For Clinical Research and Forensic Toxicology Use Only Exactive LC- MS for Quantitation in Clinical and Forensic Toxicology Research For Clinical Research and Forensic Toxicology Use Only

For Clinical Research and Forensic Toxicology Use Only Overview Exactive As a Quantitative Analysis Tool Clinical and Forensic Toxicology Research Methods Drugs Supported in the Methods Quantitation Workflow What You Don’t Have To Do Why it is Easy To Use User Testimonials Proof Statements and Support Summary Appendix For Clinical Research and Forensic Toxicology Use Only

Why Consider Exactive For Quantitation LOQ, Precision and Dynamic Range ~ Mid to High End QQQ QQQ Assays Plus More Easiest to Use Large Panel of Drugs in Single Assay Ideal for Analytes with Poor Fragmentation Sample Prep Requirement Same As QQQ Good Screening Capabilities Unbeatable Price/Performance For Clinical Research and Forensic Toxicology Use Only

Why Consider Exactive For Quantitation Methods Clinical Research Forensic Tox Research Immunosuppressant Drugs in Whole Blood ETG/ETS in Urine Opioids Benzodiazepines in Urine Vitamin D3 in Plasma Opiates in Urine 10 Steroid Panel in Plasma Drugs of Abuse Screening For Clinical Research and Forensic Toxicology Use Only

Why Consider Exactive For Quantitation Clinical Research Forensic Research Immunosuppressant Drugs Serolimus Tacrolimus Everolimus Cyclosporin A ETG/ETS Benzodiazepines Lorazepam Diazepam Nordiazepam Temazepam Hydroxytriazolam Aminoclonazepam Aminotriazepam Hydroxyalprazolam Aminoflunitrazepam Desalkylflurazepam Hydroxyethylflurazepam Oxazepam Opioids Norketamine Ketamine Nalbuphine Norfentanyl Fentanyl Butorphanol Alfentanil Sufentanil Vitamin D3 25 OH Vit D3 25 OH Vit D2 10 Steroid Panel Aldosterone Androstenedione Corticosterone Cortisol Deoxycortisol Dehydroepiandrosterone sulphate Progesterone 17 alpha-hydroxyprogesterone Testosterone Opiates Morphine, Hydromorphone, Oxymorphone Codeine, Dihydrocodeine, Hydrocodone Oxycodone, Meperidine, Normeperidine 6-Acetylmorphine Drugs of Abuse Screening For Clinical Research and Forensic Toxicology Use Only

Exactive Quantitation Workflow What You Do Sample Preparation Urine Dilute n Shoot or Turbo Flow LLE SPE Data Acquisition LC - Exactive Full Scan MS@ 50 - 100K resolution using HESI or APCI source Mass Range 50-4000m/z Data Processing Extract data for compounds and internal standards using better than 2ppm mass accuracy As Easy It Can Get Quantitate Quantitate using peak area of extracted ion chromatograms for samples and calibrators There is no more universal detection method for drugs-of-abuse or toxicants than electrospray LC-MS involving the simultaneous detection of positively and negatively charged ions. This is exactly the type of method used by ToxSpec where all ion source and analyzer parameters have been fully optimized for simultaneous positive and negative ion detection by mass spectrometry, for molecular weight determination, followed by data-dependent tandem mass spectrometry for fragment ion library identification. This methodology is summarized graphically in this slide. Each drug and toxicant in the ToxSpec library has it’s unique molecular ion and fragment ion spectrum; during the LC-MS run, ions are automatically chosen for tandem mass analysis based upon their relative intensities and molecular weights. The orthogonal metrics of relative retention time, molecular weight and unique ion fragmentation spectral fingerprint are more than enough to positively identify compounds of interest. A +ve MS scan is performed, the m/z of ions detected over the background are compared against the expected m/z and Rt of the drugs in the method and tentative match recorded. MS2 scan is performed on up to 5 most intense ions and data recorded. A -ve MS scan is performed, the m/z of ions detected over the background are compared against the expected m/z and Rt of the drugs in the method and tentative match recorded. MS2 scan is performed on up to 2 most intense ions and data recorded. Report Results What You Don’t Have To Do (next slide) For Clinical Research and Forensic Toxicology Use Only

Exactive Quantitation Workflow What You Don’t Have To Do Optimize SRM’s Infusion or FIA optimization Painstaking process of product ion selection for best sensitivity and specificity Collision gas optimization Optimization of ion path lens voltages Optimization of Timed SRM For Clinical Research and Forensic Toxicology Use Only

Exactive Quantitation Workflow Why it is Ease to Use Easiest to operate No SRM optimization Quantitate large panels without additional optimization considerations Easiest to expand assay Easiest to interpret data Easiest to maintain For Clinical Research and Forensic Toxicology Use Only

What Do Users Say “The beauty of the Exactive MS is that it allows us to look for unknown metabolites at the same time that we quantitate known metabolites.  In our laboratory, we have been able to identify four different fentanyl metabolites with no need for UV detection, fraction collection with NMR, or MS/MS fragmentation patterns” Uwe Christians, MD, PhD, Professor, Department of Anesthesiology, University of Colorado and iSiS Clinical Research & Development Lab Director “We historically used triple quadrupole mass spectrometers but were very impressed with the Exactive demo. After acquiring the system, we had to do little to no method development. It’s flexibility in analyzing both dilute-and-shoot and SPE prepared samples and the ease of addition of new compounds to our drug panels helped us keep things simple. Overall, it turned out to be a wise choice for our Drugs of Abuse and Pain Management drug analyses” Kent Johnson, Director Fortes Lab, Portland Oregon, USA For Clinical Research and Forensic Toxicology Use Only

Proof Statements and Support Performance and Method Details (PPT files for each method) Library of Compounds for Screening in ToxID Assay Installation and Training Customer Reference Sites For Clinical Research and Forensic Toxicology Use Only

For Clinical Research and Forensic Toxicology Use Only Summary All Things Considered, Ultrahigh Resolution Exactive Bench Top LC/MS System Is a Very Efficient and Sensitive Alternative Quantitation Tool for Use in Clinical and Forensic Research Including Pain Management. For Clinical Research and Forensic Toxicology Use Only

For Clinical Research and Forensic Toxicology Use Only Appendix For Clinical Research and Forensic Toxicology Use Only

Exactive Quantitation Workflow Why is it easy to use Easiest to operate Full scan MS operation No SRM optimization Monitor m/z of interest at ultrahigh resolution and mass accuracies Monitor large panels without additional optimization considerations Just extract m/z of interest in any full scan MS acquisition Easiest to expand assay Full scan acquisition means all ions all the time Easiest to interpret data Just need to know the m/z of the compound of interest Easiest to maintain Less complex ion path, no collision cell and the necessary gas. For Clinical Research and Forensic Toxicology Use Only