Xiang He (Presenter)
SCIEX
Bio: 2002: Ph.D in Chemistry, Stony Brook University, Stony Brook, NY 2002-2005: Post-Doctoral Fellow, Univ. California San Francisco (UCSF), San Francisco, CA 2005-2010: Scientist, PPD, Menlo Park, CA 2010-2012: Marketing Specialist, Thermo Scientific, San Jose, CA 2012 to present: Global Application Lead, Forensics, SCIEX, Redwood City, CA
Authorship: Xiang He, Casey Burrows, Matthew Noestheden, Michael Jarvis, Adrian Taylor, and Alexandre Wang
SCIEX, 1201 Redwood City, CA
| Short Abstract In this poster, we describe a rapid and sensitive analysis of a comprehensive forensic compound panel in human urine using ExionLCTM AC and QTRAP®/Triple QuadTM 4500 LC/MS/MS system. This forensic panel contains 93 compounds with both ionization polarities therefore the method involves fast polarity switching. There are a total of 212 MRM transitions in the method; monitoring 2 transitions per analyte and 1 transition for each internal standard used and the total LC runtime is 6.5 minutes (can be shortened for smaller panel). Sample preparation is based on enzymatic hydrolysis and a simple “dilute and shoot” methodology. |
Long Abstract
Introduction:
Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) is a widely used analytical tool for simultaneous quantitation of multiple compounds in forensic samples. Multiple Reaction Monitoring (MRM) detection is the gold standard for quantitation purpose because of its speed, specificity and sensitivity. As the number of analytes in a panel increases and total cycle time remains, the scanning time of each individual MRM will inevitably decrease, affecting data quality. Therefore, an algorithm of intelligently distributing the total scan time across the panel for maximum data quality is needed. Scheduled MRM™ algorithm decreases the number of concurrent MRMs monitored at any point in time, allowing both the cycle time and dwell time to remain optimal at higher levels of MRM multiplexing.
In this study we present a rapid, robust and sensitive analysis of a comprehensive forensic compound panel with 93 compounds in human urine using QTRAP®/Triple QuadTM 4500 LC/MS/MS system. Because of the presence of several barbiturates in the panel which ionize better in negative mode, polarity switching is implemented. Due to a high number of total MRM transitions (212 MRMs; monitoring 2 transitions per analyte and 1 transition for each internal standard used) and a short LC runtime (6.5 min), a newly optimized Scheduled MRM™ algorithm is used.
Methods:
Blank human urine was used to prepare calibrators. Four levels of calibrators were prepared. Actual concentrations for each compound at any calibrator level may be different but the concentration ratio between these calibrators was always (in descending order): 20:6:2:1. For instance, the four different concentrations (in descending order) for fentanyl in calibrators were: 20, 6, 2 and 1 ng/mL.
Urine sample was hydrolyzed at 55C. After hydrolysis, methanol and water were added to the mixture. The mixture was then centrifuged and the supernatant was transferred to glass vial for LC-MS/MS analysis.
HPLC separation was performed with SCIEX ExionLCTM AC HPLC system. Phenomenex Kinetex Phenyl-hexyl column were used. Mobile phase A (MPA) was ammonium formate in water and mobile phase B (MPB) was formic acid in methanol. The LC flowrate was 1 mL/min and the LC runtime was 6.5 min. Gradient starts at 20% MPB and increases to 95% MPB where it stays for 1 min before re-equilibrating at 20% MPB. Injection volume was 5 uL.
Data acquisition was done with Analyst 1.6.3. Source condition was as follows: Curtain gas: 30; collision gas (CAD): Medium; IonSpray Voltage (IS): 2500 V (positive) and -4500 V (negative); Temperature: 650C; GS1: 60; GS2: 50.
Target scan time was 0.2 sec and 0.05 sec for positive and negative modes, respectively. Pause time between MRM transitions were set to 5 msec. Detection window was 20 sec for both ionization modes. There are 212 MRMs in total in the method (will be shown in poster).
Quantitation was performed with MultiQuantTM software 3.0.
Preliminary Results:
1. We achieved fast separation of various isobaric compounds in the panel despite short LC runtime.
2. We achieved a minimum of 10 data points across the LC peak when the majority of the MRM transitions had over 15 or more data points.
3. We observed strong MRM signal at lowest calibrator level, suggesting the possibility of reaching even lower LOQ. Excellent linearity and reproducibility was observed throughout the dynamic range assessed in this effort.
4. It was essential to utilize polarity switching to accommodate more than 200 MRMs within the one short data acquisition method. In the current panel with 93 compounds, the LC runtime is 6.5 minutes. With a small panel (e.g. 72 compounds), we can easily reduce the LC runtime to 5.5 minutes (data will be shown in poster).
Conclusion:
A rapid and sensitive method for the LC-MS/MS analysis of 93-compound forensic panel in human urine was developed on the SCIEX ExionLCTM AC HPLC and QTRAP®/Triple QuadTM 4500 LC/MS/MS system. Scheduled MRMTM algorithm and polarity switching were utilized. This method utilized a dilute-and-shoot procedure. Excellent linearity and precision were observed for all the compounds in the listed calibration range.
For Research Use Only. Not for use in diagnostic procedures.
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Document number: RUO-MKT-01-2873-A.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | SCIEX |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |