CN117030918A - Method for determining concentration of progesterone in human plasma by using liquid chromatography-tandem mass spectrometry - Google Patents
Method for determining concentration of progesterone in human plasma by using liquid chromatography-tandem mass spectrometry Download PDFInfo
- Publication number
- CN117030918A CN117030918A CN202311293042.4A CN202311293042A CN117030918A CN 117030918 A CN117030918 A CN 117030918A CN 202311293042 A CN202311293042 A CN 202311293042A CN 117030918 A CN117030918 A CN 117030918A
- Authority
- CN
- China
- Prior art keywords
- progesterone
- concentration
- plasma
- human plasma
- methanol
- 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.)
- Granted
Links
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 title claims abstract description 263
- 239000000186 progesterone Substances 0.000 title claims abstract description 131
- 229960003387 progesterone Drugs 0.000 title claims abstract description 130
- 238000000034 method Methods 0.000 title claims abstract description 92
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 58
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 93
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims abstract description 53
- 229940050526 hydroxyethylstarch Drugs 0.000 claims abstract description 53
- 239000003058 plasma substitute Substances 0.000 claims abstract description 50
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 38
- 238000004458 analytical method Methods 0.000 claims abstract description 37
- 239000012086 standard solution Substances 0.000 claims abstract description 34
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000002360 preparation method Methods 0.000 claims abstract description 28
- RJKFOVLPORLFTN-PQIPVKAESA-N (8s,9s,10r,13s,14s,17s)-2,2,4,6,6,17-hexadeuterio-10,13-dimethyl-17-(2,2,2-trideuterioacetyl)-7,8,9,11,12,14,15,16-octahydro-1h-cyclopenta[a]phenanthren-3-one Chemical compound C([C@]1(C)[C@@]([2H])(C(=O)C([2H])([2H])[2H])CC[C@H]1[C@@H]1CC2([2H])[2H])C[C@@H]1[C@]1(C)C2=C([2H])C(=O)C([2H])([2H])C1 RJKFOVLPORLFTN-PQIPVKAESA-N 0.000 claims abstract description 18
- 239000000203 mixture Substances 0.000 claims abstract description 17
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 11
- 238000005119 centrifugation Methods 0.000 claims abstract description 9
- 239000012074 organic phase Substances 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- 239000013558 reference substance Substances 0.000 claims abstract description 7
- 238000001819 mass spectrum Methods 0.000 claims abstract description 6
- 238000000132 electrospray ionisation Methods 0.000 claims abstract description 5
- 238000010828 elution Methods 0.000 claims abstract description 5
- 210000002381 plasma Anatomy 0.000 claims description 111
- 239000008354 sodium chloride injection Substances 0.000 claims description 31
- 238000012360 testing method Methods 0.000 claims description 24
- 239000007901 soft capsule Substances 0.000 claims description 17
- 238000009472 formulation Methods 0.000 claims description 16
- 239000011550 stock solution Substances 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 9
- 238000004811 liquid chromatography Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 238000004445 quantitative analysis Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- 238000003556 assay Methods 0.000 claims description 5
- 239000002775 capsule Substances 0.000 claims description 5
- 239000008213 purified water Substances 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 229940036998 hypertonic sodium chloride Drugs 0.000 claims description 4
- 238000012417 linear regression Methods 0.000 claims description 4
- 238000012544 monitoring process Methods 0.000 claims description 4
- 239000008186 active pharmaceutical agent Substances 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 3
- 239000000583 progesterone congener Substances 0.000 claims description 3
- 230000001681 protective effect Effects 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000005096 rolling process Methods 0.000 claims description 2
- 238000003260 vortexing Methods 0.000 claims description 2
- 238000011156 evaluation Methods 0.000 abstract description 9
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 abstract description 3
- 239000008194 pharmaceutical composition Substances 0.000 abstract 1
- 238000004885 tandem mass spectrometry Methods 0.000 abstract 1
- 239000011159 matrix material Substances 0.000 description 36
- 239000000523 sample Substances 0.000 description 35
- 238000000605 extraction Methods 0.000 description 32
- 238000011084 recovery Methods 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000002904 solvent Substances 0.000 description 15
- 238000003908 quality control method Methods 0.000 description 13
- 239000012472 biological sample Substances 0.000 description 12
- 238000005259 measurement Methods 0.000 description 12
- 229940079593 drug Drugs 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 230000000291 postprandial effect Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000012491 analyte Substances 0.000 description 4
- 238000012937 correction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- AURFZBICLPNKBZ-YZRLXODZSA-N 3alpha-hydroxy-5beta-pregnan-20-one Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H](C(=O)C)[C@@]2(C)CC1 AURFZBICLPNKBZ-YZRLXODZSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 3
- AURFZBICLPNKBZ-UHFFFAOYSA-N Pregnanolone Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(=O)C)C1(C)CC2 AURFZBICLPNKBZ-UHFFFAOYSA-N 0.000 description 3
- 230000036470 plasma concentration Effects 0.000 description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
- DOMWKUIIPQCAJU-LJHIYBGHSA-N Hydroxyprogesterone caproate Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)CCCCC)[C@@]1(C)CC2 DOMWKUIIPQCAJU-LJHIYBGHSA-N 0.000 description 2
- 206010021137 Hypovolaemia Diseases 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- SYGWHBDCTWDQJT-UHFFFAOYSA-N dichloromethane;hexane;propan-2-ol Chemical compound ClCCl.CC(C)O.CCCCCC SYGWHBDCTWDQJT-UHFFFAOYSA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- 229950000801 hydroxyprogesterone caproate Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000013062 quality control Sample Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000013215 result calculation Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 description 1
- DBPWSSGDRRHUNT-UHFFFAOYSA-N 17alpha-hydroxy progesterone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C(=O)C)(O)C1(C)CC2 DBPWSSGDRRHUNT-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 description 1
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 1
- 101100289061 Drosophila melanogaster lili gene Proteins 0.000 description 1
- 206010013908 Dysfunctional uterine bleeding Diseases 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 208000008899 Habitual abortion Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000019255 Menstrual disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 1
- 206010027514 Metrorrhagia Diseases 0.000 description 1
- 102000014961 Protein Precursors Human genes 0.000 description 1
- 108010078762 Protein Precursors Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003263 anabolic agent Substances 0.000 description 1
- 229940070021 anabolic steroids Drugs 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 229960004544 cortisone Drugs 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 229950007402 eltanolone Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 235000020937 fasting conditions Nutrition 0.000 description 1
- 238000010579 first pass effect Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000002546 full scan Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- DNZMDASEFMLYBU-RNBXVSKKSA-N hydroxyethyl starch Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O.OCCOC[C@H]1O[C@H](OCCO)[C@H](OCCO)[C@@H](OCCO)[C@@H]1OCCO DNZMDASEFMLYBU-RNBXVSKKSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000001172 liquid--solid extraction Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229960001207 micronized progesterone Drugs 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 229940022719 progesterone 200 mg Drugs 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000012795 verification 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method for determining the concentration of progesterone in human plasma by using liquid chromatography-tandem mass spectrometry, which comprises the following steps: providing a pharmaceutical composition comprising progesterone, progesterone-d 9 Reagent reagents such as a reference substance, a hydroxyethyl starch-based blood volume expander and the like; providing a tandem mass spectrometer and a high performance liquid chromatography system; using C 18 The chromatographic column is subjected to gradient elution by a water-methanol system; the mass spectrum adopts an electrospray ionization source, human plasma is taken and placed in a centrifuge tube, internal standard solution, normal hexane and vortex are sequentially added, mixed and centrifuged, an upper organic phase is taken and placed in the centrifuge tube which is added with isopropanol in advance, the mixture is concentrated to dryness by centrifugation at 35 ℃, and after residues are dissolved by 50% methanol, LC-MS/MS analysis is carried out; additionally preparing a standard curve; substituting the result obtained according to the chromatogram of the human plasma sample into a standard curve, and calculating to obtain the concentration of the progesterone in the human plasma. The chromatographic tandem mass spectrometry method can effectively determine the concentration of the progesterone in human plasma, and can be applied to bioequivalence evaluation of the progesterone preparation.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a method for detecting the concentration of a drug in a human biological sample such as human blood plasma, in particular to a method for detecting the concentration of progesterone in a human biological sample such as human blood plasma, more particularly relates to a method for detecting the concentration of progesterone in human blood plasma by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and the method can be applied to bioequivalence evaluation of a progesterone preparation.
Background
Progesterone (progestrone, also known as progesterone) is a natural progestogen secreted by the corpus luteum of the ovary, which, together with estrogens, is involved in the regulation of the hypothalamic-pituitary ovarian axis [ Kawaguchi M, fujii SI, itoh N, et al Development of vial wall sorptive extraction and its application to determination of progesterone in human serum.J Chromatogr A, 2009, 1216: 7553-7557]. Clinically used for treating menstrual disorder, habitual abortion, dysfunctional uterine bleeding and other diseases [ Athanasiadou I, angelis YS, lyris E, et al Chemical derivatization to enhance ionization of anabolic steroids in LC-MS for doping-control analysis ].Trends in Analytical Chemistry, 2013, 42: 137-156]. At present, new applications of progesterone (including new administration routes, new indications and the like) are continuously developed, and physiological effects and in-vivo metabolic laws of the progesterone are continuously studied in depth [ Wang HB, liu MZ, fu Q, et al Pharmacokinetics of hard micronized progesterone capsules via vaginal or oral route compared with soft micronized capsules in healthy postmenopausal women: a randomized open-label clinical study [ J ] ]. Drug Des Devel Ther, 2019, 13: 2475–2482]。
The progesterone content in the body is very low, so that the concentration in the body is difficult to detect by adopting the traditional HPLC-UV, GC-MS and other methods. Clinically, the content of progesterone is determined by a Radioimmunoassay (RIA) with higher sensitivity, but the method is extremely susceptible to various steroid hormones with similar structures in the body due to cross-immune reaction, so that a false positive result [ Andreen L, spigset O, andersson A, et al Pharmacokinetics of progesterone and its metabolites allopregnanolone and pregnanolone after oral administration of low-dose progesterone ] appears.Maturitas2006, 54 (44): 238-244; sun Lili determination of the concentration of Progesterone in rat plasma by liquid chromatography-tandem Mass Spectrometry [ J]International journal of pharmaceutical research, 2015, 42 (1): 107-111]。
LC-MS/MS method is considered as a gold standard for quantitative analysis and is widely used for the determination of drugs in biological samplesThere are several documents currently reporting LC-MS/MS methods for determining progesterone concentrations [ Mayne 1G, bloom Ede, dabelea D, et al Development and validation of an LC-MS/MS assay for the quantification of allopregnanolone and its progesterone-determined isomers, pre-cursors, and cotriol/cortisone in pregnancy in humans or animals. Anal Bioanal Chem, 2021, 413: 5427-5438；Rajeswara RP, Someswara RK. Intra subject variability of progesterone 200 mg soft capsules in indian healthy adult postmenopausal female subjects under fasting conditions. J Bioequiv Availab, 2014, 6: 139-143；Zhang S, Mada SR, Sharma S, Torch M, et al. Simultaneous quantitation of 17α-hydroxyprogesterone caproate, 17α-hydroxyprogesterone and progesterone in human plasma using high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). J Pharm Biomed Anal2008, 48:1174-1180; li Rui, chen Ying, chen Xun, zhang Zijiang. Pharmacokinetic studies of Progesterone formulations in Beagle dogs [ J]Pharmaceutical research, 2022, 41:709-712; gu Linu L. Taxifei Murr Zhao Wenhui, gao Xiaoli liquid chromatography-tandem mass spectrometry for determining progesterone concentration in Beagle canine plasma and pharmacokinetics [ J ]]Journal of pharmaceutical analysis, 2011, 31:2040-2045; zhang Li high performance liquid chromatography for determining concentration of progesterone and its metabolites in rat plasma and uterus [ J ]]Pharmaceutical journal, 2004, 39:613-617]. However, progesterone is taken as an endogenous substance, a certain baseline value exists in a blank matrix, so that the establishment of a standard curve by adopting a proper blank matrix and how to prepare a quality control sample to evaluate the accuracy of a measurement result are difficulties and important points of detection, but the above documents do not mention the influence of the problem, and do not attempt to overcome the related problems. In addition, in the prior art, solid phase extraction with high cost or a secondary extraction pretreatment technology is often adopted to improve the sensitivity, so that the operation is complicated, and the flux during biological sample analysis cannot be improved. And the dosage of the plasma is relatively large, the sensitivity is relatively low, and the method is not suitable for evaluating the bioequivalence of the human body.
Thus, it would be highly desirable for those skilled in the art to be able to establish a sensitive and reliable bioassay method for accurately quantifying the concentration of progesterone in plasma, for example, as a key method for evaluating the bioequivalence of a progesterone formulation.
Disclosure of Invention
The object of the present invention is to provide a sensitive and reliable bioassay method for accurately quantifying the concentration of progesterone in blood plasma, for example as a key method for evaluating the bioequivalence of progesterone preparations. For example, the present invention aims to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method by which the concentration of progesterone in human plasma is determined, enabling the method to be applied to the bioequivalence evaluation of progesterone formulations. It has been found that the object of one or more aspects of the invention can be achieved by using the method of the invention, and the invention has been completed based on such findings.
To this end, a first aspect of the present invention provides a method for determining the concentration of progesterone in human plasma using liquid chromatography tandem mass spectrometry, the method comprising the steps of:
s1: providing reagent reagents
Comprises progesterone control, progesterone-d 9 Reference substance, chromatographic grade methanol, n-hexane and isopropanol, purified water for chromatography, hydroxyethyl starch-based blood volume expander;
S2: providing a test instrument
Comprises a tandem mass spectrometer and a high performance liquid chromatography system;
s3: providing liquid chromatography conditions and mass spectrometry conditions:
the chromatographic column used in the liquid chromatography is C 18 Column, protective column C 18 The column was eluted with the following gradient elution procedure: a is water, B is methanol, 0-0.8 min,70% -95% of B; 0.8-1.8 min,95% B; 1.8-2.0 min,95% -70% B; 2.0-3.0 min,70% B;
the mass spectrum is detected by adopting an electrospray ionization source and a positive ion mode; the spray voltage is 5000V; source temperature of 600 o C, performing operation; the curtain rolling gas is 20psi; the collision gas is 8psi; the scanning mode is multi-reaction monitoring, and the ion reaction for quantitative analysis is as follows: progesteronem/z 315.2→m/z97.0 with CE of 27V, progesterone-d 9 Internal standardm/z 324.3→m/z100.0 with CE of 27V, the scanning time is 150msec;
s4: preparation of stock solution
The progesterone control series standard solution is prepared: precisely weighing progesterone reference substance, dissolving with appropriate amount of methanol to obtain 1.0mg/mL stock solution, and freeze-preserving at-20deg.C; precisely transferring a certain volume of stock solution, diluting with 50% methanol to obtain serial standard solutions with the concentration of serial a of 0.400, 0.800, 2.00, 10.0, 50.0, 180, 480, 600ng/mL and serial b of 1.00, 2.00, 10.0, 40.0, 200, 600, 1800 and 2000ng/mL, and storing in a refrigerator at 4 ℃ for standby;
Progesterone-d 9 Preparing an internal standard solution: progesterone-d 9 Dissolving the control with methanol to obtain 1.0mg/mL progesterone-d 9 Freezing and preserving the stock solution at-20 ℃ for standby; precisely transferring a certain volume of stock solution, diluting with 50% methanol to obtain internal standard solutions with the concentrations of 25.0ng/mL and 100ng/mL respectively, and storing in a refrigerator at 4 ℃ for later use;
preparation of plasma substitutes: measuring hydroxyethyl starch-based blood volume expander and mixing with methanol according to a volume ratio of 4:1, and uniformly mixing by vortex for later use;
s5: plasma sample pretreatment and LC-MS/MS analysis
For human plasma with a progesterone concentration in the range of 0.0200-30.0 ng/mL, i.e., linear range 1: 200 mu L of human plasma is taken and placed in a 2mL centrifuge tube, and 20 mu L of internal standard solution, namely 25.0ng/mL progesterone-d, is added in sequence 9 In (2) and 500. Mu.L of n-hexane, vortex mixing, centrifuging at 10000rpm for 3min, placing 400. Mu.L of the upper organic phase into a 1.5mL centrifuge tube with 20. Mu.L of isopropanol added in advance, centrifuging at 35 ℃ for concentration to dryness, dissolving the residue with 100. Mu.L of 50% methanol, sucking 10. Mu.L for LC-MS/MS analysis, and recording a chromatogram;
for human plasma with a progesterone concentration in the range of 0.0500 to 100ng/mL, i.e. linear range 2: taking 100 mu L of human plasma, placing into a 1.5mL centrifuge tube, sequentially adding 20 mu L of internal standard solution (100 ng/mL progesterone-d) 9 Is mixed with 200 mu L of n-hexane by vortexing for 2min and centrifuged at 10000rpm for 3min, 150 mu L of the upper organic phase is taken and placed in a 1.5mL centrifuge tube into which 20 mu L of isopropanol is added in advance, and concentrated to dryness by centrifugation at 35 ℃, and the residue is dissolved with 100 mu L of 50% methanolThen, 10 μL is absorbed for LC-MS/MS analysis, and a chromatogram is recorded;
s6: preparation of a Standard Curve
Taking 190 mu L of blank plasma substitutes, adding 10 mu L of progesterone control series standard solution of the series a obtained in the step S4, and preparing a standard curve sample with 5-10 concentration points, which is equivalent to the concentration in the range of 0.0200-30.0 ng/mL, namely the linear range of 1; taking 95 mu L of blank plasma substitutes, adding 5 mu L of progesterone control series standard solution of a series b obtained in the step S4 to prepare standard curve samples with 5-10 concentration points, wherein the concentration is in a range of 0.0500-100 ng/mL, namely a linear range of 2, and then performing the following steps of' S5: plasma sample pretreatment and LC-MS/MS analysis "under the conditions of 10 μL, LC-MS/MS analysis, and chromatogram recording; the concentration of an object to be detected in the plasma substitute is taken as an abscissa, the peak area ratio of the object to be detected to an internal standard is taken as an ordinate, regression operation is carried out by a weighted least square method, and a obtained linear regression equation is taken as a standard curve, so that standard curves in two linear ranges of 0.0200-30.0 ng/mL and 0.0500-100 ng/mL are obtained;
S7: result calculation
Substituting the result obtained by calculating the chromatogram in the step S5 into the standard curve obtained in the step S6, and calculating to obtain the concentration of the progesterone in the human blood plasma.
According to the method of the first aspect of the invention, the hydroxyethyl starch-based blood volume expander is selected from: hydroxyethyl starch 40 sodium chloride injection, hydroxyethyl starch 200/0.5 sodium chloride injection, hydroxyethyl starch 20 sodium chloride injection, hydroxyethyl starch 130/0.4 sodium chloride injection, hypertonic sodium chloride hydroxyethyl starch 40 injection, and hypertonic hydroxyethyl starch 200/0.5 sodium chloride injection.
According to the method of the first aspect of the invention, the tandem mass spectrometer is provided with an ionization source and a data processing system.
According to the method of the first aspect of the invention, the tandem mass spectrometer is an API 4500 type tandem mass spectrometer equipped with a Turbo Ionspray ionization source and an analysis 1.6.3 data processing system.
According to the method of the first aspect of the invention, the high performance liquid chromatography system comprises a binary gradient pump, a degasser and an autosampler
According to the method of the first aspect of the invention, the high performance liquid chromatography system is an LC-20AD type high performance liquid chromatography system provided with a binary gradient pump, a degasser and an autosampler.
According to the method of the first aspect of the invention, the chromatographic column is an Accore TM C 18 The column has a specification of 2.6 μm,30×4.6 mm.
According to the method of the first aspect of the invention, the guard column is Phenomenex C 18 The column had a specification of 4×3.0. 3.0 mm.
According to the method of the first aspect of the invention, the liquid chromatography uses a mobile phase flow rate of 0.5 mL/min.
According to the method of the first aspect of the invention, the sample injection amount used by the liquid chromatograph is 10 mu L.
According to the method of the first aspect of the present invention, the column temperature used for the liquid chromatography is 40 ℃.
According to the method of the first aspect of the present invention, the human plasma is human plasma obtained after the use of a soft progesterone capsule or a progesterone gel.
According to the method of the first aspect of the invention, the r-value of the standard curve, i.e. the regression equation, is greater than 0.99, e.g. greater than 0.999.
Further, the second aspect of the present invention provides a method for evaluating human bioequivalence of a progesterone formulation, comprising the steps of: human plasma obtained from human after the use of a soft progesterone capsule or a progesterone gel is assayed for the concentration of progesterone by the method of any one of the first aspects of the present invention, and the human bioequivalence of the test formulation and the reference formulation is evaluated by comparing the mean plasma concentration versus time profile of the test formulation and the reference formulation based on the results of the assay of the concentration.
In describing the method steps of the present invention, the specific steps described therein may be distinguished in some details or in language description from the steps described in the examples of the detailed description section below, however, the above-described method steps may be summarized by one skilled in the art in light of the detailed disclosure of the present invention as a whole.
Any of the embodiments of any of the aspects of the invention may be combined with other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.
The hydroxyethyl starch/sodium chloride injection is a blood volume expander, is clinically used as a replacement matrix of blood plasma for preventing and treating hypovolemia, and adopts the hydroxyethyl starch 130/0.4 sodium chloride injection as the replacement matrix in the experiment. To increase the solubility of progesterone in the surrogate matrix, surrogate matrix/methanol was formulated as a surrogate plasma at 4/1 (v/v) for preparation of standard curve samples. The quality control samples are intended to simulate research samples formulated by adding known concentrations of the analyte to the plasma substitutes and the real matrix, respectively, to assess the effectiveness of the analytical method. The established method is verified to meet the related requirements of biological sample analysis (the 2020 edition of the pharmacopoeia of the people's republic of China): four 9012 "biological sample quantitative analysis method verifies the guidelines" ], and the verified method is successfully applied to bioequivalence evaluation of progesterone soft capsules and vaginal sustained-release gel in postmenopausal female subjects.
The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method established by the invention can be used for measuring the concentration of the progesterone in human plasma and evaluating the bioequivalence of the progesterone preparation. The method of the invention typically operates by extracting a plasma sample with n-hexane followed by a subsequent extraction with Accumore TM C 18 (2.6µm，30mm×4.6mm I.D.) For the analytical column, methanol-water was eluted with a mobile phase gradient at a flow rate of 0.5mL/min. Positive ion detection is carried out by adopting an ESI source in a multi-reaction monitoring mode, and the ion reaction for quantitative analysis is thatm/z315.2 cubic 97.0 (progesterone) andm/z324.3 cubic 100.0 (progesterone-d) 9 Internal standard). Since progesterone is an endogenous substance of the human body, blank human plasma free of the analyte is not available at present, so that a standard curve is prepared by adopting an alternative matrix, and a quality control sample is prepared by adopting a real matrix. The plasma concentration of the progesterone is greatly different after administration of different preparations (such as soft capsules and gel), even if the same preparations such as soft capsules are administered in a fasting state or a postprandial state, two standard curves with different linear ranges are established to quantify the plasma samples. The linear range of the two standard curves is 0.0200-30.0 ng/mL and 0.0500-100 ng/mL, the precision (CV) between the batch and the batch is less than 15%, and the accuracy (RE) is between-13.2% and 9.0%. The test result shows that the method of the invention has the advantages of sensitivity, good repeatability, small required sample volume and short analysis time, and is suitable for human bioequivalence research.
The oral preparation of progesterone has large first pass effect and low bioavailability (about 7%), and the food has large influence on absorption, so that the individual variation of the soft capsule of progesterone after oral administration is large. In order to meet the requirement of progesterone bioequivalence evaluation, the test designs two standard curves (0.0200-30.0 ng/mL and 0.0500-100 ng/mL) with different linear ranges, and is suitable for evaluating the progesterone bioequivalence. The LC-MS/MS method established by the research has the advantages of high sensitivity, small dosage of plasma samples, good reproducibility and short analysis time, can realize high-throughput analysis, and is successfully used for measuring a large number of biological samples in the evaluation of the biological equivalence of progesterone.
Drawings
Fig. 1: progesterone (A) and internal standard progesterone-d 9 (B) Is a secondary mass spectrum of (c).
Fig. 2: LC-MS/MS determines chromatograms (linear range 1) of progesterone A (I) and internal standard A (II) in human plasma, blank plasma samples.
Fig. 3: LC-MS/MS determines chromatograms (linear range 1) of progesterone B (I) and internal standard B (II) in human plasma, with the addition of only internal standard plasma substitute samples.
Fig. 4: LC-MS/MS determines the chromatograms (linear range 1) of progesterone C (I) and internal standard C (II) in human plasma, with the addition of only the plasma substitute sample of ULOQ.
Fig. 5: LC-MS/MS determines chromatograms (linear range 1) of progesterone D (I) and internal standard D (II) in human plasma, LLOQ plasma substitutes.
Fig. 6: LC-MS/MS determination of chromatograms (linear range 2) of progesterone a (I) and internal standard a (II) in human plasma, a: blank plasma samples.
Fig. 7: LC-MS/MS determines chromatograms (linear range 2) of progesterone B (I) and internal standard B (II) in human plasma, with the addition of only internal standard plasma substitute samples.
Fig. 8: LC-MS/MS determines the chromatograms (linear range 2) of progesterone C (I) and internal standard C (II) in human plasma, with the addition of only the plasma substitute sample of ULOQ.
Fig. 9: LC-MS/MS determines chromatograms (linear range 2) of progesterone D (I) and internal standard D (II) in human plasma, LLOQ plasma substitutes.
Fig. 10: mean plasma concentration-time profile after fasting oral progesterone soft capsule Test and Reference formulations (references) for 40 healthy menopausal female subjects.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. The following examples further illustrate the invention, but do not limit it.
Example 1: liquid chromatography tandem mass spectrometry for determining concentration of progesterone in human plasma
The present example provides a liquid chromatography-tandem mass spectrometry (LC-MS/MS, or liquid chromatography tandem mass spectrometry) method for determining the concentration of progesterone in human plasma, which can be used for bioequivalence evaluation of progesterone formulations. The method of this embodiment includes the following steps, and it is well known that the sequencing of certain steps may be adjustable:
S1: providing reagent reagentsSuch as but not limited to progesterone control, progesterone-d 9 Reference substance, chromatographic grade methanol, n-hexane and isopropanol, purified water for chromatography, hydroxyethyl starch 130/0.4 sodium chloride injection.
In this example, a progesterone control (structural formula shown in FIG. 1A) was purchased from China food and drug assay institute, progesterone-d 9 The control (internal standard, structural formula shown in FIG. 1B) was purchased from Canadian TLC company. In this example, the specific chromatographic grade methanol, n-hexane and isopropanol used were all purchased from Fisher, USA; the purified water for chromatography is Waha purified water, purchased from Hangzhou Waha group Co., ltd. In this example, a specific injection of hydroxyethyl starch 130/0.4 sodium chloride was purchased from Nanjing Datianqing pharmaceutical Co., ltd (H20065430), and the specification thereof was 30g of hydroxyethyl starch 130/0.4 and 4.5g of sodium chloride in 500ml of the injection per bottle, wherein the hydroxyethyl starch 130/0.4 was hydroxyethyl starch having a molecular weight of 13 ten thousand and a substitution degree of 0.4, and all the specifications and types of hydroxyethyl starch were similarly defined herein.
S2: providing a test instrumentSuch as, but not limited to, tandem mass spectrometers (e.g., equipped with an ionization source and a data processing system), high performance liquid chromatography systems (preferably, chromatography systems include binary gradient pumps, deaerators, and autosamplers to increase the efficiency of the detection operation).
In this example, a specifically used tandem mass spectrometer was an API 4500 type tandem mass spectrometer (AB Sciex, usa, which is equipped with a Turbo ion source and an analysis 1.6.3 data processing system); in this example, the high performance liquid chromatography system specifically used was an LC-20AD type high performance liquid chromatography system (shimadzu corporation, japan, which is equipped with a binary gradient pump, a deaerator, and an autosampler).
S3: providing liquidPhase chromatography conditions and mass spectrometry conditions:
the chromatographic column used in the liquid chromatography is C 18 Column, protective column C 18 The column was eluted with the following gradient elution procedure: a is water, B is methanol, 0-0.8 min,70% -95% of B; 0.8-1.8 min,95% B; 1.8-2.0 min,95% -70% B; 2.0-3.0 min,70% B;
the mass spectrum is detected by adopting an electrospray ionization source (Turbo ion) and a positive ion mode; the spray voltage is 5000V; source temperature of 600 o C, performing operation; curtain gas (CUR) at 20psi; collision gas (CAD) at 8psi; the scanning mode is multi-reaction monitoring (MRM), and the ion reaction for quantitative analysis ism/z 315.2→m/z97.0 (Progesterone, CE 27V) andm/z 324.3→m/z100.0 (Progesterone-d) 9 Internal standard, CE 27V), the scan time was 150msec.
In this example, the chromatographic column specifically used is Accore TM C 18 Columns (2.6 [ mu ] m, 30X 4.6 mm, thermo, USA), other chromatographic columns of similar type and/or size may be used; in this embodiment, the protection column specifically used is Phenomnex C 18 Columns (4 x 3.0 mm, phenomenex, USA), other similar types and/or sizes of guard columns may also be used; in this example, the flow rate of the mobile phase specifically used was 0.5 mL/min; in this embodiment, the sample injection amount specifically used is 10 μl; in this example, the column temperature used was 40 ℃.
In this embodiment, the electrospray ionization source specifically used is a Turbo ion source. Progesterone (A) and internal standard progesterone-d 9 (B) The second-order full-scan mass spectrum of (2) is shown in figure 1.
S4: preparation of stock solution
The progesterone control series standard solution is prepared: precisely weighing progesterone reference substance, dissolving with appropriate amount of methanol to obtain 1.0mg/mL stock solution, and freeze-preserving at-20deg.C; a certain volume of stock solution is precisely removed, and 50% methanol is used for dilution into serial standard solutions with the concentration of serial a, namely 0.400, 0.800, 2.00, 10.0, 50.0, 180, 480, 600ng/mL and serial b, namely 1.00, 2.00, 10.0, 40.0, 200, 600, 1800 and 2000ng/mL, and the serial standard solutions are stored in a refrigerator at the temperature of 4 ℃ for standby.
Progesterone-d 9 Preparing an internal standard solution: progesterone-d 9 Control 1 bottle (1 mg) was dissolved in 1mL of methanol to give 1.0mg/mL of progesterone-d 9 Freezing and preserving the stock solution at-20 ℃ for standby; a certain volume of stock solution is precisely removed, and 50% methanol is used for dilution to obtain internal standard solutions with the concentration of 25.0ng/mL and 100ng/mL respectively, and the internal standard solutions are stored in a refrigerator at the temperature of 4 ℃ for standby.
Preparation of plasma substitutes: and (3) measuring and mixing the hydroxyethyl starch 130/0.4 sodium chloride injection with methanol according to the volume ratio of 4:1, and uniformly mixing by vortex for later use.
S5: plasma sample pretreatment and LC-MS/MS analysis
Linear range 1 (0.0200-30.0 ng/mL): 200. Mu.L of human plasma (which was treated according to the present method based on preliminary measurement that the concentration of progesterone in human plasma falls within linear range 1) was placed in a 2mL centrifuge tube, and 20. Mu.L of an internal standard solution (25.0 ng/mL progesterone-d) was sequentially added 9 ) 500. Mu.L of n-hexane was vortexed for 2min, centrifuged for 3min (10000 rpm), 400. Mu.L of the upper organic phase was placed in a 1.5mL centrifuge tube into which 20. Mu.L of isopropanol had been previously added, concentrated to dryness by centrifugation at 35℃and the residue was dissolved in 100. Mu.L of 50% methanol, and 10. Mu.L was aspirated for LC-MS/MS analysis and the chromatogram was recorded.
Linear range 2 (0.0500 to 100 ng/mL): 100. Mu.L of human plasma (which was treated according to the present method based on preliminary measurement that the concentration of progesterone in the human plasma falls within the linear range 2) was placed in a 1.5mL centrifuge tube, and 20. Mu.L of an internal standard solution (100 ng/mL progesterone-d) was sequentially added 9 ) 200. Mu.L of n-hexane was vortexed for 2min, centrifuged for 3min (10000 rpm), 150. Mu.L of the upper organic phase was placed in a 1.5mL centrifuge tube into which 20. Mu.L of isopropanol had been previously added, concentrated to dryness by centrifugation at 35℃and the residue was dissolved in 100. Mu.L of 50% methanol, and 10. Mu.L was aspirated for LC-MS/MS analysis and the chromatogram was recorded.
S6: preparation of a Standard Curve
Taking 190 [ mu ] L of blank plasma substitute, adding 10 [ mu ] L of progesterone control series standard solution of the series a obtained in the step S4, and preparing a standard curve sample (or referred to as a linear range 1) with concentration ranging from 0.0200 to 30.0ng/mL and equivalent to 5 to 10 concentration points (8 concentration points are arranged in the embodiment: 0.0200, 0.0400, 0.100, 0.500, 2.50, 9.00, 24.0 and 30.0 ng/mL); taking 95 mu L of blank plasma substitutes, adding 5 mu L of progesterone control series standard solution of the series b obtained in the step S4 to prepare standard curve samples (linear range 2) which are equivalent to 5-10 concentration points (8 concentration points are arranged in the embodiment: 0.0500, 0.100, 0.500, 2.00, 10.0, 30.0, 90.0 and 100 ng/mL) with the concentration ranging from 0.0500 to 100ng/mL, and then performing the following steps of' S5: plasma sample pretreatment and LC-MS/MS analysis "under the conditions of 10 μL, LC-MS/MS analysis, and chromatogram recording; and (3) taking the concentration of the object to be measured in the plasma substitute as an abscissa, taking the peak area ratio of the object to be measured to the internal standard as an ordinate, and carrying out regression operation by using a weighted least square method, wherein the obtained linear regression equation is a standard curve, and obtaining the standard curves in two linear ranges of 0.0200-30.0 ng/mL and 0.0500-100 ng/mL.
In view of the fact that different preparations are taken or the difference of the concentration of the progesterone in human blood plasma is large when different preparations are taken at different taking times, the method for measuring the concentration of the progesterone in human blood plasma in the embodiment provides two standard curves with linear ranges of 0.0200-30.0 ng/mL and 0.0500-100 ng/mL respectively, and can meet the quantitative detection requirement of the progesterone in human blood plasma for bioequivalence evaluation of different preparations (such as soft capsules and gel) or the same preparation (such as soft capsules) under fasting and postprandial states.
S7: result calculation
Substituting the result obtained by calculating the chromatogram in the step S5 into the standard curve obtained in the step S6, and calculating to obtain the concentration of the progesterone in the human blood plasma.
The method for determining the concentration of progesterone in human plasma by using the liquid chromatography-tandem mass spectrometry provided in the embodiment 1 has excellent methodological performance, and completely accords with the relevant national regulations for biological sample determination (pharmacopoeia of the people's republic of China 2020 edition): four 9012 "biological sample quantitative analysis method validation guidelines" ]. These superior methodological properties are demonstrated by the examples below.
Example 2: method specificity
According to the method of example 1, 200 μl or 100 μl of plasma is replaced, respectively, except that no internal standard solution is added, according to "S5: plasma sample pretreatment and LC-MS/MS analysis, 10 [ mu ] L of sample is injected, and chromatograms are recorded, as shown in figures 2 and 6; the internal standard solution (25.0 ng/mL or 100ng/mL progesterone-d) 9 ) Plasma substitutes were added separately and chromatograms were recorded by the same procedure as shown in fig. 3 and 7; adding progesterone standard solution with upper limit of quantification (ULOQ) concentration into plasma substitute respectively, performing the same method, and recording chromatograms as shown in fig. 4 and 8; the lower limit of quantitation (LLOQ) concentration of progesterone standard solution and internal standard solution were added to the plasma substitutes separately, and the chromatograms were recorded as shown in fig. 5 and 9. The result shows that the plasma substitute does not interfere with the measurement of the object to be measured and the internal standard, and the object to be measured and the internal standard do not interfere with each other.
Example 3:linear range and minimum quantitative limit
According to "S6" of example 1: preparing a standard curve in a concentration range of 0.0200-30.0 ng/mL and 0.0500-100 ng/mL, specifically, taking 190 mu L of blank plasma substitute, adding 10 mu L of progesterone control series standard solution of series a obtained in the step S4, and preparing a standard curve sample (linear range 1) with the concentration of 0.0200, 0.0400, 0.100, 0.500, 2.50, 9.00, 24.0 and 30.0 ng/mL; taking 95 mu L of blank plasma substitute, adding 5 mu L of progesterone control series standard solution of series b obtained in the step S4, preparing standard curve samples (linear range 2) with concentrations of 0.0500, 0.100, 0.500, 2.00, 10.0, 30.0, 90.0 and 100ng/mL, operating the rest under the pretreatment item of 1.4 plasma samples, sampling 10 mu L, and recording a chromatogram. And (3) taking the concentration of the object to be detected in the plasma substitute as an abscissa, taking the peak area ratio of the object to be detected to the internal standard as an ordinate, and carrying out regression operation by using a weighted least square method, wherein the obtained linear regression equation is the standard curve. According to a standard curve, the linear range of the progesterone plasma concentration measuring method is 0.0200-30.0 ng/mL and 0.0500-100 ng/mL respectively, so that the quantitative detection requirement of progesterone in human plasma for bioequivalence evaluation under fasting and postprandial states of different preparations (such as soft capsules and gel) or the same preparation (such as soft capsules) can be met. The regression equations of the two typical standard curves obtained according to the present embodiment, namely, the two standard curves of the linear range 1 and the linear range 2 are respectively: y=0.361 x-0.000436 (r=0.9997) and y= 0.0437x-0.000147 (r=0.9993), and the parameter summary data of the two standard curves are shown in tables 1 and 2.
Table 1: standard Curve 1 parameter summary (LLOQ and ULOQ units are ng/mL)
Table 2: standard Curve 2 parameter summary (LLOQ and ULOQ units are ng/mL)
Example 4: precision and accuracy
Quality Control (QC) samples 0.0200,0.0600,0.225,2.25, 22.5ng/mL (linear range 1) and 0.0500,0.150,0.750,7.50, 75.0ng/mL (linear range 2) were prepared from blank plasma substitutes for precision and accuracy as described in example 1. In addition, the precision and accuracy of the real substrate (human plasma) were also examined: taking 200 mu L of human blank plasma, and performing a process of' S5: plasma sample pretreatment and LC-MS/MS analysis "under linear range 1 operation, the measured concentration is the concentration of endogenous progesterone (baseline concentration) in human blank plasma, the concentration is taken as the real matrix quantitative lower limit QC sample concentration of linear range 1, and then progesterone quality control solution is added to prepare the real matrix QC sample. For each concentration, 6 sample analyses were performed, 3 analysis batches were determined, and together with the standard curve, QC sample concentrations of plasma and real substrate were calculated from the standard curve, and precision (CV) and accuracy (RE) of plasma and real substrate were calculated, and the results are shown in tables 3 and 4, respectively.
Table 3: LC-MS/MS method for determining precision and accuracy of progesterone in plasma substitutes (n=18)
Table 4: LC-MS/MS method for determining precision and accuracy of progesterone in real matrix (n=18)
According to the results, the method of the embodiment 1 of the invention has excellent precision and accuracy, and can completely meet the drug concentration measurement requirements of common biological samples.
Example 5: extraction recovery and matrix effects
Following the procedure of example 1, 3 parts of empty human plasma were taken according to "S5: after plasma sample pretreatment and LC-MS/MS analysis, LC-MS/MS measurement is directly carried out to obtain a blank plasma baseline value (peak area average value), the ratio of the peak area measured by using human plasma QC samples with low, medium and high concentrations to the peak area of the blank human plasma added with the corresponding concentration solution after pretreatment (the peak area is deducted from the baseline value), and the extraction recovery rate of the to-be-detected substance in the real matrix is calculated; and calculating the extraction recovery rate of the to-be-detected substances in the plasma by using the ratio of the peak area measured by the low, medium and high 3 concentration plasma QC samples to the peak area of the blank plasma which is pretreated and added with the solution with the corresponding concentration. The extraction recovery rates of the real matrix of the human plasma with 3 concentrations of low, medium and high (0.0920, 2.28, 22.5ng/mL and 0.182, 7.53, 75.0 ng/mL) are respectively 113.6+/-11.9%, 100.0+/-4.6%, 101.5+/-2.3% and 108.4+/-10.1%, 105.8+/-3.7% and 103.1+/-4.5% after internal standard correction. The extraction recovery rates of the low, medium and high (0.0600, 2.25, 22.5ng/mL and 0.150, 7.50, 75.0 ng/mL) 3 concentration plasma substitutes are respectively 101.0+/-15.6%, 102.4+/-2.0%, 102.8+/-2.6% and 99.5+/-5.2%, 100.0+/-6.1% and 101.5+/-2.8% after internal standard correction.
Taking blank plasma of 6 subjects, adding low-concentration QC solution and high-concentration QC solution after pretreatment, and calculating the matrix effect of a real matrix by subtracting the ratio of the corresponding blank plasma baseline value to the solution peak area of the corresponding concentration; after pretreatment of 1 batch of blank plasma, the ratio of the peak area of low-concentration QC solution to the peak area of solution with corresponding concentration is added to calculate the matrix effect of the plasma. The internal standard normalized matrix effector variation Coefficient (CV) of 2 concentrations of low and high (0.0920, 22.5ng/mL and 0.182, 75.0 ng/mL) progesterone in the real matrix of human plasma is 4.4%, 1.6% and 10.0%, 1.5%; the concentration of 2 internal standard normalized matrix effector CV at low and high (0.0600, 22.5ng/mL and 0.162, 75.0 ng/mL) concentrations of progesterone in the plasma substitutes was 5.9%, 1.0% and 2.8%, 1.2%.
The above results indicate that the method of example 1 of the present invention has excellent extraction recovery; the effect of matrix effects on the assay was negligible under the analytical conditions selected for the experiment.
Example 6: stability of
The stability of low and high 2 concentration human plasma authentic substrate samples (0.0920, 22.5ng/mL and 0.182, 75.0 ng/mL) and low and high 2 concentration plasma substitute samples (0.0600, 22.5ng/mL and 0.150, 75.0 ng/mL) of progesterone were examined in this experiment according to the method of example 1. The results showed that the progesterone was truly a matrix sample: standing at room temperature for 24h (RE is between-13.7% and 1.3%), freezing-thawing for 5 cycles (RE is between 1.1% and 4.1%), standing at room temperature for 24h (RE is between-9.9% and 0.9%) after treatment, standing at-80 ℃ for 110 days (RE is between 1.5% and 13.2%), standing at-20 ℃ for 110 days (RE is between 1.1% and 14.7%), and standing at room temperature for 2h (RE is between-5.1% and 0.4%). Progesterone plasma samples: the method comprises the steps of standing at room temperature for 24 hours (RE is between-5.6% and 2.7%), freezing-thawing for 5 cycles (RE is between-4.7% and 7.1%), standing at room temperature for 24 hours (RE is between-4.3% and 5.3%) after treatment, standing at-80 ℃ for 110 days (RE is between-3.8% and 11.6%), and standing at-20 ℃ for 62 days (RE is between-0.7% and 2.7%).
Example 7: application of the method
The LC-MS/MS method established in example 1 of the present invention, through the above detailed methodological verification, meets the relevant regulations for biological sample measurement (pharmacopoeia of the people's republic of China 2020 edition: four 9012 "biological sample quantitative analysis method verifies the guiding principle" ], then, the method of the invention uses the method of the example 1 to complete the analysis and detection of the 7000 samples of the hollow and postprandial bioequivalence test of the progesterone soft capsule and the 3000 samples of the bioequivalence test of the progesterone vaginal slow-release gel.
Since the blood concentration of progesterone after administration of different formulations (e.g., soft capsule and gel) is greatly different, even if the same formulation such as soft capsule is administered in a fasting or postprandial state, two standard curves with different linear ranges are needed to quantify the plasma sample. Wherein the empty stomach test sample and the slow release gel sample of the progesterone soft capsule are quantified by a standard curve with a linear range of 1, and the postprandial test sample of the progesterone soft capsule is quantified by a standard curve with a linear range of 2. Fig. 10 provides a graph of mean blood concentration versus time for 40 healthy menopausal female subjects using the method of example 1 of the present invention after empty-web oral progesterone soft capsule Test (Test, homemade) and Reference formulation (Reference commercially available from angel, besins Healthcare Benelux company), from which it can be seen that the Test formulation and Reference formulation mean blood concentration versus time graph substantially match.
Example 8: study of measurement methods
1. Selection of mobile phase systems
In previous studies by the group of the present inventors, it has been found that methanol can significantly promote ionization of progesterone by comparing the ionization degree of different flows with respect to progesterone, improving the mass spectral response. As described in the examples above, the final invention selected a water-methanol system as the mobile phase for gradient elution, resulting in good chromatographic peak profile with an analysis time of 3min for each sample.
2. Selection of plasma surrogate matrices
The invention aims to measure the concentration of progesterone in human blood plasma, but an object to be measured is an endogenous substance of a human body, and the blank human blood plasma without the progesterone cannot be obtained at present, so that an alternative matrix method is adopted. Hydroxyethyl starch 130/0.4 sodium chloride injection is a blood volume expander, and is clinically used as a replacement matrix for blood plasma for preventing and treating hypovolemia. The invention therefore uses hydroxyethyl starch 130/0.4 sodium chloride injection as a replacement matrix. To increase the solubility of progesterone in the surrogate matrix, surrogate matrix/methanol was formulated as 4/1 (v/v) plasma substitutes for the preparation of calibration curve standards, and the final QC results demonstrated that hydroxyethylstarch 130/0.4 sodium chloride injection as surrogate matrix was viable to establish a standard curve, the detailed results being described in the examples above.
Hydroxyethyl starch 130/0.4 sodium chloride injection (typical specification: 500ml:30g hydroxyethyl starch 130/0.4 and 4.5g sodium chloride) is a blood volume extender approved by the national drug administration, and the same types of varieties include the following 5 types: hydroxyethyl starch 40 sodium chloride injection (typical specification 500ml:30g hydroxyethyl starch 40 and sodium chloride 4.5g, e.g. H11021164), hydroxyethyl starch (200/0.5) sodium chloride injection (typical specification 500ml: hydroxyethyl starch (200/0.5) 30g and sodium chloride 4.5g, e.g. H20050886), hydroxyethyl starch 20 sodium chloride injection (typical specification 500ml:30g hydroxyethyl starch 20 and sodium chloride 4.5g, e.g. H20033771), hypertonic sodium chloride hydroxyethyl starch 40 injection (typical specification 250ml:10.5g sodium chloride and 19g hydroxyethyl starch 40, e.g. H20041554), hypertonic hydroxyethyl starch 200/0.5 sodium chloride injection (typical specification 250ml:15g hydroxyethyl starch 200/0.5 and 18g sodium chloride, e.g. H20173260).
The inventors used the method of example 1 except that the hydroxyethyl starch 130/0.4 sodium chloride injection used therein was replaced with the 5 similar blood volume expanders described above, and the results showed excellent methodological performance in both linear ranges of 0.0200 to 30.0ng/mL and 0.0500 to 100 ng/mL. For example, in the method-specific test performed in reference to example 2, it was found that none of the 5 types of plasma substitutes interfered with the measurement of the analyte and the internal standard, and that none of the analyte and the internal standard interfered with each other; in the precision and accuracy test performed in reference example 3, it was found that the precision (CV) and accuracy (RE) of 5 plasma substitutes were as good as each other, and all of them were capable of fully satisfying the drug concentration measurement requirements of general biological samples, for example, the hydroxyethyl starch 40 sodium chloride injection as the plasma substitute in table 1 had intra-batch CV (%) of less than 9.7%, inter-batch CV (%) of less than 7.6%, and relative deviation RE (%) of within ±5.2%; in the extraction recovery rate test performed in reference example 4, the extraction recovery rates of the to-be-detected substances in 5 types of plasma substitutes are found to be within the range of 90-110%, for example, when hydroxyethyl starch 40 sodium chloride injection is used as the plasma substitutes, the extraction recovery rates of 3 types of low, medium and high (0.0600, 2.25, 22.5ng/mL and 0.150, 7.50 and 75.0 ng/mL) concentration plasma substitutes are respectively 103.2+/-8.5%, 99.6+/-2.7%, 101.2+/-4.8% and 101.3+/-6.1%, 98.7+/-4.7% and 102.4+/-4.3% after internal standard correction; in the matrix effect test performed with reference to example 4, the 2 low and high (0.0600, 22.5ng/mL and 0.162, 75.0 ng/mL) concentrations of the internal standard normalization matrix effect factor CV were all less than 7%, for example, when hydroxyethyl starch 40 sodium chloride injection was used as the plasma substitute, the 2 low and high (0.0600, 22.5ng/mL and 0.162, 75.0 ng/mL) concentrations of the internal standard normalization matrix effect factor CV were 4.2%, 2.1% and 3.4%, 1.6%.
Thus, in the present invention, the plasma surrogate matrix used may be selected from: hydroxyethyl starch 40 sodium chloride injection, hydroxyethyl starch (200/0.5) sodium chloride injection, hydroxyethyl starch 20 sodium chloride injection, hydroxyethyl starch 130/0.4 sodium chloride injection, hypertonic sodium chloride hydroxyethyl starch 40 injection, and hypertonic hydroxyethyl starch 200/0.5 sodium chloride injection.
3. Extraction method and selection of extraction solvent
The present inventors have conducted a great deal of research on the measurement of plasma drug concentration of progesterone for many years, and among these, an important one is the selection of extraction method and extraction solvent.
In previous studies of some extraction methods and extraction solvent selections, "S5" of example 1 was used: plasma sample pretreatment and LC-MS/MS analysis "using no isopropanol, but" 400. Mu.L of the upper organic phase was placed in a 1.5mL centrifuge tube, and concentrated to dryness by centrifugation at 35 ℃, based on the operation of not adding isopropanol, the inventors examined various extraction solvents for use in the plasma drug concentration measurement of the present invention during the pretreatment of the sample, such as ethyl acetate, t-butyl methyl ether, acetone, n-hexane, n-heptane, cyclohexane, n-hexane-dichloromethane-isopropanol (100:50:5, the inventors have succeeded in the measurement of progesterone in rat plasma), etc., but the extraction recovery rates of each of the test solvents including the above 7 solvents were less than 40%, in the range of about 30% (all in the range of 23% -34%). For example, using n-hexane-dichloromethane-isopropanol (100:50:5) as the extraction solvent, the extraction recovery rates of 3 concentrations of human plasma real matrix were 31.2.+ -. 5.4%, 28.6.+ -. 3.8%, 32.6.+ -. 2.3% and 25.6.+ -. 6.2%, 31.2.+ -. 3.2%, 29.3.+ -. 4.2%, and 33.6.+ -. 7.3%, 25.9.+ -. 2.6%, 32.2.+ -. 6.6% and 29.3.+ -. 5.6%, 29.3.+ -. 3.2%, respectively, of 3 concentrations of low, medium, high (0.0600, 2.25, 22.5ng/mL and 0.150, 7.50, 75.0 ng/mL) plasma were 33.6.+ -. 7.3%, 25.9.+ -. 2.6%, 32.2.+ -. 6.6% and 29.3.+ -. 5.7%, 27.8.+ -. 3.2%, respectively; for another example, when n-hexane was used as the extraction solvent, the extraction recovery rates of 3 human plasma real matrices at low, medium, and high (0.0920, 2.28, 22.5ng/mL and 0.182, 7.53, 75.0 ng/mL) were 28.6.+ -. 3.7%, 31.2.+ -. 4.4%, 34.5.+ -. 2.7% and 28.4.+ -. 3.7%, 30.5.+ -. 3.1%, 28.6.+ -. 5.7%, and the extraction recovery rates of 3 plasma substitutes at low, medium, and high (0.0600, 2.25, 22.5ng/mL and 0.150, 7.50, 75.0 ng/mL) were 29.5.+ -. 6.7%, 34.4.+ -. 5.1%, 30.7.+ -. 4.1% and 30.3.+ -. 3.4%, 27.8.+ -. 4.2%, 29.4.+ -. 3.7%, respectively.
However, it has been found that the treated samples are cleaner and less noisy in background when n-hexane is used as the extraction solvent, which is advantageous for LC-MSMS assays, relative to other solvents that have been tried, including the 7 solvents described above.
In addition, it was unexpectedly found that, in the subsequent centrifugation concentration step, when n-hexane was used as the extraction solvent, it was found that by adding an appropriate amount of isopropyl alcohol at the time of sample concentration, as in "S5" of example 1: the pretreatment of plasma samples and LC-MS/MS analysis can lead the recovery rate of the samples after internal standard correction to reach more than 90%, and can obviously and effectively reduce the loss of progesterone in the sample treatment process, and the specific test results are shown in the example 5. However, even as in "S5" of example 1: plasma sample pretreatment and LC-MS/MS analysis "uses isopropyl alcohol in the centrifugation concentration step, but when the other 6 extraction solvents described above are used, the extraction recovery rate of human plasma real matrix and plasma substitute is less than 50%, for example, refer to" S5 "of example 1: plasma sample pretreatment and LC-MS/MS analysis "low, medium, high (0.0920, 2.28, 22.5ng/mL and 0.182, 7.53, 75.0 ng/mL) 3 concentrations of human plasma true matrix extraction recovery were 46.4±3.4%, 49.6±7.1%, 37.4±5.3% and 49.2±4.1%, 48.2±3.8%, 46.4±3.6%, respectively, using cyclohexane as extraction solvent and isopropanol in the centrifugation concentration step. Experiments also show that the isopropanol not only can improve the recovery rate, but also can make the extraction more stable, and further improves the detection sensitivity. Finally, n-hexane is selected as an extraction solvent to treat the plasma sample, and an appropriate amount of isopropanol is added in a sample centrifugal concentration link to improve the recovery rate, which is not reported in other documents, and the result is not foreseeable in the prior art at all.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A method for determining the concentration of progesterone in human plasma using liquid chromatography tandem mass spectrometry, comprising the steps of:
s1: providing a reagent: comprises progesterone control, progesterone-d 9 Reference substance, chromatographic grade methanol, n-hexane and isopropanol, purified water for chromatography, hydroxyethyl starch-based blood volume expander;
s2: providing a test instrument: comprises a tandem mass spectrometer and a high performance liquid chromatography system;
s3: providing liquid chromatography conditions and mass spectrometry conditions:
the chromatographic column used in the liquid chromatography is C 18 Column, protective column C 18 The column was eluted with the following gradient elution procedure: a is water, B is methanol, 0-0.8 min,70%~95%B；0.8~1.8 min，95%B；1.8~2.0 min，95% ~70%B；2.0~3.0 min，70%B；
The mass spectrum is detected by adopting an electrospray ionization source and a positive ion mode; the spray voltage is 5000V; source temperature of 600 o C, performing operation; the curtain rolling gas is 20psi; the collision gas is 8psi; the scanning mode is multi-reaction monitoring, and the ion reaction for quantitative analysis is as follows: progesterone m/z315.2→m/z97.0 with CE of 27V, progesterone-d 9 Internal standardm/z 324.3→m/z100.0 with CE of 27V and scanning time of 150msec;
s4: preparation of stock solution
The progesterone control series standard solution is prepared: precisely weighing progesterone reference substance, dissolving with appropriate amount of methanol to obtain 1.0mg/mL stock solution, and freeze-preserving at-20deg.C; precisely transferring a certain volume of stock solution, diluting with 50% methanol to obtain serial standard solutions with the concentration of serial a of 0.400, 0.800, 2.00, 10.0, 50.0, 180, 480, 600ng/mL and serial b of 1.00, 2.00, 10.0, 40.0, 200, 600, 1800 and 2000ng/mL, and storing in a refrigerator at 4 ℃ for standby;
progesterone-d 9 Preparing an internal standard solution: progesterone-d 9 Dissolving the control with methanol to obtain 1.0mg/mL progesterone-d 9 Freezing and preserving the stock solution at-20 ℃ for standby; precisely transferring a certain volume of stock solution, diluting with 50% methanol to obtain internal standard solutions with the concentrations of 25.0ng/mL and 100ng/mL respectively, and storing in a refrigerator at 4 ℃ for later use;
preparation of plasma substitutes: measuring hydroxyethyl starch-based blood volume expander and mixing with methanol according to a volume ratio of 4:1, and uniformly mixing by vortex for later use;
s5: plasma sample pretreatment and LC-MS/MS analysis
For human plasma with a progesterone concentration in the range of 0.0200-30.0 ng/mL, i.e., linear range 1: 200 mu L of human plasma is taken and placed in a 2mL centrifuge tube, and 20 mu L of internal standard solution, namely 25.0ng/mL progesterone-d, is added in sequence 9 Is mixed with 500 mu L of n-hexane by vortexing for 2min and centrifuged at 10000rpm for 3min, 400 mu L of the upper organic phase is taken and placed in a 1.5mL centrifuge tube into which 20 mu L of isopropanol is added in advance, and concentrated to dryness by centrifugation at 35 ℃, and after the residue is dissolved with 100 mu L of 50% methanol, 10 mu L is sucked upPerforming LC-MS/MS analysis and recording a chromatogram;
for human plasma with a progesterone concentration in the range of 0.0500 to 100ng/mL, i.e. linear range 2: taking 100 mu L of human plasma, placing into a 1.5mL centrifuge tube, sequentially adding 20 mu L of internal standard solution (100 ng/mL progesterone-d) 9 Mixing with 200 μl of n-hexane, centrifuging at 10000rpm for 3min, collecting 150 μl of the upper organic phase, placing into 1.5mL centrifuge tube with 20 μl isopropanol, centrifuging at 35deg.C, concentrating to dryness, dissolving the residue with 50% methanol 100 μl, sucking 10 μl, performing LC-MS/MS analysis, and recording chromatogram;
s6: preparation of a standard curve: taking 190 mu L of blank plasma substitutes, adding 10 mu L of progesterone control series standard solution of the series a obtained in the step S4, and preparing a standard curve sample with 5-10 concentration points, which is equivalent to the concentration in the range of 0.0200-30.0 ng/mL, namely the linear range of 1; taking 95 mu L of blank plasma substitutes, adding 5 mu L of progesterone control series standard solution of a series b obtained in the step S4 to prepare standard curve samples with 5-10 concentration points, wherein the concentration is in a range of 0.0500-100 ng/mL, namely a linear range of 2, and then performing the following steps of' S5: plasma sample pretreatment and LC-MS/MS analysis "under the conditions of 10 μL, LC-MS/MS analysis, and chromatogram recording; the concentration of an object to be detected in the plasma substitute is taken as an abscissa, the peak area ratio of the object to be detected to an internal standard is taken as an ordinate, regression operation is carried out by a weighted least square method, and a obtained linear regression equation is taken as a standard curve, so that standard curves in two linear ranges of 0.0200-30.0 ng/mL and 0.0500-100 ng/mL are obtained;
S7: and (3) calculating results: substituting the result obtained by calculating the chromatogram in the step S5 into the standard curve obtained in the step S6, and calculating to obtain the concentration of the progesterone in the human blood plasma.
2. The method of claim 1, wherein the hydroxyethyl starch-based blood volume expander is selected from the group consisting of: hydroxyethyl starch 40 sodium chloride injection, hydroxyethyl starch 200/0.5 sodium chloride injection, hydroxyethyl starch 20 sodium chloride injection, hydroxyethyl starch 130/0.4 sodium chloride injection, hypertonic sodium chloride hydroxyethyl starch 40 injection, and hypertonic hydroxyethyl starch 200/0.5 sodium chloride injection.
3. The method of claim 1, wherein the tandem mass spectrometer is equipped with an ionization source and a data processing system.
4. The method of claim 1, wherein the tandem mass spectrometer is an API 4500 type tandem mass spectrometer equipped with a Turbo ion pray ionization source and an analysis 1.6.3 data processing system.
5. The method of claim 1, wherein the high performance liquid chromatography system comprises a binary gradient pump, a degasser, and an autosampler.
6. The method of claim 1, wherein the chromatographic column is an Accucore TM C 18 A column with a specification of 2.6 μm,30×4.6 mm; the guard column is Phenomenex C 18 The column had a specification of 4×3.0. 3.0 mm.
7. The method according to claim 1, wherein the mobile phase used for the liquid chromatography has a flow rate of 0.5 mL/min, a sample injection amount of 10 μl and a column temperature of 40 ℃.
8. The method of claim 1, wherein the human plasma is human plasma obtained from a progestin soft capsule or a progestin gel.
9. The method according to claim 1, wherein the r-value of the standard curve, the regression equation, is greater than 0.999.
10. A method for evaluating human bioequivalence of a progesterone formulation, comprising the steps of: human plasma obtained from the use of a soft progesterone capsule or a progesterone gel is assayed for concentration of progesterone by the method of any one of claims 1 to 9, and the human bioequivalence of the test preparation and the reference preparation is evaluated by comparing the mean plasma concentration-time curve of the test preparation and the reference preparation based on the result of the assay of concentration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311293042.4A CN117030918B (en) | 2023-10-08 | 2023-10-08 | Method for determining concentration of progesterone in human plasma by using liquid chromatography-tandem mass spectrometry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311293042.4A CN117030918B (en) | 2023-10-08 | 2023-10-08 | Method for determining concentration of progesterone in human plasma by using liquid chromatography-tandem mass spectrometry |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117030918A true CN117030918A (en) | 2023-11-10 |
| CN117030918B CN117030918B (en) | 2023-12-08 |
Family
ID=88641544
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311293042.4A Active CN117030918B (en) | 2023-10-08 | 2023-10-08 | Method for determining concentration of progesterone in human plasma by using liquid chromatography-tandem mass spectrometry |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117030918B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103336065A (en) * | 2013-01-15 | 2013-10-02 | 东南大学 | A method for simultaneously extracting and detecting a plurality of hormones and metabolites thereof in biology mediums |
| CN108169370A (en) * | 2017-12-19 | 2018-06-15 | 万舒（北京）医药科技有限公司 | Measure the method for ethyl pyruvate and its metabolin pyruvic acid in biological sample |
| CN110208442A (en) * | 2019-05-24 | 2019-09-06 | 国科卓越(北京)医药科技研究有限公司 | A kind of method of the drug concentration of progesterone in detection biological sample |
| CN114414810A (en) * | 2021-11-18 | 2022-04-29 | 上海中医药大学 | Application of beta-carboline alkaloid harmine in diagnosing Alzheimer's disease |
-
2023
- 2023-10-08 CN CN202311293042.4A patent/CN117030918B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103336065A (en) * | 2013-01-15 | 2013-10-02 | 东南大学 | A method for simultaneously extracting and detecting a plurality of hormones and metabolites thereof in biology mediums |
| CN108169370A (en) * | 2017-12-19 | 2018-06-15 | 万舒（北京）医药科技有限公司 | Measure the method for ethyl pyruvate and its metabolin pyruvic acid in biological sample |
| CN110208442A (en) * | 2019-05-24 | 2019-09-06 | 国科卓越(北京)医药科技研究有限公司 | A kind of method of the drug concentration of progesterone in detection biological sample |
| CN114414810A (en) * | 2021-11-18 | 2022-04-29 | 上海中医药大学 | Application of beta-carboline alkaloid harmine in diagnosing Alzheimer's disease |
Non-Patent Citations (6)
| Title |
|---|
| M. SASAKI 等: "Development and Validation of LC-MS/MS Assay for the Quantification of Progesterone in Rat Plasma and its Application to Pharmacokinetic Studies", DRUG RESEARCH, vol. 55, no. 09, pages 484 - 489 * |
| NICO C. VAN DE MERBEL: "Quantitative determination of endogenous compounds in biological samples using chromatographic techniques", TRENDS IN ANALYTICAL CHEMISTRY, vol. 27, no. 10, pages 924 - 933, XP025642457, DOI: 10.1016/j.trac.2008.09.002 * |
| R.M.T. FERNANDES 等: "LC-MS/MS quantitation of plasma progesterone in cattle", THERIOGENOLOGY, vol. 76, no. 07, pages 1266 - 1274 * |
| YUYONG KE: "Impact of sample extraction on the accurate measurement of progesterone in human serum by liquid chromatography tandem mass spectrometry", OURNAL OF CHROMATOGRAPHY B: ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, vol. 1052, pages 110 - 120, XP085006880, DOI: 10.1016/j.jchromb.2017.03.025 * |
| 古丽努尔・塔西铁木尔 等: "液相色谱-串联质谱法测定Beagle犬血浆中黄体酮浓度及其药动学", 药物分析杂志, vol. 31, no. 11, pages 2040 - 2045 * |
| 孙丽丽 等: "液相色谱-串联质谱法测定大鼠血浆中黄体酮的浓度", 国际药学研究杂志, vol. 42, no. 01, pages 107 - 111 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117030918B (en) | 2023-12-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ye et al. | Determination and pharmacokinetics of engeletin in rat plasma by ultra-high performance liquid chromatography with tandem mass spectrometry | |
| Ma et al. | Simultaneous determination of tetrahydropalmatine, protopine, and palmatine in rat plasma by LC-ESI-MS and its application to a pharmacokinetic study | |
| Dong et al. | Simultaneous determination of major phytocannabinoids, their main metabolites, and common synthetic cannabinoids in urine samples by LC⿿ MS/MS | |
| Fan et al. | Comparative pharmacokinetics of ginsenoside Rg3 and ginsenoside Rh2 after oral administration of ginsenoside Rg3 in normal and walker 256 tumor-bearing rats | |
| Chen et al. | A novel UPLC/MS/MS method for rapid determination of metapristone in rat plasma, a new cancer metastasis chemopreventive agent derived from mifepristone (RU486) | |
| CN107064389A (en) | The UPLC MS/MS detection methods of free paclitaxel in a kind of blood plasma | |
| Shen et al. | Simple and sensitive liquid chromatography–tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera | |
| Gandhi et al. | High-sensitivity simultaneous liquid chromatography–tandem mass spectrometry assay of ethinyl estradiol and levonorgestrel in human plasma | |
| He et al. | Simultaneous quantification of ginsenoside Rg1 and its metabolites by HPLC–MS/MS: Rg1 excretion in rat bile, urine and feces | |
| Li et al. | Therapeutic monitoring of serum digoxin for patients with heart failure using a rapid LC-MS/MS method | |
| Qiu et al. | Simultaneous determination of bosentan and glimepiride in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study | |
| Yu et al. | Simultaneous determination of eight flavonoids in plasma using LC–MS/MS and application to a pharmacokinetic study after oral administration of Pollen Typhae extract to rats | |
| Zhang et al. | Validated LC-MS/MS method for the determination of rosuvastatin in human plasma: application to a bioequivalence study in Chinese volunteers | |
| Weng et al. | Determination of licochalcone A in rat plasma by UPLC–MS/MS and its pharmacokinetics | |
| Yaroshenko et al. | Development and validation of a LC-MS/MS method for d-cycloserine determination in human plasma for bioequivalence study | |
| CN117030918B (en) | Method for determining concentration of progesterone in human plasma by using liquid chromatography-tandem mass spectrometry | |
| Ma et al. | Development of a simple and rapid UPLC-MS/MS method for determination of 8-deacetyl-yunaconitine in rat plasma and its application to pharmacokinetics | |
| Zuo et al. | Integrated UPLC-MS/MS and UHPLC-Q-orbitrap HRMS analysis to reveal pharmacokinetics and metabolism of five terpenoids from Alpiniae oxyphyllae fructus in rats | |
| Song et al. | Rapid and sensitive liquid chromatography–tandem mass spectrometry: Assay development, validation and application to a human pharmacokinetic study | |
| Wen et al. | A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of hydroxysafflor yellow A in human plasma: Application to a pharmacokinetic study | |
| Hu et al. | A rapid and sensitive UHPLC–MS/MS assay for the determination of trelagliptin in rat plasma and its application to a pharmacokinetic study | |
| Li et al. | Development and full validation of a liquid chromatography-tandem mass spectrometry method for determination of carbinoxamine in beagle plasma and its application to a pharmacokinetic study | |
| Li et al. | Development of an LC–MS/MS method for the quantitation of deoxyglycychloxazol in rat plasma and its application in pharmacokinetic study | |
| Fan et al. | Determination of TJ0711 hydrochloride in rat plasma by high performance liquid chromatography with fluorescence detection and its application to pharmacokinetics | |
| Duan et al. | Rapid determination of anastrozole in plasma by gas chromatography with electron capture detection and its application to an oral pharmacokinetic study in healthy volunteers |
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 |