THE IMPLEMENTATION OF A SCREENING WORKFLOW FOR ION MOBILITY QUADRUOPOLE TIME-OF-FLIGHT MASS SPECTROMETRIC ANALYSIS OF PFOS ISOMERS

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1 THE IMPLEMENTATION O A SCREENING WORKLOW OR ION MOBILITY QUADRUOPOLE TIME-O-LIGHT MASS SPECTROMETRIC ANALYSIS O POS ISOMERS Ken Rosnack ken_rosnack@waters.com Ingrid Ericson Jogsten and Bert van Bavel (MTM Research Centre) Mike McCullagh, Leonard Dillon, Mike Hodgkinson, Lauren Mullin, & Jennifer Burgess (Waters Corporation) 2015 Waters Corporation 1

2 Overview POS analysis collaboration between Waters Corporation and Örebro University Challenges of POS analysis Instrumentation: Synapt G2-S and HDMS E Ion mobility Analysis of environmental samples for POS using HDMS E Experimental Results Screening approach vs Software enabled identification (Development of prototype software) Conclusions 2015 Waters Corporation 2

3 Introduction: Poly and perfluoroalkyl substances (PASs) 2015 Waters Corporation 3

4 PCs research Houde M, Martin JW, Letcher RJ, Solomon KR, Muir DC (June 2006). "Biological monitoring of polyfluoroalkyl substances: A review". Environ. Sci. Technol. 40 (11): doi: /es052580b. PMID Supporting Information(PD) Waters Corporation 4

5 POS Analysis Challenges Matrix effects, retention time shifts. Correct POS isomer identification: The physical, chemical and biological properties may be affected by perfluoromethyl branching. Source elucidation. Response factors of individual isomers. Increased scientific interest in toxicity, environmental transport, degradation and bioaccumulation of isomers. POS and TDCA as well as other cholic acids have similar isomeric profiles, retention times and MRM transitions (499 m/z 80m/z). Interferences can be mistaken for POS and lead to a positive bias Waters Corporation 5

6 Chemical Structures POS and Cholic Acid Interference s TDCA POS TCDCA Taurodeoxycholate Perfluorooctane sulfonate Taurochenodeoxycholic acid C 26 H 45 NO 6 S= [M-H] - = C 8 H 17 O 3 S= [M-H] - = Waters Corporation 6

7 ragmentation Series for POS 2015 Waters Corporation 7

8 HDMS E Uses high resolution MS and high efficiency ion mobility based measurements and separations. Both precursor ion and fragment ion information can be acquired in a single HDMS E experiment. This technique offers some unique advantages to profiling complex matrices. HDMS E can provide a route to specific and unambiguous identification, enabling the distinction of POS isomers. UPLC_SynaptG2-S_ _IEJ.raw : Waters Corporation 8

9 Ion Mobility Spectrometry Ion mobility spectrometry (IMS) is a rapid, orthogonal, gas phase separation technique which allows another dimension of separation. Separation is driven by electric fields not under vacuum. Compounds can be differentiated based on size, shape and charge Waters Corporation 9

10 Ion Mobility Spectrometry (IMS) Small Compact Slide courtesy of Severine Goscinny, ISP-WIV, Belgium Large Extended 2015 Waters Corporation 10

11 SYNAPT G2-S High Definition MS - Ion Mobility Explained 1. Increased sensitivity 2. Ion mobility 3. Accurate mass Size Shape Charge 2015 Waters Corporation 11

12 Ion Mobility: an Orthogonal Dimension of Separation TO MS LC IMS n seconds 2015 Waters Corporation n 12

13 MS E ragmentation in Trap or transfer Alternate Low and High Energy Scans 2015 Waters Corporation 13

14 Environmental samples SLU project Anna Rotander, Sara Persson Mink 2015 Waters Corporation 14

15 Extraction of environmental samples 1 g homogenized liver sample, Add labeled IS Repeated ACN extraction Vortex & ultrasonication Condition: MeOH and water Load samples Wash: 4 ml NaAc (ph 4) 4 ml 20% MeOH SPE, Oasis WAX Dry cartridges ENVI-carb Elute: 4 ml MeOH (discarded) 4 ml NH 4 OH iltration, performance standards added Instrumental analysis 2015 Waters Corporation 15

16 Experimental UPLC: Waters ACQUITY UPLC I-Class (equipped with PC kit) Column: Waters ACQUITY UPLC BEH C18 (100 mm x 2.1 mm, 1.7 µm) Column temperature: 50 o C Mobile phase low: 0.30 ml/min A: H 2 O:MeOH/ACN 70:30 (80/20, 2 mm Ammonium Acetate) B: MeOH:ACN 80:20 (2 mm Ammonium Acetate) Gradient Time(min) low Rate %A %B Initial Waters Corporation 16

17 Experimental MS: Waters SYNAPT G2-S Ionisation Mode: ES- Desolvation Temperature: 550 C Acquisition Modes: IMS MS E M/Z Range: Acquisition rate: 10 spectra/second Capillary Voltage: 2.3 kv Cone Voltage: 15 V Drift Gas: CO 2 and N 2 Collision Energy Ramp: ev IMS Wave Velocity Range: 400 m/s to 550 m/s IMS Wave Height: 40 V Mobility Drift Gas Mass Polarisability (10e -24 cm 3 ) Nitrogen N Carbon Dioxide Waters Corporation 17

18 BPI Chromatogram for HDMS Analysis of Extract of Mink Liver for POS Components Detected 2015 Waters Corporation 18

19 LC-HDMS E means... Increased Peak Capacity IMS PEAK CAPACITY Peak capacity 2015 Waters Corporation 19

20 Extracted ion chromatograms for matrix interferences and the POS isomers DL :205A ng/ul UPLC_SynaptG2-S_ _IEJ 100 A : TO MS ES _ Da 4.14e6 Isobaric Interference s (A,B) D G POS Isomers (C-G) % C E B Time 2015 Waters Corporation 20

21 Mobility plots for the isobaric interferences and POS isomers using N 2 drift gas. Ion Mobility Separation TDCA Interference s (N 2 Drift Gas) UPLC_SynaptG2-S_ _IEJ.raw : 1 Ion Mobility Separation POS Isomers (N 2 Drift Gas) UPLC_SynaptG2-S_ _IEJ.raw : 1 Dt = 6 ms = 2 ms Dt = 4 ms 2015 Waters Corporation 21

22 Mobility plots for the isobaric interferences and POS isomers using CO 2 drift gas. Ion Mobility Separation TDCA/TCDCA Interference s (CO 2 Drift Gas) UPLC_SynaptG2-S_ _IEJ.raw : 1 A POS Isomer Ion Mobility Separations (CO 2 Drift Gas) POS isomers UPLC_SynaptG2-S_ _IEJ.raw : 1 UPLC_SynaptG2-S_ _IEJ.raw : 1 Dt = 9.2 ms = 2.7 ms Dt = 6.5 ms B 2015 Waters Corporation 22

23 Ion Mobility Separation Component drift plot showing drift times vs retention time for nominally isobaric interferences (A) and POS isomers (B) 2015 Waters Corporation 23

24 Time and Mobility Aligned ragmentation m/z m/z Drift time Drift time Precursor ions Precursor ions Precursor and products are time aligned 2015 Waters Corporation 24

25 Ion Mobility Resolved TDCA (Interference A) with MS E Precursor and ragmentation Spectra 2015 Waters Corporation 25

26 POS (C) isomer, ion mobility resolved from isobaric interference TCDCA (A) at retention time mins O OH S O C Waters Corporation 26

27 POS (D) isomer, ion mobility resolved from isobaric interference TCDCA (A) at retention time mins O OH S O 2015 Waters Corporation 27

28 Unassigned POS (E), ion mobility resolved from isobaric interference TDCA (A) at retention time mins 2015 Waters Corporation 28

29 POS ISOMER C 2015 Waters Corporation 29

30 POS ISOMER D 2015 Waters Corporation 30

31 POS ISOMER E 2015 Waters Corporation 31

32 POS ISOMER 2015 Waters Corporation 32

33 POS ISOMER G 2015 Waters Corporation 33

34 Summary for major POS isomers and matrix interferences POS ISOMER IDENTIICATION POS Isomers C 5mPOS J 3mPOS D IsoPOS E 2,2- perfluoro- methyl POS (tentative) 1mPOS G npos Drift Time (ms) Mass Measurement Error ppm ppm ppm ppm ppm (2.68ppm HE) Retention Time (mins) TDCA Interferences Drift Time (ms) Mass Measurement Error A TDCA ppm B TCDCA ppm Retention Time (mins) A summary of drift times, retention times and isomer assigments for major POS isomers and co-eluting matrix Waters Corporation 34

35 Screening database A screening database was created for branched POS isomers based on observed retention times and unique product ions 1m-POS, 3m-POS, 4m-POS, 5m-POS, 6m-POS, 4,4dim- POS, 3,5-dim-POS, 4,5-dim-POS, 5,5-dim-POS Environmental samples and a technical blend was analysed and compared against the database. The software filtered the results to include only isomers having a positively identified product ion Waters Corporation 35

36 Component summary for POS screening of mink liver extracts 2015 Waters Corporation 36

37 Software enabled identification Extracted ion chromatogram and spectrum for both low energy (top) and high energy (bottom) collision states. Using the structure, proposed product ions were deduced from the observed spectral peaks, as indicated by the blue molecule icons Waters Corporation 37

38 Drift time corrected spectrum Spectrum for the same chromatographic peak, 1-POS, with interference from TDCA evident in the top spectrum. The bottom spectrum is from the same chromatographic peak and injection, but with the removal of any ions that do not share the same drift time as 1-POS Waters Corporation 38

39 Conclusions Co-eluting isobaric biological interference s TDCA and TCDCA have been resolved from POS isomers using ion mobility. Utilising CO 2 as a mobility drift gas further enhanced the mobility resolution between POS isomers and isobaric TDCA / TCDCA interference isomers. The enhanced mobility resolution induced using CO 2 has a drift gas also enabled distinctive drift times to be obtained for the POS isomers. Single component precursor ion and fragmentation spectra have been generated for POS isomers and TDCA / TCDCA interference POS isomers can be characterised without contribution from isobaric interference isomers of TDCA / TCDCA. UPLC IMS MS E facilitates accurate and informative data generation by isomerspecific analysis. This will be increasingly important as regulatory, scientific, and public awareness of the environmental impact of perflouralkylsubstances increases Waters Corporation 39

40 Acknowledgements BENGT LUNDQVIST MINNE Waters Centre of Innovation Contact: 2015 Waters Corporation 40

41 Thank You!!! Questions??? Ken Rosnack Ingrid Ericson Jogsten and Bert van Bavel (MTM Research Centre) Mike McCullagh, Leonard Dillon, Mike Hodgkinson, Lauren Mullin, & Jennifer Burgess (Waters Corporation) 2015 Waters Corporation 41