Accucore. Ultimate Core Performance LC Column Technology to Maximize Your Investment. Dave Jarzinski. October 2011

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1 Accucore Ultimate Core Performance LC Column Technology to Maximize Your Investment Dave Jarzinski Thermo Fisher Scientific Account Manager Greater Boston/Cambridge & North Phone: October 2011

2 Fast Chromatography - Understanding the Drivers Analytical Scientist want Speed 62% Resolution 23% Sensitivity 7% Other 8% FAST CHROMATOGRAPHY 2

3 Accucore - Core Enhanced Technology TM Solid Core Particles 2.6µm diameter particles with a solid core generate high speed, high resolution separations without excessive backpressure Advanced Bonding Technology Optimised phase bonding creates a series of high coverage, robust phases Tight Control of Particle Diameter Enhanced selection process keeps particle size distribution to a minimum and produces high efficiency columns Automated Packing Process - Enhanced automated procedures ensure that all columns are packed with the highest quality 3

4 Particle Evolution Higher Efficiency Pellicular particles, 50μm 4

5 Resolution Equation R s = N α α 1 1 k' + k' Efficiency Particle size / packing Selectivity Retention (Chemistry) Factor (Surface area) 5

6 The Theory Van Deemter Equation HETP = A + B u + C u m + C u s Eddy Diffusion Longitudinal Diffusion 6

7 A Term - Eddy Diffusion or Multiple Paths The A term depends on; Quality of the packing (better packing smaller A term) Particle size (smaller the particle the smaller is the A term) 7

8 Core Enhanced Technology Eddy Diffusion Fully Porous Particles D 90/10 ~ 1.5 Core Enhanced Technology D 90/10 ~ 1.1 8

9 C Term - Resistance to Mass Transfer (s) The C term depends on: Differences in diffusion path in the silica pores Fully Porous Particle Core Enhanced Technology Particle 9

10 Core Enhanced Technology Packed Bed Porous Silica Core Enhanced Technology 10

11 Core Enhanced Technology - Efficiency 20.0 Height Equivalent Theoretical Plate Linear velocity of mobile phase (mm/s) Accucore 2.6µm 5µm 3µm <2µm Highest efficiency and lowest rate of efficiency loss with flow rate with Core Enhanced Technology 11

12 Core Enhanced Technology Low Pressure 1000 Pressure (bar) bar limit HPLC pressure limit Columns: 100 x 2.1 mm Mobile phase: H 2 O / ACN (1:1) Temperature: 30 C Flow rate (µl/min) Accucore RP-MS 2.6µm <2µm 3µm 5µm Pressures below 600 bar over optimum flow rate range with Core Enhanced Technology 12

13 Kinetic Plots Allows for fairer comparisons of analytical systems Van Deemter just compares pure separation ability Incorporates time of analysis Analysts want FASTER chromatography Van Deemter plots do not specify the time of analysis Incorporates pressure limitations of systems Van Deemter does not account for a pressure limitation on system 13

14 Kinetic Plot Efficiency & Speed t 0 /N (s) Time required to obtain a set efficiency Efficiency increasing N opt , , , ,000, Accucore RP-MS 2.6um 5um 3um <2um Highest plate generation rate with Core Enhanced Technology N (/) 14

15 Impedance Devised by Knox and Bristow in 1977 Defines the resistance a compound has to moving down a column relative to the performance of that column Allows for pressure to be incorporated Often plotted with a reverse axis Mimics van Deemter plot Minimum value optimum conditions Often plotted as a dimensional form t/n 2 t 0 or t r both used E = Pt N 2 η 15

Core Enhanced Technology Low Impedance 100,000 10,000 Impedance 1,000 0.10 1.00 10.00 Linear velocity of mobile phase (mm/s) Accucore RP-MS 2.

16 Core Enhanced Technology Low Impedance 100,000 10,000 Impedance 1, Linear velocity of mobile phase (mm/s) Accucore RP-MS 2.6µm 5µm 3µm <2µm Lowest impedance (best combination speed, efficiency and low pressure) of with Core Enhanced Technology 16

17 Core Enhanced Technology Features & Benefits Features More uniform particle sizing Better packing of particles Reduced pore depth Reduced mass transfer effects in mobile phase Benefits More Efficient Chromatography Allows the use of low pressure systems Competitive Edge Bar for bar gives better separations than porous materials 17

18 Faster than Fully Porous 5 and 3µm Rs = µL injection Fully porous 5µm, 150 x 4.6 mm ΔP = 59 bar Method Transfer Calculator: Gradient and flow rate: Fully porous 5 μm 150 x 4.6 mm Rs =1.96 Rs = 2.50 Rs = µL injection 1µL injection 1µL injection ΔP = 23 bar Fully porous 5µm, 100 x 2.1 mm ΔP = 97 bar Fully porous 3µm, 100 x 2.1 mm ΔP = 218 bar %B in 10.0 min 1000 µl/min solvent used 17 ml Fully porous 5 μm, 100 x 2.1 mm %B in 6.7 min 210 µl/min solvent used 2.4 ml Fully porous 3 μm, 100 x 2.1 mm 35 60%B in 4.0 min 350 µl/min solvent used 2.4 ml Accucore RP-MS 2.6 μm, 100 x 2.1 mm %B in 3.5 min 400 µl/min solvent used 2.4 ml ACCUCORE 2.6µm, 100 x 2.1 mm Minutes -100 Reduced analysis time and solvent costs 18

More Peak Capacity than Fully Porous 5 or 3µm 5µm,100 x 2.1 mm 240 Gradient: 65 95%B in 2.1 min, 220 95% B for 0.4 min Flow rate: 400 μl/min 3µm, 100 x 2.1 mm ACCUCORE 2.6µm, 100 x 2.

19 More Peak Capacity than Fully Porous 5 or 3µm 5µm,100 x 2.1 mm 240 Gradient: 65 95%B in 2.1 min, % B for 0.4 min Flow rate: 400 μl/min 3µm, 100 x 2.1 mm ACCUCORE 2.6µm, 100 x 2.1 mm Normalised peak capacity Accucore 2.6µm 3µm 5µm Minutes Higher peak capacity more peaks can be separated per injection 19

20 More Sensitive than Fully Porous 5 and 3µm S/N = 368 S/N = 399 S/N = 169 5µm, 100 x 2.1 mm 3µm, 100 x 2.1 mm mau Gradient and flow rate: 5μm, 100 x 2.1mm %B in 6.7 min 210 µl/min 3μm, 100 x 2.1mm %B in 4.0 min 350 µl/min Accucore RP-MS 2.6μm, 100 x 2.1mm %B in 3.5 min 400 µl/min ACCUCORE 2.6µm, 100x2.1mm Minutes Higher S/N ratios detection and quantitation of low level impurities 20

21 Lower Backpressure than Fully Porous sub-2µm Sub 2µm, 100 x 2.1 mm Flow rate: 500 μl/min Mobile phase: A - Water; B Acetonitrile Accucore RP-MS 2.6µm, 100 x 2.1 mm Accucore RP-MS 2.6 µm, 100 x 2.1 mm Sub 2 µm, 100x2.1mm Maximum pressure (bar) Minutes Equivalent performance, lower pressure (50% lower) 21

22 UHPLC Systems Not Required Dwell volume: 100 µl Column: Accucore RP-MS 2.6µm, 100 x 2.1 mm Dwell volume: 800 µl Accela 1250 Gradient: % B in 2.1 min 95 % B for 0.4 min Flow rate: 400 µl/min Surveyor Minutes Dwell volume: 1000 µl Accela 1250 Surveyor Agilent 1100 Agilent 1100 Run time (min) Accucore can deliver performance on a number of different systems min Average PW (1/2 Height)

23 Equivalent Loading Capacity to Fully Porous sub-2µm 2,500,000 Columns: Accucore RP-MS 2.6µm,100 x 2.1mm <2µm,100 x 2.1mm R² = ,000,000 R² = Effect of Loading - Accucore Peak area 1,500,000 1,000, ,000 R² = Normalised Value As N Tr A s T r Load on column (µg) <2µm Accucore 2.6μm Competitor 0 Load on Column (µg) No loss in performance with 2µg loaded on a 2.1mm ID Accucore column 23

24 Accucore Evaluation for Metabolite Profiles Courtesy of: Anila Desai - Scientist

25 ESI+ LC-MS Profile of a Drug Hypersil Gold, 50 x 2.1, 1.9 micron ESI + LC-MS Profile of a Drug / Hypersil Gold, 50 x 2.1, 1.9 micron ESI + LC-MS Profile of a Drug / Hypersil Gold, 50 x 2.1, 1.9 micron RT: TIC (RT 5.22) Relative Abundance M1 Oxidation (RT 4.55) M2 Hydrolysis of X (RT 4.36) M3 Glucuronidation (RT 4.61) Time (min) M4 O+Glucuronidation (RT 4.52) M5 O+SO 3 H (RT 4.39) 25

26 RT: Relative Abundance ESI+ LC-MS Profile of a Drug Accucore RP-MS, 50 x 2.1, 2.6 micron Time (min) Decrease in retention time while keeping same resolution TIC (RT 4.64) M1 Oxidation (RT 4.02) M2 Hydrolysis of X (RT 3.69) 7.18 M3 Glucuronidation (RT 4.15) M4 O+Glucuronidation (RT 4.18) M5 O+SO 3 H (RT 4.05)

27 RT: ESI+ LC-MS Profile of a Drug Accucore C18, 50 x 2.1, 2.6 micron 4.44 TIC (RT 4.44) Relative Abundance M1 Oxidation (RT 3.84) M2 Hydrolysis of X (RT 3.48) M3 Glucuronidation (RT 4.02) Time (min) Decrease in retention time while keeping same resolution 27 M4 O+Glucuronidation (RT 4.05) M5 O+SO 3 H (RT 3.95)

28 Retention Time Comparison Metabolite Profiles Retention Time HypersilGOLD 50x2.1; 1.9um Accucore RP-MS 50x2.1; 2.6um Accucore C18 50x2.1; 2.6um 3 28

29 Long Column Lifetime Stability at extremes of ph Stability at elevated temperature Packed bed ruggedness 29

30 Reproducible Chromatography - Batch-to-Batch Accucore C18 Test 1 - Hydrophobic Interactions HS SS HBC EV EV EV EV EV EV Average %RSD Fully characterised primary and secondary interactions 30

31 Reproducible Chromatography Run-to-Run Rosuvastatin Retention M i n u t e s Injection ~2,400 injections on Accucore column with TLX system no change in retention 31

32 Accucore Column Stability Low ph Column Stability at ph < 2 ph 1.8 (0.1% TFA) Retention Factor Acetaminophen p-hba o-hba Amitriptyline Nortriptyline DIPP DNPP 30,000 column volumes (5.5 days) Column Volumes Accucore columns are stable at low ph 32

33 Accucore Column Stability High ph ph 10.5 (0.1% ammonia) Retention Factor 30,000 column volumes (5.5 days) Accucore columns are stable at high ph 33

34 Accucore Column Stability Elevated Temperature 9 8 Column Stability at 70 C Mobile phase: MeOH/H 2 O (65:35) Flow rate: 0.4 ml/min Column temperature: 70 C 7 Run time: 5 min Retention Factor Phenol Butylbenzene o-terphenyl Pentylbenzene Column Volumes 9,000 column volumes (400 injections) Accucore columns are stable at high temperature 34

Accucore Column Stability Ruggedness Accucore RP-MS 2.6µm 100x 2.1mm ID Mobile Phase: 60/40 ACN/H 2 O Flow Rate: 400 µl/min Injection Volume: 1 µl Column Temp: 30 C Accucore RP-MS 2.6µm 100x 2.1mm ID Mobile Phase A: Water (0.

35 Accucore Column Stability Ruggedness Accucore RP-MS 2.6µm 100x 2.1mm ID Mobile Phase: 60/40 ACN/H 2 O Flow Rate: 400 µl/min Injection Volume: 1 µl Column Temp: 30 C Accucore RP-MS 2.6µm 100x 2.1mm ID Mobile Phase A: Water (0.05% TFA) Mobile Phase B: Acetonitrile (0.05% TFA) Wash: H 2 O (0.05% TFA) Injection Volume: 1 µl Column Temperature: 30 C Efficiency (o-xylene) Asymmetry (o-xylene) 4,000 + isocratic test injections with no decrease in performance 6,000 + fast gradient injections with no change in retention 35

36 Accucore HPLC Columns Proof of Concept Ultimate Core Performance Fast Separations High Peak Capacity Increased Sensitivity Lower Pressure Loading Capacity Wide Selectivity Reproducible Chromatography Long Lifetime Maximize Your Investment Increase sample throughput and decrease solvent costs Separate and identify more peaks Detect trace amounts Use existing HPLC instruments or prolong the life of UHPLC instruments Analyze a wide range of concentrations Use the right column for your analysis Have confidence in your separations Use columns for longer 36

37 Theory & Practice Conclusion Ultimate Core Performance to Maximize Your Investment Rugged and reproducible solid core particles Fast separations with superb resolution Low backpressures Core Enhanced Technology 37

38 Accucore Evaluation Chris Singleton Biogen Idec 14 Oct 2011

39 Outline Isocratic and gradient evaluation on C18 Multiple analyte evaluation on aq Temperature effect on aq separations Selectivity comparison between columns 39

40 Accucore C18, 2.1mm*50mm A: 20mM ammonium formate +0.1% formic acid B: Acetonitrile +0.1% formic acid TIC of +MRM (3 pairs): from Sample 1 (isocratic_accucore_01) of AccuCore eval FINAL.wiff (Turbo Spray) Max. 7.3e5 cps. 0.65ml/min 1µL injection 7.3e5 7.0e5 6.5e5 6.0e5 Protryptyline Dibucaine Shimadzu HPLC 5.5e5 5.0e5 MS/MS detection Intensity, cps 4.5e5 4.0e5 3.5e5 3.0e5 Mebendazole 2.5e5 2.0e5 1.5e seconds 1.0e5 5.0e Time, min 40

41 Accucore C18, 2.1mm*50mm 0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 30 C Time %B Same mobile phases as previous slide Chlorpheniramine Mebendazole Amitryptyline Loperamide Intensity, cps 2.9e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 TIC of +MRM (5 pairs): from Sample 2 (gradient_accucore_21) of AccuCore eval FINAL.wiff (Turbo Spray) 1.00 Max. 2.9e5 cps Time, min DPCPX min 27 sec 41

42 Outline Isocratic and gradient evaluation on C18 Multiple analyte evaluation on aq Temperature effect on aq separations Selectivity comparison between columns 42

43 Accucore aq, 2.1mm*50mm 0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 30 C Time %B Same mobile phases as previous slide 1.09e6 1.05e6 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 Intensity, cps 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e4 TIC of +MRM (6 pairs): from Sample 3 (gradient_accucore_aq_30c_01) of AccuCore eval FINAL.wiff (Turbo Spray) 0.00 Chlorpheni -ramine Mebendazole 0.98 Protryptyline Amitryptyline Max. 1.1e6 cps Time, min 1.27 DPCPX Loperamide 43

44 Outline Isocratic and gradient evaluation on C18 Multiple analyte evaluation on aq Temperature effect on aq separations Selectivity comparison between columns 44

45 0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 30 C Time %B Accucore aq, 2.1mm*50mm 1.09e6 1.05e6 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 Intensity, cps 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e4 XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 3 (gradient_accucore_aq_30c_01) of AccuCore Max. eval 8.1e5 FINAL.wiff cps. ( Time, min

46 0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 35 C Time %B Accucore aq, 2.1mm*50mm 1.10e6 1.05e6 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 Intensity, cps 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e4 XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 4 (gradient_accucore_aq_35c_01) of AccuCore Max. eval 8.3e5 FINAL.wiff cps. ( Time, min

47 0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 40 C Time %B Accucore aq, 2.1mm*50mm XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 5 (gradient_accucore_aq_40c_01) of AccuCore Max. eval 8.4e5 FINAL.wiff cps. ( e6 1.15e6 1.10e6 1.05e6 1.00e6 9.50e5 9.00e5 8.50e e5 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e Time, min Intensity, cps 47

48 0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 45 C Time %B Accucore aq, 2.1mm*50mm XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 6 (gradient_accucore_aq_45c_01) of AccuCore Max. eval 9.4e5 FINAL.wiff cps. ( e6 1.25e6 1.20e6 1.15e6 1.10e6 1.05e6 1.00e6 9.50e e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e Time, min Intensity, cps 48

49 0.65ml/min 1µL injection Shimadzu HPLC MS/MS detect 50 C Time %B Accucore aq, 2.1mm*50mm XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 7 (gradient_accucore_aq_50c_01) of AccuCore Max. eval 9.0e5 FINAL.wiff cps. ( e6 1.25e6 1.20e6 1.15e6 1.10e6 1.05e6 1.00e6 9.50e e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e Time, min Intensity, cps 49

50 Accucore aq, 2.1mm*50mm 0.65ml/min 1µL injection 1.4e6 1.3e6 1.2e6 XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 8 (gradient_accucore_aq_55c_01) of AccuCore Max. eval 9.6e5 FINAL.wiff cps. (... Shimadzu HPLC 1.1e6 MS/MS detect 1.0e6 9.0e C Time %B Intensity, cps 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e Time, min 50

51 Accucore aq, 2.1mm*50mm 0.65ml/min 1µL injection 1.5e6 1.4e6 1.3e6 XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 9 (gradient_accucore_aq_60c_01) of AccuCore Max. eval 9.4e5 FINAL.wiff cps. (... Shimadzu HPLC MS/MS detect 60 C Time %B Intensity, cps 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e Time, min

52 1ml/min 1µL injection Shimadzu HPLC MS/MS detect 60 C Time %B Accucore aq, 2.1mm*50mm XIC of +MRM (6 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 11 (gradient_accucore_aq_60c_1ml-min_01) of Max. AccuCore 8.6e5 cps. eval F e6 1.20e6 1.15e6 1.10e6 1.05e6 1.00e6 9.50e5 9.00e e5 8.00e5 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e Time, min Intensity, cps 52

53 Accucore aq, 2.1mm*50mm 1ml/min 1µL injection Shimadzu HPLC MS/MS detect 60 C XIC 305.0/221.1 Time %B Intensity, cps 4.2e5 4.0e5 3.8e5 3.6e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 XIC of +MRM (6 pairs): 305.0/221.1 Da ID: CPDPX from Sample 11 (gradient_accucore_aq_60c_1ml-min_01) of AccuCore Max. 4.2e5 eval cps. FINAL second peak width at baseline Time, min 53

54 Outline Isocratic and gradient evaluation on C18 Multiple analyte evaluation on aq Temperature effect on aq separations Selectivity comparison between columns 54

Phenyl, Phenyl-Hexyl, perfluorinated Dipole-dipole, π-π, hydrogen-bonding interactions

55 Typical bonded phases for Reversed-Phase LC Alkyl chain: C18, C8, C4, C1 same selectivity, decreasing degree of hydrophobicity/retention Dispersive interactions Aromatic groups Phenyl, Phenyl-Hexyl, perfluorinated Dipole-dipole, π-π, hydrogen-bonding interactions Other Cyanos, aminos, polar embedded Dipole-dipole, hydrogen-bonding interactions Perfluorinated 55

56 Phase Characteristics HR HS SS HBC BA C IEX(7.6) AI IEX(2.7) K Pentylbenzene α Butylbenzene / Pentylbenzene α Triphenylene / o-terphenyl α Caffeine / Phenol tf Amitripyline tf Quinizarin α Benzylamine / Phenol tf 4-Chlorocinnamic acid α Benzylamine / Phenol Hydrophobic Interactions Hydrophobic retention Secondary Interactions Base activity Acidic Interactions Acid interaction HR BA AI Hydrophobic Chelation Ion exchange selectivity capacity ph 2.7 HS C IEX (2.7) Steric selectivity Ion exchange capacity (ph 7.6) SS IEX (7.6) Hydrogen bonding capacity HBC 56

57 Accucore HPLC Columns Phase Details RP-MS Optimized for MS detection, excellent combination of speed and quality of separation Phenyl-Hexyl Unique selectivity for aromatic and moderately polar analytes L11 C18 Optimum retention for non-polar analytes PFP Alternative selectivity to C18, particularly for halogenated analytes aq Compatible with 100% aqueous mobile phases, special selectivity for polar analytes HILIC Enhanced Retention of polar and hydrophilic analytes Guard Columns Available! 57

58 Accucore Phenyl-Hexyl, 2.1mm*50mm 0.65ml/min 1.7e6 XIC of +MRM (7 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 2 (Accucore_phenyl-hexyl_001) of Accurcore_selectivity.wiff Max. 2.7e5 cps. (Turb... Loperamide 4µL injection Shimadzu HPLC MS/MS detect 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 Mebendazole 30 C Time %B Intensity, cps 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 Protryptyline Amitryptyline 1.15 DPCPX Time, min 58

59 Accucore PFP, 2.1mm*50mm 0.65ml/min 4µL injection Shimadzu HPLC MS/MS detect 30 C Time %B Intensity, cps 1.7e6 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 XIC of +MRM (7 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 4 (Accucore_PFP_001) of Accurcore_selectivity.wiff Max. (Turbo Spray... cps. 0.0 DPCPX Mebendazole Protryptyline, amitryptyline and loperamide are highly retained and elute in subsequent injections Time, min 59

60 Accucore C18, 2.1mm*50mm 0.65ml/min 4µL injection Shimadzu HPLC MS/MS detect 30 C Time %B Intensity, cps 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 XIC of +MRM (7 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 6 (Accucore_C18_001) of Accurcore_selectivity.wiff Max. (Turbo 2.7e5 Spray) cps. 0.0 Mebendazole Protryptyline Amitryptyline 1.24 Loperamide DPCPX Time, min 60

61 Accucore aq, 2.1mm*50mm 0.65ml/min 4µL injection Shimadzu HPLC 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 XIC of +MRM (7 pairs): 278.0/117.2 Da ID: Amitryptyline from Sample 8 (Accucore_aQ_001) of Accurcore_selectivity.wiff Max. (Turbo 4.3e5 Spray) cps. Mebendazole Loperamide is a late eluter and elutes well after the gradient MS/MS detect 30 C Time %B Intensity, cps 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 0.0 DPCPX Protryptyline Amitryptyline Time, min

62 Quest ions 62

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