Pesticides e-seminar Series - Pesticide Analysis Workflow Using Hyphenated Mass Spectrometry Techniques Date: 30 April 2009 Time : 10am Singapore Time For audio, please dial one of the phone numbers listed: International Telephone Number for Singapore: +65-6622 1044 or Toll Free 8008 523 396 International Telephone Number for Hong Kong: +852 3006 8101 Toll Free International Numbers: Australia 1800 999 130 New Zealand 0800 450 755 China 800 876 5011 Malaysia 1800 807 180 Indonesia 0018 038 526 350 Taiwan 0080 185 5735 India* 0008 008 521 136 South Korea 0079 885 214 717 India (Backup)* 0008 001 006 542 Philippines 1800 185 50065 Thailand* 0018 008 526 361 Vietnam 120 650 065 * Phone line needs to be IDD enabled. Not accessible from Mobile Phones
Pesticide Analysis Workflow Using Hyphenated Mass Spectrometry Techniques E-Seminar, April 30, 2009 Chinkai (Kai) Meng, Ph.D. Senior Applications Chemist Wilmington, Delaware USA chin_meng@agilent.com Page 2
MS Techniques Outline MS Analyzers Selection GC/MS deconvolution QQQ (MS/MS) LC/Q-TOF Summary Page 3
Analytical Instrumentations Chromatography (Separation the best friend of any detector) GC volatile and semi-volatile non-polar compounds LC polar or moderately polar or thermally labile compounds Detectors GC (element selective, FPD, NPD, ECD) high sensitivity, poor specificity LC (UV, FLD) general purpose, limited sensitivity MS SQ: high confidence from spectral confirmation, limited MDL QQQ: low detection limits of target compounds in dirty matrices (MRM) TOF/Q-TOF: always full spectrum and accurate mass for screening TOF-TOF, Q-Trap, and others (generally not for ROUTINE use) Page 4
Guiding Principles in MS Analyzer Selection The correct choice of analyzer depends on whether one is LOOKING for UNKNOWNS or whether one is CONFIRMING & MEASURING KNOWNS. The correct choice of analyzer depends on the COMPLEXITY of the MATRIX. Page 5
Basic Questions Which MS Solution Target analysis only? Scan or SIM or MS/MS Analysis of unknowns? Scan MS with Quad, IT or TOF How much chemical noise from the matrix? How much sample prep? Deconvolution or MS/MS MS/MS Page 6
General Workflow: Screen, Confirm and Quantify S C Q Representative Sample GC/MS (PTV) SIM/Scan for known and unknown Deconvolution Final Report S C (+backflush) Q QuEChERS Extraction 1 3 GC/QQQ MRM for known targets S C LC/QQQ MRM for known targets S C Q Q 1 3 LC/QTOF or TOF Full Spectrum for unknown compounds Exact Mass Database Search Clean-up 1 3 S Molecular Formula Generation Q Another injection for MS/MS (QQQ or QTOF) C C16 H19 N3 P Cl C15 H25 O P S Cl C18 H21 O P Cl Page 7
MS Techniques Outline MS Analyzers Selection GC/MS deconvolution QQQ (MS/MS) LC/Q-TOF Summary Page 8
TICs of Surface Water Extracts How many pesticides (drugs, allergens etc.) are in these samples and how long does it take you to confirm? 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Page 9
17 Surface Water TICs (Scan): Pesticide Analysis Using DRS with Pesticide Database (927 entries) Targets Found (not counting ISTD) CDFA* 37 Agilent DRS Same 37 +99 more False Positives 1 0 Processing Time ~8 hrs 32 min DRS: Deconvolution Reporting Software Saving 7.5 hours *CDFA is the California Department of Food and Agriculture Page 10
How Does Deconvolution Work? 170 280 50 185 75 160 310 Page 11
Eliminate Ions Don t Fit the Criteria 170 280 50 185 75 160 310 Ion grouping criteria: 1. Same RetTime at apex 2. Same peak width Page 12 160 50 170 280 185 75 310 Extracted Ion Chromatograms (EIC)
Spectrum is Deconvoluted/Cleaned 170 280 50 A component in AMDIS 50 170 280 Related ions are grouped together as a component. Page 13
TIC of Spinach Extract More than 370 peaks found 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Deconvolution Page 14 Library Search
QEdit, p,p -DDT selected 5 ion overlay MSD ion AMDIS ion MSD & AMDIS areas & amounts 3 spectra x and A indicators Page 15
Pesticides in Red Wine - Fenpropathrin Found by AMDIS 250 ppb 100 250 ppb Raw scan Not found 1 ppm MF = 65 83 Deconvoluted MF = 783 Fenpropathrin 5 ppm Library entry MF = 877 Fenpropathrin Page 16
MS Techniques Outline MS Analyzers Selection GC/MS deconvolution QQQ (MS/MS) LC/Q-TOF Summary Page 17
Analyzers for Trace Target Compounds Who should use QQQ (MS/MS)? User doing Selected Ion Monitoring (SIM) for target compound analysis in traditional markets, needing additional sensitivity and selectivity with less sample prep, to meet more demanding analytical requirements. GC/QQQ MRM for known targets S LC/QQQ MRM for known targets S C C Q Q Page 18
Advantages of a QQQ as a Chromatographic Detector - Multiple Reaction Monitoring (MRM) Source Quad Mass Filter (Q1) Collision Cell Quad Mass Filter (Q3) Spectrum with background ions (from EI) 210 222 Q1 lets only target ion 210 pass through 210 Collision cell breaks ion 210 apart Q3 monitors only characteristic fragments 158 and 191 from ion 210 for quant and qual. 165 268 280 158 191 210 158 191 170 210 250 290 190 210 150 170 190 210 160 190 no chemical background Page 19
Why MS/MS? Greater Selectivity Than SIM EI-SIM selectivity proportional to spectral resolution no selectivity against ions with same m/z interference analyte EI-MS/MS Precursor selectivity same as SIM High probability that at least one product ion will be a unique dissociation product of the precursor BUT not the interference Product 2 unit mass resolution Product 3 Product 1 interference Precursor Ion The precursor ion should NOT be used for ion ratios or quantitation since the interferences will be the same as the SIM ion Page 20
Consistent MS/MS Results, Regardless of Matrices Analysis of spiked p,p -DDE at 10 ppb (All injections = 1 µl) SIM - EIC (246) MRM (2 transitions) Apple Cabbage 246.0 -> 176.1, 246.0 -> 175.1 x10 4 Ratio=23.5 Counts 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0-0.1 11.8 11.9 12 12.1 12.2 12.3 A c q u isitio n T im e (m in ) 246.0 -> 176.1, 246.0 -> 175.1 x10 3 Ratio=22.9 Counts 8 7 6 5 4 3 2 1 0 S/N = 448 S/N = 241 Ginseng 11.8 11.9 12 12.1 12.2 12.3 A c q u is itio n T im e (m in ) 246.0 -> 176.1, 246.0 -> 175.1 Counts x10 4 Ratio=23.3 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 S/N = 446 Orange 246.0 -> 176.1, 246.0 -> 175.1 x10 3 Ratio=23.6 8 7 6 5 4 3 2 1 Counts 11.8 11.9 12 12.1 12.2 12.3 Acquisition Time (min) S/N = 456 0 Spinach 11.8 11.9 12 12.1 12.2 12.3 A c q uisitio n T ime (min ) 246.0 -> 176.1, 246.0 -> 175.1 x10 4 Ratio=24.0 1.2 Counts 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0-0.1 11.8 11.9 12 12.1 12.2 12.3 A c q u is itio n T im e (m in ) S/N = 260 AppNote: 5990-3578; 175 pesticides Page 21
Carrot Extract - 1 µl with GC/QQQ is Much Better than 5 µl GC/Q GC/Q GC/QQQ 5 µl (Multi-mode Inlet) 1 µl Pesticide Cold SL Scan + DRS Cold SL SIM Hot SL (ppb) Diclobenil 0.38* Pentachlorobenzene 0.75* Trifluralin 2.3* Tefluthrin 0.53* 4,4'-Dichlorobenzophenone 1.2* Chlorpyrifos 24.7 o,p'-dde 3.7 p,p'-dde X X 240 o,p'-ddd 9 p,p'-ddd X o,p'-ddt X Sum = 45 p,p'-ddt X X 130 Fenazaquin X Not in Method * = Below Calibration Level Page 22
p,p -DDE in Carrot Extract (240 ppb) 5 µl GC/Q vs. 1 µl GC/QQQ 5 µl Cold SL Scan EIC S/N = 35 Same as left, after deconvolution 5 µl Cold SL SIM EIC S/N = 375 1 µl Hot SL QQQ MRM 246.0 -> 176.1, 246.0 -> 175.1 x10 5 Ratio=23.8 Counts 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0-0.1 S/N = 434 11.8 11.9 12 12.1 12.2 12.3 Acquisition Time (min) Page 23
Why a GC/MS/MS System? Allows for the selective quantitation of target compounds in high chemical background samples Better S/N in complex matrices than can be achieved by single quadrupole scan or SIM approaches. Newer regulations in some markets specify analytical power commensurate with GC/MS/MS Page 24
The Limitations of Converting an LC/MS/MS to GC/MS/MS ESI, APCI and APPI - Atmospheric pressure sources Designed to separate ions from molecules before entering the vacuum manifold Higher m/z metal rods are typically temperature compensated for 100ºC operation (some only 50ºC) no problem since only ions enter the analyzer EI, PCI and NCI - Vacuum sources All neutral molecules (carrier gas, analytes and matrix) enter the vacuum manifold BEFORE ionization Most molecules are NOT ionized and must be pumped away High boiling molecules common to GC will contaminate all cooler surfaces Contamination causes loss of sensitivity Cool (< 100ºC) metal quadrupoles are easily contaminated under GC/MS Page 25
Why Heated Quartz Gold Quads? Higher temperature reduces potential for contamination 200 C max Virtually eliminates the need to clean quads Low coefficient for thermal expansion for quartz Stable structure during maintenance cycles (hot-cool-hot) for source or detector More stable tunes and methods over a longer period of time in real world sample environments Page 26
MS Technique Outline MS Analyzers Selection GC/MS deconvolution QQQ (MS/MS) LC/Q-TOF Summary Page 27
Agilent Q-TOF Fundamentals Two key concepts for TOF: 1. Exact Mass 2. Mass Error Collision cell Common with QQQ Ion optics Common with Q & QQQ Flight tube Common with TOF Ion Mirror Octopole 1 Quad Mass Filter (Q1) Octopole 2 Detector Lens 1 and 2 Collision Cell DC Quad Ion Pulser Rough Pump Turbo Turbo Turbo Turbo Page 28 September, 2008
Mass Analysis for TOF v = d/t E = ½ m v 2 = ½ m (d/t) 2 m = (2E/d 2 ) t 2 Energy (E) and Distance (d) are fixed The measured mass is proportional to the flight time (time-of-flight). Page 29
What does Exact Mass mean? 0.2874 amu Element Atomic Number Exact Mass H 1 1.007825 C 6 12.000000 N 7 14.003074 O 8 15.994915 C6H6Cl6 287.8600665 Lindane C10H12N2O6S 288.0416000 Carbasulam C9H21O2PS3 288.0441285 Terbufos C13H21O3PS 288.0949000 Iprobenfos C15H17N4Cl 288.1141743 Myclobutanil C12H21N2O4P 288.1238937 Diazoxon C11H20N4O3PS 288.1256000 Epronaz C11H21N4O3P 288.1351000 Pirimetaphos C16H20N2O3 288.1473925 Imazamethabenz The Key for getting useful TOF results is good mass accuracy. Uses accurate mass on TOF/Q-TOF to identify all of them. Page 30 September, 2008
Calculation of Error in Measured Mass (MH + ) of Reserpine Atom Mass of Atom # of Atoms Sum Hydrogen 1.00783 40 40.31300 Carbon 12.00000 33 396.00000 Nitrogen 14.00307 2 28.00615 Oxygen 15.99492 9 143.95424 Total 608.27338 Plus H 1.00783 1 1.00783 Total 609.28121 Minus e- 0.00055 1 0.00055 609.28066 Calculated = exact CH 3 O CH 3 (Measured - Calculated) Calculated X 1,000,000 = ppm 0.9027038 ppm Error if the electron was not omitted! O CH 3 N H H O H O N CH 3 O O H CH 3 Reserpine (C 33 H 40 N 2 O 9 ) O O O CH 3 Page 31 September, 2008
TOF/Q-TOF Unlimited Number of Compound Screening 5556.D (Grape) MS1 Full Spectrum Molecular Feature Extractor found 510 compounds in the TIC. 15 out of 510 compounds had hits from EXACT MASS database search, i.e., these 15 matched entries in the database within 3 ppm mass accuracy. The three highlighted compounds were further confirmed by MS/MS. Page 32 September, 2008
Terbuconazole The compound name Terbuconazole came up in the MS1 mode search. Use formula results and MS/MS results to confirm the hit. Cl 125.0158 H 3 C CH 3 x10 4 7 6 5 4 3 2 Cpd 11:+ Scan (13.974-14.248 min, 5 scans) 5556.d 121.05086 MS1 Full Spectrum 308.15216 C16H23ClN3O N N N CH 3 OH 1 0 x10 4 6 5 4 3 2 1 0 Cpd 11:+ Product Ion (14.121 min) (308.15240[z=1] -> **) 5556.d 70.04052 C4H6O MS/MS Full Spectrum 125.01448 C7H6Cl 308.15199 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 Counts vs. Mass-to-Charge (m/z) x10 4 1 0.8 0.6 0.4 0.2 Cpd 11:+ Scan (13.974-14.248 min, 5 scans) 5556.d 308.15216 C16H23ClN3O 310.14951 311.15253 0 307 308 309 310 311 Counts vs. Mass-to-Charge (m/z) Boxes represent theoretical isotope ratios Page 33
Screen Pesticides with LC-TOF/Q-TOF Accurate Mass provides added compound selectivity Higher resolution provides added interference selectivity Always full spectral data Unlimited number of compounds can be screened (search exact mass compound database for identification) Sensitivity is the same regardless of number of compounds screened* MS/MS (Q-TOF) assists compound confirmation *Triple quadrupole (QQQ) mass spectrometer can be more sensitive up to a limited number of compounds. That limit has not been definitively determined. Page 34
MS Technique Outline MS Analyzers Selection GC/MS deconvolution QQQ (MS/MS) LC/Q-TOF Summary Page 35
Summary GC/MS and Deconvolution to Screen, Confirm and Quantify QQQ for routine Targeted trace analysis in complex matrix MRM Sensitivity unsurpassed (up to a few hundred compounds) Complex matrix with less clean-up TOF/Q-TOF for Targeted and Unknown Screening Sensitive full scan analysis Searching exact mass database leads to identification Unlimited number of compounds Sensitivity is the same regardless of number of compounds screened Quantitative Accurate mass MS/MS for identification of fragments and structure elucidation (hotlink available to search on-line databases) Page 36
References Application Note 5989-7670: Replacing Multiple 50-Minute GC and GC- MS/SIM Analyses with One 15-Minute Full-Scan GC-MS Analysis for Nontargeted Pesticides Screening and >10x Productivity Gain Pesticide Brochure 5989-8652 GC-QQQ Brochure 5990-3152 Current Trends in Mass Spectrometry, pp 33-38, Nov. 2008 (Supplement to LC/GC North America): Comprehensive Screening, Confirmation, and Quantification of Organic Pesticides in Foods by GC-MS and LC-MS The Applications Book, LC/GC Europe, pp 9-11, Dec. 2008: The Benefits of Incorporating GC/QQQ into Pesticide Analysis Methods Page 37
Thank you for your attention. Questions? Jan. in Amsterdam Page 38
Technology Agilent took the best technologies from their industry leading 597X Series GC/MSD: Heated monolithic gold plated quartz quadrupole Proven reliable high performance source design AUTOTUNE and the 6410A LC/QQQ: Linear acceleration enhanced Collision Cell Wide Mass-Bandwidth QQQ ion optics MassHunter software Page 39
Identify Dichlofenthion in MSD ChemStation -Red wine hidden 250 ppb No match 500 ppb No match 1 ppm MF = 630/670 Dichlofenthion Page 40
Identify Dichlofenthion in AMDIS hidden 250 ppb 250 ppb Raw scan No match 500 ppb MF = 82 Deconvoluted No match 1 ppm Library entry MF = 630/670 Dichlofenthion Page 41
Agilent Has the TOTAL Portfolio for Pesticide hidden Analysis G A S phase GC FPD/ECD GC/QQQ MRM (semi)-volatile or non-polar GC/Q DRS L I Q U I D phase LC UV, FLD LC/QQQ MRM Routine Analysis (moderate)-polar or thermally labile LC/Q TOF Q-TOF Exact Mass Database Search Identify Unknowns Page 42