1. 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

2. 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

3. 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

4. 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

5. Step 1 Preparation of Calibration levels and QC’s 100 uL of Urine Spike 7 internal standards (50-100 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)

6. 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

7. List of Multi-Class Drugs

8. LC Separation of Compounds with Same Exact Masses Time (min) 0 50 100 4.24 5.63 RT:0.00 - 15.28SM:13G 0 50 100 5.865.19 0 50 100 4.47 5.82 0123456789101112131415 0 50 100 5.30 5.71 NL: 3.12E5 m/z= 328.1531-328.1564 F: FTMS {1,1} + p APCI corona Full ms [50.00-500.00] MS 8e NL: 6.00E5 m/z= 302.1351-302.1409 F: FTMS {1,1} + p APCI corona Full ms [50.00-500.00] MS 8e NL: 1.53E6 m/z= 286.1402-286.1463 F: FTMS {1,1} + p APCI corona Full ms [50.00-500.00] MS 9b NL: 1.05E6 m/z= 300.1555-300.1623 F: FTMS {1,1} + p APCI corona Full ms [50.00-500.00] MS 8b Codeine hydrocodone morphine hydromorphone Oxymorphone Noroxycodone Naloxone 6-acetylmorphine Time [min]

9. Compounds Detected in Negative Ion Mode RT:0.00 - 13.21SM:11G 012345678910111213 Time (min) 0 20 40 60 80 100 Relative Abundance 0 20 40 60 80 100 Relative Abundance 0 20 40 60 80 100 Relative Abundance 0 20 40 60 80 100 Relative Abundance BUTALBITAL 8.76 9.72 11.7912.620.131.864.704.183.801.338.122.270.696.752.79 BUTABARBITAL 8.69 9.6110.34 11.0911.3912.593.475.003.171.523.801.862.314.330.626.335.97 PENTOBARBITAL 9.04 9.54 11.4611.8212.592.834.071.783.322.421.295.560.544.974.526.156.577.667.237.98 PHENOBARBITAL 8.44 9.769.34 11.1311.9013.134.032.940.773.322.681.895.684.675.416.251.227.316.54 NL: 7.97E5 m/z= 223.10660-223.10884 F: FTMS {1,2} - p APCI corona Full ms [50.00-400.00] MS mix of all-3 NL: 3.66E5 m/z= 211.10666-211.10878 F: FTMS {1,2} - p APCI corona Full ms [50.00-400.00] MS mix of all-3 NL: 3.76E5 m/z= 225.12224-225.12450 F: FTMS {1,2} - p APCI corona Full ms [50.00-400.00] MS mix of all-3 NL: 2.04E5 m/z= 231.07524-231.07750 F: FTMS {1,2} - p APCI corona Full ms [50.00-400.00] MS mix of all-3 Butalbital Butabarbital Pentobarbital Phenobarbital

10. Calibration Curves of Some Compounds R 2 = 0.9957 Oxymorphone R 2 = 0.9968 Estazolame R 2 = 0.9978 Alfentanil R 2 = 0.9984 7-Aminoclonazepam

11. CompoundFormula Theoretical Mass (m/z) Measured Mass (m/z) (  ppm) LOQ [ng/ml] R2R2 11-nor-9-Carboxy-  9-THC C21H28O4345.2066345.20781.21000.9991 HydroxyethylflurazepamC17H14ClFN2O2333.0806333.08201.4100.9970 Cis-Tramadol HCLC16H25NO2264.1964264.1957-0.6500.9937 Citalopram HBRC20H21FN2O325.1716325.17251250.9970 ClomipramineC19H23N2Cl315.1628315.16381.1100.9960 ClonazepamC15H10ClN3O3316.0489316.05001.2100.9953 CocaineC17H21NO4304.1549304.15570.9250.9954 CodeineC18H21NO3300.1600300.16080.8250.9943 Cyclobenzaprine HClC20H21N276.1752276.17621500.9945 Delta-9-THCC21H30O2315.2324315.23260.2500.9929 DesipramineC18H22N2267.1861267.18690.8100.9932 DesmethyldoxepinC18H19NO266.1545266.15530.9100.9978 DiazepamC16H13ClN2O285.0795285.08041100.9956 Doxepin HCLC19H21NO280.1701280.17090.9100.9967 EDDP PerchlorateC20H24N279.1987279.18991.5500.9954 EstazolamC16H11ClN4295.0750295.07601100.9924 FentanylC22H28N2O337.2280337.22951.6100.9934 FlunitrazepamC16H12FN3O3314.0941314.09561.5100.9978 FlurazepamC21H23ClFN3O388.1592388.16091.7100.9925 GabapentinC9H17NO2172.1338172.13370.11000.9928 GGECC11H15NO5242.1028242.1023-0.5250.9963

12. Q0 IQ2IQ3 Q0 Sensitivity

13. Resolution: 10k, 30k, 50k, 100k 279.12279.14279.16279.18279.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance Ethinyl-Estradiol, 279.17434 Butyl-Phthalate, 279.15909 (ubiquitous background ion)

14. 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.

15.  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

16. Ross EA, Watson M, Goldberger B. "Bath salts" intoxication. N Engl J Med. 2011 Sep 8;365(10):967-8.