Using Deconvolution to Improve GC/MS Detection and Quantitation E-Seminar, October 19th, 2010 2 pm, EST (11 am PST) Chinkai (Kai) Meng, Ph.D. Senior Applications Chemist Wilmington, Delaware USA [email protected] Outline • Introduction • Deconvolution - Find Trace Compounds in Complex Matrices • Deconvolution - Quantitation • Summary E-seminar, October 2010 Page 2 Introduction • Instrument sensitivity (e.g., signal-to-noise ratio) is usually determined by the amount of sample injected and the responses from the detector. We will discuss sensitivity from a different view. • In a multi-residue analysis, the data reviewing process is also very important in confirming the hits found by the software and reviewing the integration/quantitation for accuracy. • Deconvolution has been proven to be a powerful data processing tool in finding trace compounds in complex matrices. In this study, results from Deconvolution (AMDIS) is closely looked at and compared to the results from ChemStation. The goal is to determine if Deconvolution can provide better results (sensitivity) than the routine ChemStation data processing. E-seminar, October 2010 Page 3 What is Deconvolution? Deconvolution is a process (tool) to extract ions from a complex full-scan total ion chromatogram (TIC), even with the target compound signal at trace level. The software used for this technique is AMDIS (Automated Mass spectral Deconvolution and Identification System) developed by NIST (National Institute of Standards and Technology). Magic Eye 3-D movie Information is there, you just need the right tool to see it. E-seminar, October 2010 Page 4 GC/MSD Deconvolution – What it can do. From here… GC/MSD in Scan Mode Deconvolution Reporting Software Quant on Deconvoluted Peaks …to here in 2 - 3 min E-seminar, October 2010 Page 5 Extracted Ion Chromatograms of a Pear Extract Ion 41 Ion 42 Ion 43 Ion 55 Ion 56 Ion 57 Ion 98 Ion 99 Ion 116 Ion 131 Ion 154 Ion 248 10 15 20 25 30 35 40 E-seminar, October 2010 Page 6 How Does Deconvolution Work? Eliminate Ions that Do Not Fit the Criteria 160 shape 50 same shape and 17 same retention 0 28 time 0 185 shape & early retention 75time late retention time 310 early retention time Deconvolution Spectrum at dotted line 50 75 17 18 160 5 28 0 0 31 0 Two criteria: 1. Same RT 2. Same peak shape Spectrum at dotted line (a component) 50 17 0 28 0 These deconvoluted ions are group together 50 17 0 28 0 E-seminar, October 2010 Page 7 More than 370 peaks found TIC of Spinach Extract 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Deconvolution Components Search RTL Pesticide Library for hits Page 8 E-seminar, October 2010 Comparison of Raw, Deconvoluted and Library Spectra Scan at 10.776 min Deconvoluted/extracted spectrum A component in the scan above. Library spectrum Fenbuconazole E-seminar, October 2010 Page 9 Sample and Instruments • Spinach extract prepared using QuEChERS protocol • 35 pesticides spiked into spinach extract at 50 ppb (pg/uL) each • 7890 GC, Multimode Inlet (MMI), 2-µL cold splitless injection • 7693 Automatic Liquid Sampler (ALS) • 5975 MSD in full-scan mode (45 - 550 amu) Acknowledgement The author would like to thank Dr. Jon Wong (FDA-CFSAN, College Park, Maryland) for graciously providing samples for this study. E-seminar, October 2010 Page 10 Typical TIC of Spinach Extracts (in Toluene, 35 pesticides spiked at 50 ppb) 3.8e+07 3.4e+07 3e+07 2.6e+07 2.2e+07 1.8e+07 1.4e+07 1e+07 6000000 2000000 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 TIC: Grp1_spinach_50ppb_cold_SL_ramp3_1.D\data.ms E-seminar, October 2010 Page 11 MSD ChemStation “Edit Compound” Screen This screen shows the quantitation database for locating and then confirming a compound. In ChemStation, the target compound identification is based on 4 ions and three ion ratios. However, the target compound identification in AMDIS was based on the full spectral library match which is more dependable. E-seminar, October 2010 Page 12 AMIDS “Identification” Settings The minimum match factor was set to 30 and the retention time window was limited to ±30 seconds to qualify the hits from the retention-time library search. Typical RT window is ±10-15 seconds. E-seminar, October 2010 Page 13 AMIDS “Deconvolution” Settings (1 of 2) Assumed component width in scans. Increase this if all peaks are wider. Masses listed here will not be used as models but can still be included in a component. A closely eluting large ion will be subtracted to allow more models to be considered. None yields the fastest processing, two the slowest. E-seminar, October 2010 Page 14 AMIDS “Deconvolution” Settings (2 of 2) Higher resolution will separate closer eluting peaks finding more components – runs slower Higher sensitivity will find smaller, noisier components but maybe more false positives – runs slower Higher requires that EICs have exactly the same shape – fewer components found and more “uncertain” peaks may be present. E-seminar, October 2010 Page 15 Comparison of Match Factors with AMDIS Settings 100 Adjacent peak subtraction (1 or 2) and Sensitivity (VH or H) 90 80 70 60 50 1 H VH M 40 2 H VH M 1HHM 2HHM 30 20 10 0 E-seminar, October 2010 Page 16 Resolution = H, Sensitivity = H, Peak Shape = M Chlorpyrifos Methyl Net = 94 Methyl Parathion Net = 92 25 standards mixture E-seminar, October 2010 Page 17 Resolution = M, Sensitivity = H, Peak Shape = M Chlorpyrifos Methyl Net = 89 Methyl Parathion Net = 37 25 standards mixture E-seminar, October 2010 Page 18 Comparison of Number of Compounds Found with Various AMDIS Settings (Spinach Extract, MF = 30) Changing Resolution only M 31 H M 35 Changing Sensitivity only H 35 VH M H H 33 H H H M Changing Shape Requirement only H M 33 M H H L 32 E-seminar, October 2010 Page 19 35 Targets @ 50 ppb in Spinach ChemStation Results 120 AMDIS Results 110 100 88 80 83 72 73 60 49 40 20 11 35 17 6 12 20 35 35 19 35 0 50% Relative 30% Relative 50% Absolute 30% 1 H VH M 2 H VH M Absolute ChemStation Settings False Positive 1HH M AMDIS Settings 2HH M Actual Targets Found E-seminar, October 2010 Page 20 35 Targets @ 50 ppb in Spinach ChemStation Results 120 AMDIS Results 110 100 88 80 83 72 73 60 49 40 20 11 35 17 6 12 20 35 35 19 35 0 50% Relative 30% Relative 50% Absolute 30% 1 H VH M 2 H VH M Absolute ChemStation Settings False Positive 1HH M AMDIS Settings 2HH M Actual Targets Found E-seminar, October 2010 Page 21 Deconvolution (AMDIS) Helps to Find all Compounds in a Complex Matrix. Will Deconvolution Help in Quantitation? E-seminar, October 2010 Page 22 Deconvolution Advantage – no baseline drift, noise-free, easier to integrate for more reliable quantitation results Ion 123 Ion 171 Ion 128 Ion 143 AMDIS 14000 12000 | 10000 | 8000 | 14.079 14.078 6000 | 4000 | 2000 | 0 13.60 13.70 13.80 13.90 14.00 14.10 14.20 14.30 Deconvolution shows a flat baseline Deconvolution is fully integrated in MSD ChemStation. E-seminar, October 2010 Page 23 Deconvolution Advantage – finds the correct peak 3500 Ion 147 Ion 76 Ion 104 Ion 103 AMDIS 3000 2500 2000 | 3.873 4.067 | | 1500 | 1000 | 500 0 | 3.50 | 3.60 3.70 3.80 Depending on the ion-ratio criteria (relative or absolute), peaks might be incorrectly identified by ChemStation. 3.90 4.00 4.10 (79) Phthalimide 4.069min (+0.079) 0.07 response 62142 Ion Exp% Act% 147.00 100 100 76.00 60.50 48.95 104.00 57.30 14.64 103.00 28.80 35.45 4.20 AMDIS: 0.04 AMDIS: 36450 E-seminar, October 2010 Page 24 ChemStation could not discriminate co-eluting signals, Deconvolution isolates the target signal from the matrix 12.482 50000 | 40000 | 30000 | 20000 | 10000 | 12.303 0 | 11.70 Ion 175 Ion 177 Ion 258 Ion 260 AMDIS | | 11.90 12.10 12.30 Zoom in 7000 12.50 12.70 A factor of 150x ! 12.482 6000 5000 4000 3000 12.303 2000 1000 0 || 11.70 | | 11.90 12.10 12.30 12.50 12.70 (604) Oxadiazon 12.486min (+0.277) 9.89 response 8806390 Ion Exp% Act% 175.00 100 100 177.00 64.50 73.85 258.00 53.10 0.96# 260.00 35.20 6.05 AMDIS: 0.06 AMDIS: 55432 E-seminar, October 2010 Page 25 Summary • Deconvolution (AMDIS) finds more target compounds than ChemStation does with fewer false positives in a complex matrix. (Improved sensitivity?) This minimizes the time an analyst must spend reviewing results. • Deconvolution provides a cleaned peak to be integrated properly for more reliable results. (Improved sensitivity?) Using Deconvolution to Improve GC/MS Detection and Quantitation E-seminar, October 2010 Page 26 References • Christopher P. Sandy, “A Blind Study of Pesticide Residues in Spiked and Unspiked Fruit Extracts Using Deconvolution Reporting Software”Agilent Technologies publication, 59891654EN, October 2006 • M. Anastassiades, S. J. Lehotay, D. Stajnbaher, and F. J. Schenck, “Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and ‘Dispersive SolidPhase Extraction’ for the Determination of Pesticide Residues in Produce,” 2003, J. AOAC Int, 86:412–431 • S. J. Lehotay, K. Maštovská, and A.R. Lightfield, “Use of Buffering and Other Means to Improve Results of Problematic Pesticides in a Fast and Easy Method for Residue Analysis of Fruits and Vegetables,” 2005, J. AOAC Int, 88:615–629 • Philip L. Wylie, “Screening for 926 Pesticides and Endocrine Disruptors by GC/MS with Deconvolution Reporting Software and a New Pesticide Library,” Agilent Technologies publication, 5989-5076EN, April 2006 • Chin-Kai Meng and Mike Szelewski, “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” Agilent Technologies publication, 5989-7670EN, December 2007 • Chin-Kai Meng and Mike Szelewski , “Can Deconvolution Improve GC/MS Detectability?”, Agilent Technologies publication, 5990-5052EN, January 2010. E-seminar, October 2010 Page 27
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