1. LASER DIODE THERMAL DESORPTION IONIZATION SOURCE FOR MASS SPECTROMETRY Patrice Tremblay, Ph.D.

2. Pharmaceutical, CRO, environmental and food industries need to improve productivity of high throughput screening and analysis. Actual techniques are often limited by :  Extensive samples preparation;  Risk of cross contamination between samples;  Background noise induced by mobile phase or enhancement matrix;  Analysis time. In order to eliminate these problems, a new ionization source has been developed. The LDTD technology (Laser Diode Thermal Desorption) coupled to a mass spectrometer offers the same analytical performances as any LC- MS/MS system and is an alternative to the problems encountered with usual techniques. Motivations

3. LDTD Ionization Source

4. IR Laser Beam LazWell Sample Plate Carrier Gas Corona Discharge Needle Mass Spectrometer Inlet Piston Transfer Tube Piston head LDTD Ionization Source Sample is dried onto the bottom of a well from a standard 96-well plate with a metal sheet insertion. Thermal desorption induced by a laser at 980 nm (no photon-sample interactions). Gaseous neutral species transferred by a carrier gas. Ionization occurs into the corona discharge region.

5. The ionization process that occurs in the LDTD source is an Atmospheric pressure chemical (APC) type of ionization without the presence of solvent (no mobile phase or enhancement matrix.) LC APCI H2OH2O O2O2 N2N2 H2O+H2O+ H2OH2O H3O+H3O+ N2N2 N2+N2+ + + + + + + + + e-e- e-e- e-e- e-e- N4+N4+ N2N2 H2OH2O N2+N2+ N2+N2+ (H 2 O) n H + + Solvent (Solvent+H) + transfers charge onto analyte (if possible) HV Corona discharge Theoretical Aspects of the Ionization

6. The ionization process that occurs in the LDTD source is an Atmospheric pressure chemical (APC) type of ionization without the presence of solvent (no mobile phase or enhancement matrix.) H2OH2O O2O2 N2N2 H2O+H2O+ H2OH2O H3O+H3O+ N2N2 N2+N2+ + + + + + + + + e-e- e-e- e-e- e-e- N4+N4+ N2N2 H2OH2O N2+N2+ N2+N2+ (H 2 O) n H + + LDTD APCI Analyte Analyte is forced to react with the cluster ion HV Corona discharge

7. Low volume sample analysis (1 to 10 µL) 96-well plates are designed to be compatible with conventional sample preparation systems. No extra sample pre-treatment needed The absence of enhance matrix and mobile phase lower the noise signal. Elimination of cross contamination due to LC. Each well are individually isolated during the thermal desorption. The thermal desorption process takes seconds Key Features

8. Plug-and-play device Key Features Sciex Source Housing for API 3000, 4000 and 5000 Also available on Thermo, Waters and Agilent systems

9. LazSoft and GLP environment : Thermo mass spectrometers (Xcalibur) LazSoft fully integrated into Xcalibur Operated under GLP Sciex mass spectrometers (Analyst) Actually in discussion with Sciex to have access to the programmation code for LazSoft integration Log Book created to trace the launch batch (LazSoft and Analyst) Key Features

10. Application – Drugs Analysis in Plasma ParacetamolMifepristoneMidazolam

11. LDTD Method and Plasma Sample Preparation (Paracetamol analysis) Sample Preparation (crashed plasma) 100 µ L of Human Plasma Spike Paracetamol and Paracetamol-d4 (40 ng/mL) 500 µ L of acetonitrile (precipitation agent) Vortex 4 min. Centrifuge at 14000 RPM for 10 min. Transfer Manually 4 µ L onto LazWell to perform LDTD-MS/MS analysis LDTD Parameters Carrier gas flow :3 L/min APCI (+) Laser Pattern Increase laser power to 25 % in 1.0 s Hold at 25 % for 0.5 sec. Decrease laser power to 0 % MS Parameters MS/MS transition :152.0 – 110.1 amu 156.0 – 114.1 amu Collision gas pressure :1.5 mTorr (Ar) Collision energy :16 eV Scan time :0.050 s Q1 width :0.30 amu Q3 width :0.70 amu M.W. 151.17 g/mol

12. High-Throughput LDTD-MS/MS Analysis of Paracetamol in Human Plasma Analyte Desorption in 1.8 seconds Paracetamol raw signal ISTD signal 96-replicates

13. Robustness and Repeatability (Paracetamol in Human Plasma) Run time of 75 minutes Area Ratio

14. Linearity (Paracetamol in Human Plasma) 0.6 to 5000 ng/mL

15. LDTD Method and Plasma Sample Preparation (Mifepristone analysis) Sample Preparation (liquid-liquid extraction) 50 µ L of Mouse Heparin Plasma Spiked Mifepristone (10 to 2000 ng/mL) 20 µ L IS(d 4 ) + 50 µ L NH 4 OH 4% 2 mL of each MTBE and Hexane Vortex 15 min. Centrifuge at 2500 RMP for 10 min. Evaporate organic phase to dryness at 40 o C Reconstitute in 200 µ L of Water:ACN:Formic acid (75:25:0.1 v/v/v) Transfer Manually 2 µ L onto LazWell to perform LDTD-MS/MS analysis LDTD Parameters Carrier gas flow :2 L/min APCI (+) Laser Pattern Increase laser power to 60 % in 3 sec. Hold at 60 % for 2 sec. Decrease laser power to 0 % MS Parameters MS/MS transition :430.14 – 372.25 amu Collision gas pressure :1.5 mTorr (Ar) Collision energy :18 eV Scan time :0.02 sec. Q1 width :0.70 amu Q3 width :0.70 amu M.W. 429.59 g/mol

16. Calibration Curve (Mifepristone analysis) Concentration (ng/mL) Area ratio w = 1/x Calibration range : 10 to 2000 ng/mL Sample-to-sample run time 9 sec.

17. Back-Calculation and QC’s (Mifepristone analysis) Standard concentrations back-calculated from calibration curve QC ’ s performance

18. LDTD Method and Plasma Sample Preparation (Midazolam) Sample Preparation (liquid-liquid extraction) 100 µ L of Human Plasma Spiked Midazolam (0.5 to 250 ng/mL) 10 µ L IS(d 4 ) + 50 µ L NH 4 OH 4% 3 mL of MTBE and 1 mL of Hexane Vortex 15 min. Centrifuge at 2500 RMP for 10 min. Evaporate organic phase to dryness at 40 o C Reconstitute in 500 µ L of Water:ACN:Formic acid (75:25:0.1 v/v/v) Transfer Manually 2 µ L onto LazWell to perform LDTD-MS/MS analysis LDTD Parameters Carrier gas flow :2 L/min APCI (+) Laser Pattern Increase laser power to 40 % in 2 sec. Hold at 40 % for 2 sec. Decrease laser power to 0 % MS Parameters MS/MS transition :430.14 – 372.25 amu Collision gas pressure :1.5 mTorr (Ar) Collision energy :18 eV Scan time :0.02 sec. Q1 width :0.70 amu Q3 width :0.70 amu M.W. 325.78 g/mol

19. Calibration Curve (Midazolam analysis) Concentration (ng/mL) Calibration range : 0.5 to 250 ng/mL Sample-to-sample run time 8 sec.

20. Back-Calculation, QC’s and Unknown (Midazolam analysis) Standard concentration back-calculated from calibration curve QC ’ s performance Unknown : LDTD-MS/MS vs LC-MS/MS * Calculated as LC-MS/MS provides the true values * Calculated as if LC-MS/MS provides the true values…

21. Application – Drugs Analysis in Dried Blood Spot Paracetamol

22. LDTD Method and Blood Spot Sample Preparation (Paracetamol analysis) Sample Preparation Dried blood spot (with Paracetamol) Punch out a 3 mm disc 100 µ L 50/50 Meoh/H 2 O + 250 ng/mL IS(D 4 ) Vortex for 20 sec. Allow to stand for 30 min. Centrifuge at 14000 RPM for 1 min Transfer Manually 2 µ L onto LazWell to perform LDTD-MS/MS analysis LDTD Parameters Carrier gas flow :2 L/min APCI (+) Laser Pattern Increase laser power to 25 % in 2.0 sec. Hold at 25 % for 2 sec. Decrease laser power to 0 % MS Parameters MS/MS transition :152.0 – 110.15 amu Collision gas pressure :1.5 mTorr (Ar) Collision energy :16 eV Scan time :0.02 s Q1 width :0.70 amu Q3 width :0.70 amu M.W. 151.17 g/mol

23. Calibration Curve : Paracetamol in blood Concentration (ng/mL) Calibration range : 3.6 to 909 ng/mL 4 replicates CV lower then 4.6 % Sample-to-sample run time 8 sec.

24. LDTD-MS/MS Signal Limit of detection : 2.6 ng/mL (3-times the blank value) Blank 3.6 ng/mL909 ng/mL

25. Application – Sulfonamide Residues in Dairy Milk Analysis How to obtain specificity without LC

26. Sulfonamides Isomers and related structures APCI(+), isomers show the same MRM transitions APCI (-), specific MRM transitions Specificity achieve by : Right APCI mode MRM mode

27. LDTD-MS/MS Specificity No chromatographic separation to analyze 16 compounds in 10 seconds Specificity achieve using MRM in (-)APCI Sulfadoxine 309  251 Sulfadimethoxine 309  131 TIC Isomer analysis without chromatographic separation 2 extracted samples with Sulfadoxine or Sulfadimethoxine (isomers) TIC signal and extract signal for 2 MS/MS transitions Excellent specificity

28. LDTD Method and Dairy Milk Sample Preparation Sample Preparation 100 µ L of Whole dairy milk Spiked 16 sulfonamides (2 ng/mL to 1000 ng/mL) Add Indapamide as internal standard 500 µ L of acetonitrile (precipitation agent) Vortex 4 min. Centrifuge at 14000 RPM for 10 min. Filtrate supernatant on Nanosep 0.2 µ m Transfer 2 µ L onto LazWell to perform LDTD-MS/MS analysis LDTD Parameters Carrier gas flow :2 L/min APCI (-) Laser Pattern Increase laser power to 25 % in 2 sec. Hold at 25 % for 3 sec. Decrease laser power to 0 % MS Parameters Collision gas pressure :1.5 mTorr (Ar) Scan time :0.02 sec. Q1 width :0.70 amu Q3 width :0.70 amu MS/MS transition and Collision energy :

29. Analytical Performance Excellent linearity for all sulfonamides (> 0.99) Extraction recovery from 85 % to 100 % LOD of 2 ng/mL (4 pg loaded into well) from blank analysis Blank 1000 ng/mL

30. Application – Phase I and Phase II Metabolite Back Conversion Evaluation

31. Back-Conversion LDTD-MS/MS does not have any chromatographic separation All sample constituent may thermally desorbed, ionized and be introduced into the MS Phase I and Phase II metabolites may back-convert (thermally or in the APCI region) into the corresponding drugs which may affect the quantification Experiment Sample containing high metabolite quantities LDTD setup at the drug operating conditions Monitoring the MS/MS transition of the drug and the metabolite Evaluation of the back-conversion of the metabolite

32. OH-Midazolam Back-Conversion MS/MS Midazolam signal 326.04 – 291.04 amu MS/MS OH-Midazolam signal 2.5 µ g/mL solution 340.0 – 305.0 amu Observed back- conversion of 0.2 %

33. Testosterone and Testosterone Glucuronide Experiment : 100 µ L Stripped Human EDTA Plasma 500 µ L Ethyl Acetate Vortex agitation for 4 min. Spiked supernatant with Testosterone (3 ng/mL) Spiked supernatant with Testosterone glucuronide (200 ng/mL) Analyze 2 µ L in LDTD-MS/MS, following Testosterone transition (289.26 – 109.19 uma, 21 eV) From Peng et al. Clinical Chemistry, 46:4, 515-522 (2000) Testosterone glucuronide in healthy Caucasian subject lower then 5 nM Testosterone oral dose of 120-mg 1 hour after administration blood Testosterone glucuronide increase at 310 nM

34. Testosterone and Testosterone Glucuronide Real-life (Peng et al.) [5 nM of Testosterone glucuronide in blood] Liquid-liquid extraction with organic hydrophobic solvent Testosterone extracted in organic phase Testosterone glucuronide stays in aqueous phase 0.25 % of back-conversion will be negligible on the Testosterone signal (less then 0.01 ng/mL) Results Extract with Testosterone and no Testosterone glucuronide : 23254 count Extract with Testosterone + Testosterone glucuronide : 27021 count Back-conversion : 0.25 %

35. Human Liver Microsomes

36. CYP3A4 inhibition study (Midazolam signal) 3 Inhibition studies 4 replicates 2 µL directly spotted into well Sample-to-sample run time of 10 seconds No internal standard correction

37. CYP3A4 inhibition study (Midazolam signal) Area count Inhibition time

38. CYP3A4 inhibition study (OH-Midazolam signal) No internal standard correction CV lower then 15 %

39. CYP3A4 : OH-Midazolam (with ISTD) Sample number Signal ratio (Analyte/ISTD)

40. CYP2D6 : OH-Bufurolol (with ISTD) Signal ratio (Analyte/ISTD) Sample number

41. CYP2C9 : OH-Diclofenac (with ISTD) Signal ratio (Analyte/ISTD) Sample number

42. Pooled-CYP and CYP-Cocktail Samples LDTD allows to analyzed Pooled-CYP and CYP-cocktail samples Run-time of 9 seconds per samples List of CYP studies available until now :

43. Thermal desorption in induced indirectly by laser diode. No photon-sample interactions There is no need for an enhancement matrix. There is no liquid mobile phase. Ionization is produced by corona discharge. Sample-to-sample run time as low as 4.5 seconds. CONCLUSIONS

44. Picogram sensitivity can be achieved using 2-5 μL of sample. No carryover or memory effect is observed during the process of 960 samples batch (and more). Excellent linearity and accuracy achieve with the LDTD-MS/MS system. Comparable performance to LC-MS/MS with higher throughput. CONCLUSIONS

45. QUESTIONS