The world leader in serving science An Ultrahigh

The world leader in serving science An Ultrahigh Resolution Accurate Mass LC/MS solution to Forensic Toxicology Screening in Serum

Presentation overview § Introduction to Exactive benchtop LC/MS system § List of drugs monitored in serum § Sample preparation § LC/MS method overview, HPLC and UPLC § Resolution and mass accuracy data § Identification performance § LOD vs resolution & concentration § Conclusions § Appendix 2

Exactive™ a short introduction Collision cell x Octapole C-Trap Split Lens Flatapole Skimmer Tubelens Orbitrap 1. 2. 3. 4. Ions are injected through the source …and trapped in the C-trap and squeezed into a smaller cloud …then a voltage pulse across C-trap ejects ions towards the Orbitrap …where they are trapped and detected 3 Capillary

Orbitrap – Principle of Operation r z φ Hyper-logarithmic potential distribution: “ideal Kingdon trap” § Characteristic frequencies: • Frequency of rotation ωφ • Frequency of radial oscillations ωr • Frequency of axial oscillations ωz Makarov A. Anal. Chem. 2000, 72, 1156 -1162. 4

The 40 drugs in serum Concentrations : 0. 4 µg/L - 1. 25 mg/L for most drugs § Isobars § Isomers Amitriptyline 278. 1903 LSD 324. 2070 Phenobarbital 231. 0764 Bromazepam 316. 0080 Maprotiline 278. 1903 Prazepam 325. 1102 Buprenorphine 468. 3108 Methadone 310. 2165 Quinine 325. 1911 Citalopram 325. 1711 Nadolol 310. 2012 Quinidine 325. 1911 Clobazam 301. 0738 Norbuprenorphine 414. 2638 THC COOH 345. 2060 Clomipramine 315. 1623 Norclobazam 287. 0582 THC Delta 9 315. 2319 Clonazepam 316. 0483 Norcyamemazine 310. 1372 Thiopental 241. 1062 Cyamemazine 324. 1529 Nordiazepam 271. 0633 Tramadol 264. 1958 Declomipramine 301. 1466 Norfluoxetine 296. 1257 Venlafaxine 278. 2115 Diazepam 285. 0789 Nor. LSD 310. 1914 Verapamil 455. 2904 EDDP 278. 1903 Normaprotiline 264. 1747 Zolpidem 308. 1757 Fluoxetine 310. 1413 Nortritpryline 264. 1747 Zopiclone 389. 1123 Glibenclamide 494. 1511 Norvenlafaxine 264. 1958 Hydroxyzine 375. 1834 Oxazepam 287. 0582 Note: Lowest concentration samples prepared by dilutions with a mixture 80/20 A/B 5

Serum sample preparation protocol Liquid Extraction 1000 µL human serum Add 200 µL 20% Na 2 CO 3 Add 5 m. L ether Vortex 1 minute Separate organic layer dry at 40 deg C under N 2 Reconstitute in 400 µL 80/20 A/B Inject 10 ul 6

LC/MS method overview HRMS method • ESI source • Mass range 100 – 800 amu • Res: 10 K, 50 K, 100 K • Pos/Neg switching • HCD MS/MS after every MS scan Alternate Scan Polarity Switching Pos. Full Scan MS 0. 1 s Pos. HCD 1 Second Neg. HCD R =10, 000 7 Pos. Full Scan MS 100, 000 1 Second Neg. Full Scan MS 1 Second

Mass accuracy for identification Mass accuracy not affected by concentration or resolution 1000 mg/l 10 mg/l 100µg/l 100 k 50 k 10 k 100 k 50 k 10 k Imipramine 2. 5 2. 4 1. 4 2. 9 1. 7 2. 5 3 1. 7 2. 9 1. 1 3. 4 2. 2 1. 4 Levomepromazine 3. 5 3. 6 3. 7 3. 3 3. 6 3. 8 3 3. 9 3 3. 2 4. 4 3. 5 Lidocaine 2. 2 1. 8 1. 7 1. 9 1. 4 2. 6 1. 7 1. 1 2. 3 1. 2 2. 1 1. 4 2. 9 2. 5 2. 2 2. 8 3. 0 2. 3 2. 9 3. 1 2. 7 3. 0 2. 8 2. 4 3. 0 2. 7 2. 3 …. . All molecules (ppm) Note: Data on standards using external mass calibration 8

Impact of concentration and resolution on identification 35 drugs identified with exact mass and RT upto 10 µg/L Selectivity example Extra confirmation with at least one fragment ion for most drugs 45 40 35 30 Zolpidem 0. 4 µg/L 25 20 15 10 5 0 100 k 1. 25 mg/L 100 k 10 k 250 µg/L 100 k 10 k 10 µg/L 100 k 10 k 2 µg/L 100 k 10 k 0. 4 µg/L Buprenorphine 1 µg/L Note: Identification with 5 ppm accuracy 9

Impact of resolution on isotope ratio matching for confirmation Resolution ~ 100, 000 FWHM provided reliable isotope ratio confirmation 34 S Resolution 100. 000 Olanzapine C 17 H 20 N 4 S Resolution 10. 000 10

Accuracy of experimental isotope pattern - Bromazepam Experimental and theoretical pattern match well EXPERIMENTAL m/z Intensity Relative 316. 00692 10055447. 0 100. 00 317. 01021 1337341. 6 13. 30 318. 00479 9632045. 0 95. 79 319. 00810 1243849. 1 12. 37 THEORITICAL m/z Intensity Relative 316. 00800 429640. 4 100. 00 317. 01136 65056. 2 15. 14 318. 00595 417943. 8 97. 28 319. 00931 63285. 1 14. 73 11

Additional confirmation using fragment ions MS chromatogram Maprotiline and Amitryptiline • identical mass • different structure • different RT • different fragment ions Maprotiline C 20 H 23 N Amitryptiline C 20 H 23 N Unique fragment ion additionally confirms Maprotiline Fragment Maprotiline XIC of fragment ion 12

Benefit of resolution for co-eluting compounds Quinine and Bisprolol co-elute; 13 C isotope mass of one matches with the other HPLC data Quinine C 20 H 24 N 2 O 2 MW : 325. 1911 Bisoprolol C 18 H 31 NO 4 326. 2326 13

Benefit of resolution for co-eluting compound Sample with both drugs - confirmation compromised @10, 000 Quinine 12 C Quinine 13 C Quinine + Bisoprolol 12 C Resolution 100, 000 Resolution 10, 000 Mass accuracy Bisoprolol 3 ppm Mass accuracy Bisoprolol 9 ppm 14

Sensitivity increase with U-HPLC 80. 0 Sensitivity gain with U-HPLC Dynamic range 2 to 3 orders of magnitude (0. 4 to 250 ug/L) % of drugs 5 X for ~70% drugs 68. 4 70. 0 68. 4 60. 0 50. 0 40. 0 26. 3 23. 7 30. 0 20. 0 10. 0 7. 9 5. 3 0. 0 Resolution 100 k No gain in Sensitivity (%) Resolution 10 k Increase by 1 to 5 x (%) LSD 15 Increase above 5 x (%)

HPLC/UPLC – 1. 25 mg/L in serum HPLC UPLC 16

Identification range comparison - HPLC vs UPLC % drugs Best results with UPLC at 100, 000 resolution 17

Automated reporting with Tox. ID Software Compound ID Formula. Mass. Adducts and fragments Mass Retention Time Accuracy Intensity 18

Conclusions § Ultra high resolution >50, 000 needed for specificity in screening § Better than 5 ppm mass accuracy identified all drugs § Isotope ratio matching is reliable at 100, 000 resolution § Additional confirmation using at least one fragment ion § UHPLC improved sensitivity by 5 x over HPLC § Reduced run time by half to 13 minutes § LOD for most drugs ~ 2 ug/L § Tox. ID software is ideally suited for screening 19

Thanks To…. § CHU Reims, Pharmacology/Toxicology • Denis Lamiable, Guillaume Hoizey, Olivier Oget, Laurent Binet § Thermo. Fisher Scientific • Eric Génin, Lab manager. 20

Analysis of Benzodiazepines in urine using Exactive benchtop LC/MS system 21

Standards, QC and sample preparation QC sample Sample preparation 6 drugs at 250 ng/m. L from El. Sohly Labs 2 m. L urine Standard preparation Negative standard urine Add 12 Benzodiazepines Add 7 deuterated IS Standards Hydrolysis SPE IS Lorazepam-D 4 Nordiazepam-D 5 Oxazepam-D 5 Temazepam-D 5 Hydroxyalprazolam Hydroxytriazolam-D 4 Desalkylflurazepam Hydroxyalprazolam-D 5 Aminoclonazepam Hydroxyalprazolam-D 5 Aminonitrazepam Aminoflunitrazepam-D 7 Hydroxyalprazolam Aminoflunitrazepam Desalkylflurazepam Hydroxyethylflurazepam Diazepam Note: Blanks were negative standard urine spiked with IS at 1000 ng/ml 22 200 ul of extract 5 u. L inject

LC/MS Method § LC method • Mobile Phase • A: water containing 0. 1% formic acid • B: ACN containing 0. 1% formic acid • Column • Thermo Scientific Hypersil GOLD PFP 50 x 2. 1 mm, 5 µm • Injection volume: 5 µL • Flow rate: 400 µL/min • Gradient Time (min) % A 0 95 0. 5 95 3. 0 5 4. 1 95 4. 2 95 6. 0 95 %B 5 5 95 9 5 5 5 § Exactive MS method • Resolution: 100, 000 • Source: ESI 23

12 Benzodiazepines with 7 IS Total ion chromatogram (TIC) QC sample 200 ng/m. L std Benzodiazepines elution window (0. 9 min) Note: 4 X throughput with LX – 4 multiplexing system 24

Blank – All IS elute within 0. 9 min window Very low background, low 0 -3 e 3 for blank vs 2 - 5 e 6 for IS@1000/ng/ml Internal standards Standards Hydroxyethylflurazepam Temazepam Hydroxytriazolam Lorazepam-D 4 Nordiazepam-D 5 Lorazepam Oxazepam-D 5 Diazepam Hydroxyalprazolam Temazepam-D 5 Desalkylflurazepam Aminoclonazepam Hydroxytriazolam-D 4 Nordiazepam Oxazepam Aminonitrazepam Aminoflunitrazepam Note: Blanks were negative standard urine spiked with IS at 1000 ng/ml 25 Hydroxyalprazolam-D 5 Aminoflunitrazepam-D 7

200 ng/m. L standard – All elute in 0. 5 min window Hydroxyethylflurazepam Lorazepam-D 4 Temazepam Nordiazepam-D 5 Lorazepam Hydroxytriazolam Oxazepam-D 5 Diazepam Hydroxyalprazolam Temazepam-D 5 Desalkylflurazepam Aminoclonazepam Hydroxytriazolam-D 4 Nordiazepam Oxazepam Aminonitrazepam Hydroxyalprazolam-D 5 Aminoflunitrazepam-D 7 26

1000 ng/m. L standard Hydroxyethylflurazepam Temazepam Lorazepam-D 4 Nordiazepam-D 5 Lorazepam Hydroxytriazolam Oxazepam-D 5 Diazepam Hydroxyalprazolam Temazepam-D 5 Aminoclonazepam Desalkylflurazepam Hydroxytriazolam-D 4 Nordiazepam Aminonitrazepam Hydroxyalprazolam-D 5 Oxazepam Aminoflunitrazepam 27 Aminoflunitrazepam-D 7

2000 ng/m. L standard Temazepam Lorazepam-D 4 Hydroxyethylflurazepam Lorazepam Hydroxytriazolam Nordiazepam-D 5 Oxazepam-D 5 Diazepam Hydroxyalprazolam Temazepam-D 5 Desalkylflurazepam Aminoclonazepam Hydroxytriazolam-D 4 Nordiazepam Oxazepam Aminonitrazepam Aminoflunitrazepam 28 Hydroxyalprazolam-D 5 Aminoflunitrazepam-D 7

Carryover - Much less than 1% Me. OH after 2000 ng/m. L std Hydroxyethylflurazepam Lorazepam-D 4 Temazepam No analyte signal Low background signal in all channels Nordiazepam-D 5 Lorazepam Hydroxytriazolam Oxazepam-D 5 Diazepam Hydroxyalprazolam Temazepam-D 5 Desalkylflurazepam Aminoclonazepam Hydroxytriazolam-D 4 Nordiazepam Oxazepam Aminonitrazepam Hydroxyalprazolam-D 5 Aminoflunitrazepam-D 7 29

QC containing 6 drugs – 250 ng/m. L( expected and found) IS Hydroxyethylflurazepam Lorazepam-D 4 Temazepam Nordiazepam-D 5 Lorazepam Diazepam Hydroxytriazolam Oxazepam-D 5 Hydroxyalprazolam Temazepam-D 5 Desalkylflurazepam Aminoclonazepam Hydroxytriazolam-D 4 Nordiazepam Oxazepam Aminonitrazepam Hydroxyalprazolam-D 5 Aminoflunitrazepam-D 7 30

QC sample – Quant performance statistics 31

Conclusions § The method for Benzodiazepines analysis in urine meets all the criteria set by the forensic toxicology lab customer • • • Run time - 6 min Linearity range 200 -2000 ng/m. L No carryover Use of only 7 internal standards Works on customer’s hydrolysis and SPE protocol Recovery in high 90% for most drugs § The specificity of the system for this assay is equivalent or better than MRM on triple quadrupole LC/MS/MS system Note: MRM data not shown here 32

Quantitation of Urinary Ethyl Glucuronide using Ultra High Resolution LC-MS System 33

Goal § Develop a method to quantitate urinary Ethyl glucoronide (ETG) in Fortes lab (forensic toxicology lab customer) with the following characteristics • • • LOQ 500 ng/m. L or better Linearity range : 500 -10, 000 ng/m. L Accuracy >15% Carryover <1% of the highest concentration standard Good correlation with reference lab method 34

LC/MS system § Accela LC System § Exactive System § Hypercarb 50 x 2. 1 mm 35

ETG calibrator and sample preparation Calibrator Concentration ETG ng/m. L Sample preparation Cal 1 100 1 m. L urine Cal 2 250 Cal 3 500 Cal 4 5000 Cal 5 10000 Add 100 u. L IS@5000 ng/m. L Dilute 10 X with H 2 O Note: Internal Standard ETG-D 5 stock@5000 ng/m. L 10 u. L inject 36

Calibration standards chromatograms XIC@5 ppm mass accuracy, res. 100, 000 250 ng/m. L 100 ng/m. L* ETG IS IS *not included in calibration curve 37

Calibration standards chromatograms XIC@5 ppm mass accuracy 10000 ng/m. L 5000 ng/m. L ETG IS IS 38

Calibration curve - 250 to 10000 ng/m. L Good linearity and accuracy Note: 500 ng/m. L standard analyzed in triplicate 39

No carryover Blank urine after 10, 000 ng/m. L standard No ETG peak IS 40

Urine samples 22665 21891 ETG > 10 000 ng/m. L ETG: 4200 ng/m. L IS IS 41

Urine samples 22287 ETG > 10, 000 ng/m. L ETG: 380 ng/m. L IS IS 42

Comparison with reference lab results 21828 Comparison summary ETG < 250 ng/m. L *Sample was injected twice IS 43

Conclusion § The method developed on Exactive LC/MS system meets all the criteria for ETG analysis set by Fortes lab (forensic toxicology lab customer) • • • LOQ better than 500 ng/m. L Good linearity between 500 – 10, 000 ng/m. L Accuracy better than 15% (mostly better than 5%) No carryover Excellent correlation with established reference lab method 44

Appendix 45

LC-MS method § Column • Hypercarb 50 x 2. 1, 5 mm § Mobile phase • A: 20 m. M ammonium acetate in DI water • B: ACN § Gradient method § Autosampler method • • • Injection volume: 20 u. L Injection mode: no waste Wash solution: 80% Me. OH Wash volume: 1000 m. L Flash volume: 1000 m. L LC-gradient Time (min) %A %B Flow rate (m. L/min) 0 98 2 200 3. 1 5 95 600 4. 01 98 2 600 10. 0 98 2 600 46

MS method § Source parameters • • ESI source Sheet gas (units): 15 Aux gas (units): 10 Sweep gas (units): 0 Spray voltage (k. V): 5 Capillary temperature (C 0): 320 Capillary voltage (V): -82. 5 Tube lens voltage (V): -135 § Data acquisition parameters • • • Polarity: negative Microscans: 1 Resolution: 100, 000 Max inject time (ms): 100 Scan range (mu): 221 -227 Probe position: C 47

Human urine data 48