How Can Orbitrap Technology Help My Food Safety Analysis? Larry Burchfield Mass Spectrometry Product Manager Latin America Region August 27, 2012

EFS Target or Non-target analysis workflow SAMPLE Target Analysis Non-Target Analysis Target screening ? Profiling Fingerprinting Authenticity Quantitation Quantitation Food Safety Environmental

Which LCMS Analyzer Do I Choose? Pure Quantitative Pure Qualitative Determine structure Detect & Quantify Structural ID Compound Confirmation Reaction Monitoring Process Monitoring Metabolism Proteomics Metabolomics Targeted screening Targeted & Unknowns screening 1 – >40 cpds 40 – 600 cpds Multiple cpds ORBITRAP QTRAPs Q-TOF Triple Quads Ion Traps Exactive & Q-Exactive (Orbitrap)

Triple Quadrupoles for Target Quan : Traditional Workhorse and still 75% of EFS Instruments Sold in 2011 TSQ Quantum Access Max TSQ Quantum Access TSQ Quantum Ultra TSQ Vantage The following is what Thermo Scientific has to offer in the TSQ line. For the ultimate value we have the TSQ Quantum Access Max. For the utmost detection needed TSQ Vantage which was introduce in 2008. Ultimate Value Ultimate Performance

However, conventional thinking is evolving… Food Safety: International Requirements and Innovative Analytical Solutions

EU Commission directive on HRAM One monitored m/z at R=10K, gives 2 IPs (but One QQQ SRM = 1.5 IP) However, this R refers to Sector instruments R @ 10% FWHM ToFs/Orbis measure R @ 50% FWHM Thus, R = 10K (sector) corresponds to R= 20K @ 50% FWHM (ToF/Orbi) There`s still no specific clarity in EU directives about the use of HRAM (Orbi) for multi-residue work

EU - Q Exactive exceeds IP requirements (min = 4) Score Max Possible 6.5 points    EC Directive 96/23/EC for analytical methods and the interpretation of results

How does this translate to LCMS instrument selection in EFS labs today ?

Shift towards HRAM : Crossover Point Official Food Control Authority of the Canton of Zurich

Shift towards HRAM : Crossover Point (cont.)

Quantitative Performance : Orbitrap vs TOF vs Triple Quad Note: Orbi and TOF not MS/MS Kaufmann et al, Rapid Comm Mass Spectrom, 2011, 25, 1-14

Presentation of Dr. Hans Mol

Visualizing the matrix…(TICs) compound feed honey solvent 0.25 g matrix/ml extract 5 µl injection

Detection: selectivity Pirimicarb m/z 239.15028 ± 12 mDa [0.012 Da/239*106] What mass extraction window is required for adequate selectivity in single stage MS? Pirimicarb 10 ng/g in horse feed (R = 100,000 FWHM @ m/z 200)

Requirements for narrow mass extraction windows 1) high mass accuracy intrinsic property of mass analyzer (after adequate mass calibration) but: in real life analyte may co-elute with compounds with same nominal mass. This may lead to wrong mass assignment 2) full separation of analyte from other compounds with same nominal mass chromatographic resolution mass spectrometric resolution

How much resolving power is required ? XIC m/z 246.16009 ±5 ppm mass profiles at rt = 3.69 min Isopyrin, 25 ng/g in animal feed

Quantitative aspects: linearity Solvent standards, R= 25,000; mass extr. window 5 ppm

Conclusions Detectability/selectivity: resolving power key parameter needed for selectivity (narrow mass extraction windows) higher degree of initial confirmation more reliable quantification minimum resolving power? => depends on rel. response analyte/matrix* Identification: HCD (or IS-CID) fragmentation very useful for identification and data generated suited for confirmation according to EU legislation Quantitative aspects: suited for quantitative analysis Single stage full scan hr/hma MS is a very promising option for comprehensive screening, identification and determination of toxicants in food/feed *M. Kellman, H. Muenster, P. Zomer, H. Mol, J Am Soc Mass Spectrom. 20(8) 1464-76 (2009)

Q Exactive EFS Data CDFA, Sacramento US FDA, DC

Q Exactive for EFS Targeted Screening & GUS High Confidence Confirmation Targeted screening and GUS Goal How Q Exactive addresses the needs Mode/s of operation Software Data collected in HRAM MS and MS/MS Software capable of identifying all unique components in the chromatographic data Database searches for HRAM MS and library searches with MS/MS spectra Highest specificity with 140K resolution Software that supports multiple orthogonal ways of confirmation including elemental composition determination, isotope matching, DB search, spectral library matching. Xcalibur; ExactFinder Highest confidence in Targeted screening & GUS Full Scan MS w AIF Precursor ion selection w HCD MS/MS

Q Exactive Quan capabilities for EFS Quantitation Performance Similar to High End QqQ Goal How Q Exactive addresses the needs Mode/s of operation Software Unit mass resolution precursor ion selection like QqQ Full scan HRAM MS/MS with stable fragment ion ratio Higher specificity than QqQ due to HRAM fragmment ions Ability to confirm with many fragment ions seen in full scan MS/MS Four to five orders of dynamic range UHPLC compatible Targeted MS/MS similar to SRM; Data dependent MS/MS where quant done using precursor ion in full scan MS and confirmation using HRAM fragment ions in MS/MS; Full scan MS; HRAM SIM Quant capability of high-end QqQ ExactFinder or Xcalibur

Q Exactive TM Hardware Innovations Hardware Demonstration

Q Exactive RSLC < 1.5 m

California Dept of Food & Ag Sample : 60+ pesticides in crop extracts In neat In Green Bell Peppers In avocado Calibration levels vary depending on analytes 500 ppb – 1 ppt Mass tolerance set at 5ppm Sample injection size = 5uL Column = Hypersil Gold aQ 100 x 2.1mm 1.9u

Q Exactive Workflows for EFS : Full Scan : Screening (target and non-target) Quan Full scan Targeted Data Dependent MS/MS : Full scan screening Targeted precursor ion selection DD MS/MS of precursor ions UHPLC

Full Scan Targeted DD MS/MS Quadrupole scans from a mass range of 100-1000 Sends the ions to Ctrap, compress and send into orbitrap for analysis From a targeted list (Data Dependent MS2 Quadrupole selects the ion of interest from list Pass it to Ctrap Then pass it to HCD for fragmentation Then sends data packet back to Ctrap and injects to Orbitrap to analyze for MS2 Full Scan Data Dependent (MS2) (TopN) – Ions are passed from a selected mass range, then triggered of a targeted listed (precursor) to collect ms/ms data for confirmation – result is 2 scans (FS, MS2) – This type of experiment is Quan followed by Confirmation. You can also run this experiment with “No Targeted” and than instrument will trigger of most intense ion in the spectrum and give you MS2 scan (great for metabolism)

CDFA samples (targeted list) Parent selection list of 62 pesticide analyzed by CDFA of Sacramento Method transferred from TSQ Ultra

Q Exactive Full Scan Quan (Azoxystrobin at 1.5ppb) Direct comparison of neat vs matrix

Q Exactive Quantitation Full Scan Targeted DD MS/MS (Azoxystrobin at 1 Q Exactive Quantitation Full Scan Targeted DD MS/MS (Azoxystrobin at 1.5ppb) Direct comparison of neat vs matrix Quan IP = 2

Quanfirmation Boscalid 1.5ppb HRAM IP = 2 Boscalid Diuron Azoxystrobin Methomyl Indoxacarb Spinosad D Number of scans across a peak doing MS/MS Quantitation Confirmation Boscalid – MS/MS Accurate Mass Fragmentation HR MS(n) IP = 2.0

Quanfirmation Azoxystrobin 1.5ppb Quantitation Number of scans across a peak doing MS/MS Diuron Azoxystrobin Boscalid Methomyl Spinosad D Indoxacarb Azoxystrobin MH+ = 404.1242 Confirmation

Q Exactive Quan performance : Repeat 3x injections at LOD/LOQ in Matrix (Green Bell Pepper) 0.1 – 1.1 ppb data represented from FS/ Targeted DD MS2 QE

Comparison of CDFA pesticides in matrix : Q Exactive vs TSQ Vantage LOD/LOQ (ppb)

UHPLC compatibility : 60+ pesticides in 2 mins, 4s wide peaks, Full scan Accela 1250 + Q Exactive R = 70,000 All compound elute in 2mins, with peak width of 0.04mins = 9scans under the peak

Target Screen Method Select database, Identify with mz and confirm with isotope, library match

Unknown Screen Method Select database, Identify with mz and confirm with isotope, library match

Library/Database Searching Capabilities- Spectral Library Included List selected based on customer feedback and from the major worldwide regulated lists Over 1000 compounds Over 6000 spectra HCD CID Positive Negative Multiple CE Pesticides, hormones, POPs, PPCP Created on LTQ Orbitrap Velos HRAM library courtesy : Eric Genin, Les Ulis

Q Exactive Quanfirmation High performance HRAM Quantitation and Confirmation bench top LCMS system, capable of : Multi-residue quan performance similar to mid-high end Triples Ideal for targeted and general unknown screening Highest confidence confirmation with R = 140K, and MS/MS UHPLC compatible

History and Advances in Orbitrap Technology Salón EBRO 11:00 -12:00 Tuesday, 28 August History and Advances in Orbitrap Technology Larry A. Burchfield Latin America Manager for Chromatography and Mass Spectrometry

The End Thank You! See y’all tomorrow…