Toxicological Screening of 80 Drugs in Urine Using the High Resolution Exactive LC/MS Orbitrap Mass Spectrometer Coupled to Online Extraction and Turbulent Flow Chromatography Touraj Shokati, Paul Simmons, Baharak Davari, Keith Hoffman, James Byrd, Marta Kozack, Jeffrey Zonderman, Jeffrey Galinkin, and Uwe Christians iC42, University of Colorado Denver, Clinical Research and Development and Thermo Fisher Scientific, Fremont, CA Presented by: Touraj Shokati ASMS June 5 th 2011

Introduction Currently Established Workflows: (1)Immunoassay  if positive  verification/ quantification by LC-MS/MS (2)GC-MS  data base search (3)Targeted LC-MS/MS assays

Exactive Bench-Top LC/MS Advantages: High accuracy and precision. Ability to scan up to 100,000 (Ultra high resolution). Switching between positive and negative polarity. Easy to develop the screening method based on exact mass and RT of compounds. Good sensitivity

Step 1 Prepare Samples Extract analytes with TurboFlow method Step 3 Analyze Samples Detect the targeted compounds using MS method Step 4 Analyze Data Fast, accurate, automated; ToxID™ software report LCQuan software Step 2 Separation of Compounds Elute Analytes with LC method 5-10 min/100 samples 17 min/Sample based on our assay

Step 1 Preparation of Calibration levels and QC’s 100 uL of Urine Spike 7 internal standards ( ng/ml) Spike Compounds dissolved in MeOH Dilute with HPLC water (1:10) 100 uL Injection and online extraction using TFC (2.5 – 1000 ng/ml) 9 calibration levels (Std) 8 quality controls (QC’s)

Step 2 LC Method TurboFlow Columns Cyclone-P and C18 XL ( 0.5x50mm) Thermo Scientific LC ColumnBetasil Phenyl-Hexyl, (3.0x100mm), 3µm Thermo Scientific Loading PumpQuaternary Pump Solvent AWater:ACN (1:1) + 10mM Ammonium bicarbonate + 0.1% FA Solvent BWater + 1% FA Solvent DACN:IPA:Acetone 7:2:1 Eluting PumpQuaternary Pump Solvent AWater + 5 mM Ammonium acetate + 0.1% FA Solvent BMethanol + 0.1% Formic Acid Solvent DACN:IPA:Acetone 7:2:1

List of Multi-Class Drugs

LC Separation of Compounds with Same Exact Masses Time (min) RT: SM:13G NL: 3.12E5 m/z= F: FTMS {1,1} + p APCI corona Full ms [ ] MS 8e NL: 6.00E5 m/z= F: FTMS {1,1} + p APCI corona Full ms [ ] MS 8e NL: 1.53E6 m/z= F: FTMS {1,1} + p APCI corona Full ms [ ] MS 9b NL: 1.05E6 m/z= F: FTMS {1,1} + p APCI corona Full ms [ ] MS 8b Codeine hydrocodone morphine hydromorphone Oxymorphone Noroxycodone Naloxone 6-acetylmorphine Time [min]

Compounds Detected in Negative Ion Mode RT: SM:11G Time (min) Relative Abundance Relative Abundance Relative Abundance Relative Abundance BUTALBITAL BUTABARBITAL PENTOBARBITAL PHENOBARBITAL NL: 7.97E5 m/z= F: FTMS {1,2} - p APCI corona Full ms [ ] MS mix of all-3 NL: 3.66E5 m/z= F: FTMS {1,2} - p APCI corona Full ms [ ] MS mix of all-3 NL: 3.76E5 m/z= F: FTMS {1,2} - p APCI corona Full ms [ ] MS mix of all-3 NL: 2.04E5 m/z= F: FTMS {1,2} - p APCI corona Full ms [ ] MS mix of all-3 Butalbital Butabarbital Pentobarbital Phenobarbital

Calibration Curves of Some Compounds R 2 = Oxymorphone R 2 = Estazolame R 2 = Alfentanil R 2 = Aminoclonazepam

CompoundFormula Theoretical Mass (m/z) Measured Mass (m/z) (  ppm) LOQ [ng/ml] R2R2 11-nor-9-Carboxy-  9-THC C21H28O HydroxyethylflurazepamC17H14ClFN2O Cis-Tramadol HCLC16H25NO Citalopram HBRC20H21FN2O ClomipramineC19H23N2Cl ClonazepamC15H10ClN3O CocaineC17H21NO CodeineC18H21NO Cyclobenzaprine HClC20H21N Delta-9-THCC21H30O DesipramineC18H22N DesmethyldoxepinC18H19NO DiazepamC16H13ClN2O Doxepin HCLC19H21NO EDDP PerchlorateC20H24N EstazolamC16H11ClN FentanylC22H28N2O FlunitrazepamC16H12FN3O FlurazepamC21H23ClFN3O GabapentinC9H17NO GGECC11H15NO

Q0 IQ2IQ3 Q0 Sensitivity

Resolution: 10k, 30k, 50k, 100k m/z Relative Abundance Ethinyl-Estradiol, Butyl-Phthalate, (ubiquitous background ion)

Conclusion  A fast, sensitive, and reliable TurboFlow chromatography- Exactive Orbitrap high-resolution MS method for clinical drug screening as relevant for pain clinics was developed and validated.  LLODs for the majority of measured drugs were in a range of 5 ng/mL or below and LLOQs for most of tested drugs were less than 50 ng/ml.  Based on our experience, the Exactive LC/MS platform is well-suited for the routine screening of legal and illegal drugs.

 Broad Screen not limited by antibody availability or preset MRMs (QQQ) Avoid false negative results  High specificity and molecular verification ID (High Resolution) Avoid false positive results  Superior sensitivity compared to immunoassays  Quantification  Data re-analysis All in one assay

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