curve equation, wherein the standard curve equation comprises the following specific steps: drawing a standard curve by taking the marked concentration of the 6 calibrator as an abscissa (x) and taking the ratio of the actual detected peak area of the 6 calibrator to the peak area of each internal standard as an ordinate (y); linear regression of the indicated concentration (x) with peak area ratio (y) yields the regression equation: y=ax+b, wherey is the ordinate, x is the abscissa, a is the slope of the curve, and the fitting correlation coefficient (r 2 ) Requirement r 2 Should not be less than 0.990;
s3, processing to obtain a sample to be tested, wherein the step comprises the following steps: taking plasma to be measured, and obtaining a sample to be measured after pretreatment;
s4, detecting a sample to be detected, including: and detecting the sample to be detected by using a high performance liquid chromatography mass spectrometer, and substituting the detection result into a standard curve equation to obtain the concentration of the immunosuppressant in the sample to be detected.
In S2 and S3, the preprocessing includes: taking a sample solution, adding an internal standard substance, wherein the sample solution comprises a whole blood sample to be tested, a calibrator and a quality control substance; adding the protein precipitant I, mixing uniformly, adding the protein precipitant II, mixing uniformly, centrifuging, taking supernatant, centrifuging, taking the supernatant as a sample to be detected for chromatographic analysis.
As an improvement of the technical scheme of the embodiment of the invention, in S1, the mobile phase A obtained by dilution is an aqueous solution containing 0.05-0.2% formic acid, preferably 0.1% formic acid, 0.5-2 mM, preferably 1mM ammonium acetate; the mobile phase B is a methanol solution containing formic acid with the volume percentage concentration of 0.05-0.2%, preferably 0.1%.
The preparation method specifically comprises the following steps: adding 5mL of mobile phase stock solution A into 200mL of deionized water, fully and uniformly mixing, and performing ultrasonic treatment for 3-5 minutes; mobile phase B phase: and 5mL of mobile phase stock solution B is taken, methanol is added to 300mL, and the mixture is fully and uniformly mixed and then is subjected to ultrasonic treatment for 3-5 minutes to obtain the preparation.
As an improvement of the technical scheme of the embodiment of the invention, in the pretreatment process, the volume ratio of the sample solution to the internal standard substance is 1:0.5 to 2, preferably 1:1, a step of; the volume ratio of the sample solution to the protein precipitant I was 1:0.5 to 2, preferably 1:1, a step of; the volume ratio of the sample solution to the protein precipitant II is 1:1 to 3, preferably 1:2.
as an improvement of the technical scheme of the embodiment of the invention, in the pretreatment process, the implementation process of the whole blood sample is as follows:
the 96-well plate treatment method comprises the following steps: precisely sucking 100 mu L of sample solution into a 96-well plate, adding 100 mu L of internal standard substance, adding 100 mu L of protein precipitant I, mixing for 1 min at 500r/min, adding 200 mu L of protein precipitant II, coating, mixing for 5 min at 2000r/min, centrifuging for 10 min at 4000r/min, taking 300 mu L of supernatant, coating, centrifuging for 10 min at 4000r/min, and collecting supernatant for chromatographic analysis.
EP tube treatment method: precisely sucking 100 mu L of sample solution into a 1.0mL centrifuge tube, adding 100 mu L of internal standard substance, adding 100 mu L of protein precipitant I, mixing for 2 minutes at 2000r/min, adding 200 mu L of protein precipitant II, mixing for 5 minutes at 2000r/min, centrifuging for 10 minutes at 14000r/min, taking 300 mu L of supernatant, centrifuging for 5 minutes at 14000r/min, and taking 100 mu L of on-machine inspection.
As an improvement of the technical scheme of the embodiment of the invention, the detection system is a high performance liquid chromatography tandem mass spectrometry detection system, the detection method is a liquid chromatography tandem mass spectrometry method, triple quadrupole tandem mass spectrometry is used, ESI positive ion detection mode is used, and multi-reaction detection MRM scanning mode is adopted. Specific examples: SCIEX tripleQuad 4500 series liquid chromatography tandem mass spectrometry system.
As an improvement of the technical scheme of the embodiment of the invention, the specific conditions of the high performance liquid chromatography are as follows: the chromatographic column is selected from C18 column with particle diameter of 2-5 μm; the specific optional chromatographic column comprises: waters, e.g., AWaters, XBridge C18 column, (2.1X100 mm,5 μm); waters, atlantis dC18 column, (2.1X10 mm,5 μm); waters, atlantis T3 column (2.1X10 mm,3 μm); preferably a 2.1X100 mm,5 μm or equivalent chromatography column.
As an improvement of the technical scheme of the embodiment of the invention, the conditions of the high performance liquid chromatography are as follows:
the column temperature is 25-45 ℃, preferably 40 ℃;
the flow rate is 0.3-0.5 mL/min, preferably 0.4mL/min;
the sample amount is 10 to 30. Mu.L, preferably 20. Mu.L.
As an improvement of the technical scheme of the embodiment of the invention, the elution of the high performance liquid chromatography adopts gradient elution:
0 to 0.5 minutes, mobile phase A: 30-50%, mobile phase B: 50-70%;
0.51-2 minutes, mobile phase A: 1-5%, mobile phase B: 5-99%;
2.1 to 4 minutes, mobile phase A: 30-50%, mobile phase B: 50-70%;
as an improvement of the technical scheme of the embodiment of the invention, the elution conditions of the high performance liquid chromatography are shown in table 1:
TABLE 1
| Time/minute | Water (0.1% formic acid+1 mM ammonium acetate)/% | Methanol (0.1% formic acid)% | Flow rate/(mL/min) |
| 0 | 40 | 60 | 0.4 |
| 0.5 | 3 | 97 | 0.4 |
| 2.0 | 3 | 97 | 0.4 |
| 2.1 | 40 | 60 | 0.4 |
| 3.0 | 40 | 60 | 0.4 |
| 4.0 | 40 | 60 | 0.4 |
The conditions of mass spectrometry were:
the Ionspray voltage is 0-5500V, preferably 4000V; gas 1: 40-60 psi, preferably 50psi; gas 2:0 to 90psi, preferably 50psi; air curtain gas: 20 to 50psi, preferably 25psi; the heating gas temperature is 150 to 550 ℃, preferably 400 ℃.
ESI ion source recommended parameters, ion pair information is shown in Table 2:
TABLE 2
The technical scheme of the embodiment of the invention is further described by the following specific embodiments, and all reagents adopted in the specific embodiments are commercially available.
Example 1
The specific composition of the kit is shown in the following table 3:
table 3: kit (96 person/box)
Wherein, the volume percentage concentration of sheep red blood cells is 50%, diluted with phosphate buffer salt with ph=7.4.
Example 2
The embodiment is used for explaining a kit using method and a detection method:
s1, preparing a solution:
1. dissolving the calibrator freeze-dried powder and the quality control freeze-dried powder in the kit with 0.5mL of purified water respectively for later use;
2. mobile phase a phase: adding deionized water to 200mL into 5mL of mobile phase stock solution A in the kit, fully and uniformly mixing, and performing ultrasonic treatment for 3 minutes to obtain the kit; mobile phase B phase: adding 5mL of mobile phase stock solution B in the kit into 300mL of methanol, fully and uniformly mixing, and performing ultrasonic treatment for 3 minutes to obtain the kit;
s2, preparing a standard curve equation: pretreating a calibrator, precisely sucking 100 mu L of the calibrator into a 1.0mL centrifuge tube, adding 100 mu L of an internal standard, adding 100 mu L of a protein precipitant I, mixing for 2 minutes at 2000r/min, adding 200 mu L of a protein precipitant II, mixing for 5 minutes at 2000r/min, centrifuging for 10 minutes at 14000r/min, taking 300 mu L of supernatant, centrifuging for 5 minutes at 14000r/min, and taking 100 mu L of the supernatant for on-machine inspection.
Detecting by adopting a high performance liquid chromatography mass spectrometer, and drawing a standard curve by taking the marked concentration of 6 calibrators as an abscissa (x) and the ratio of the actual detection peak area of the 6 calibrators to the peak area of each internal standard peak as an ordinate (y); linear regression of the indicated concentration (x) with peak area ratio (y) yields the regression equation: y=ax+b, where y is the ordinate, x is the abscissa, a is the slope of the curve, and the fitting correlation coefficient (r 2 ) Requirement r 2 Should not be less than 0.990; the standard curve equation is obtained as shown in fig. 1;
s3, obtaining a sample to be tested: precisely sucking 100 mu L of whole blood sample into a 1.0mL centrifuge tube, adding 100 mu L of internal standard substance, adding 100 mu L of protein precipitant I, mixing for 2 minutes at 2000r/min, adding 200 mu L of protein precipitant II, mixing for 5 minutes at 2000r/min, centrifuging for 10 minutes at 14000r/min, taking 300 mu L of supernatant, centrifuging for 5 minutes at 14000r/min, and taking 100 mu L of sample for machine inspection;
s4, detecting a sample to be detected, injecting the processed sample supernatant into a high performance liquid tandem mass spectrometer for detection, and recording the chromatograms, peak areas of tacrolimus, cyclosporine A, rapamycin and mycophenolic acid in the sample and the peak areas of isotope internal standards thereof. Substituting the ratio of the actual peak area of the sample to be detected to the internal standard peak area into the equation, and calculating the concentration of each immunosuppressant in the sample to be detected.
The specific chromatographic conditions are as follows:
a SCIEX tripleQuad 4500 series liquid chromatography tandem mass spectrometry system is adopted, and a chromatographic column is adopted: waters XBiridge C18 column (2.1X100 mm,5 μm); column temperature: 40 ℃; sample injection amount: 20. Mu.L; the gradient elution mode is the same as that of table 1; the Ionspray voltage is 4000V; gas 1:50psi; gas 2:50psi; air curtain gas: 25psi; heating air temperature is 400 ℃; ion pair information is as in table 2.
According to the chromatographic conditions and the mass spectrum conditions, a liquid chromatography-mass spectrometer is arranged, a sample is injected into the instrument for detection and analysis, so that chromatograms of the standard immunosuppressant and the isotope thereof can be obtained, main information of the chromatograms is shown in a table 4 as shown in fig. 2-6, four immunosuppressants and isotope thereof can be seen, the peak type is symmetrical, the response is moderate, and the interference of impurities is basically avoided, thereby providing conditions for accurate quantification of the immunosuppressant to be detected.
Table 4: standard chromatogram information
| Target substance | Area(cps) | RT(min) | IS Area |
| FK-506 | 1565000 | 2.37 | 972600 |
| CsA | 3030000 | 2.5 | 8768000 |
| RPM | 3546000 | 2.01 | 1566000 |
| MPA | 327900 | 2.37 | 58680 |
Four immunosuppressant chromatograms in the whole blood samples are shown in fig. 7-11, main chromatograms are shown in table 5, and the main chromatograms have fewer impurities and partial impurities, but have no influence on target peaks.
Table 5: whole blood sample chromatogram information
| Target substance | Area(cps) | RT(min) | IS Area |
| FK-506 | 936200 | 2.4 | 530400 |
| CsA | 60710 | 2.5 | 4373000 |
| RPM | 80640 | 2.01 | 1100000 |
| MPA | 10010 | 2.4 | 28810 |
Example 3
This example is used to illustrate the method of kit preparation:
1. preparation method of calibration material and quality control material
(1) Preparing: tacrolimus, cyclosporine A, rapamycin, mycophenolic acid and pharmaceutical excipients in the calibrator and quality control at each final concentration were mixed uniformly according to Table 3 in example 1, wherein the concentrations of Tacrolimus and rapamycin were sequentially 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, cyclosporine A was sequentially 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL, and the concentrations of mycophenolic acid were sequentially 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL, 10000ng/mL. During the period, the calibrator and the quality control semi-finished product solution are obtained by taking care of light-shielding and airtight stirring.
(2) And (5) subpackaging: and (3) using a yellow light lamp in the clean area environment, subpackaging each calibrator and quality control semi-finished product solution in a brown ampoule, adding a plug in the second half, and putting into a freeze dryer.
(3) And (3) freeze drying:
lyophilizing the semi-finished product to obtain lyophilized powder reagent in the kit: calibrator, quality control, lyophilization procedure is:
precooling: precooling the equipment for 2 hours at the temperature of-20 ℃;
pre-freezing: placing the sample into equipment, pre-freezing for 4 hours at the temperature of minus 35 ℃;
sublimation: sublimation temperature is minus 25 ℃ and time is 10 hours;
and (5) analysis and drying: maintaining at 15 ℃ for 3 hours;
enhanced drying: maintained at 20℃for 4 hours.
(4) And (3) packaging: and the freeze-drying stage is finished, and the processes of pressing the plug, rolling the cover and packaging are started, and the whole process is finished in a light-proof or yellow light environment.
Experimental example 1
This experimental example is used to illustrate the stability of the kit of the invention:
(1) Placing the semi-finished product prepared in the embodiment 3 in a refrigerator with the temperature of 2-8 ℃ for investigating the stability of the semi-finished product; randomly taking a semi-finished product sample after sub-packaging; the relative deviation between the actual concentration and the theoretical charged concentration was calculated by using the mean value for three samples at each concentration point at five time points of 0 day, 1 day, 3 days, 5 days, and 7 days, and the results are shown in tables 8 to 11:
Table 8: stability investigation of tacrolimus semi-finished product
| C1 | C2 | C3 | C4 | C5 | C6 | Q7 | Q8 | Q9 | |
| Day 0 | 101％ | 100％ | 102％ | 101％ | 102％ | 99％ | 98％ | 101％ | 102％ |
| For 1 day | 103％ | 103％ | 103％ | 103％ | 102％ | 97％ | 100％ | 99％ | 103％ |
| For 3 days | 104％ | 98％ | 101％ | 103％ | 100％ | 98％ | 99％ | 103％ | 102％ |
| For 5 days | 104％ | 102％ | 103％ | 103％ | 102％ | 97％ | 100％ | 99％ | 103％ |
| For 7 days | 106％ | 103％ | 104％ | 97％ | 102％ | 100％ | 101％ | 101％ | 106％ |
Table 9: stability investigation of cyclosporine A semi-finished product
| C1 | C2 | C3 | C4 | C5 | C6 | Q7 | Q8 | Q9 | |
| Day 0 | 98％ | 102％ | 102％ | 101％ | 102％ | 100％ | 99％ | 103％ | 101％ |
| For 1 day | 98％ | 101％ | 101％ | 103％ | 102％ | 98％ | 96％ | 104％ | 100％ |
| For 3 days | 100％ | 104％ | 103％ | 104％ | 102％ | 98％ | 101％ | 104％ | 103％ |
| For 5 days | 99％ | 102％ | 101％ | 102％ | 102％ | 99％ | 101％ | 105％ | 100％ |
| For 7 days | 103％ | 104％ | 103％ | 101％ | 103％ | 102％ | 101％ | 103％ | 104％ |
Table 10: stability investigation of rapamycin semifinished products
| C1 | C2 | C3 | C4 | C5 | C6 | Q7 | Q8 | Q9 | |
| Day 0 | 102％ | 102％ | 102％ | 101％ | 98％ | 102％ | 98％ | 101％ | 100％ |
| For 1 day | 103％ | 104％ | 102％ | 102％ | 101％ | 99％ | 99％ | 98％ | 98％ |
| For 3 days | 103％ | 99％ | 101％ | 102％ | 100％ | 99％ | 99％ | 99％ | 99％ |
| For 5 days | 102％ | 105％ | 101％ | 101％ | 100％ | 99％ | 100％ | 99％ | 99％ |
| For 7 days | 106％ | 103％ | 102％ | 97％ | 102％ | 101％ | 101％ | 96％ | 97％ |
Table 11: stability investigation of mycophenolic acid semi-finished product
| C1 | C2 | C3 | C4 | C5 | C6 | Q7 | Q8 | Q9 | |
| Day 0 | 103％ | 100％ | 101％ | 103％ | 99％ | 101％ | 100％ | 97％ | 101％ |
| For 1 day | 102％ | 98％ | 100％ | 100％ | 100％ | 99％ | 98％ | 99％ | 101％ |
| For 3 days | 103％ | 100％ | 101％ | 104％ | 102％ | 103％ | 98％ | 98％ | 102％ |
| For 5 days | 105％ | 99％ | 103％ | 100％ | 100％ | 99％ | 99％ | 99％ | 100％ |
| For 7 days | 103％ | 101％ | 100％ | 100％ | 101％ | 103％ | 99％ | 98％ | 102％ |
The statistics can be obtained, the semi-finished product is placed in a refrigerator at the temperature of 2-8 ℃ for 5 days, the relative deviation of tacrolimus, cyclosporine A, rapamycin and mycophenolic acid is not more than 3 percent, and the semi-finished product is placed for 7 days and not more than 5 percent, so that the semi-finished product has better stability.
(2) Inspecting the moisture content and the appearance of the freeze-dried product;
4 boxes of the kit prepared in the example 3 are randomly extracted, and a Karl Fischer moisture meter is used for detecting the moisture of the freeze-dried product by a volume titration method. Each sample was measured in duplicate 2 times and its mean was calculated. The experimental results are shown in table 12:
table 12: investigation of the moisture stability of the lyophilized finished product
| C1 | C2 | C3 | C4 | C5 | C6 | Q7 | Q8 | Q9 | |
| 0 month | 1.45％ | 1.50％ | 1.38％ | 1.48％ | 1.45％ | 1.52％ | 1.45％ | 1.58％ | 1.56％ |
| 1 month | 1.50％ | 1.52％ | 1.40％ | 1.43％ | 1.44％ | 1.48％ | 1.49％ | 1.49％ | 1.55％ |
| 3 months of | 1.46％ | 1.52％ | 1.47％ | 1.50％ | 1.48％ | 1.53％ | 1.52％ | 1.54％ | 1.58％ |
| 5 months of | 1.62％ | 1.58％ | 1.52％ | 1.54％ | 1.59％ | 1.56％ | 1.52％ | 1.58％ | 1.62％ |
| 6 months of | 1.58％ | 1.62％ | 1.60％ | 1.57％ | 1.55％ | 1.61％ | 1.58％ | 1.61％ | 1.65％ |
Taking a finished product, storing the finished product in a dark environment at 2-8 ℃ for 6 months, sampling 5 boxes at 0, 1, 3, 5 and 6 months respectively, and examining the appearance change, wherein the experimental result is shown in table 13:
table 13: inspection of the appearance of freeze-dried finished products
/>
The packaged finished product has the moisture content of not more than 3% when stored for 6 months at room temperature in dark place, good packaging tightness and good finished product stability.
(3) And (3) finished product stability inspection: and (3) taking a finished product, storing the finished product in a dark environment at 2-8 ℃ for 6 months, sampling 5 boxes at 0, 1, 3, 5 and 6 months respectively, processing samples according to the requirements on the instruction book of the kit, and calculating the relative deviation between each concentration point and the theoretical concentration. The experimental results are shown in tables 14 to 17:
table 14: tacrolimus finished product stability investigation
/>
Table 15: stability investigation of cyclosporine A finished product
| C1 | C2 | C3 | C4 | C5 | C6 | Q7 | Q8 | Q9 | |
| Day 0 | 100％ | 100％ | 99％ | 100％ | 99％ | 98％ | 101％ | 104％ | 100％ |
| 1 month | 101％ | 102％ | 96％ | 96％ | 95％ | 95％ | 104％ | 99％ | 97％ |
| 3 months of | 103％ | 105％ | 103％ | 103％ | 103％ | 100％ | 102％ | 100％ | 100％ |
| 5 months of | 100％ | 98％ | 98％ | 99％ | 97％ | 97％ | 100％ | 102％ | 99％ |
| 6 months of | 98％ | 96％ | 98％ | 97％ | 96％ | 95％ | 100％ | 100％ | 97％ |
Table 16: stability investigation of rapamycin finished products
| C1 | C2 | C3 | C4 | C5 | C6 | Q7 | Q8 | Q9 | |
| Day 0 | 103％ | 99％ | 99％ | 100％ | 98％ | 99％ | 103％ | 103％ | 102％ |
| 1 month | 101％ | 102％ | 99％ | 98％ | 96％ | 99％ | 100％ | 97％ | 97％ |
| 3 months of | 99％ | 101％ | 99％ | 98％ | 97％ | 98％ | 103％ | 100％ | 102％ |
| 5 months of | 100％ | 98％ | 98％ | 99％ | 96％ | 99％ | 99％ | 100％ | 98％ |
| 6 months of | 100％ | 97％ | 98％ | 97％ | 97％ | 97％ | 100％ | 100％ | 99％ |
Table 17: examination of stability of mycophenolic acid product
| C1 | C2 | C3 | C4 | C5 | C6 | Q7 | Q8 | Q9 | |
| Day 0 | 103％ | 99％ | 99％ | 101％ | 98％ | 99％ | 99％ | 101％ | 104％ |
| 1 month | 97％ | 102％ | 98％ | 100％ | 100％ | 97％ | 96％ | 100％ | 101％ |
| 3 months of | 97％ | 104％ | 100％ | 99％ | 97％ | 98％ | 99％ | 98％ | 104％ |
| 5 months of | 97％ | 95％ | 95％ | 97％ | 96％ | 99％ | 97％ | 98％ | 100％ |
| 6 months of | 96％ | 95％ | 94％ | 96％ | 95％ | 95％ | 97％ | 97％ | 98％ |
The experimental results show that the freeze-dried powder has good stability at the temperature of 2-8 ℃.
Experimental example 2:
the experimental example is used for explaining the linear range and the quantitative lower limit of the kit and the detection method in the embodiment of the invention:
the 6 concentrations of calibrator shown in table 3 were performed to obtain chromatograms according to the method of example 2. The chromatographic peak area-concentration was plotted to give a standard curve, and the results indicate that the linear range and quantitative limit of the 4 immunosuppressants are shown in table 18.
TABLE 18
| Compounds of formula (I) | Detection limit | Quantitative limit | Linear range |
| FK-506(ng/mL) | 0.1 | 0.2 | 1～100 |
| CsA(ng/mL) | 2 | 5 | 10～1000 |
| RPM(ng/mL) | 0.1 | 0.3 | 1～100 |
| MPA(ng/mL) | 1 | 3 | 100～1000 |
The experimental results show that the kit and the detection method provided by the embodiment of the invention have the advantages of wide linear range and low detection limit.
Experimental example 3:
the experimental example is used for explaining the uniformity of the kit and the detection method in the embodiment of the invention:
the experimental method comprises the following steps: the 15-box kit prepared in example 3 was randomly extracted, and three samples were tested for each concentration point of each box of calibrator according to a certain ordering rule, and the in-bottle variation Coefficient (CV) of each concentration point of C1, C2, C3, C4, C5 and C6 In the bottle ) And coefficient of variation between bottles (CV) Bottle room )。
Table 19: calibrator uniformity
As is clear from the above experimental results, (CV) In the bottle ) And coefficient of variation between bottles (CV) Bottle room ) The kit is less than 6% and less than 15% specified by the standard, so that the kit has better uniformity.
Experimental example 4:
the experimental example is used for explaining the precision of the kit and the detection method according to the embodiment of the invention:
the experimental method comprises randomly extracting 1 batch of reagent kit and normal human whole blood sample, synchronously processing 10 human whole blood samples and quality control products at a time, determining the concentrations of four immunosuppressants in the whole blood and the quality control products according to the quantitative method, wherein the quality control products are in the given target value range, and calculating the precision range in batch to be 1.10% -2.54%, as shown in table 20; in addition, 3 batches of kits and normal human whole blood samples are randomly extracted, and the calibrator, the quality control product and the human whole blood samples are synchronously processed in batches;
Table 20: precision in batch
Table 21: precision between batches
And (3) determining the concentration of each immunosuppressant in the quality control product and the serum sample according to the quantitative method, wherein the quality control product is in the given target value range, and the precision range between calculated batches is within 10%.
Experimental example 5:
the experimental example is used for explaining the recovery rate of the kit and the detection method in the embodiment of the invention:
the experimental method comprises the steps of selecting a normal whole blood sample, dividing the normal whole blood sample into 4 equal parts, adding standard solutions of to-be-detected substances with different concentrations and the same volume into 3 samples, preparing 3 samples to be recovered and analyzed with different added concentrations, and ensuring that the final concentration of the samples does not exceed a linear range; the same volume of solvent without the analyte is added to the remaining sample to make a base sample. The recovery rate was calculated using formula (1).
Wherein:
r: recovery rate;
v: adding the volume of standard solution of the object to be detected or solvent without the object to be detected;
V 0 : the volume of the sample;
c: detecting the concentration of the sample after the sample is added into the standard solution;
C label (C) : concentration of standard solution.
Note that: concentration=standard solution concentration× [ standard solution addition volume/(sample volume+standard solution volume) ]
Table 22: recovery rate
As shown by the experimental results, the recovery rate of the kit and the experimental method is 90-110%.
Experimental example 6:
the experimental example is used for explaining the technical effect of adding the types of the pharmaceutical excipients in the embodiment of the invention.
A kit was prepared according to the procedure of example 3, except that:
comparative example 1: the calibrator C4 was prepared by measuring the stability of the lyophilized products without sheep erythrocytes according to the method of experimental example 1, and obtaining the content of each immunosuppressant (obtained by converting the percentage of the theoretical charge to 100%, the measurement result and the theoretical value) as shown in Table 23:
table 23
As can be seen from the data in table 23, the stability of the sample without sheep red blood cells was poor.
Comparative example 2: the calibrator C4 is prepared by adopting bovine whole blood to replace sheep red blood cells, and experiments show that the sample solution is easy to be flocculated after freeze-drying and re-dissolving the bovine whole blood, so that the sample adding accuracy is affected.
Comparative example 3: other substances are adopted in the pharmaceutical excipients of the calibrator C4 to be matched with bovine serum albumin, and the pharmaceutical excipients are specifically shown in Table 24. The appearance, moisture and content (100% of theoretical charge, and the percent conversion between the detection result and the theoretical value) of the freeze-dried product were then detected according to the method of experimental example 1, and the experimental results are shown in table 24:
table 24
As can be seen from Table 24, sucrose, PVP and trehalose are used to replace mannitol, and the product has a atrophy appearance after freeze-drying, has higher moisture content and has poorer freeze-drying effect. If mannitol is not added, only serum albumin is used as a filling agent, and the product is frozen and dried to have appearance fragmentation.
Experimental example 7:
the experimental example is used for explaining the technical effect of the adding proportion of the pharmaceutical excipients in the embodiment of the invention.
Comparative example 4: lyophilized powder was prepared using the pharmaceutical excipients shown in table 25, and the remaining formulation was the same as in example 1 to prepare calibrator C4. The contents (100% of theoretical charge, the percent conversion between the detection result and the theoretical value, and the preservation in the environment of 2-8 ℃ in the dark for 72 hours) and the solution state after the freeze-dried product is redissolved were detected according to the method of experimental example 1, and the experimental results are shown in table 25:
table 25
Wherein, the red blood cells are diluted by phosphate buffer salt with pH7.4 to obtain the red blood cells with the volume percent concentration of 80%, 50% and 20% respectively.
As can be seen from the data in Table 25, the 50% and 80% erythrocyte contents and the stability can be satisfied, and 50% erythrocyte is more preferable from the viewpoints of cost and test workability (80% erythrocyte products are liable to be flocculated).
Comparative example 5: using the excipients shown in table 26, the remaining formulation was the same as in example 1, and calibrator C4 was prepared according to the method of example 3: the content (100% of theoretical charge, and the percent of the detection result and the theoretical value were converted) and the appearance (the state after reconstitution of the lyophilized product) and the moisture were detected according to the method of experimental example 1, and the experimental results were shown in table 26:
Table 26
/>
As shown in Table 26, if the ratio of bovine serum albumin to mannitol is too high, which affects lyophilization water too high, the pre-lyophilization and analytical drying must be performed for an extended period of time during the lyophilization process, increasing the lyophilization cost. And after freeze-drying and re-dissolving, the sample is easy to be flocculated, and the sample loading accuracy is affected.
Example 8:
the experimental example is used for explaining the technical effect of the freeze-drying curve of the embodiment of the invention;
calibration material C4 was prepared as in example 3, except that the lyophilization curve shown in table 27 was used.
Comparative example 6: and (3) in the precooling process, putting the product into a freeze-drying box at the temperature of minus 35 ℃ for 2 hours.
Comparative example 7: analytical drying, temperature and conditions are shown in Table 27.
The content of each immunosuppressant (obtained by converting the percentage of the detection result and the theoretical value based on the theoretical charge) and the experimental result of the pre-frozen state are shown in table 27:
table 27
As can be seen from table 27, the pre-frozen effect product of fast freeze-drying was less good in appearance and content than the slow freeze effect. The increase in the analytical drying temperature and the prolonged time do not improve the moisture and increase the lyophilization cost.
Example 9:
the present example is used to illustrate the technical effects of a protein precipitant and a pretreatment method:
comparative example 9: when the volume ratio of the calibrator C4 to the protein precipitant II is 1: 1. 1: 2. 1:3, the other conditions were the same as in example 2, and the content (obtained by converting the percentage of the theoretical value to the theoretical feed of 100%) was measured in accordance with the method of experimental example 1. The experimental results obtained are shown in table 28:
Table 28
From the experimental results in table 28, the volume ratio of the sample solution to the protein precipitant II was 1:2, the erythrocytes are fully cracked, and the recovery rate of the to-be-detected substance is better.
Comparative example 10: the content of each immunosuppressant in the calibrator C4 (obtained by converting the percentage of the theoretical value to the numerical value of the detection result with the theoretical value by taking the theoretical charge as 100%) was measured by the method of experimental example 1, with only the change of the concentration of the protein precipitant I. The experimental results are shown in table 29.
Table 29
As shown in the experimental results of Table 29, the concentration of the protein precipitant I was 0.2mol/L, and erythrocytes were sufficiently lysed, so that the recovery rate of the analyte was good.
Comparative example 11: only the type of protein precipitant II was changed, and the effect of the protein precipitant II on centrifugal precipitation during the treatment of the calibrator C4 sample was examined, as shown in Table 30.
Table 30
| Protein precipitant II species | Centrifugal sedimentation effect |
| Acetonitrile | Precipitation is complete |
| N-hexane | Poor sedimentation effect, floating small particles |
As is clear from the experimental results in Table 30, acetonitrile has a better effect of acetonitrile precipitation than n-hexane.
Comparative example 12: only the volume ratio of the calibrator C4 sample solution to the protein precipitant II was changed, and the content of each immunosuppressant in the calibrator C4 (obtained by converting the percentage of the theoretical batch to 100% and the detection result value and the theoretical value) was detected according to the method of experimental example 1. The experimental results are shown in table 31.
Table 31
From the experimental results in table 31, the volume ratio of the sample solution to the protein precipitant II was 1:2, the content of the object to be detected is better, and the sample after the category treatment is cleaner.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. The kit for detecting the immunosuppressant in the whole blood by using the high performance liquid chromatography tandem mass spectrometry is characterized by comprising a calibrator, an internal standard, a mobile phase stock solution and a protein precipitant;
the calibrator contains immunosuppressant and pharmaceutical adjuvant with serial concentrations; the immunosuppressant comprises tacrolimus, cyclosporine A, rapamycin and mycophenolic acid;
the kit also comprises a quality control product, wherein the quality control product contains low, medium and high-concentration immunosuppressants and pharmaceutic adjuvants; the immunosuppressant comprises tacrolimus, cyclosporine A, rapamycin and mycophenolic acid;
The calibrator and the quality control product are freeze-dried powder; the freeze-dried powder contains serial concentration of the immunosuppressant and pharmaceutical excipients,
the pharmaceutical excipients comprise:
7-12 parts of excipient;
100-800 parts by volume of sheep red blood cells;
100-700 parts by volume of phosphate buffer salt;
the volume percentage concentration of the red blood cells is 50% -70%;
the excipient is selected from bovine serum albumin and mannitol;
the pH value of the phosphate buffer salt is 7.0-7.4;
when the unit of parts by weight is g, the unit of parts by volume is mL.
2. The kit according to claim 1, wherein,
the series of concentrations of immunosuppressant in the calibrator comprises 6 concentrations of immunosuppressant, the 6 concentrations of immunosuppressant being:
calibrator C1: tacrolimus 0.9-1.1 ng/mL, cyclosporine A9-11 ng/mL, rapamycin 0.9-1.1 ng/mL, mycophenolic acid 90-110 ng/mL;
calibrator C2: tacrolimus 4.5-5.5 ng/mL, cyclosporine A45-55 ng/mL, rapamycin 4.5-5.5 ng/mL, mycophenolic acid 450-550 ng/mL;
calibrator C3: tacrolimus 9-11 ng/mL, cyclosporine A90-110 ng/mL, rapamycin 9-11 ng/mL, mycophenolic acid 900-1100 ng/mL;
Calibrator C4: tacrolimus 18-22 ng/mL, cyclosporine A180-220 ng/mL, rapamycin 18-22 ng/mL, mycophenolic acid 1800-2200 ng/mL;
calibrator C5: tacrolimus 45-55 ng/mL, cyclosporine A450-550 ng/mL, rapamycin 45-55 ng/mL, mycophenolic acid 4500-5500 ng/mL;
calibrator C6: tacrolimus 90-110 ng/mL, cyclosporine A900-1100 ng/mL, rapamycin A90-110 ng/mL, mycophenolic acid 9000-11000 ng/mL;
the series of concentrations of immunosuppressant in the quality control product comprises 3 concentrations of immunosuppressant, including:
low value quality control Q7: tacrolimus 1.8-2.2 ng/mL, cyclosporine A18-22 ng/mL, rapamycin 1.8-2.2 ng/mL, mycophenolic acid 180-220 ng/mL;
median quality control Q8: tacrolimus 9-11 ng/mL, cyclosporine A180-220 ng/mL, rapamycin 9-11 ng/mL, mycophenolic acid 900-1100 ng/mL;
high value quality control Q9: tacrolimus 72-88 ng/mL, cyclosporine A720-880 ng/mL, rapamycin 72-88 ng/mL, mycophenolic acid 7200-8800 ng/mL.
3. The kit according to claim 1, wherein,
the mobile phase comprises a mobile phase A stock solution and a mobile phase B stock solution, wherein the mobile phase A stock solution is an aqueous solution containing formic acid with the volume percentage concentration of 2-8% and ammonium acetate with the concentration of 0.5-5 mM; the mobile phase B stock solution is a methanol solution containing formic acid with the volume percentage concentration of 2-8%;
The inner standard is acetonitrile solution containing the inner standard, and the inner standard is ascomycin, cyclosporin A-d4, rapamycin-d 3 and mycophenolic acid-d 3; the concentration of ascomycin in the inner standard substance is 0.009-0.011 [ mu ] g/mL, the concentration of cyclosporin A-d4 is 0.4-0.6 [ mu ] g/mL, the concentration of rapamycin-d 3 is 0.01-0.03 [ mu ] g/mL, and the concentration of mycophenolic acid-d 3 is 0.8-1.2 [ mu ] g/mL;
the protein precipitant comprises a protein precipitant I and a protein precipitant II, wherein the protein precipitant I is an aqueous solution of zinc sulfate with the concentration of 0.1-0.3 mol/L; the protein precipitant II is acetonitrile solution containing 0-5% of methanol by volume percent.
4. A method of preparing the kit of any one of claims 1 to 3, comprising the steps of preparing the calibrator and the quality control material, comprising at least:
s1, taking the immunosuppressant and the pharmaceutic adjuvant according to a proportion, and preparing to obtain a solution;
s2, subpackaging the solution;
s3, freeze drying, wherein the freeze drying at least comprises the following steps:
s31, precooling: precooling the equipment for 1-3 hours at-20 ℃ to-50 ℃;
s32, pre-freezing: pre-freezing for 2-5 hours at the temperature of minus 30 ℃ to minus 50 ℃;
S33, sublimating: sublimating for 8-12 hours at the temperature of-20 ℃ to-35 ℃;
s34, analysis and drying: the time is 3-5 hours, and the temperature is 0-20 ℃;
s35, reinforcing drying: the time is 3-10 hours, and the temperature is 15-25 ℃;
s36, packaging.
5. A method for detecting immunosuppressant in whole blood using the kit of claim 3, comprising at least the steps of:
s1, preparing a solution: comprising the following steps:
dissolving the calibrator and the quality control product with water respectively for standby; diluting the mobile phase A stock solution with water to obtain a mobile phase A; diluting the mobile phase B stock solution with methanol to obtain a mobile phase B for later use;
s2, preparing a standard curve equation, which comprises the following steps:
pretreating the calibrator, and detecting by adopting a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation;
s3, obtaining a sample to be tested, including: taking a whole blood sample to be tested or a quality control product, and obtaining the sample to be tested after pretreatment;
s4, detecting the sample to be detected, including: detecting the sample to be detected by using a high performance liquid chromatography-mass spectrometer, and substituting a detection result into the standard curve equation to obtain the concentration of the immunosuppressant in the sample to be detected;
In S2 and S3, the preprocessing includes: taking a sample solution, and adding an internal standard substance, wherein the sample solution comprises a whole blood sample to be tested, a calibrator and a quality control substance; adding a protein precipitator I, uniformly mixing, adding a protein precipitator II, uniformly mixing, centrifuging, and taking supernatant as a sample to be detected for chromatographic analysis;
the method also comprises the step of adopting the quality control quality evaluation detection method to evaluate the precision.
6. The method of claim 5, wherein, in S2 and S3,
the volume ratio of the sample solution to the internal standard is 1:0.5 to 2; the volume ratio of the sample solution to the protein precipitant I is 1:0.5 to 2; the volume ratio of the sample solution to the protein precipitant II is 1: 1-3.
7. The method of claim 5, wherein the conditions of the high performance liquid chromatography are: the chromatographic column is selected from C18 columns, and the particle size is 2-5 mu m; the column temperature is 25-45 ℃; the flow rate is 0.3-1 mL/min;
the gradient elution conditions were:
0-0.5 min, mobile phase A: 30-50%, mobile phase B: 50-70%;
0.51-2 minutes, mobile phase A: 1-5%, mobile phase B: 5-99%;
2.1 to 4 minutes, mobile phase A: 30-50%, mobile phase B: 50-70%;
The conditions of the mass spectrum are:
an Ionspray voltage; 0-5500V; gas 1: 40-60 psi; gas 2: 0-90 psi; air curtain gas: 20-50 psi; heating air temperature: 150-550 ℃;
ion pair information is:
tacrolimus: parent ion 821.6, child ion 768.5, collision energy 85 eV;
rapamycin: parent ion 931.7, child ion 864.5, collision energy 80 eV;
cyclosporin a: parent ion 1220, child ion 1202.9, collision energy 40 eV;
mycophenolic acid: parent ion 321.1, child ion 207, collision energy 75 eV.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210638740.2A CN115236216B (en) | 2022-06-07 | 2022-06-07 | Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210638740.2A CN115236216B (en) | 2022-06-07 | 2022-06-07 | Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115236216A CN115236216A (en) | 2022-10-25 |
| CN115236216B true CN115236216B (en) | 2024-03-01 |
Family
ID=83668729
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210638740.2A Active CN115236216B (en) | 2022-06-07 | 2022-06-07 | Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115236216B (en) |
Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1270529A (en) * | 1997-09-18 | 2000-10-18 | 人类Rt·公司 | Pharmaceutical compositions containing plasma protein |
| CN101953807A (en) * | 2010-10-09 | 2011-01-26 | 山西普德药业有限公司 | Mycophenolate mofetil lyophilized powder injection for injection and preparation method thereof |
| CN102357077A (en) * | 2011-09-30 | 2012-02-22 | 中国药科大学 | Protein nanometer particle for wrapping slightly soluble medicines and preparation method thereof |
| CN104160273A (en) * | 2012-03-07 | 2014-11-19 | 西门子医疗保健诊断公司 | Sandwich assay for immunosuppressant drugs |
| CN107266563A (en) * | 2017-08-01 | 2017-10-20 | 四川沃文特生物技术有限公司 | A kind of preparation method of hemoglobin quality-control product |
| CN109030673A (en) * | 2018-07-04 | 2018-12-18 | 易达精准（杭州）科技有限公司 | The detection method and detection kit of immunosuppressor in dried blood spot |
| CN109187839A (en) * | 2018-10-25 | 2019-01-11 | 美康生物科技股份有限公司 | The kit and detection method of four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
| CN109406650A (en) * | 2018-10-25 | 2019-03-01 | 美康生物科技股份有限公司 | Kit and detection method for four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
| CN110470753A (en) * | 2019-07-24 | 2019-11-19 | 天津国科医工科技发展有限公司 | Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake |
| CN110554123A (en) * | 2019-09-11 | 2019-12-10 | 深圳华大临床检验中心 | Method and kit for rapidly detecting immunosuppressant in whole blood and application of kit |
| CN110849694A (en) * | 2019-12-02 | 2020-02-28 | 北京丹大生物技术有限公司 | Tacrolimus whole blood sample pretreatment liquid and use method and application thereof |
| CN111678874A (en) * | 2019-01-09 | 2020-09-18 | 北京九强生物技术股份有限公司 | 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of cyclosporine A detection reagent |
| CN111856044A (en) * | 2019-10-30 | 2020-10-30 | 美康生物科技股份有限公司 | Quality control substance of whole blood type freeze-dried powder immunosuppressant and preparation method and application thereof |
| CN112213417A (en) * | 2020-09-16 | 2021-01-12 | 苏州药明泽康生物科技有限公司 | Kit and method for detecting concentration of mycophenolic acid medicine in dried blood spots |
| CN113533555A (en) * | 2021-06-17 | 2021-10-22 | 杭州凯莱谱精准医疗检测技术有限公司 | Detection kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry and detection method thereof |
| CN114354820A (en) * | 2021-11-30 | 2022-04-15 | 苏州药明泽康生物科技有限公司 | Kit and method for detecting concentration of cyclosporine drug in dried blood slice sample |
-
2022
- 2022-06-07 CN CN202210638740.2A patent/CN115236216B/en active Active
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1270529A (en) * | 1997-09-18 | 2000-10-18 | 人类Rt·公司 | Pharmaceutical compositions containing plasma protein |
| CN101953807A (en) * | 2010-10-09 | 2011-01-26 | 山西普德药业有限公司 | Mycophenolate mofetil lyophilized powder injection for injection and preparation method thereof |
| CN102357077A (en) * | 2011-09-30 | 2012-02-22 | 中国药科大学 | Protein nanometer particle for wrapping slightly soluble medicines and preparation method thereof |
| CN104160273A (en) * | 2012-03-07 | 2014-11-19 | 西门子医疗保健诊断公司 | Sandwich assay for immunosuppressant drugs |
| CN107266563A (en) * | 2017-08-01 | 2017-10-20 | 四川沃文特生物技术有限公司 | A kind of preparation method of hemoglobin quality-control product |
| CN109030673A (en) * | 2018-07-04 | 2018-12-18 | 易达精准（杭州）科技有限公司 | The detection method and detection kit of immunosuppressor in dried blood spot |
| CN109187839A (en) * | 2018-10-25 | 2019-01-11 | 美康生物科技股份有限公司 | The kit and detection method of four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
| CN109406650A (en) * | 2018-10-25 | 2019-03-01 | 美康生物科技股份有限公司 | Kit and detection method for four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood |
| CN111678874A (en) * | 2019-01-09 | 2020-09-18 | 北京九强生物技术股份有限公司 | 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of cyclosporine A detection reagent |
| CN110470753A (en) * | 2019-07-24 | 2019-11-19 | 天津国科医工科技发展有限公司 | Four kinds of immunosuppressor detection methods and detection kit in a kind of dry blood cake |
| CN110554123A (en) * | 2019-09-11 | 2019-12-10 | 深圳华大临床检验中心 | Method and kit for rapidly detecting immunosuppressant in whole blood and application of kit |
| CN111856044A (en) * | 2019-10-30 | 2020-10-30 | 美康生物科技股份有限公司 | Quality control substance of whole blood type freeze-dried powder immunosuppressant and preparation method and application thereof |
| CN110849694A (en) * | 2019-12-02 | 2020-02-28 | 北京丹大生物技术有限公司 | Tacrolimus whole blood sample pretreatment liquid and use method and application thereof |
| CN112213417A (en) * | 2020-09-16 | 2021-01-12 | 苏州药明泽康生物科技有限公司 | Kit and method for detecting concentration of mycophenolic acid medicine in dried blood spots |
| CN113533555A (en) * | 2021-06-17 | 2021-10-22 | 杭州凯莱谱精准医疗检测技术有限公司 | Detection kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry and detection method thereof |
| CN114354820A (en) * | 2021-11-30 | 2022-04-15 | 苏州药明泽康生物科技有限公司 | Kit and method for detecting concentration of cyclosporine drug in dried blood slice sample |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115236216A (en) | 2022-10-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113533555B (en) | Detection kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry and detection method thereof | |
| CN109406650A (en) | Kit and detection method for four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood | |
| CN110554123A (en) | Method and kit for rapidly detecting immunosuppressant in whole blood and application of kit | |
| CN109187839A (en) | The kit and detection method of four kinds of immunosuppressant drug concentrations in Accurate Determining people's whole blood | |
| KR20090115930A (en) | Serum proteomics system and associated methods | |
| CN110441515B (en) | Fecal occult blood detection kit | |
| CN111856044A (en) | Quality control substance of whole blood type freeze-dried powder immunosuppressant and preparation method and application thereof | |
| CN114935619A (en) | Method and kit for detecting water-soluble vitamins | |
| CN113933419A (en) | Method for determining concentration of 5 immunosuppressive agents in human whole blood | |
| CN115248272A (en) | Method for detecting alpha-ketoglutaric acid and chiral 2-hydroxyglutaric acid | |
| CN115236216B (en) | Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof | |
| CN110940742A (en) | Method for detecting concentration of blood disease related medicine and application | |
| CN116500158A (en) | Blood sample antiepileptic drug detection method and kit | |
| CN112798708A (en) | Kit for detecting fat-soluble vitamins | |
| CN115494182A (en) | Method and kit for detecting 18 antibiotics in blood by liquid chromatography-tandem mass spectrometry | |
| CN114509526A (en) | LC-MS/MS-based pure solvent type whole blood immunosuppressant drug concentration joint inspection kit and detection method | |
| CN113049726A (en) | Method for detecting immunosuppressant drug in whole blood | |
| KR20180103961A (en) | Biological analysis of insulin analogues | |
| CN111638285A (en) | Method for detecting concentration of abamectin and ceftazidime in blood plasma | |
| CN113156027A (en) | Derivatization method of carboxyl metabolites and efficient non-targeted metabonomics analysis method | |
| CN113109493A (en) | Method for measuring rifampicin in plasma by high performance liquid chromatography-mass spectrometry | |
| CN111551649A (en) | Kit for detecting concentration of avibactam and ceftazidime in blood plasma and application thereof | |
| CN109298081B (en) | Method for determining impurity A biological sample in Cetilistat | |
| CN112067709A (en) | Method for determining cefodizime sodium related substance for injection and application | |
| CN116026949A (en) | Method for detecting four immunosuppressants in whole blood |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |