血浆中候选药物混合物定量分析检测方案(液质联用仪)

thermoscientific APPLICATION NOTE 64977 Sensitive， robust quantitative analysis of amixture of drug candidates in plasma using aTSQ Altis triple quadrupole mass spectrometer Authors Keeley Murphy， Simon Szwandt，and Claudia Martins Thermo Fisher Scientific. San Jose， CA Keywords Antidepressants， LC-MS/MS， TSQ Altis MS， pharmaceutical， small molecules， bioanalysis， bioequivalence To develop a sensitive， robust， reproducible LC-MS/MS assay fordetermination and quantitation of amixture of compounds of pharmaceuticainterest in human plasma and plasma from preclinical animal species. Introduction Small molecules represent a significant proportion of drug discoveryand development in the search for new chemical entities， in addition tothe extensive work involved in the regulatory filings of generics. Targetedquantitation assays are a critical part of an optimal workflow， which isrequired to successfully develop a small molecule drug. These targetedlquantitation analyses must be done in biological matrices， which can oftencreate analytical challenges. In this study， we report the development of asensitive， robust， reliable， and reproducible LC-MS/MS assay for multipledrug standards in rat plasma. ExperimentalSample preparation Crashed plasma stock solutions were prepared usingan acetonitrile (ACN) crash at a ratio of 3：1， ACN toplasma. The resulting solution was centrifuged at10，000 rpm for 10 minutes. The supernatant wasremoved and added to an equivalent volume of waterto make the final crashed plasma stock solution. Stocksolutions of each standard compound at 1 mg/mL werediluted in a pooled mix in the crashed plasma stockto concentration ranges of 1 pg/mL to 25，000 pg/mLand 10 pg/mL to 100，000 pg/mL. Isotopically labeledinternal standards were added to each calibration levelto produce a final internal standard concentration of0.5 ng/mL. All reagents were obtained from CerilliantCorporation， Round Rock， Texas， at 1 mg/mL inmethanol. Liquid chromatography Chromatographic separation was performed using aThermo ScientificVanquish" Horizon HPLC system. Thecolumn used was a Thermo ScientificHypersil GOLDaQ C18 Polar Endcapped LC column (100×2.1 mm，1.9 um particle size). Mobile phases A and B consisted of10 mM ammonium formate in Fisher Chemical"Optimagrade water and 0.1% formic acid in Fisher ChemicalOptima"grade acetonitrile， respectively. The columntemperature was 50 C. The total run time was 3.5minutes (Table 1). Table 1.Chromatography gradient for analysis. Time(min) Flow Rate(mL/min) %A %B 0 0.6 95 5 0.4 0.6 95 5 0.5 0.6 65 35 1.5 0.6 64 36 1.6 0.6 55 45 2.2 0.6 53 47 2.3 0.6 5 95 2.95 0.6 5 95 2.995 0.6 95 5 3.5 0.6 95 5 Mass spectrometry Mass spectrometry analysis was carried out on aThermo ScientificTSQ Altistriple quadrupole massspectrometer equipped with the Thermo Scientific"OptaMaxNG source housing. Tables 2 and 3 showmass spectrometer source and SRM parameters used inthe experimental setup. Table 2. Mass spectrometer set-up. Parameter Setting Run Time 3.5 min lon Source HESI Spray Voltage 3500 V Sheath Gas 40 Arb Auxiliary Gas 15 Arb Sweep Gas 0 Arb Ion Transfer Tube Temperature 350°℃ Vaporizer Temperature 325°C Experiment Type SRM Cycle Time 0.3 s Chromatography Peak Width 6s Collision Gas Pressure 1.5 mTorr Q1 Resolution 0.7 FWHM Q3 Resolution 0.7 FWHM Data analysis Data was acquired and processed using Thermo Scientific TraceFinder software. Compound Name Collision Start Time End Time (min) (min) Polarity Precursor m/z Product m/z Energy (V) RF Lens Desomorphine 0.760 1.060 Positive 272.062 215.054 26 69 Desmethyldoxepin 1.230 1.530 Positive 266.062 107.000 23 56 Flecainide 1.310 1.610 Positive 415.050 398.054 24 84 Midazolam 1.410 1.710 Positive 326.012 291.054 28 87 Imipramine 1.660 1.960 Positive 281.462 86.054 17 48 Amitriptyline 1.800 2.100 Positive 278.075 233.111 18 53 Fluoxetine 1.890 2.190 Positive 310.362 43.889 11 39 Diazepam 2.230 2.530 Positive 285.012 193.071 33 78 Results and discussion Table 4 shows the lower limits of quantitation (LLOQ)obtained with the TSQ Altis MS for each of the drugcandidates， which were significantly lower than thoseobtained from previous generation MS systems. Inaddition， significantly lower %CV values for the IS alsoimplies increased robustness and reproducibility for theTSQ Altis MS. The representative chromatogram ofQC 2 at 300 pg/mL is show in Figure 1. Further detailson linearity and reproducibility of the QCs are shown inTable 5. Table4. Limits of quantitation for the drug candidates in plasmaand %cV (n=3) for the internal standards. Compound LOQ (pg/mL) IS %cv Desomorphine 5 3.5 Desmethyldoxepin 2.5 3.5 Flecainide 1 3.5 Midazolam 2.5 4.4 Imipramine 2.5 4.4 Amitriptyline 2.5 4.4 Fluoxetine 5 5.1 Diazepam 2.5 3.4 Table 5. Linearity and reproducibility data for four QC points per calibration curve. Compound QC 1%CV QC2%CV QC 3%CV QC4%CV R2 30 pg/mL 300pg/mL 3000 pg/mL 15，000 pg/mL Linear Fit Desomorphine 7.93 4.72 3.47 1.15 0.9945 Desmethyldoxepin 5.28 1.55 0.67 1.01 0.9904 Flecainide 4.20 4.88 2.46 2.97 0.9924 Midazolam 2.96 1.52 1.71 2.77 0.9917 Imipramine 2.50 1.26 0.38 1.24 0.9913 Amitriptyline 7.04 3.16 0.68 0.83 0.9908 Fluoxetine 3.15 2.80 2.03 2.87 0.9901 Diazepam 5.77 3.15 0.53 2.69 0.9927 Conclusion The method referenced in this application note showsexcellent linearity and reproducibility over the dynamicrange of the assay. This method demonstrates that theTSQ Altis MS provides the sensitivity and reproducibilityrequired in the analysis of pharmaceutical compounds. For Research Use Only. Not for use in diagnostic procedures. Find out more at thermofisher.com/Altis-Quantis @2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. andits subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It isnot intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.Specifications， terms and pricing are subject to change. Not all products are available in all countries. Please consult your localsales representative for details.AN64977-EN 0517S ThermoFisherSCIENTIFIC To develop a sensitive, robust, reproducible LC-MS/MS assay for determination and quantitation of a mixture of compounds of pharmaceutical interest in human plasma and plasma from preclinical animal species.Small molecules represent a significant proportion of drug discovery and development in the search for new chemical entities, in addition to the extensive work involved in the regulatory filings of generics. Targeted quantitation assays are a critical part of an optimal workflow, which is required to successfully develop a small molecule drug. These targeted quantitation analyses must be done in biological matrices, which can often create analytical challenges. In this study, we report the development of a sensitive, robust, reliable, and reproducible LC-MS/MS assay for multiple drug standards in rat plasma.The method referenced in this application note shows excellent linearity and reproducibility over the dynamic range of the assay. This method demonstrates that the TSQ Altis MS provides the sensitivity and reproducibility required in the analysis of pharmaceutical compounds.