DEVELOPMENT OF AN AUTOMATED HIGH THROUGHPUT …TO DOWNLOAD A COPY OF THIS POSTER VISIT ©2006 Waters...
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TO DOWNLOAD A COPY OF THIS POSTER VISIT WWW.WATERS.COM/POSTERS ©2006 Waters Corporation LL-PDF-2626 DEVELOPMENT OF AN AUTOMATED HIGH THROUGHPUT UPLC/MS/MS PROTOCOL WITH MULTI-MODE IONIZATION FOR IN-VITRO METABOLIC STABILITY ASSAY Kate Yu 1 , Peter Alden 1 , Rob Plumb 1 , Li Di 2 , Susan Li 2 , Edward Kerns 2 , Paul Chilvers 3 1. Waters Corporation, Milford, MA; 2. Wyeth Research, Princeton, NJ; 3. Waters Micromass, Manchester, UK INTRODUCTION Highlights Multi-mode ionization: ESCiTM • No need to physically change ionization sources for ESI/ APCI switching. Automated LC/MS/MS analytical protocol (from method development to report generation) • Automated MS and MSMS scans for optimization • Automated MRM MS acquisition method generation • Automated data acquisition • Automated quantification and report generation Application example presented for microsome-stability test in drug discovery ESCi Multi-mode Ionization • “ESCi™ multi-mode ionization source” refers to a combination ESI & APCI capability available on Waters mass spectrometers. • The enabling of the ESCi source is driven by specifically designed elec- tronics capitalizing on unique attrib- utes of the source geometry. • In ESCi, both ESI and the APCI modes can function by alternating at high MS switching speeds in both positive & negative polarities 4 . Project Goal To develop an automated ESCi-LC/MS/MS analytical protocol for the in vitro metabolic stability test using the 96 well plate to demonstrate the ad- vantages of ESCi™ multimode ionization and the Waters ACQUITY UPLC™ for higher throughput and broader compound coverage in drug discovery. Background LC/MS analysis typically utilizes atmospheric pressure ionization (API) as the ionization source with two options: • Electrospray (ESI): favor polar compounds • Atmospheric pressure chemical ionization (APCI): favors non-polar compounds and relative small molecules. The best ionization mode for any compound is determined by optimizing MS conditions in both ESI and APCI. • Generally, about 80% compounds can be analyzed by ESI 1 . • APCI is the common alternative option for non=ESI compounds. Traditionally, to change between ESI and APCI • Analysts need to physically change the ionization source • All ion source parameters need to be re-optimized • This results in lost analysis time and reduces analytical throughput sig- nificantly 1 . • This results in lost analysis time, hence significantly reduces analytical throughput 1 . Challenges for ESCi Applied to Drug Discovery The characteristics for analyses carried out in drug discovery are: • Many new compounds to be analyzed for screening purpose • Typically, each sample solution will contain a single compound for the analysis • Many injections will be required. Each injection will contain a single target analyte. The bottle necks are: • MS optimization including tuning each analyte for MRM transition, cone voltage (CV), and collision energy (CE) Method creation: • MRM MS acquisition method: each analyte requires a separate MS acquisition method MS quantification method for post run quantifica- tion The instrument feasibility as well as the advantages of ESCi Multi-mode ionization have been previously demonstrated 5 . In reality, to apply ESCi in a drug discovery lab, an automated protocol is crucial to minimize the instrument time required for analysts. EXPERIMENTAL CONDITIONS In-Vitro Metabolic Stability Screening The test method is a routine method in the Wyeth Research Lead Optimi- zation Lab • Rat liver microsomes • Single time point incubation: 15 min • Physiological level: 1 μ M Samples ready for LC/MS/MS analysis after the microsome incubation • Samples are in 96 well plate format 48 different compounds per plate, different RT and MS conditions for each compound • One compound per well • Each compound appeared in two wells for duplicated injections • Typically, 45 new analytes and 3 reference standards UPLC Conditions Waters ® ACQUITY UPLC™ Binary Solvent Manager Mobile Phase: A: 10 mM NH4OAc in ACN/H2O 10/90 B: 10 mM NH4OAc in ACN/MeOH 80/20 Column: ACQUITY UPLC™ C18 BEH Column 1.7 mm, 2.1 x 50 mm, 40oC Sample Temperature: 5 o C Flow Rate: 0.6 ml/min. Optimization: 90%B Isocratic elution Quantification Gradient: Time (min.) %A Curve 0.0 95% 6 0.8 5% 6 1.0 5% 1 3.0 95% 1 MS Conditions Waters Micromass ® Quattro Premier TM Triple Quadrupole MS Tune Page Parameters: ESCi Multi-Mode Ionization enabled ESI capillary voltage (kV): 3.00 APCI corona current (μ A): 4.0 Source Temperature ( o C): 130 Desolvation Temperature ( o C): 420 Desolvation Flow (L/Hr): 980 Cone Gas Flow (L/Hr): 50 Optimization and Quantification performed by QuanOptimize MS and MSMS scans for optimization MRM for quantification Microsomal Incubation: Plate Layout Di, J Biomolec Screening (2003) 8, 453 Di, J Pharm Sci (2004) 93, 1537 Incubation Plates Plate for Quantitation Stock Plate Reagent & Microsome Reservoirs MS OPTIMIZATION QuanOptimize in MassLynx TM • QuanOptimize is a MassLynx optional application package. It en- ables a complete automatic MS quantification process. • Once the LC conditions for the target analytes were determined, only one analyte needs to be infused into the mass spectrometer (T with the LC mobile phase at the operating LC flow rate) to optimize the tune page parameters (everything except the cone voltages for parent ions and collision energies for daughter ions). • QuanOptimize can be set up with all necessary methods and sample lists specified, the LC/MS/MS run can be initiated. QuanOptimize will then perform the following tasks: • Perform MS scan and Daughter scan to obtain MS data acquisition conditions for each analyte by injecting individual standard solutions. • Create MS acquisition method based on the optimization injections QuanOptimize Method Set Up • Acquire quantification data for sample analysis • Create MS quantification method for post run processing • Complete post run quantification and generate report in QuanLynx browser MS Scan in 4 Ionization Modes in a Single Injection Daughter Scan with the Optimized Ionization Mode During a QuanOptimize run, two sets of samples are required per analyte: the first set is the pure standard solution at high concentration, which is to be used for the MS the scan optimizations. The second set is the calibration solutions of the analyte, they can be a mixture if the project requires the analysis of a mix- ture with certain concentration series. They are to be injected for the calibration. The number of injections required per compound during the optimization stage will depend on the QuanOptimize method. For non-split optimization, there will be two injections total: The first injection is for MS scan of all four mode (ESI+/ESI-/APCI+/APCI-), and the second injection will be the daughter ion scan at the optimum ionization mode. For split optimization, there will be three injections total: The first injection is for MS scan in positive mode (ESI+/ APCI+), the second injection is for MS scan in negative mode (ESI-/APCI-), and the third injection is for daughter ion scan at the optimum ionization mode. MS Optimization Results MS and Daughter Scan Spectra There are a few different ways that optimization to be carried on by Quan- Optimize.In this project, the optimization was performed this way: • A single injection for MS scan in all four ionization mode (ESI+/ESI-/ APCI+/APCI-). • The optimum ionization mode was decided based on the integration of the peak area. The ionization mode and cone voltage that resulted in the highest peak area was chosen to be the optimum MS condition. In the example demonstrated, the ESI- at function 7 (CV 20) was cho- sen to be the optimum MS condition. • A second injection was made for daughter ion scan at ESI– with the MS cone voltage set at 20v. • The best fragment ion and collision energy was again chosen based on the integration of peak area. In the example demonstrated, colli- sion energy of 5v and daughter ion 161 was chosen as the optimum condition. 19 30 281.36 > 85.76 ESP+ 280 Imipramine 20 5 20 205.18 > 161.83 ESP- 206 Ibuprofen 19 12 20 256.28 > 167.49 APcI+ 255 Diphenhydramine 18 26 40 415.33 > 178.12 ESP+ 414 Diltiazem 17 19 10 319.18 > 163.36 ESP+ 318 Diclofenac Sodium 16 40 10 390.53 > 293.62 ESP- 392 Dexamethasone 15 12 30 267.33 > 72.18 ESP+ 266 Desipramine 14 12 20 176.01 > 98.45 ESP+ 175 Debrisoquine Sulfate 13 19 40 327.27 > 270.57 ESP+ 326 Clozapine 12 26 30 253.26 > 95.24 ESP+ 252 Cimetidine 11 19 40 167.92 > 132.25 ESP- 169 Chlorzoxazone 10 19 30 319.27 > 85.79 ESP+ 318 Chlorpromazine 9 40 40 322.22 > 121.30 APcI- 323 Chlorampehnicol 8 12 30 348.26 > 158.34 ESP+ 347 Cephalexin 7 26 30 289.43 > 139.90 ESP+ 288 Bupivacaine 6 12 10 311.33 > 243.59 ESP+ 310 Bifonazole 5 26 30 267.33 > 145.40 ESP+ 266 Atenolol 4 40 50 459.46 > 135.28 ESP+ 458 Astemizole 3 26 20 366.38 > 126.84 APcI+ 365 Amoxicillin 2 40 50 309.27 > 205.37 ESP+ 308 Alprazolam 1 CE (V) CV (V) MRM Mode MW Name No 19 30 281.36 > 85.76 ESP+ 280 Imipramine 20 5 20 205.18 > 161.83 ESP- 206 Ibuprofen 19 12 20 256.28 > 167.49 APcI+ 255 Diphenhydramine 18 26 40 415.33 > 178.12 ESP+ 414 Diltiazem 17 19 10 319.18 > 163.36 ESP+ 318 Diclofenac Sodium 16 40 10 390.53 > 293.62 ESP- 392 Dexamethasone 15 12 30 267.33 > 72.18 ESP+ 266 Desipramine 14 12 20 176.01 > 98.45 ESP+ 175 Debrisoquine Sulfate 13 19 40 327.27 > 270.57 ESP+ 326 Clozapine 12 26 30 253.26 > 95.24 ESP+ 252 Cimetidine 11 19 40 167.92 > 132.25 ESP- 169 Chlorzoxazone 10 19 30 319.27 > 85.79 ESP+ 318 Chlorpromazine 9 40 40 322.22 > 121.30 APcI- 323 Chlorampehnicol 8 12 30 348.26 > 158.34 ESP+ 347 Cephalexin 7 26 30 289.43 > 139.90 ESP+ 288 Bupivacaine 6 12 10 311.33 > 243.59 ESP+ 310 Bifonazole 5 26 30 267.33 > 145.40 ESP+ 266 Atenolol 4 40 50 459.46 > 135.28 ESP+ 458 Astemizole 3 26 20 366.38 > 126.84 APcI+ 365 Amoxicillin 2 40 50 309.27 > 205.37 ESP+ 308 Alprazolam 1 CE (V) CV (V) MRM Mode MW Name No 36 30 307.92>235.16 ESP+ 306 Zolpidem 46 19 40 307.37 > 161.18 ESP- 308 Wafarin 45 19 40 353.29 > 86.33 ESP+ 352 Triflupromazine 44 33 30 271.26 > 91.25 ESP+ 270 Tolbutamide 43 33 40 241.24 > 174.09 ESP- 242 Thymidine 42 33 20 355.18 > 194.70 ESP- 356 Thiamphenicol 41 26 30 472.51 > 436.92 ESP+ 471 Terfenadine 40 19 30 226.20 > 152.03 ESP+ 225 Terbutaline 39 12 20 336.22 > 272.54 ESP- 337 tenoxicam 38 19 20 319.15>276.58 APCI-- 320 Tenidap 37 33 40 254.15 > 92.17 ESP+ 253 Sufamethoxazole 36 33 50 325.33 > 81.43 ESP+ 324 Quinidine 35 19 40 260.31 > 115.76 ESP+ 259 Propranolol 34 19 30 285.32 > 85.83 APcI+ 284 Promethazine 33 19 30 284.36 > 240.66 ESP- 285 Probenecid 32 12 30 359.36>147.28 ESP+ 358 Prednisone 31 19 30 332.20 > 95.18 ESP+ 331 Piroxicam 30 19 40 362.27 > 318.70 ESP+ 361 Ofloxacin 29 26 30 264.33 > 90.99 ESP+ 263 Nortriptyline 28 19 50 320.27 > 302.62 ESP+ 319 Norfloxacin 27 26 40 326.25 > 291.58 ESP+ 325 Midazolam 26 26 50 417.14 > 159.19 ESP+ 416 Miconazole 25 26 40 371.33 > 125.85 ESP+ 370 Lorcainide 24 19 50 327.40 > 309.78 ESP- 328 Labetalol 23 33 30 238.20 > 124.75 ESP+ 237 Ketamine 22 12 30 334.25 > 270.57 ESP- 335 Isoxicam 21 36 30 307.92>235.16 ESP+ 306 Zolpidem 46 19 40 307.37 > 161.18 ESP- 308 Wafarin 45 19 40 353.29 > 86.33 ESP+ 352 Triflupromazine 44 33 30 271.26 > 91.25 ESP+ 270 Tolbutamide 43 33 40 241.24 > 174.09 ESP- 242 Thymidine 42 33 20 355.18 > 194.70 ESP- 356 Thiamphenicol 41 26 30 472.51 > 436.92 ESP+ 471 Terfenadine 40 19 30 226.20 > 152.03 ESP+ 225 Terbutaline 39 12 20 336.22 > 272.54 ESP- 337 tenoxicam 38 19 20 319.15>276.58 APCI-- 320 Tenidap 37 33 40 254.15 > 92.17 ESP+ 253 Sufamethoxazole 36 33 50 325.33 > 81.43 ESP+ 324 Quinidine 35 19 40 260.31 > 115.76 ESP+ 259 Propranolol 34 19 30 285.32 > 85.83 APcI+ 284 Promethazine 33 19 30 284.36 > 240.66 ESP- 285 Probenecid 32 12 30 359.36>147.28 ESP+ 358 Prednisone 31 19 30 332.20 > 95.18 ESP+ 331 Piroxicam 30 19 40 362.27 > 318.70 ESP+ 361 Ofloxacin 29 26 30 264.33 > 90.99 ESP+ 263 Nortriptyline 28 19 50 320.27 > 302.62 ESP+ 319 Norfloxacin 27 26 40 326.25 > 291.58 ESP+ 325 Midazolam 26 26 50 417.14 > 159.19 ESP+ 416 Miconazole 25 26 40 371.33 > 125.85 ESP+ 370 Lorcainide 24 19 50 327.40 > 309.78 ESP- 328 Labetalol 23 33 30 238.20 > 124.75 ESP+ 237 Ketamine 22 12 30 334.25 > 270.57 ESP- 335 Isoxicam 21 MS Scan for Ibuprofen m/z 201 202 203 204 205 206 207 208 209 210 211 17: Scan AP+ 9.45e5 202.1 12: Scan AP- 9.45e5 205.2 7: Scan ES- 9.45e5 205.2 206.2 1: Scan ES+ 9.45e5 207.1 210.7 Daughter Scan for Ibuprofen m/z 60 80 100 120 140 160 180 200 4: Daughters of 205ES- 8.10e5 3: Daughters of 205ES- 8.10e5 2: Daughters of 205ES- 8.10e5 161.3 1: Daughters of 205ES- 8.10e5 205.5 161.8 Daughter ion scan spectra in the optimized ionization mode (ESI–) at different colli- sion energies. Example MS spectra for Ibuprofen . One in each of the 4 ionization ESI+ ESI- APCI- APCI+ CE 10V CE 20V CE 30V CE 40V Ibuprofen, Extracted MS Chromatograms at m/z 205 Time 0.20 0.40 0.60 0.80 10: Scan ES- 9.45e5 9: Scan ES- 9.45e5 8: Scan ES- 9.45e5 7: Scan ES- 9.45e5 6: Scan ES- 9.45e5 5: Scan ES+ 9.45e5 4: Scan ES+ 9.45e5 3: Scan ES+ 9.45e5 2: Scan ES+ 9.45e5 1: Scan ES+ 9.45e5 Time 0.20 0.40 0.60 0.80 20: Scan AP+ 2.53e5 19: Scan AP+ 2.53e5 18: Scan AP+ 2.53e5 17: Scan AP+ 2.53e5 16: Scan AP+ 2.53e5 15: Scan AP- 2.53e5 14: Scan AP- 2.53e5 13: Scan AP- 2.53e5 12: Scan AP- 2.53e5 11: Scan AP- 2.53e5 Time 0.20 0.40 0.60 0.80 10: Scan ES- 9.45e5 9: Scan ES- 9.45e5 8: Scan ES- 9.45e5 7: Scan ES- 9.45e5 6: Scan ES- 9.45e5 5: Scan ES+ 9.45e5 4: Scan ES+ 9.45e5 3: Scan ES+ 9.45e5 2: Scan ES+ 9.45e5 1: Scan ES+ 9.45e5 Time 0.20 0.40 0.60 0.80 20: Scan AP+ 2.53e5 19: Scan AP+ 2.53e5 18: Scan AP+ 2.53e5 17: Scan AP+ 2.53e5 16: Scan AP+ 2.53e5 15: Scan AP- 2.53e5 14: Scan AP- 2.53e5 13: Scan AP- 2.53e5 12: Scan AP- 2.53e5 11: Scan AP- 2.53e5 ESI- APCI- ESI+ APCI+ * RESULTS AND DISCUSSION QuanOptimize ESCi Application for in-vitro Microsome Stability Test • The application of ESCi places significant demands on data acquisi- tion of the mass spectrometer. This is specifically true for MS and MSMS scan experiments during the optimization stage. • However, the actual requirements for any experiment are dependent on the fitness of purpose. “All factors bearing on any analysis must be consistent with the overall purpose of the task” 6 . • For ESCi MS and MSMS scan experiments, the purpose was to choose the optimum ionization mode as well as the optimum MRM pa- rameters. This type of experiment is typically qualitative. Even though it is always better to obtain more data point across the peak, for quali- tative work, a few data points across the peak should be sufficient for the purpose. As shown in the sample MS and daughter ion scan spec- tra, we were able to obtain spectra with sufficient quality to allow opti- mization to be completed automatically. • For ESCi MRM experiments, the number of data points obtained var- ied depending on the number of channels to be monitored in a single injection. Less data points were obtained when more MRM channels were to be monitored, especially when ionization mode switching is required during a single injection. • Mixture analysis can be useful for cassette dosing experiments. In drug discovery, the analytical requirement may be semi-quantitative estimation of the ADME properties. This is less demanding in figure of merit allowing more compounds to be analyzed simultaneously. Even with mixture and 4 mode simultaneous switching, Quattro Premier still has the capability for mixture analysis 5 . • For the microsomal stability test, there is no need for multi-analyte analysis. A typical experiment is carried out for a single unknown compound per well. Therefore, the need is simply to analyze a single compound per injection. With the ESCi capability, compounds in a 96 well plate can be analyzed in a single analytical batch with both ESI and APCI. Example MRM MS Acquisition Methods Analyte MRM Chromatograms Microsome Stability Test Results CONCLUSIONS We have developed an automatied HT ESCi-LC/MS/MS analytical proto- col for the in vitro metabolic stability test: • HT separation enabled by the Waters ACQUITY UPLC™ Technology. • High speed capability of the Waters Quattro Premier allowing simulta- neous four mode ESCi switching during a single injection This method can be applied in screening a large number of compounds with high speed and covering diverse structures for drug discovery and development. The protocol will allow the analytical test to be run completely automated so that • The instrument time required for analyst is minimized • Some bottle necks are eliminated • Method transfer is made easy • The protocol is easily adapted into the current lab routine Reference 1. R. Gallagher et al. Anal. Chem., Vol. 75, 973-977, 2003 2. E. Kerns, J. Pharm. Sci., Vol. 90, No. 11, November 2001 3. L. Di, and E. Kerns, Current Opinion in Chemical Biology, 7:402-408, 2003 4. M. Balogh, US patent WO 2003/102537 5. K. Yu, P. Alden, L. Di, S. Li, E. Kerns, Poster Presentation, Montreux 2004 6. P. Jane Gale, LC/MS Quantification Training Class, ASMS, 1999 Daspone ESI+ Tolbutamide APCI- APCI+ Ibuprofen ESI- Daspone ESI+ Tolbutamide APCI- APCI+ Ibuprofen ESI- Alprazolam, ESI+ Chlorzoxazone, ESI- Promethazine, APCI+ Tenidap, APCI- These MRM methods were generated automatically by QuanOptimize after Athe MS optimization was completed. The MRM ionization mode was chosen based on the integration of peak area. The scan conditions that resulted in the highest peak area were chosen to be the MRM condi- Alprazolam, ESI+ Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 % 0 100 MRM of 1 Channel ES+ 309.2 > 205.3 0.78 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 % 0 100 MRM of 1 Channel ES+ 309.2 > 205.3 0.78 Chlorzoxazone, ESI- Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 % 0 100 MRM of 1 Channel ES- 167.9 > 132.2 0.69 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 % 0 100 MRM of 1 Channel ES- 167.9 > 132.2 0.69 Promethazine, APCI+ Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 % 0 100 MRM of 1 Channel AP+ 285.322 > 85.832 0.76 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 % 0 100 MRM of 1 Channel AP+ 285.322 > 85.832 0.76 Tenidap, APCI- Time 0.20 0.60 1.00 1.40 1.80 2.20 2.60 % 0 100 MRM of 1 Channel AP- 319.152 > 276.583 0.64 Time 0.20 0.60 1.00 1.40 1.80 2.20 2.60 % 0 100 MRM of 1 Channel AP- 319.152 > 276.583 0.64 2 0.22 ESP+ 280 Imipramine 20 >30 86.89 ESP- 206 Ibuprofen 19 4 9.01 APcI+ 255 Diphenhydramine 18 3 3.10 ESP+ 414 Diltiazem 17 5 14.09 ESP+ 318 Diclofenac Sodium 16 9 29.85 ESP- 392 Dexamethasone 15 6 16.08 ESP+ 266 Desipramine 14 <1 118.58 ESP+ 175 Debrisoquine Sulfate 13 2 0.53 ESP+ 326 Clozapine 12 >30 83.66 ESP+ 252 Cimetidine 11 <1 110.42 ESP- 169 Chlorzoxazone 10 2 0.52 ESP+ 318 Chlorpromazine 9 16 53.02 APcI- 323 Chlorampehnicol 8 26 66.93 ESP+ 347 Cephalexin 7 1 0.03 ESP+ 288 Bupivacaine 6 3 1.83 ESP+ 310 Bifonazole 5 >30 91.95 ESP+ 266 Atenolol 4 4 8.24 ESP+ 458 Astemizole 3 >30 90.32 APcI+ 365 Amoxicillin 2 10 37.06 ESP+ 308 Alprazolam 1 Metabolic Half-life (min.) %Remaining Mode MW Name No. 2 0.22 ESP+ 280 Imipramine 20 >30 86.89 ESP- 206 Ibuprofen 19 4 9.01 APcI+ 255 Diphenhydramine 18 3 3.10 ESP+ 414 Diltiazem 17 5 14.09 ESP+ 318 Diclofenac Sodium 16 9 29.85 ESP- 392 Dexamethasone 15 6 16.08 ESP+ 266 Desipramine 14 <1 118.58 ESP+ 175 Debrisoquine Sulfate 13 2 0.53 ESP+ 326 Clozapine 12 >30 83.66 ESP+ 252 Cimetidine 11 <1 110.42 ESP- 169 Chlorzoxazone 10 2 0.52 ESP+ 318 Chlorpromazine 9 16 53.02 APcI- 323 Chlorampehnicol 8 26 66.93 ESP+ 347 Cephalexin 7 1 0.03 ESP+ 288 Bupivacaine 6 3 1.83 ESP+ 310 Bifonazole 5 >30 91.95 ESP+ 266 Atenolol 4 4 8.24 ESP+ 458 Astemizole 3 >30 90.32 APcI+ 365 Amoxicillin 2 10 37.06 ESP+ 308 Alprazolam 1 Metabolic Half-life (min.) %Remaining Mode MW Name No. 14 46.52 ESP+ 306 Zolpidem 46 23 63.86 ESP- 308 Wafarin 45 3 2.01 ESP+ 352 Triflupromazine 44 >30 97.26 ESP+ 270 Tolbutamide 43 26 66.98 ESP- 242 Thymidine 42 25 65.95 ESP- 356 Thiamphenicol 41 4 8.73 ESP+ 471 Terfenadine 40 >30 98.45 ESP+ 225 Terbutaline 39 >30 74.44 ESP- 337 tenoxicam 38 14 46.52 APCI- 320 Tenidap 37 >30 87.67 ESP+ 253 Sufamethoxazole 36 3 2.26 ESP+ 324 Quinidine 35 2 0.79 ESP+ 259 Propranolol 34 2 0.13 APcI+ 284 Promethazine 33 17 53.39 ESP- 285 Probenecid 32 2 76.23 ESP+ 358 Prednisone 31 >30 72.96 ESP+ 331 Piroxicam 30 >30 96.81 ESP+ 361 Ofloxacin 29 3 2.60 ESP+ 263 Nortriptyline 28 <1 105.41 ESP+ 319 Norfloxacin 27 2 0.53 ESP+ 325 Midazolam 26 4 9.74 ESP+ 416 Miconazole 25 1 0.06 ESP+ 370 Lorcainide 24 2 0.57 ESP- 328 Labetalol 23 2 0.39 ESP+ 237 Ketamine 22 27 68.39 ESP- 335 Isoxicam 21 14 46.52 ESP+ 306 Zolpidem 46 23 63.86 ESP- 308 Wafarin 45 3 2.01 ESP+ 352 Triflupromazine 44 >30 97.26 ESP+ 270 Tolbutamide 43 26 66.98 ESP- 242 Thymidine 42 25 65.95 ESP- 356 Thiamphenicol 41 4 8.73 ESP+ 471 Terfenadine 40 >30 98.45 ESP+ 225 Terbutaline 39 >30 74.44 ESP- 337 tenoxicam 38 14 46.52 APCI- 320 Tenidap 37 >30 87.67 ESP+ 253 Sufamethoxazole 36 3 2.26 ESP+ 324 Quinidine 35 2 0.79 ESP+ 259 Propranolol 34 2 0.13 APcI+ 284 Promethazine 33 17 53.39 ESP- 285 Probenecid 32 2 76.23 ESP+ 358 Prednisone 31 >30 72.96 ESP+ 331 Piroxicam 30 >30 96.81 ESP+ 361 Ofloxacin 29 3 2.60 ESP+ 263 Nortriptyline 28 <1 105.41 ESP+ 319 Norfloxacin 27 2 0.53 ESP+ 325 Midazolam 26 4 9.74 ESP+ 416 Miconazole 25 1 0.06 ESP+ 370 Lorcainide 24 2 0.57 ESP- 328 Labetalol 23 2 0.39 ESP+ 237 Ketamine 22 27 68.39 ESP- 335 Isoxicam 21 %Remaining = (Peak Area T 20 /Peak Area T 0 )x100 In-Vitro Metabolite Half-life = (ln2 x Incubation Time)/ln(%Remaining/100) Ibuprofen, ESI- Time 0.20 0.40 0.60 0.80 1.00 8: Daughters of 205ES- 1.31e6 7: Daughters of 205ES- 1.31e 6 6: Daughters of 205ES- 1.31e6 5: Daughters of 205ES- 1.31e6 4: Daughters of 205ES- 1.31e6 3: Daughters of 205ES- 1.31e6 2: Daughters of 205ES- 1.31e6 0.37 1: Daughters of 205ES- 1.31e6 0.37 Ibuprofen, ESI- Time 0.20 0.40 0.60 0.80 1.00 8: Daughters of 205ES- 1.31e6 7: Daughters of 205ES- 1.31e 6 6: Daughters of 205ES- 1.31e6 5: Daughters of 205ES- 1.31e6 4: Daughters of 205ES- 1.31e6 3: Daughters of 205ES- 1.31e6 2: Daughters of 205ES- 1.31e6 0.37 1: Daughters of 205ES- 1.31e6 0.37 720001935EN
Transcript of DEVELOPMENT OF AN AUTOMATED HIGH THROUGHPUT …TO DOWNLOAD A COPY OF THIS POSTER VISIT ©2006 Waters...
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DEVELOPMENT OF AN AUTOMATED HIGH THROUGHPUT UPLC/MS/MS PROTOCOL WITH MULTI-MODE IONIZATION FOR IN-VITRO METABOLIC STABILITY ASSAY
Kate Yu1, Peter Alden1, Rob Plumb1, Li Di2, Susan Li2, Edward Kerns2, Paul Chilvers3 1. Waters Corporation, Milford, MA; 2. Wyeth Research, Princeton, NJ; 3. Waters Micromass, Manchester, UK
INTRODUCTION Highlights
Multi-mode ionization: ESCiTM • No need to physically change ionization sources for
ESI/ APCI switching. Automated LC/MS/MS analytical protocol (from method development to report generation) • Automated MS and MSMS scans for optimization • Automated MRM MS acquisition method generation • Automated data acquisition • Automated quantification and report generation Application example presented for microsome-stability test in drug discovery
ESCi Multi-mode Ionization
• “ESCi™ multi-mode ionization source” refers to a combination ESI & APCI capability available on Waters mass spectrometers.
• The enabling of the ESCi source is driven by specifically designed elec-tronics capitalizing on unique attrib-utes of the source geometry.
• In ESCi, both ESI and the APCI modes can function by alternating at high MS switching speeds in both positive & negative polarities4.
Project Goal To develop an automated ESCi-LC/MS/MS analytical protocol for the in vitro metabolic stability test using the 96 well plate to demonstrate the ad-vantages of ESCi™ multimode ionization and the Waters ACQUITY UPLC™ for higher throughput and broader compound coverage in drug discovery.
Background
LC/MS analysis typically utilizes atmospheric pressure ionization (API) as the ionization source with two options: • Electrospray (ESI): favor polar compounds • Atmospheric pressure chemical ionization (APCI): favors non-polar
compounds and relative small molecules. The best ionization mode for any compound is determined by optimizing MS conditions in both ESI and APCI. • Generally, about 80% compounds can be analyzed by ESI1. • APCI is the common alternative option for non=ESI compounds. Traditionally, to change between ESI and APCI • Analysts need to physically change the ionization source • All ion source parameters need to be re-optimized • This results in lost analysis time and reduces analytical throughput sig-
nificantly1. • This results in lost analysis time, hence significantly reduces analytical
throughput1.
Challenges for ESCi Applied to Drug Discovery
The characteristics for analyses carried out in drug discovery are: • Many new compounds to be analyzed for screening purpose • Typically, each sample solution will contain a single compound for
the analysis • Many injections will be required. Each injection will contain a single
target analyte.
The bottle necks are: • MS optimization including tuning each analyte for MRM transition,
cone voltage (CV), and collision energy (CE) Method creation: • MRM MS acquisition method: each analyte requires a separate MS
acquisition method MS quantification method for post run quantifica-tion
The instrument feasibility as well as the advantages of ESCi Multi-mode ionization have been previously demonstrated5. In reality, to apply ESCi in a drug discovery lab, an automated protocol is crucial to minimize the instrument time required for analysts.
EXPERIMENTAL CONDITIONS In-Vitro Metabolic Stability Screening
The test method is a routine method in the Wyeth Research Lead Optimi-zation Lab • Rat liver microsomes • Single time point incubation: 15 min • Physiological level: 1 μ M Samples ready for LC/MS/MS analysis after the microsome incubation • Samples are in 96 well plate format 48 different compounds per plate, different RT and MS conditions for each compound • One compound per well • Each compound appeared in two wells for duplicated injections • Typically, 45 new analytes and 3 reference standards
UPLC Conditions Waters ® ACQUITY UPLC™ Binary Solvent Manager Mobile Phase: A: 10 mM NH4OAc in ACN/H2O 10/90
B: 10 mM NH4OAc in ACN/MeOH 80/20 Column: ACQUITY UPLC™ C18 BEH Column
1.7 mm, 2.1 x 50 mm, 40oC Sample Temperature: 5oC Flow Rate: 0.6 ml/min. Optimization: 90%B Isocratic elution Quantification Gradient: Time (min.) %A Curve
0.0 95% 6 0.8 5% 6 1.0 5% 1 3.0 95% 1
MS Conditions Waters Micromass® Quattro PremierTM Triple Quadrupole MS Tune Page Parameters:
ESCi Multi-Mode Ionization enabled ESI capillary voltage (kV): 3.00 APCI corona current (μ A): 4.0 Source Temperature (oC): 130 Desolvation Temperature (oC): 420 Desolvation Flow (L/Hr): 980 Cone Gas Flow (L/Hr): 50
Optimization and Quantification performed by QuanOptimize MS and MSMS scans for optimization MRM for quantification
Microsomal Incubation: Plate Layout
Di, J Biomolec Screening (2003) 8, 453Di, J Pharm Sci (2004) 93, 1537
IncubationPlates
Plate forQuantitation
StockPlate
Reagent &MicrosomeReservoirs
MS OPTIMIZATION QuanOptimize in MassLynxTM • QuanOptimize is a MassLynx optional application package. It en-
ables a complete automatic MS quantification process. • Once the LC conditions for the target analytes were determined, only
one analyte needs to be infused into the mass spectrometer (T with the LC mobile phase at the operating LC flow rate) to optimize the tune page parameters (everything except the cone voltages for parent ions and collision energies for daughter ions).
• QuanOptimize can be set up with all necessary methods and sample lists specified, the LC/MS/MS run can be initiated. QuanOptimize will then perform the following tasks: • Perform MS scan and Daughter scan to obtain MS data acquisition
conditions for each analyte by injecting individual standard solutions. • Create MS acquisition method based on the optimization injections
QuanOptimize Method Set Up
• Acquire quantification data for sample analysis • Create MS quantification method for post run processing • Complete post run quantification and generate report in QuanLynx
browser
MS Scan in 4 Ionization Modes in a Single Injection
Daughter Scan with the Optimized Ionization Mode During a QuanOptimize run, two sets of samples are required per analyte: the first set is the pure standard solution at high concentration, which is to be used for the MS the scan optimizations. The second set is the calibration solutions of the analyte, they can be a mixture if the project requires the analysis of a mix-ture with certain concentration series. They are to be injected for the calibration.
The number of injections required per compound during the optimization stage will depend on the QuanOptimize method. For non-split optimization, there will be two injections total: The first injection is
for MS scan of all four mode (ESI+/ESI-/APCI+/APCI-), and the second injection will be the daughter ion scan at the optimum ionization mode. For split optimization, there will be three injections total: The first injection is for MS scan in positive mode (ESI+/APCI+), the second injection is for MS scan in negative mode (ESI-/APCI-), and the third injection is for daughter ion scan at the optimum ionization mode.
MS Optimization Results
MS and Daughter Scan Spectra
There are a few different ways that optimization to be carried on by Quan-Optimize.In this project, the optimization was performed this way:
• A single injection for MS scan in all four ionization mode (ESI+/ESI-/APCI+/APCI-).
• The optimum ionization mode was decided based on the integration of the peak area. The ionization mode and cone voltage that resulted in the highest peak area was chosen to be the optimum MS condition. In the example demonstrated, the ESI- at function 7 (CV 20) was cho-sen to be the optimum MS condition.
• A second injection was made for daughter ion scan at ESI– with the MS cone voltage set at 20v.
• The best fragment ion and collision energy was again chosen based on the integration of peak area. In the example demonstrated, colli-sion energy of 5v and daughter ion 161 was chosen as the optimum condition.
1930281.36 > 85.76ESP+280Imipramine20
520205.18 > 161.83ESP-206Ibuprofen19
1220256.28 > 167.49APcI+255Diphenhydramine18
2640415.33 > 178.12ESP+414Diltiazem17
1910319.18 > 163.36ESP+318Diclofenac Sodium16
4010390.53 > 293.62ESP-392Dexamethasone15
1230267.33 > 72.18ESP+266Desipramine14
1220176.01 > 98.45ESP+175Debrisoquine Sulfate13
1940327.27 > 270.57ESP+326Clozapine12
2630253.26 > 95.24ESP+252Cimetidine11
1940167.92 > 132.25ESP-169Chlorzoxazone10
1930319.27 > 85.79ESP+318Chlorpromazine9
4040322.22 > 121.30APcI-323Chlorampehnicol8
1230348.26 > 158.34ESP+347Cephalexin7
2630289.43 > 139.90ESP+288Bupivacaine6
1210311.33 > 243.59ESP+310Bifonazole5
2630267.33 > 145.40ESP+266Atenolol4
4050459.46 > 135.28ESP+458Astemizole3
2620366.38 > 126.84APcI+365Amoxicillin2
4050309.27 > 205.37ESP+308Alprazolam1
CE (V)CV (V)MRMModeMWNameNo
1930281.36 > 85.76ESP+280Imipramine20
520205.18 > 161.83ESP-206Ibuprofen19
1220256.28 > 167.49APcI+255Diphenhydramine18
2640415.33 > 178.12ESP+414Diltiazem17
1910319.18 > 163.36ESP+318Diclofenac Sodium16
4010390.53 > 293.62ESP-392Dexamethasone15
1230267.33 > 72.18ESP+266Desipramine14
1220176.01 > 98.45ESP+175Debrisoquine Sulfate13
1940327.27 > 270.57ESP+326Clozapine12
2630253.26 > 95.24ESP+252Cimetidine11
1940167.92 > 132.25ESP-169Chlorzoxazone10
1930319.27 > 85.79ESP+318Chlorpromazine9
4040322.22 > 121.30APcI-323Chlorampehnicol8
1230348.26 > 158.34ESP+347Cephalexin7
2630289.43 > 139.90ESP+288Bupivacaine6
1210311.33 > 243.59ESP+310Bifonazole5
2630267.33 > 145.40ESP+266Atenolol4
4050459.46 > 135.28ESP+458Astemizole3
2620366.38 > 126.84APcI+365Amoxicillin2
4050309.27 > 205.37ESP+308Alprazolam1
CE (V)CV (V)MRMModeMWNameNo
3630307.92>235.16ESP+306Zolpidem46
1940307.37 > 161.18ESP-308Wafarin45
1940353.29 > 86.33ESP+352Triflupromazine44
3330271.26 > 91.25ESP+270Tolbutamide43
3340241.24 > 174.09ESP-242Thymidine42
3320355.18 > 194.70ESP-356Thiamphenicol41
2630472.51 > 436.92ESP+471Terfenadine40
1930226.20 > 152.03ESP+225Terbutaline39
1220336.22 > 272.54ESP-337tenoxicam38
1920319.15>276.58APCI--320Tenidap37
3340254.15 > 92.17ESP+253Sufamethoxazole36
3350325.33 > 81.43ESP+324Quinidine35
1940260.31 > 115.76ESP+259Propranolol34
1930285.32 > 85.83APcI+284Promethazine33
1930284.36 > 240.66ESP-285Probenecid32
1230359.36>147.28ESP+358Prednisone31
1930332.20 > 95.18ESP+331Piroxicam30
1940362.27 > 318.70ESP+361Ofloxacin29
2630264.33 > 90.99ESP+263Nortriptyline28
1950320.27 > 302.62ESP+319Norfloxacin27
2640326.25 > 291.58ESP+325Midazolam26
2650417.14 > 159.19ESP+416Miconazole25
2640371.33 > 125.85ESP+370Lorcainide24
1950327.40 > 309.78ESP-328Labetalol23
3330238.20 > 124.75ESP+237Ketamine22
1230334.25 > 270.57ESP-335Isoxicam21
3630307.92>235.16ESP+306Zolpidem46
1940307.37 > 161.18ESP-308Wafarin45
1940353.29 > 86.33ESP+352Triflupromazine44
3330271.26 > 91.25ESP+270Tolbutamide43
3340241.24 > 174.09ESP-242Thymidine42
3320355.18 > 194.70ESP-356Thiamphenicol41
2630472.51 > 436.92ESP+471Terfenadine40
1930226.20 > 152.03ESP+225Terbutaline39
1220336.22 > 272.54ESP-337tenoxicam38
1920319.15>276.58APCI--320Tenidap37
3340254.15 > 92.17ESP+253Sufamethoxazole36
3350325.33 > 81.43ESP+324Quinidine35
1940260.31 > 115.76ESP+259Propranolol34
1930285.32 > 85.83APcI+284Promethazine33
1930284.36 > 240.66ESP-285Probenecid32
1230359.36>147.28ESP+358Prednisone31
1930332.20 > 95.18ESP+331Piroxicam30
1940362.27 > 318.70ESP+361Ofloxacin29
2630264.33 > 90.99ESP+263Nortriptyline28
1950320.27 > 302.62ESP+319Norfloxacin27
2640326.25 > 291.58ESP+325Midazolam26
2650417.14 > 159.19ESP+416Miconazole25
2640371.33 > 125.85ESP+370Lorcainide24
1950327.40 > 309.78ESP-328Labetalol23
3330238.20 > 124.75ESP+237Ketamine22
1230334.25 > 270.57ESP-335Isoxicam21
MS Scan for Ibuprofen
m/z201 202 203 204 205 206 207 208 209 210 211
17: Scan AP+ 9.45e5
202.1
12: Scan AP-9.45e5
205.2
7: Scan ES-9.45e5
205.2
206.2
1: Scan ES+ 9.45e5
207.1 210.7
Daughter Scan for Ibuprofen
m/z60 80 100 120 140 160 180 200
4: Daughters of 205ES-8.10e5
3: Daughters of 205ES-8.10e5
2: Daughters of 205ES-8.10e5
161.3
1: Daughters of 205ES-8.10e5205.5
161.8
MS Scan for Ibuprofen
m/z201 202 203 204 205 206 207 208 209 210 211
17: Scan AP+ 9.45e5
202.1
12: Scan AP-9.45e5
205.2
7: Scan ES-9.45e5
205.2
206.2
1: Scan ES+ 9.45e5
207.1 210.7
Daughter Scan for Ibuprofen
m/z60 80 100 120 140 160 180 200
4: Daughters of 205ES-8.10e5
3: Daughters of 205ES-8.10e5
2: Daughters of 205ES-8.10e5
161.3
1: Daughters of 205ES-8.10e5205.5
161.8
Daughter ion scan spectra in the optimized ionization mode (ESI–) at different colli-sion energies.
Example MS spectra for Ibuprofen . One in each of the 4 ionization
ESI+
ESI-
APCI-
APCI+
CE 10V
CE 20V
CE 30V
CE 40V
Ibuprofen, Extracted MS Chromatograms at m/z 205
Time0.20 0.40 0.60 0.80
10: Scan ES-9.45e5
9: Scan ES-9.45e5
8: Scan ES-9.45e5
7: Scan ES-9.45e5
6: Scan ES-9.45e5
5: Scan ES+ 9.45e5
4: Scan ES+ 9.45e5
3: Scan ES+ 9.45e5
2: Scan ES+ 9.45e5
1: Scan ES+ 9.45e5
Time0.20 0.40 0.60 0.80
20: Scan AP+ 2.53e5
19: Scan AP+ 2.53e5
18: Scan AP+ 2.53e5
17: Scan AP+ 2.53e5
16: Scan AP+ 2.53e5
15: Scan AP-2.53e5
14: Scan AP-2.53e5
13: Scan AP-2.53e5
12: Scan AP-2.53e5
11: Scan AP-2.53e5
Time0.20 0.40 0.60 0.80
10: Scan ES-9.45e5
9: Scan ES-9.45e5
8: Scan ES-9.45e5
7: Scan ES-9.45e5
6: Scan ES-9.45e5
5: Scan ES+ 9.45e5
4: Scan ES+ 9.45e5
3: Scan ES+ 9.45e5
2: Scan ES+ 9.45e5
1: Scan ES+ 9.45e5
Time0.20 0.40 0.60 0.80
20: Scan AP+ 2.53e5
19: Scan AP+ 2.53e5
18: Scan AP+ 2.53e5
17: Scan AP+ 2.53e5
16: Scan AP+ 2.53e5
15: Scan AP-2.53e5
14: Scan AP-2.53e5
13: Scan AP-2.53e5
12: Scan AP-2.53e5
11: Scan AP-2.53e5
ESI-
APCI-ESI+
APCI+
*
RESULTS AND DISCUSSION QuanOptimize ESCi Application for in-vitro Microsome Stability Test • The application of ESCi places significant demands on data acquisi-
tion of the mass spectrometer. This is specifically true for MS and MSMS scan experiments during the optimization stage.
• However, the actual requirements for any experiment are dependent on the fitness of purpose. “All factors bearing on any analysis must be consistent with the overall purpose of the task”6.
• For ESCi MS and MSMS scan experiments, the purpose was to choose the optimum ionization mode as well as the optimum MRM pa-rameters. This type of experiment is typically qualitative. Even though it is always better to obtain more data point across the peak, for quali-tative work, a few data points across the peak should be sufficient for the purpose. As shown in the sample MS and daughter ion scan spec-tra, we were able to obtain spectra with sufficient quality to allow opti-mization to be completed automatically.
• For ESCi MRM experiments, the number of data points obtained var-ied depending on the number of channels to be monitored in a single injection. Less data points were obtained when more MRM channels were to be monitored, especially when ionization mode switching is required during a single injection.
• Mixture analysis can be useful for cassette dosing experiments. In drug discovery, the analytical requirement may be semi-quantitative estimation of the ADME properties. This is less demanding in figure of merit allowing more compounds to be analyzed simultaneously. Even with mixture and 4 mode simultaneous switching, Quattro Premier still has the capability for mixture analysis5.
• For the microsomal stability test, there is no need for multi-analyte analysis. A typical experiment is carried out for a single unknown compound per well. Therefore, the need is simply to analyze a single compound per injection. With the ESCi capability, compounds in a 96 well plate can be analyzed in a single analytical batch with both ESI and APCI.
Example MRM MS Acquisition Methods
Analyte MRM Chromatograms
Microsome Stability Test Results
CONCLUSIONS We have developed an automatied HT ESCi-LC/MS/MS analytical proto-col for the in vitro metabolic stability test: • HT separation enabled by the Waters ACQUITY UPLC™ Technology. • High speed capability of the Waters Quattro Premier allowing simulta-
neous four mode ESCi switching during a single injection This method can be applied in screening a large number of compounds with high speed and covering diverse structures for drug discovery and development.
The protocol will allow the analytical test to be run completely automated so that • The instrument time required for analyst is minimized • Some bottle necks are eliminated • Method transfer is made easy • The protocol is easily adapted into the current lab routine
Reference 1. R. Gallagher et al. Anal. Chem., Vol. 75, 973-977, 2003 2. E. Kerns, J. Pharm. Sci., Vol. 90, No. 11, November 2001 3. L. Di, and E. Kerns, Current Opinion in Chemical Biology, 7:402-408, 2003 4. M. Balogh, US patent WO 2003/102537 5. K. Yu, P. Alden, L. Di, S. Li, E. Kerns, Poster Presentation, Montreux 2004 6. P. Jane Gale, LC/MS Quantification Training Class, ASMS, 1999
Daspone
ESI+
Tolbutamide
APCI-
Sulfadimethoxine
APCI+
Ibuprofen
ESI-
Alprazolam, ESI+
Chlorzoxazone, ESI-
Promethazine, APCI+
Tenidap, APCI-
Daspone
ESI+
Tolbutamide
APCI-
Sulfadimethoxine
APCI+
Ibuprofen
ESI-
Alprazolam, ESI+
Chlorzoxazone, ESI-
Promethazine, APCI+
Tenidap, APCI-
These MRM methods were generated automatically by QuanOptimize after Athe MS optimization was completed. The MRM ionization mode was chosen based on the integration of peak area. The scan conditions that resulted in the highest peak area were chosen to be the MRM condi-
Alprazolam, ESI+
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100MRM of 1 Channel ES+
309.2 > 205.30.78
Chlorzoxazone, ESI-
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100 MRM of 1 Channel ES-167.9 > 132.2
0.69
Promethazine, APCI+
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100 MRM of 1 Channel AP+ 285.322 > 85.832
0.76
Tenidap, APCI-
Time0.20 0.60 1.00 1.40 1.80 2.20 2.60
%
0
100 MRM of 1 Channel AP-319.152 > 276.583
0.64
Alprazolam, ESI+
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100MRM of 1 Channel ES+
309.2 > 205.30.78
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100MRM of 1 Channel ES+
309.2 > 205.30.78
Chlorzoxazone, ESI-
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100 MRM of 1 Channel ES-167.9 > 132.2
0.69
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100 MRM of 1 Channel ES-167.9 > 132.2
0.69
Promethazine, APCI+
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100 MRM of 1 Channel AP+ 285.322 > 85.832
0.76
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100 MRM of 1 Channel AP+ 285.322 > 85.832
0.76
Tenidap, APCI-
Time0.20 0.60 1.00 1.40 1.80 2.20 2.60
%
0
100 MRM of 1 Channel AP-319.152 > 276.583
0.64
Time0.20 0.60 1.00 1.40 1.80 2.20 2.60
%
0
100 MRM of 1 Channel AP-319.152 > 276.583
0.64
20.22ESP+280Imipramine20
>3086.89ESP-206Ibuprofen19
49.01APcI+255Diphenhydramine18
33.10ESP+414Diltiazem17
514.09ESP+318Diclofenac Sodium16
929.85ESP-392Dexamethasone15
616.08ESP+266Desipramine14
<1118.58ESP+175Debrisoquine Sulfate13
20.53ESP+326Clozapine12
>3083.66ESP+252Cimetidine11
<1110.42ESP-169Chlorzoxazone10
20.52ESP+318Chlorpromazine9
1653.02APcI-323Chlorampehnicol8
2666.93ESP+347Cephalexin7
10.03ESP+288Bupivacaine6
31.83ESP+310Bifonazole5
>3091.95ESP+266Atenolol4
48.24ESP+458Astemizole3
>3090.32APcI+365Amoxicillin2
1037.06ESP+308Alprazolam1
Metabolic Half-life (min.)%RemainingModeMWNameNo.
20.22ESP+280Imipramine20
>3086.89ESP-206Ibuprofen19
49.01APcI+255Diphenhydramine18
33.10ESP+414Diltiazem17
514.09ESP+318Diclofenac Sodium16
929.85ESP-392Dexamethasone15
616.08ESP+266Desipramine14
<1118.58ESP+175Debrisoquine Sulfate13
20.53ESP+326Clozapine12
>3083.66ESP+252Cimetidine11
<1110.42ESP-169Chlorzoxazone10
20.52ESP+318Chlorpromazine9
1653.02APcI-323Chlorampehnicol8
2666.93ESP+347Cephalexin7
10.03ESP+288Bupivacaine6
31.83ESP+310Bifonazole5
>3091.95ESP+266Atenolol4
48.24ESP+458Astemizole3
>3090.32APcI+365Amoxicillin2
1037.06ESP+308Alprazolam1
Metabolic Half-life (min.)%RemainingModeMWNameNo.
1446.52ESP+306Zolpidem46
2363.86ESP-308Wafarin45
32.01ESP+352Triflupromazine44
>3097.26ESP+270Tolbutamide43
2666.98ESP-242Thymidine42
2565.95ESP-356Thiamphenicol41
48.73ESP+471Terfenadine40
>3098.45ESP+225Terbutaline39
>3074.44ESP-337tenoxicam38
1446.52APCI-320Tenidap37
>3087.67ESP+253Sufamethoxazole36
32.26ESP+324Quinidine35
20.79ESP+259Propranolol34
20.13APcI+284Promethazine33
1753.39ESP-285Probenecid32
276.23ESP+358Prednisone31
>3072.96ESP+331Piroxicam30
>3096.81ESP+361Ofloxacin29
32.60ESP+263Nortriptyline28
<1105.41ESP+319Norfloxacin27
20.53ESP+325Midazolam26
49.74ESP+416Miconazole25
10.06ESP+370Lorcainide24
20.57ESP-328Labetalol23
20.39ESP+237Ketamine22
2768.39ESP-335Isoxicam21
1446.52ESP+306Zolpidem46
2363.86ESP-308Wafarin45
32.01ESP+352Triflupromazine44
>3097.26ESP+270Tolbutamide43
2666.98ESP-242Thymidine42
2565.95ESP-356Thiamphenicol41
48.73ESP+471Terfenadine40
>3098.45ESP+225Terbutaline39
>3074.44ESP-337tenoxicam38
1446.52APCI-320Tenidap37
>3087.67ESP+253Sufamethoxazole36
32.26ESP+324Quinidine35
20.79ESP+259Propranolol34
20.13APcI+284Promethazine33
1753.39ESP-285Probenecid32
276.23ESP+358Prednisone31
>3072.96ESP+331Piroxicam30
>3096.81ESP+361Ofloxacin29
32.60ESP+263Nortriptyline28
<1105.41ESP+319Norfloxacin27
20.53ESP+325Midazolam26
49.74ESP+416Miconazole25
10.06ESP+370Lorcainide24
20.57ESP-328Labetalol23
20.39ESP+237Ketamine22
2768.39ESP-335Isoxicam21
%Remaining = (Peak Area T20/Peak Area T0)x100
In-Vitro Metabolite Half-life = (ln2 x Incubation Time)/ln(%Remaining/100)
Ibuprofen, ESI-
Time0.20 0.40 0.60 0.80 1.00
8: Daughters of 205ES-1.31e6
7: Daughters of 205ES-1.31e6
6: Daughters of 205ES-1.31e6
5: Daughters of 205ES-1.31e6
4: Daughters of 205ES-1.31e6
3: Daughters of 205ES-1.31e6
2: Daughters of 205ES-1.31e60.37
1: Daughters of 205ES-1.31e6
0.37
Ibuprofen, ESI-
Time0.20 0.40 0.60 0.80 1.00
8: Daughters of 205ES-1.31e6
7: Daughters of 205ES-1.31e6
6: Daughters of 205ES-1.31e6
5: Daughters of 205ES-1.31e6
4: Daughters of 205ES-1.31e6
3: Daughters of 205ES-1.31e6
2: Daughters of 205ES-1.31e60.37
1: Daughters of 205ES-1.31e6
0.37
Ibuprofen, ESI-
Time0.20 0.40 0.60 0.80 1.00
8: Daughters of 205ES-1.31e6
7: Daughters of 205ES-1.31e6
6: Daughters of 205ES-1.31e6
5: Daughters of 205ES-1.31e6
4: Daughters of 205ES-1.31e6
3: Daughters of 205ES-1.31e6
2: Daughters of 205ES-1.31e60.37
1: Daughters of 205ES-1.31e6
0.37
720001935EN
