For Forensic Toxicology use Only Quantitative Screening of 46 Illicit Drugs in Urine using Ultrahigh Resolution and Accurate Mass Exactive System For Forensic Toxicology use Only Research Use Only

For Forensic Toxicology use Only Presentation overview Exactive system introduction Principle of operation Method overview and salient features Urine analysis results Conclusions Appendix   What’s different about this Orbitrap compared to the one in the LTQ Orbitrap? Orbitrap itself is very similar but its mounting and wiring have been made more compact. How do you get faster scan speed? Why is it faster than the LTQ Orbitrap? We operate orbitrap mass analyzer at higher voltage which results in higher frequencies. In parallel, we have reduced overheads on ion transfer (as there is no LTQ) and thus we need less time for a spectrum and less time for inter-spectrum delay. What’s the ultimate resolution? 100,000 at m/z 200 and 1 sec acquisition. What’s the dynamic range At least similar to that in LTQ Orbitrap, Full MS mode. Pumping system? There is a novel split-flow pump which allows to keep vacuum chamber smaller and make it from aluminium. Final vacuum is similar to that in a standard LTQ Orbitrap. C Trap is this the same as the current LTQ Orbitrap Yes, C-trap and ion optics are the same though their mounting is different (from a flange) HCD Scan – how does this work and – what’s new? Ions get accelerated through the C-trap into a quadrupolar collision cell where they fragment and get stored. After that, voltages are ramped and ions are transferred back to the C-trap from which they are injected into the orbitrap. There is no mass selection so only a narrow mass range of ions fragment with optimal sequence coverage, with lower m/z fragmenting excessively and higher m/z fragmenting only weakly. Is the mass accuracy the same at the LTQ Orbitrap?  (based on work in Bremen, it was better than the LTQ Orbitrap) Actually, we have a better layout and components of central electrode power supply so we expect mass accuracy at least as good as on LTQ Orbitrap XL. For Forensic Toxicology use Only 2

For Forensic Toxicology use Only Exactive Benchtop LC-MS Resolution 100,000 at 1 scan per second 10,000 at 10 scans per second Mass accuracy Sub ppm Sensitivity 500 fg Buspirone with S/N >10:1 Dynamic range >10,000 within a spectrum Scan speed Up to 10 scans per second Mass range m/z 50 - 4000 Polarity switching One positive and one negative scan < 1 second (25K Resolution)   What’s different about this Orbitrap compared to the one in the LTQ Orbitrap? Orbitrap itself is very similar but its mounting and wiring have been made more compact. How do you get faster scan speed? Why is it faster than the LTQ Orbitrap? We operate orbitrap mass analyzer at higher voltage which results in higher frequencies. In parallel, we have reduced overheads on ion transfer (as there is no LTQ) and thus we need less time for a spectrum and less time for inter-spectrum delay. What’s the ultimate resolution? 100,000 at m/z 200 and 1 sec acquisition. What’s the dynamic range At least similar to that in LTQ Orbitrap, Full MS mode. Pumping system? There is a novel split-flow pump which allows to keep vacuum chamber smaller and make it from aluminium. Final vacuum is similar to that in a standard LTQ Orbitrap. C Trap is this the same as the current LTQ Orbitrap Yes, C-trap and ion optics are the same though their mounting is different (from a flange) HCD Scan – how does this work and – what’s new? Ions get accelerated through the C-trap into a quadrupolar collision cell where they fragment and get stored. After that, voltages are ramped and ions are transferred back to the C-trap from which they are injected into the orbitrap. There is no mass selection so only a narrow mass range of ions fragment with optimal sequence coverage, with lower m/z fragmenting excessively and higher m/z fragmenting only weakly. Is the mass accuracy the same at the LTQ Orbitrap?  (based on work in Bremen, it was better than the LTQ Orbitrap) Actually, we have a better layout and components of central electrode power supply so we expect mass accuracy at least as good as on LTQ Orbitrap XL. For Forensic Toxicology use Only 3

Orbitrap – Principle of Operation z φ Hyper-logarithmic potential distribution: “ideal Kingdon trap” r Characteristic frequencies: Frequency of rotation ωφ Frequency of radial oscillations ωr Frequency of axial oscillations ωz Makarov A. Anal. Chem. 2000, 72, 1156-1162. For Forensic Toxicology use Only

Method overview and salient features Negative urine sample Dilute with 25% ACN Spike 46 drugs at different concentrations Spike isotopically labeled int standards at 50 ng/mL Inject 10 uL Hypersil GOLD™ PFP 2.1 x 100 mm, 3 µm Operated at res. 50,000 w/ Ext. Calib. Mass accuracy better than 5 ppm Note: LC/MS method details in appendix For Forensic Toxicology use Only

For Forensic Toxicology use Only Results - XIC@ ± 5 ppm at LOQs in urine B C D E F G H I J K L M N O P Q R S T U V W A Note: Drug names and LOQs on slide 9 & 10; XIC – Extracted Ion Chromatogram For Forensic Toxicology use Only

For Forensic Toxicology use Only Results - XIC @± 5ppm at LOQs in urine. AA BB CC DD EE FF GG HH II JJ KK LL MM NN OO PP QQ RR SS TT UU VV WW Note: Drug names and LOQs on slide 9 & 10; XIC – Extracted Ion Chromatogram For Forensic Toxicology use Only

For Forensic Toxicology use Only List of drugs and mass errors @ LOQ For Forensic Toxicology use Only

For Forensic Toxicology use Only List of drugs monitored and mass errors @ LOQ For Forensic Toxicology use Only

For Forensic Toxicology use Only Need for 50,000 resolution Nicotine mass spectra at experimental (Exp) and different simulated (Sim) resolutions (FWHM) Exp: 50,000 Sim: 50,000 Sim: 20,000 Sim: 10,000 Note: Experimental Nicotine at RT 1.40 min; exact mass = 163.1230 For Forensic Toxicology use Only

For Forensic Toxicology use Only Conclusions The method is capable of easy confirmatory and quantitative analysis of 46 illicit drugs at LOQs of 0.5 – 5 ng/mL in urine. Ultrahigh mass resolution of 50,000 FWHM provided the necessary selectivity to identify 46 drugs in urine in 9 minutes with minimal sample preparation Analyte confirmation is achieved by utilizing better than 5 ppm mass accuracy with external mass calibration. Good quantitative accuracy achieved by isotopically-labeled standard. Nicotine example demonstrates the need for ≥50,000 resolution in addition to exact masses. For Forensic Toxicology use Only

For Forensic Toxicology use Only Appendix For Forensic Toxicology use Only

For Forensic Toxicology use Only LC/MS Method HPLC system Accela UHPLC™ Pump, PAL autosampler (CTC Analytics) LC Column 2.1 x 100 mm, 3 µm Hypersil GOLD™ PFP Injection Volume = 10 µL Mobile Phase (A) 5 mM NH4OOCH + 0.1% HCOOH, (B) ACN + 0.1% HCOOH Flow Rate 0.35 mL/min MS system Exactive™ (Thermo Scientific) Heated ESI; Polarity: Positive; Scan Range: m/z 110 – 800 Resolution: 50,000 FWHM; External mass calibration For Forensic Toxicology use Only