Improved Detection of Food Contamination using GC/QQQ and GC/QTOF

Transcription

1 Improved Detection of Food Contamination using GC/QQQ and GC/QTOF Philip L. Wylie, Ph.D. Sr. Applications Chemist for Food Safety Agilent Technologies December 1, 011 1

2 Some of the Food and Feed Contaminants that we Know About and Need to Analyze Acrylamide Aflatoxins Bisphenol A & F Brominated Flame Retardants Dioxins Dibenzofurans -Monochloroproane-1, Diol Esters Synthetic Colors Mycotoxins Nanoparticles PAHs Pesticides PCBs Drug residues Perfluorooctanesulfonate Perfluorooctanoic Acid Phthalates Melamine Nitrosamines Nonylphenol Octylphenol Styrene dimers & Trimers

3 What about the ones we don t know about? Acrylamide Aflatoxins Bisphenol A & F Brominated Flame Retardants Dioxins Dibenzofurans -Monochloroproane-1, Diol Esters Synthetic Colors Mycotoxins Nanoparticles PAHs Pesticides PCBs Drug residues Perfluorooctanesulfonate Perfluorooctanoic Acid Phthalates Melamine Nitrosamines Nonylphenol Octylphenol Styrene dimers & Trimers

4 Presentation Outline Advantages of using GC/QQQ Sensitive analysis of pesticides and other food contaminants New MRM database for 1074 pesticides and environmental contaminants Sample prep and backflushing New high resolution, accurate mass GC/Q-TOF Design Food applications Unknowns Analysis 4

5 Chemical Residue Analysis Today GC/MS/MS is Replacing GC/MS and Selective Detectors Agilent 7890A/7000 Series GC/QQQ Introduced in 008 5

6 Advantages of a QQQ as a Chromatographic Detector - Multiple Reaction Monitoring (MRM) Source Quad Mass Filter (Q1) Collision Cell Quad Mass Filter (Q) Spectrum with background ions (from EI) Q1 lets only target ion 10 pass through Collision cell breaks ion 10 apart Q monitors fragments 158 and 191 for quant and qual No chemical background 6

7 MS/MS Eliminates Scan and SIM Interferences Single Quad MS no selectivity against ions with same m/z interference Triple Quad MS Selectivity by selection of product ions analyte Product unit mass resolution Product Product 1 interference analyte 7

8 Dieldrin EI Spectrum Precursor ion m/z 6 8

9 GC/MS SIM vs. GC/QQQ; Dieldrin at 10 ppb GC/SIM m/z 6, 65, 77,79 Apple GC/QQQ MRM 6 &191 & 6&19 Cabbage Ginseng Orange Spinach 9

10 Lambda-Cyhalothrin I and II in Spinach using NCI-ammonia I 5 ppb spike I+II Sample spiked at 5ppb I + II I quantified at.05 ppb (R^ = ) II quantified at 1.10 (R^ = 1.0) Summed in Mass Hunter for total of.15 ppb II

11 Incurred Cypermethrin I IV in Spinach at 0.75 ppb by NH NCI GC/MS/MS

12 Building MRM Methods for Food Analysis 1

13 What is the hard part about developing a GC/QQQ method? Optimizing the GC method Identifying the best MRM transitions for each compound Purchase the standards - $$$$$ Run each one in scan mode Choose possible precursor ions Run Product Ion Scans Choose the best transitions Optimize collision energy Create the method Test the method Method development can take days or weeks 1

14 8000+ optimized MRMs for >1000 Pesticides & Pollutants -- based on >500 injections on $70,000 worth of chemical standards Extensive flexibility allows method optimization average of 8 MRM transitions with relative intensity for each compound -- provides alternatives to avoid matrix interference compound classification, CAS number etc. in Excel format -- allows easy searching and sorting for method customization three GC methods with Retention Times (RTs) and Retention Indexes (RIs) -- allows maximum freedom to follow user s workflow Tools and macros in Database -- build a MRM acquisition method, based on your compound list, in 5 mins Available Now! Agilent has built methods for you. New Comprehensive MRM Database for Pesticides and Other Food/Environmental Contaminants We bought standards and made >500 runs, so you don t have to! 14

15 Flexibility: multiple transitions; classifications; RTs and RIs Database has RTs (and RIs) to be used with three GC methods (CF-40min, CP-40min, and CF-0min) Average and exact Molecular Weight Each compound is classified in two categories 15

16 Flexibility: Excel Format, Relative and Absolute Transition Intensity MassHunter Format The absolute and relative intensities of transitions (Color Scales): Red denotes strong intensity and blue denotes weak intensity among ALL transitions. Compound names in Chinese and Japanese One Quant and several Qualification ions for each compound 16

17 Sample Prep and Method Ruggedness 17

18 QuEChERS Sample Preparation Quick Easy Cheap Effective Rugged Safe Very popular in labs all over the world Just enough sample preparation means that extracts can be dirty Agilent supplies QuEChERS Kits with preweighed reagents in sealed packets 18

19 The QuEChERS Method for Pesticide Residues Tomato Grape Spinach Strawberry 1) Shake sample with solvent and salts ) Centrifuge for 1 min Spinach Grape Tomato Strawberry 4) Centrifuge for 1 min 5) Analyze Pesticides ) Mix a portion with a sorbent Slide courtesy of Dr. Stephen Lehotay, USDA 19

20 Heavy Compounds May Be Left in Head of Column After Each Injection Inlet After Run 1 After Run After Run Carrier Flow Column These heavy materials build up and travel further into the column with each injection. This buildup of heavy materials causes retention time shifts, peak distortion, higher bleed, ghost peaks, and loss of sensitivity Page 0

21 Backflushing After Each Injection End of Run Flow Inlet Column Backflush 0 sec Flow Backflush 0 sec Flow Backflush 40 sec Flow Backflush 60 sec Flow Backflushing removes heavy materials after each injection. Page 1

22 What Happens when you Don t Backflush Dandelion root powder full scan (m/z ) analysis without backflushing x10 9 Run time = 0 min 1 x Acetonitrile blank analysis after the dandelion root powder analysis without backflushing and run time of 0 min Matrix peaks observed in two subsequent blank injections! x Run time = 5 min (additional 15 min at 90 C) At least additional 10 min at 90 C needed to elute the less volatile matrix components (e.g. sterols) Counts vs. Acquisition Time (min) Results provided by Dr. Katarina Mastovska, Covance Laboratories

23 Typical Backflush Configuration: Analysis Mode Split/Splitless Injection Port Aux EPC Z ml/min = very low flow Vent 7890A GC Z ml/min Purged Ultimate Union 7000B GC MS/MS -m x 0.5 mm deactivated fused silica (Optional Retention Gap) Y+Z ml/min Or 5975C MSD Y ml/min Purged Union 15-m HP-5ms UI (0.5mm id x0.5um) 0.65-m fused silica (0.15 mm id) Or Second column

24 Purged Union: Backflush Mode Split/Splitless Injection Port Aux EPC 7890A GC Vent Decrease inlet pressure during backflush Purged Ultimate Union 7000B GC MS/MS -m x 0.5 mm deactivated fused silica Or 5975C MSD (Optional Retention Gap) 15-m HP-5ms UI (0.5mm id x0.5um) 0.65-m fused silica (0.15 mm id) Or Second column 4

25 The Purged Ultimate Union (PUU) configurations PUU Aux EPC Purged Ultimate Union MSD or Vocabulary Post-column inlet Column 1 Column Capillary column QQQ PUU restrictor MS (Turbo Only) Mid-column inlet Capillary column PUU Capillary column MS (Turbo or Diff) Mid-column inlet PUU Short capillary column Longer capillary column MS (Turbo or Diff) 5

26 Backflushing Eliminates Less Volatile Matrix Components (Using Mid-Column Backflush) Dandelion root powder full scan (m/z ) analysis with backflushing x Last analyte RT = min Deltamethrin ( m/z 5 >17) x Run time = 0 min Counts vs. Acquisition Time (min) x Backflushing starts (after deltamethrin safely elutes from the first column) Acetonitrile blank analysis with (no backflushing extended run, additional 15 min at 90 C) after No matrix the dandelion peaks from root powder the previous analysis injection with backflushing observed! No matrix peaks from the previous injection observed! Counts vs. Acquisition Time (min) Results provided by Dr. Katarina Mastovska, Covance Laboratories 6

27 Long-term System Performance Overlays of GC-MS/MS chromatograms for selected analytes in spiked samples obtained within the sequence of 15 matrix injections Dichlorvos Malathion Ethion m/z 185>9 m/z 17>99 m/z 1>19 Phosalone m/z 67>18 Deltamethrin m/z 5>174 Ginseng Root Powder x Counts vs. Acquisition Time (min) x Counts vs. Acquisition Time (min) x Counts vs. Acquisition Time (min) x Counts vs. Acquisition Time (min) x Counts vs. Acquisition Time (min) Saw Palmetto Berry Powder x Counts vs. Acquisition Time (min) x Counts vs. Acquisition Time (min) x Counts vs. Acquisition Time (min) x Counts vs. Acquisition Time (min) x Counts vs. Acquisition Time (min) Scutellaria Powdered Extract x Counts vs. Acquisition Time (min) x10 x10 x10 x Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Results provided by Dr. Katarina Mastovska, Covance Laboratories 7

28 Backflush: Many Advantages for GC/MS(/MS) Analysis of Complex Samples ( Dirty Matrices ) Provides more consistent GC retention times Provides better, more consistent mass spectra throughout a sequence Reduces chemical noise - reduced carryover of matrix Higher quality quantitation without increase in interfering ions Reduces contamination of the source Reduces analysis time Increases lifetime of analytical column Change columns, liners, septa without venting! 8

29 Evolution of Benchtop GC-MS at HP / Agilent A MSD 5971A MSD 597A MSD 597A MSD A MSD 7000 QQQ 40 ITD 9

30 Evolution of Benchtop GC-MS at HP / Agilent A MSD 5971A MSD 597A MSD 597A MSD A MSD 7000 QQQ 40 ITD 0

31 700 Series Q-TOF for GC/MS High Resolution and Accurate Mass 7000 GC/MS QQQ based 6500 LC/MS QTOF based Ion Source Quad Mass Filter (Q1) Octopole Collision Cell DC Quad Ion Pulser Turbo 1b Turbo 1a Turbo Turbo 1 FPRW 011 FPRW GC-QTOF 011 Overview GC-QTOF Chris Sandy, July 011

32 700 Analyzer Four stages of pumping Doug King

33 Removable Ion Source Automated Gate Valve RIS Automated Retractable Transfer Line 0 min to change source and make new run FPRW 011 FPRW GC-QTOF 011 Overview GC-QTOF Chris Sandy, July 011

34 Internal Reference Mass (IRM) Compound CAS , 1,,5-Triazine,,4,6-tris(pentafluoroethyl)-, C 9 F 15 N, Accurate mass EI spectrum [M] + = Measured mass Accurate Mass Mass Accuracy ppm

35 What Will the 700 Q-TOF Do for You? TOF mode High resolution full spectral acquisition Accurate mass measurements Fast acquisition of full spectra MS/MS mode Full product ion spectral acquisition With high resolution and accurate mass Ideal tool for solving complex analytical problems 5

36 Resolving power & Mass accuracy R = 614/0.68 = 90 Δmz = 0.1/614 = 160 ppm Mz=614 SQ TQ IT Resolving Power: R=mz/FWHM Mass Accuracy: Δmz=dm/mz*10 6, parts per million (ppm) PFTBA mass 614 C1F4N= Pw=0.68 TOF Q-TOF R = 614/0.04 = 145 Δmz = /61.96 = 0.7 ppm 1 Da. 1 Da. 6

37 Ethion Spectrum C 5 H 1 O PS + = C 9 H O 4 P S 4 M*+ =

38 Enhanced Selectivity using Accurate Mass 5 ppb in Ginseng Extract Extracted Ions = and /- 10 ppm Full Scale 00,000,000 00, /- 10 ppm /- 10 ppm 8

39 Fludioxonil in Frozen Blueberry Extract 8 ppb in extract TIC EIC: /- 0.5 amu Signal/Noise = 8 EIC: /- 5 ppm Signal/Noise = 81 9

40 # Possible Chemical Formulas Many Possible Formulas with Quadrupole or Ion Trap - But only a few with Accurate Mass TOF Possible Number of Chemical Formulas at m/z 7 Octafluoronaphthalene (CAS 1-7-4) C 10 F 8 = mass uncertainty, ppm mass uncertainty ppm Formulas made of: C,H,N,O,F, & Cl # of Possible Formulas amu Accurate mass reduces risk of investing effort on the wrong molecule 40

41 The Problem Confirm Most Likely Structure Kava Extract - Compound B, C 16 H 14 O 4 (Rings + Double Bonds = 10) EI Full Spectrum (M H) Candidate structures H C 6 H 5 CH=CH C 6 H 5 CH =CH C 6 H 5 CO Experimental measurements m/z (experimental ) Formula C 16 H 1 O 4 Error (ppm) Score C 10 H 9 O C 8 H 7 O 4.0 N/A C 8 H 6 O N/A CO C 7 H 6 O CH C 6 H 6 O.0 N/A C 5 H O For the 5 candidate structures, only one fit the losses identified by CID experiments on multiple precursor ions 41

42 Agilent 700 Series GC-QTOF Identifying and quantification of S- and N-containing compounds in beverages 4

43 Problem Compounds affecting taste and flavor Example of complex matrix Coffee extract -formyl thiophene and -acetyl thiazole are common contaminants Low sensory threshold and can have negative effect on product flavor or aroma Easy to separate from each other Often requires sophisticated extraction/enrichment procedures and/or powerful D GC techniques for separation from matrix for quantitation GC-Q-TOF Method highlights : Simple Liq / Liq extraction in Dichloromethane (10:1 enrichment) 1:10 split injection with S/SL inlet DB-5MS column 0 m x 0.5 mm x 0.5 μm 4

44 Standards at 100 pg On Column formyl thiophene Formula C 5 H 4 OS MW: acetyl thiazole Formula C 5 H 5 NOS MW: 17 44

45 45

46 46 FPRW 011 GC-QTOF

47 Photodegradation Products of Beer Completely untargeted (initially) study of beer photodegradation Method highlights 0 min extraction at 0 C using manual SPME holder and conditioned 50/0 µm DVB/Carboxen/PDMS StableFlex SPME fiber (Supelco), no agitation Desorption at 00 C for min in the SSL injector; 1:10 split Agilent J&W column DB-5MS 0 m x 0.5 mm x 0.5 µm 47

48 Changes in the Chromatogram Appears following the exposure of the sample to direct sunlight. Peak height is dependent on the duration of exposure to the sun No exposure to direct sunlight hours 6 hours 48

49 Summary of MS/MS Experiments 109 C 6 H 7 NO 81 N O C 10 H 14 N C H 5 C 4 H 6 N C 5 H 6 N C 5 H 7 N C 6 H 8 N C 6 H 6 N O C 7 H 8 N C 9 H 14 N C 9 H 11 N C 10 H 15 NO Butanamine, N-(-furanylmethylene)--methyl- 109 C 6 H 7 NO 1 C 7 H 8 NO 16 C 9 H 14 N 148 C 10 H 14 N 81 C 5 H 7 N 108 C 6 H 6 NO 80 C 5 H 6 N 94 C 6 H 8 N 78 C 5 H 4 N 55 C H 5 N 5 C 4 H 5 41 C H 5 66 C 4 H 4 N 1 C 9 H 11 N 49

50 Unknowns Analysis A feature in MassHunter Quant Run after finishing Quant Analysis Deconvolutes chromatogram Library search deconvoluted components Can use retention times to filter hits Sort into target compounds and non-target compounds Easy review of results Export spectra to mass spec library Easy way to build an accurate mass library from acquired data 50

51 Unknowns Analysis A feature in MassHunter Quant TIC Deconvoluted accurate mass spectrum Library match - Ditalimfos Ditalimphos Component EICs 51

52 Fundamental Benefits (Agilent 700 Q-TOF) High resolution (> 10K, typically > 1K FWHM) Increased detector selectivity (few interferences) Accurate mass measurements (low to sub-ppm) TOF mode - Typically < ppm Q-TOF mode (MS/MS) Typically < 5 ppm Valuable qualitative information about each ion Structural elucidation: Accurate Mass MS/MS High sensitivity tool to complement NMR Hyper-selective, Hi-Res MS/MS Fast (up to 50 spectra/sec), full spectra acquisition with excellent sensitivity 5

53 5975E SQ 5975C SQ 5975T LTM SQ Thank You 0 IT 700 Q-TOF 40 IT 7000 TQ The Agilent Portfolio of Benchtop GC-MS Systems 5 FPRW 011 GC-QTOF