Simplified Workflow and Improved Confidence in Screening Methods on Time of Flight Mass Spectrometry

Transcription

1 Simplified Workflow and Improved Confidence in Screening Methods on Time of Flight Mass Spectrometry Steve Preece MS Business Manager Waters Wilmslow 2016 Waters Corporation 1

2 Overview Use of high resolution MS (Q-Tof) for suspect screening Key criteria for identification Case study with Xevo G2-XS QTof What else can I do with the data? Using ion mobility MS for screening 2016 Waters Corporation 2

3 Advantages of accurate mass screening? Over recent years high resolution mass spectrometry has gained in popularity as a screening tool in the food and environmental sector Ability to perform non-targeted analysis The freedom to measure compounds without prior compound specific tuning Ability to perform full spectral analysis Providing greater insight into the composition of a complex sample Ability to perform historical (retrospective) data review The capability of performing structural elucidations of unknowns or suspected compounds Ability to screen for larger number of compounds and adducts Compared to QqQ based screening Increased specificity & selectivity in complex matrices Sensitivity in line with Regulations. Accurate mass, diagnostic fragment ions HRMS strongly competes with classical tandem mass spectrometry in the field of quantitative multiresidue methods (e.g., pesticides and veterinary drugs). It is one of the most promising tools when moving towards nontargeted approaches... Kaufman A., Anal BioAnal Chem 2011 Dec Waters Corporation 3

4 HRMS UNIFI Screening Key Benefits Are these compounds in my sample? Screening How much is in my sample? Quantitation Scientific Library RT, MW, Structure, Fragments & CCS Workflows Simplify data review Quan/Qual Workflow MS E Single Analysis Alternately Tof MRM Ultimate Sensitivity What is the difference between my sample and another one? Binary Compare/Multivariant Analysis Statistical Comparison of Samples Comparison What else is in my sample? Elucidation Discovery Tools Anything Interesting? Elucidation tools Identify what is interesting 2016 Waters Corporation 4

5 Atrazine: TQ Identification from RT and ion ratios > > Waters Corporation 5

6 Advantages of Non-Target ToF Screening over MRM Target Screening MRM #1 MRM #4 MRM #6 MRM #3 MRM #5 MRM #7 MRM # Waters Corporation 6

7 Accurate Mass Screening Acquiring full MS spectral data See everything unlike specific MRM acquisition Using 20mDa extraction window for mass chromatograms Specificity from accurate mass data Acquiring MS E data Molecular weight and fragment data from the same run Using multiple data features to confirm or identify Accurate mass, retention time, fragments, adducts, isotopes 2016 Waters Corporation 7

8 Xevo G2-XS QTof : MS E MS E High and low energy spectra Precursor and fragment ions Single acquisition Data independent acquisition Data automatically aligned Requires partial LC peak resolution Links associated precursors and fragments 2016 Waters Corporation 8

9 Atrazine: HRMS 2016 Waters Corporation 9

10 Mass accuracy alone Number of Identified Pesticides (mass accuracy alone) ppm 3ppm 1ppm. provides insufficient specificity 2016 Waters Corporation 10

11 Retention time.. Number of Identified Pesticides (using Rt and m/z (5ppm)) min window - better than 1ppm + reduced risk of false negatives ± ± min ± 0.4 min ± 0.5 min no Rt. helps reduce false detects 2016 Waters Corporation 11

12 Fragment ions Number of Identified Pesticides (Rt, m/z and at least 1 fragment) ± 0.25 min ± 0.4 min ± 0.5 min no Rt. provide additional specificity 2016 Waters Corporation 12

13 Pesticide-Based Installation Specifications 2016 Waters Corporation 13

14 UNIFI: Raw data is processed once Tabulated in oracle database Data is componentised Spectral features are already associated Background ions are excluded Extracted Spectrum Component Spectrum 2016 Waters Corporation 14

15 Real Life Example: XEVO G2 XS Pesticide Screening Application Solution using Xevo G2-XS QTof Sample supplied as extracted by the customer Sample information approx 20 compounds >10 ppb approx 10 compounds < 10ppb What else can you see? (strawberry pomace) Identified, at least 1 fragment ion Identified, no fragment ion(s) 2016 Waters Corporation 15

16 Non-Targeted Screening with Xevo G2-XS QTof Higher instrument sensitivity means : Lower screening detection limits Better confidence in data Better isotope match scores Better ability to detect adducts Greater ability to detect fragments More pesticides being detected and they are now being detected with fragments Fragments are considered to be confirmatory Other MS information is used as additional evidence 2016 Waters Corporation 16

17 Example 3: Non targeted Screening Are these compounds in my sample? Screening How much is in my sample? Quantitation What is the difference between my sample and another one? Comparison What else is in my sample? Elucidation (strawberry pomace) January 2014 Analysis of more samples requested ( Sample information approx 20 compounds >10 ppb approx 10 compounds < 10ppb What else can you see? 2016 Waters Corporation 17

18 Qualitative Screening: Binary Compare Spinach Red = Blank/Reference Blue = Unknown Green = Difference Region of interest 2016 Waters Corporation 18

19 Qualitative Screening: Binary Compare Spinach Control sample Highlight Masses of Interest Send to Batch Elucidation Tool Reference sample 2016 Waters Corporation 19

20 Beyond Resolution 2016 Waters Corporation 20

21 Ion Mobility-Mass Spectrometry Why ion mobility for complex samples? How does it work? Orthogonal separations Calculation of collision cross section What effect does it have on analyses? Drug metabolism data Pesticide screening data 2016 Waters Corporation 21

22 Ion Mobility Spectrometry LC-IMS-MS IMS provides greater peak capacity than LC-MS alone Reveals compounds that would have been missed Provides greater coverage for sample profiling IMS generates cleaner data Removes background ions from spectra Allows easier and faster data interpretation IMS reduces errors in compound confirmation Additional collision cross section (CCS) information Reduces false positives and negatives for screening 2016 Waters Corporation 22

23 The Basic Principal Ions are driven through a tube by an electric field Velocity of ions is determined by mass and charge Motion of the ions is resisted by a drift gas Resistance is determined by size (mass) and shape of the ion N 2 Ions are separated based on their charge, mass and shape With chromatography and MS, this provides additional separation power 2016 Waters Corporation 23

24 LC-IM-MS IMS MS LC time 2016 Waters Corporation 24

25 IMS Orthogonal Separation The ability to see more Resolution of isomers Cleaner spectra Collision Cross Section IMS drift time CCS Additional identification point Matrix independent System independent 2016 Waters Corporation 25

26 What is CCS? Important differentiating characteristic of an ion Chemical structure (mass, size) Dimensional information (shape) Precise physicochemical property of an ion 2016 Waters Corporation 26

27 WIV ISP Study: SYNAPT/Vion Comparison for Pesticides Consistent CCS values Different instruments in different countries 5 Synapt WIV ISP/Vion IMS Q ToF Comparison April % CCS Difference Acetamiprid Azoxystrobin Bromuconazole II Carbetamide Carbofuran Chlorbromuron Chloridazon (Pyrazon) Clethodim I Clethodim I fgt Clethodim II Clethodim II fgt Cyromazine DEET Demeton-S-methyl-sulfone Dichlorvos Diethofencarb fgt Disulfoton-sulfone Diuron Ethoprophos Fenamiphos sulphone Fenoxycarb Fenoxycarb fgt Fenpropimorph Fenpyroximat Flutriafol Formetanate Fosthiazate Furathiocarb Heptenophos Hexazinone Indoxacarb Isoxaben Linuron Mesotrione Metalaxyl Methabenzthiazuron Metobromuron Metolachlor Metribuzin Mevinphos trans Nitenpyram Oxadiazon pethoxamid phenthoate Pirimicarb Primicarb-desmethyl fgt* Propamocarb propargite Prosulfocarb Pymetrozine Pyraclostrobin Pyridaben Pyridaben fgt 1 Pyridate Pyriproxifen Simazine Spinosad Spirodiclofen Spiroxamine II Tebuconazole tepraloxydim Thiacloprid Thiodicarb Thiophanate-methyl Triasulfuron Triflumuron Trinexapac-ethyl Triticonazole Vamidothion Pesticide Axis Title 2016 Waters Corporation 27

28 MS E HDMS E MS E High and low energy spectra Precursor and fragment ions Single acquisition Data independent acquisition Data automatically aligned Requires partial LC peak resolution Links associated precursors and fragments HDMS E IMS separation in front of MS E Can separate ions that co-elute in LC Cleaner spectra due to mobility resolution 2016 Waters Corporation 28

29 Spectral Cleanup Pentanochlor at 1 µg.kg -1 courtesy of Dr. Jens Luetjohann, GALAB, Germany, Waters Corporation 29

30 Screening Detection of low level compounds in complex samples Requirement for efficient separation Need not to miss peaks Confident confirmation of presence or absence of targets No false negatives No false positives Identification Points -Exact mass -Retention time -Fragment ions -Isotope peaks Increased likelihood of false positives with large library Matrix and system dependent Can be lost near limit of detection Use additional identifier - CCS 2016 Waters Corporation 30

31 Mandarin EU PT Sample 8 Expected to be detected 29 Observed using a wide screen 2016 Waters Corporation 31

32 Results summary for EU RL proficiency sample FV-13 8 Expected to be detected False +ve False -ve 9 observed after 10ppm accurate mass and fragment filtering 8 observed with 2% CCS filter 2016 Waters Corporation 32

33 Vion and UNIFI: Providing routine ion mobility Seamlessly from sample to answers Just press START Simply Acquire Get to the Answers 2016 Waters Corporation 33

34 UNIFI screening workflow for Vion courtesy of Dr. Jens Luetjohann, GALAB, Germany, Waters Corporation 34

35 UNIFI screening workflow for Vion courtesy of Dr. Jens Luetjohann, GALAB, Germany, Waters Corporation 35

36 Sensitivity: mg/kg pentanochlor in tomato extract HDMS E courtesy of Dr. Jens Luetjohann, GALAB, Germany, Waters Corporation 36

37 Conclusions Targeted, suspect and unknown screening require sensitivity and selectivity for successful outcomes Xevo G2-XS QTof Data processing and review have evolved Beyond 2D extracted ion chromatograms Using all of the data for confirmation CCS adds another dimension for separation and identification Matrix and system independent Additional confidence in decisions 2016 Waters Corporation 37

38 Acknowledgements US FDA Boethell Lab GALAB, Germany WIV-ISP, Belgium Thank you for your attention Any questions Waters Corporation 38