Improving Experimental Efficiency for Drug Metabolite Profiling and Identification Pengxiang Yang 1, Josef Ruzicka 1, Jessica Wang 1, Kate Comstock 1, Caroline Ding 1, Mark Sanders 1, and Michael Cameron 1 Thermo Fisher Scientific, San Jose, California, USA, Scripps Research Institute, Jupiter, Florida, USA
verview Purpose: Drug metabolite profiling and identification are a key step to promote or stop drug compound in early drug discovery stage. It has always been a challenge on how to maximize the amount of metabolite information gathered in a single analysis of drugs that were dosed in mice or incubated with mouse and human liver microsomes. In this study, several features in mass spectrometry were investigated to improve the experimental efficiency. Methods: Several approaches from using new features of the Thermo Scientific Q Exactive rbitrap mass spectrometry are presented to improve the efficiency of using HR/AM LC/MS to characterize drug metabolites in complex samples. Results: Ultra high resolution from the Q Exactive rbitrap mass spectrometry reduces interference peaks. This often improves the quantitation results. Fast polarity switching can cover positive and negative ions from a single injection. Apex-triggering of the MS/MS scans ensures that the highest quality tandem MS data are gathered on metabolites of interest. Introduction LC/MS is used routinely for drug metabolite identification and profiling of in vivo and in vitro assays. The information these assays provide on metabolic soft-spots guides the medicinal chemist in determining what modifications to make to a potential drug structural template to improve its properties. As this is a key piece of information in the development of a drug there is a continuous drive to improve efficiencies in this process. ften the goal is to reduce expensive instrument utilization and conserve precious biological samples. This poster describes several approaches to maximize the amount of metabolite information gathered in a single analysis of drugs that were dosed in mice or incubated with mouse and human liver microsomes. Methods Sample Preparation All samples were collected and prepared in Scripps Research Institute, Jupiter, Florida before shipping to Thermo Fisher Scientific demo lab, San Jose, California. Microsomal incubations were µl and contained 1 mg/ml hepatic microsomes and 1 µm compound in 1 mm potassium phosphate buffer, ph.4. The incubations were brought to 3C with shaking and started with the addition of 1 mm NADPH. µl was removed and quenched with 1 µl acetonitrile at,, 1,, 4,and 6 minutes. The samples were centrifuged to precipitate proteins. The mouse studies were IP dosed at mg/kg in CBl6 mice. Compounds were formulated at mg/ml in 1%DMS/1%tween/%water. Blood was collected in sodium EDTA tubes and plasma generated through standard centrifugation techniques. Plasma protein was removed by centrifugation after the addition of -times v:v acetonitrile. Brains were collected and processed by the addition of -times v:w acetonitrile. The tissue was disrupted with a probe tip sonicator and centrifuged to remove suspended solids and precipitated proteins. Liquid Chromatography UHPLC equipped with Thermo Scientific Accela pen AS and 1 pump. ptimized chromatography conditions were used for each compound. Column: Thermo Scientific Hypersil Gold PFP, 1 x.1 mm, 3µm Column Temp: 3 C Injection Vol : µl LC Pump: Accela 1 Pump Mobile Phase A: Water,.1% Formic Acid Mobile Phase B: Acetonitrile,.1% Formic Acid Gradient: Time / min % MP-A %MP-B Flow Rate / µl/min. 9 6 1. 9 6. 9 6 1. 9 1.1 9 1. 9 6 Improving Experimental Efficiency for Drug Metabolite Profiling and Identification
Time, min D: "" File: "Thermo_DMS6_6July11.wiff" Da"..61 4 46 44 4 4 3 36 34 3 3 6 4. 1 16 14 1 1.94 6.69 1.6 4 1.6.3 1.1...4.6. 1. 1. 1.4 1.6 1....4.6. 3. 3. 3.4 3.6 Ti i Mass Spectrometry A bench-top Q Exactive rbitrap mass spectrometry from Thermo Fisher Scientific was used with external calibration. Various MS scan modes, data dependant settings and method strategies were investigated to determine the approaches that provided maximal, high quality, metabolite information while minimizing sample consumption. === HESI II Tune Data: === Spray Voltage 3 Capillary Temperature 3 Sheath Gas Aux Gas 1 Sweep Gas Heater Temperature S-lens 4 Lock Mass: off Automatic Gain Control (AGC ) 1E6 (full-ms scan), E (SIM), 1E (MS/MS) Data Analysis Thermo Scientific Xcalibur. software and MetWorks 1.3 software. Results Ultra High Resolution to Improve Ion Selectivity In the profiling and characterization of drug metabolites both quantitative and qualitative information is needed. Full scan, single ion monitoring (SIM) and MS/MS were evaluated for quantitation in terms of both ease of instrument set up and sensitivity of measurement. This included an evaluation of the appropriate resolution to run for each method. Full scan was the most convenient in terms of instrument set up, while high resolution SIM was generally observed as the most sensitive. In very complex matrices high resolution MS/MS was sometimes needed to avoid chemical interferences. As shown in Figure 1, in lower resolution instruments, such as triple quad and ion trap, MS/MS is employed to gain selectivity by lowering baseline noise. The decrease of signal in the process of MS/MS in many cases can be compensated for by the improved selectivity. n a instrument with capable of ultra high resolution (>,), selectivity can be obtained at the MS level without the need to develop MS/MS methods in most cases, which conserves absolute signal and makes instrument set up less time consuming. Targeted analysis can be performed by extracted ion chromatogram (EIC) using a ppm window around the theoretical m/z of the compound of interest to eliminate the majority of chemical interferences (Figure and 3). This makes data review more efficient. FIGURE 1. MRM of ng/ml peptide HYLNLVTR,.4 1.4, acquired from typical triple quadrupole mass spectrometry. FIGURE. EIC of ng/ml peptide HYLNLVTR, m/z. with ppm window, acquired from Q Exactive rbitrap mass spectrometry. Intensity, cps HYLNLVTR 1.13 1.9. 1 1. HYLNLVTR.66.9.41. 3. FIGURE 3. T ½ calculation of sunitinib (SF) in mouse liver microsomes, based on the peak area of SF with ppm mass accuracy window. SF Stability in Rat Liver Microsomes T1/ 11.4 min MS response 9.. y = -.6x +.663 R² =.9 6. 6 Time / min R^.9 SF: CHFN4 Polarity Switching (Pos/Neg) to Mon Due to the difference in chemical prope metabolites, they perform differently in sometimes. For instance, oxidative met more readily detected by negative ioniz mass (HR/AM) instruments this would r the utility of HR/AM polarity switching (p proved to be especially useful for the m Thermo Scientific Poster Note PN63_E 6/1S 3
HYLNLVTR icrosomes, based 1/ 11.4 min R^.9 : CHFN4 High Mass Accuracy to Eliminate Interference Many drugs has aromatic structure, which leads to produce multiple isomers in phase-i metabolites, such as multiple oxidation sites on an aromatic ring. Itʼs very critical to get the right number of isomers and estimate the most active functional group. Triple quadrupole mass spec user has to acquire MS/MS data to study the fragmentation pattern of each isomers and needs to find the linkage from drug compound to metabolites with respect to their MS/MS data similarity. It is time consuming to understand fragmentation pattern and also highly possible to miss isomers due to low quality MS/MS data. Being benefited from the ultra high resolution of rbitrap techinique Ref 1, ions of interests can be separated from background matrix. Additionally, the excellent mass accuracy within ppm can be achieved from Q Exactive rbitrap mass spectrometry across mass range of m/z - without using lock mass. As shown in Figure 4, the number of interference peaks can be reduced significantly by narrowing mass window from ppm to ppm, but peak areas of the metabolites remained the same. Lots of efforts were saved to identify and confirm metabolite isomers. FIGURE 4. EIC of compound 4 and its metabolites with (a) ppm, (b) 3 ppm, (c) ppm mass accuracy window. (a) ppm mass accuracy window 1 1 1 1 1 1 1 1 1 1 1 1 RT: 3.49 AA: 1143191 RT:. AA: 63 RT: 4.6 AA: 66 RT: 3.13 RT: 3. AA: 3446 AA: 93 RT: 3. AA: 393 RT: 3.3 AA: 4 RT: 3.4 AA: 6346 RT: 3. AA: 1311...4.6. 3. 3. 3.4 3.6 3. 4. 4. 4.4 4.6 4. [M CH 3 ] [M + ] [M + ] RT:. AA: 63...4.6. 3. 3. 3.4 3.6 3. 4. 4. 4.4 4.6 4. M M RT:. AA: 63 RT: 3.13 RT: 3. AA: 646 AA: 999 RT: 3. AA: 393 RT: 3.3 AA: 46 RT: 3.49 AA: 1143191 RT: 3.49 AA: 113666 RT: 3.4 AA: 619 RT: 3. AA: 1311 RT: 4.6 AA: 141 RT: 3.13 RT: 3.6 RT: 3. AA: 3446 AA: 163 AA: 93 RT: 3.1 AA: 6 RT: 3. AA: 393 RT: 3.3 AA: 4 (b) 3 ppm mass accuracy window [M CH 3 ] [M + ] [M + ] (c) ppm mass accuracy window [M CH 3 ] M [M + ] [M + ] RT: 3.4 AA: 63916 RT: 3. AA: 1311 RT: 4. AA: 1996 Interference Peaks Interference Peaks Interference Free...4.6. 3. 3. 3.4 3.6 3. 4. 4. 4.4 4.6 4. Polarity Switching (Pos/Neg) to Monitor Positive and Negative Metabolite Ions Due to the difference in chemical property between the drug compound and its metabolites, they perform differently in positive ionization and negative ionization sometimes. For instance, oxidative metabolism can also form metabolites that are more readily detected by negative ionization. For traditional high resolution accurate mass (HR/AM) instruments this would require running the sample twice. Here we show the utility of HR/AM polarity switching (pos/neg) in a single LCMS run (Figure ). This proved to be especially useful for the mouse study where the sample was limited. Pos Neg Isomer1 Apex-Triggered MS/MS Scan to btai For the qualitative information, data dep were evaluated. The instrument estimat triggers an MS/MS scan (Figure 6). This gathered on metabolites of interest. FIGURE 6. EIC and Apex-Triggered M from 6 min human liver microsomal 1 1 1 1 1 1 1 EIC of [Verapamil + ] Apex-Triggered MS/MS Scan 1 16.99 1 3 191.161 16.9 191.16 16.99 1.11 11. 4 166.63 16.99 6 Apex-Triggered MS/ 16.9 16.9 11. 166.64 49.14 61.1 9.1 1 4 Improving Experimental Efficiency for Drug Metabolite Profiling and Identification
le isomers in ic ring. Itʼs very ctive functional ata to study the ge from drug ity. It is time ssible to miss e Ref 1, ions of e excellent mass mass spectrometry own in Figure 4, arrowing mass mained the same. rs. Interference Peaks FIGURE. Positive, Negative EIC and MS spectrum of [3-14 + ] metabolite from 3 min mouse liver microsomal incubation. RT: 3.9 -. SM: B RT:.4 -. 1 1 6.3 4. 4... 6. 6... 1 1 Pos Neg Isomer1.6.6 6.3..3.4..4.1.44..9..46.6.19.9 6.14 6.4..11.4 6.1 6. 6.6 6.6.6.....96 6.1 6.1.19.1.13.1.6 6. 6.3 6. 6.66.9.69.9. 6. 6..... 1 1 94.6 6.336 9 9 6 6.3.43 [M+H] + 9.136 99.13 m/z 6.13 91.99 9.11 61.14 64.91 9.19 9.13 [M-H] - 9.164 Apex-Triggered MS/MS Scan to btain Qualitative Data For the qualitative information, data dependant approaches like apex triggered MS/MS were evaluated. The instrument estimates the chromatographic peak apex and triggers an MS/MS scan (Figure 6). This ensured the highest quality data were gathered on metabolites of interest. FIGURE 6. EIC and Apex-Triggered MS/MS scan of [Verapamil + ] metabolite from 6 min human liver microsomal incubation Ref. EIC of [Verapamil + ] Apex-Triggered MS/MS Scan 6 6 Multiple Mass Defect Filter (MMDF) to MMDF is a powerful feature in the lates Software that combines the results from filter out the vast majority of the backgro preserving those related to the parent d was simplified by MMDF and it was mor compounds, especially, unusual metab compound. RT:. -.1 1 9 6 4 3.. 1. 1.. RT:. -.1 1 6 4 (a) Raw data.4.9.4.69 1.94 1.96.13 1. 1. (b) After MMDF unusual metabolite 1.. 1. 1.. F Apex-Triggered MS/MS Spectrum of [Verapamil + ] 1 1 1 16.99 191.161 16.9 319.14 41.6 41.49 Interference Free Metabolite Ions ound and its tive ionization abolites that are solution accurate twice. Here we show run (Figure ). This ple was limited. 1 1 1 1 1 1 3 319.13 191.16 49.143 16.99 41.1 1.11 11. 4 166.63 16.99 6 16.9 16.9 61.19 413.43 319.13 9.143 91.63 isomer, types 1 3 3 4 4 m/z 41.49 41.1 41. 43.43 11. 41.1 91.64 166.64 All trademarks are the property of Thermo Fisher Sc This information is not intended to encourage use of intellectual property rights of others. Thermo Scientific Poster Note PN63_E 6/1S
Multiple Mass Defect Filter (MMDF) to Discovery Unusual Metabolites MMDF is a powerful feature in the latest version of Thermo Scientific MetWorks 1.3 Software that combines the results from up to six different MDFs. MMDFs were able to filter out the vast majority of the background ions in the full scan spectra while preserving those related to the parent drug. As shown in Figure, TIC chromatograph was simplified by MMDF and it was more feasible to discover the drug-related compounds, especially, unusual metabolites generated from the dissociated drug compound. FIGURE. Total ion chromatogram (TIC) of SF incubated in mouse liver microsoms for 6 min, (a) raw data and (b) data processed by MMDF. RT:. -.1 3.3 4.3 1 4.3 9 6 4 3 (a) Raw data.3 3. 4.39 3.9 3.14 3. 4.44 3.6 3.6.4.9.9 4.46 4.9.6.4.69 1.96 1. 1.94..13 1..1.. 1. 1... 3. 3. 4. 4. RT:. -.1.3 1 6 4 (b) After MMDF SF N F F unusual metabolite 1.6.9 unusual metabolite H.. 1. 1... 3. 3. 4. 4. mil + ] Conclusion The routine study of drug metabolites in in vivo and in vitro assays can be more efficient by using new features in the Q Exactive rbitrap mass spectrometry. Ultra high resolution and accurate mass improve ion selectivity and significantly reduce the inference from biological matrix. Polarity switching (Pos/Neg scan) allows both negative and positive ions be monitored simultaneously, a great approach for metabolites mapping with a single injection. Apex-Triggered MS/MS scan allows a MS/MS spectrum be acquired at the apex of the chromatogram peak along with acquiring the quantitation data, a very efficient way for Quan/Qual analysis. Multiple Mass Defect Filter (MMDF) simplifies the full-ms spectrum and make it easier to find unusual metabolites from biological matrix. The scan speed on Q Exactive rbitrap mass spectrometry (4Hz at R=,, 1.Hz at R=1,) provides superior sampling rate allowing excellent compatibility with UHPLC. References 1. Michaela Scigelov a and Alexander Makarov, Advances in bioanalytical LC MS using the rbitrap mass analyzer, Bioanalysis, 9, Vol. 1, No. 4, 41-4.. M Walles, T Thum, K Levsen, J Borlak, Metabolism of verapamil: 4 new phase I and phase II metabolites identified in cell cultures of rat hepatocytes by liquid chromatography tandem mass spectrometry Journal of Chromatography B, 3, Vol. 9, No., 6 4. Acknowledgements We would like to thank Mark Kagan and Zhuang Miao from Novartis Institutes for BioMedical Research for supplying verapamil micorsomal incubation samples. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 6 Improving Experimental Efficiency for Drug Metabolite Profiling and Identification
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