Accelerating the Analysis of Cyanotoxins

1. Accelerating the Analysis of Cyanotoxins Sébastien Sauvé Environmental Analytical Chemistry – Université de Montréal sebastien.sauve@umontreal.ca Dipankar Ghosh Director for Environmental & Food safety Thermo Fisher Scientific Dipankar.ghosh@thermofisher.com

2. Contributors • Audrey Roy-Lachapelle • Khadija Aboulfadl • Pascal Lemoine • Sherri Macleod • Liza Viglino • Arash Zamyadi • Michèle Prévost

3. Context • Microcystins are hepatotoxins produced by cyanobacteria (Blue-green Algae) • These cyanotoxins are found in fresh waters and in drinking water reservoirs. • A bloom can occur in warm, shallow, undisturbed surface water rich in nutrients. Cyanobacterial bloom Microcystisaeruginosa http://www.aquarius-systems.com/Entries/View/349/bluegreen_algae.aspx http://www.plingfactory.de/index.html K.Sivonen, G. Jones, in: 1. Chorus, J. Bartram (Eds.), Toxic Cyanobacteria in Water: A Guide to their Public Health Consequences, London, 1999.

4. Objectives • Multi-toxin online SPE-LC-MS/MS method • Ultrafast laser diode thermal desorption methods (LDTD-APCI-MS/MS) • Anatoxin-A • Sum of microcystins

5. Online SPE-LC-MS/MS high pressure multi-toxinsmethod Sébastien Sauvé, Département de chimie

6. SPE: Enrichmment(solid phase extraction)

7. Automated SPE Extraction (Online SPE) The whole mass of analyteswithin the 1.0 ml samplewill ne injectedinto the MS detector SPE Chromatography MS/MS 1.0 ml Waste

8. LC-MS/MS Detection: Tandem mass spectrometry (selected reaction monitoring - SRM) ThermoElectron TSQ Quantum Ultra EQuan MAX System

9. Tandem Mass Spectrometry (MS/MS) Argon-induced Fragmentation m/z=156.0 m/z=108.0 Sulfamethoxazole+H+ m/z= 254.0 m/z=92.0

10. Cyanotoxins using LC-MS/MS Challenge is to combine varied compounds into a single method for the simultaneousdetermination of differentcyanotoxins.

11. Target compounds

12. Challenge • Speed!! • Eliminate off line SPE • Separate phenylanaline from anatoxin a (same SRM) http://fav.me/dsk92w

13. Anatoxine-a and phenylalanin • Separation of isobars using chromatography • Quantification of specific fragment for anatoxin-a (166.10 > 43.3) anatoxine-a 166.10 > 43.3 quantification phénylalanine 166.10 > 120.0 anatoxine-a 166.10 > 131.1 anatoxine-a 166.10 > 149.1

14. Specific conditions for cyanotoxin determinations

15. Chromatograms obtained using SPE-UPLC/MSMS-ESI, in Milli-Q water spiked @1 µg/l Chromatograms obtained using SPE-UPLC/MSMS-ESI, in real sample (lab culture)

16. Preliminary estimates of performance

17. Even faster? • LDTD

18. Primary ion formation →e- + N2→ N2+. + 2e- Secondary ion formation →N2+. + H2O → N2 + H2O+. →H2O+.+ H2O → H3O+ + HO. Proton transfer →H3O++ M → (M+H)+ + H2O Laser diode thermal desorption (LDTD) • Principles of the LDTD-APCI source: • technique that combines thermal desorption (laser diode) and APCI • sample is spotted (1-10 μL) into a 96-well plate and air-dried for 2 min • uncharged analytes are thermally desorbed into the gas phase • ionization takes place in the corona discharge region by APCI and the charged molecules will be transferred to the MS inlet Source: www.chm.bris.ac.uk/ms/theory/apci-ionisation.html

19. Laser diode thermal desorption (LDTD) LDTD Installation Auto-sampler 960 samples IR Laser (980 nm, 20 W) Corona needle position (APCI) Can ramp up to 3000oC/sec. Laser power is defined in % Normally ~100-150oC

20. Laser diode thermal desorption (LDTD)

21. LDTDProcess (2) (5) (3) (1) (4) (1) Infrared laser (980 nm, 20W) (2) LazWell Plate (96 wells): analyte desorption (1-10 µL spotted) (3) Transfer tube transporting the neutrally desorded analytes to the APCI region (4) Corona needle discharge region (APCI) (5) MS inlet

22. LDTD Optimization • No need to optimize liquid chromatography - it has been completely eliminated! • Optimization for MS (precursor) and MS/MS (SRM transitions) conditions in NI and PI mode. A minimum of 2 SRM transitions were selected + their ion ratios • Parameters of the LDTD-APCI source are optimized for signal intensity : • solvent choice for analyte deposition in the well cavities • laser power (%) • carrier gas flow rate (L/min) • mass deposition (deposition volume in µL) into plate well • laser pattern

23. Results / challenge 166.03>149.06 Onlyanatoxin-a canbevaprized and ionized by LDTD-APCI. Interferencefrom phénylalanine: Differentdesorptionpatern (signal intensity vs laser power). SRM Optimisation (main SRM identical). 166.06>149.02

24. Separation using a gradient of LDTD laser power

25. Performances (anatoxin-a)

26. Microcystins Adda MMPB Leucine (L) Microcystin-LR Arginine (R) • The are over 80 knownmicrocystins. • A unique structural feature: Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) whichplays an important role in itstoxicity. X. Wu, C. Wang, B. Xiao, Y. Wang, N. Zheng, J. Liu. Analytical Chimica Acta, 709 (2012) 66-72.

27. Context • The presence of microcystinscan pose an healthrisk for humans and animals: • Skin irritation, vomiting, diarrhea, asthma, headache, fever, and muscle weakness. • Inhibiting protein phosphatases in tissues, causing serious damage to the liver from bioaccumulation. The World Health Organisation (WHO) recomends a guideline for MC-LR of 1 mg lL-1in drinking water. K.Sivonen, G. Jones, in: 1. Chorus, J. Bartram (Eds.), Toxic Cyanobacteria in Water: A Guide to their Public Health Consequences, London, 1999.

28. Alternatives Need of robustdetectionmethods to evaluate and control the risks due to the presence of microcystins in water. D.O. Mountfort, P. Holland, J. Sprosen. Toxicon 45 (2005) 199-206 K. Kaya, T. Sano. Analytica Chimica Acta 386 (1999) 107-112.

29. Objective • Objective: Analysisof total microcystinsusing LDTD-APCI-MS/MS technology. • The methodprovides: • Instant information about risks of contamination • Information about the wholespectrum of cyanobacterial peptide toxinscongeners

30. Oxydation Experimentalworkflow: • Lemieux oxidationof microcystinsinto MMPB • Liquid-liquid extraction (Ethylacetate) • Desorption by LDTD • Negative ionisationby APCI • Detectionwith a TSQ Quantum Ultra AM triple quadrupole mass spectrometer erythro-2-Methyl-3-methoxy-4-phenylbutyric Acid MMPB X. Wu, C. Wang, B. Xiao, Y. Wang, N. Zheng, J. Liu. Analytical Chimica Acta, 709 (2012) 66-72. M-R. Neffling, E. Lance, J. Meriluoto. Environmental Pollution, 158 (2012) 948-952

31. Lemieux oxidation Adda KMnO4 +NaIO4 Microcystin MMPB • 0,05 M Potassium permanganate (KMnO4) and 0,05 M Sodium periodate (NaIO4) • Oxidation, at room temperature and pH 9 for 1 hour • Reaction quenched with saturated sodium bisulfite • Use of sulfuric acid 10% to reach pH 2 T. Sano, K. Nohara, F. Shiraishi, K. Kaya. J. Environ. Anal. Chem., 49 (1992) 163-170.

32. Microcystins Oxidation Optimisation Reagentsconcentrations KMnO4 and NaIO4 optimised at 0,05 M

33. Microcystins Oxidation Optimisation Oxidation time Optimal oxidationtime at 1h

34. LDTD parameter optimisation Laser power Best laser power at 35%

35. Microcystins Oxidation Optimisation pH during oxidation Optimised conditions at pH 9

36. Microcystin detection and quantification Quantification of MMPB by internal calibration with 4-phenylbutyric acid APCI (-) Scan time: 0,005 s Q1 width: 0,70 amu Q3 width: 0,70 amu 4-phenylbutyric acid (4-PB) (Internal standard) MMPB Optimal SelectedReaction Monitoring (SRM) parameters for the analysis of MMPB and 4-PB by MS/MS

37. Analysis of MMPB with LDTD-APCI-MS/MS Internal Calibration (MMPB / 4-PB ratio) Method Validation n=6 R2 : 0,9995 Linearity range: 1 – 500 mg/L LOD: 1 mg/L LOQ: 3 mg/L Standards Avg. RSD < 9% WHO Guideline: 1mg/L Calibration curveshowing the linearity of the LDTD experiment Oxidationreactionyield of Microcystins : 111% MMPB recoveryyield : 48%

38. Conclusions • An 8-min automated online SPE-LC-MS/MS method for many toxins (but excluding saxitoxins) • Ultrafast laser diode thermal desorption methods (LDTD-APCI-MS/MS) (15 sec per sample but with simple oxydation for MC) • Anatoxin-A • Sum of microcystins

39. Acknowledgements Parterns and funding agencies:

40. Questions?Dipankar.ghosh@thermofisher.comsebastien.sauve@umontreal.caQuestions?Dipankar.ghosh@thermofisher.comsebastien.sauve@umontreal.ca

41. Analysis with LDTD-APCI-MS/MS LDTD Source (0,5-3 L/min) (980 nm, 20 W) http://ldtd.phytronix.com/

42. Analysis with LDTD-APCI-MS/MS LDTD Source 10-plate sample loader LDTD a sample introduction method using thermal desorption • Minimal sample preparation • Small volume of sample needed (1-5 mL) • 15 sec / sample (no chromatographic separation) • No carryover • Combined with atmospheric ionisation(APCI) • High-thoughput 10 plates in the loader = 960 samples LazWell sample plate http://ldtd.phytronix.com/

43. LDTD parameter optimisation Laser desorptionparameters Laser Power: 35% Gas Flow: 3 L/min Deposition volume: 2mL Laser pattern duration: 6 s Laser pattern LDTD peakshape

44. Plate well Sampleresidue LDTD parameter optimisation Ethyl Acetate is the best deposition solvent Depositionsolvent Carrier gas flow rate Gaz flow at 3,0 L/min