Improve GC/MS 2 pm, EST (11 am PST) Detection and Quantitation · Using Deconvolution to Improve...

Using Deconvolution to Improve GC/MS

Detection and Quantitation

E-Seminar, October 19th, 20102 pm, EST (11 am PST)

Chinkai (Kai) Meng, Ph.D.Senior Applications ChemistWilmington, Delaware USA

[email protected]

Outline

• Introduction

• Deconvolution - Find Trace Compounds in Complex Matrices

• Deconvolution - Quantitation

• Summary

E-seminar, October 2010

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.


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).


3-D movieMagic Eye

Information is there, you just need the right tool to see it.

GC/MSD Deconvolution – What it can do.


From here…

Deconvolution Reporting Software

Quant on Deconvoluted Peaks

GC/MSD in Scan Mode

…to here in 2 - 3 min

10 15 20 25 30 35 40

Ion 41

Ion 42

Ion 43

Ion 154

Ion 98

Ion 55

Ion 56

Ion 57

Ion 99

Ion 116

Ion 131

Ion 248

Extracted Ion Chromatograms of a Pear Extract


How Does Deconvolution Work? Eliminate Ions that Do Not Fit the Criteria

50170

280

310

7518516

0

5017028075 late retention time

185 shape & early retention time

310 early retention time

160 shape

same shape and same retention time


Spectrum at dotted line

Deconvolution

Two criteria:1. Same RT2. Same peak shape

50170

28050

170280

Spectrum at dotted line (a component)These deconvoluted ions are group together

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

TIC of Spinach Extract

More than 370 peaks

found

Components

Deconvolution

Search RTL Pesticide Library for hits


Fenbuconazole

Scan at 10.776 min

Deconvoluted/extracted spectrum

Library spectrum

A component in the scan above.

Comparison of Raw, Deconvoluted and Library Spectra


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)

AcknowledgementThe author would like to thank Dr. Jon Wong (FDA-CFSAN, College Park, Maryland) for graciously providing samples for this study.


Typical TIC of Spinach Extracts (in Toluene, 35 pesticides spiked at 50 ppb)

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

2000000

6000000

1e+07

1.4e+07

1.8e+07

2.2e+07

2.6e+07

3e+07

3.4e+07

3.8e+07

TIC: Grp1_spinach_50ppb_cold_SL_ramp3_1.D\data.ms


MSD ChemStation “Edit Compound” Screen

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 matchwhich is more dependable.

This screen shows the quantitationdatabase for locating and then confirming a compound.


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.


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.


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.


Comparison of Match Factors with AMDIS Settings

0

10

20

30

40

50

60

70

80

90

100

1 H VH M

2 H VH M

1 H H M

2 H H M


Adjacent peak subtraction (1 or 2) and Sensitivity (VH or H)

Resolution = H, Sensitivity = H, Peak Shape = M


Chlorpyrifos Methyl Net = 94

Methyl Parathion Net = 92

25 standards mixture

Resolution = M, Sensitivity = H, Peak Shape = M


Chlorpyrifos Methyl Net = 89

Methyl Parathion Net = 37

25 standards mixture

Comparison of Number of Compounds Found with Various AMDIS Settings (Spinach Extract, MF = 30)

Changing Resolution only

Changing Sensitivityonly

Changing Shape Requirement only

HHM

MHM

HMM

HHL

HVHM

HHH

35

31

33

32

35

33


0

20

40

60

80

100

120

50% Relative

30% Relative

50% Absolute

30% Absolute

1 H VH M2 H VH M

1 H H M2 H H M

116

110

49

72

88

7383

1712

2019

35 35 35 35

False Positive Actual Targets Found

35 Targets @ 50 ppb in Spinach

AMDIS ResultsChemStation Results

AMDIS SettingsChemStation Settings


Deconvolution (AMDIS) Helps to Find all Compounds in a Complex Matrix.

Will Deconvolution Help in Quantitation?


Deconvolution Advantage – no baseline drift, noise-free, easier to integrate for more reliable quantitation results

13.60 13.70 13.80 13.90 14.00 14.10 14.20 14.30

0

2000

4000

6000

8000

10000

12000

14000

14.079|

|

|

|

|

|

14.078

Ion 123Ion 171Ion 128Ion 143AMDIS

Deconvolution shows a flat baseline


Deconvolution is fully integrated in MSD ChemStation.

Deconvolution Advantage – finds the correct peak

3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20

0

500

1000

1500

2000

2500

3000

3500

4.067|

|

|

|

|

||||

3.873


103.00 28.80 35.45 104.00 57.30 14.64 76.00 60.50 48.95 147.00 100 100

Ion Exp% Act%response 62142 AMDIS: 364504.069min (+0.079) 0.07 AMDIS: 0.04 (79) Phthalimide


Depending on the ion-ratio criteria (relative or absolute), peaks might be incorrectly identified by ChemStation.

ChemStation could not discriminate co-eluting signals, Deconvolution isolates the target signal from the matrix

260.00 35.20 6.05 258.00 53.10 0.96# 177.00 64.50 73.85 175.00 100 100

Ion Exp% Act%response 8806390 AMDIS: 5543212.486min (+0.277) 9.89 AMDIS: 0.06 (604) Oxadiazon


11.70 11.90 12.10 12.30 12.50 12.700

1000200030004000500060007000

12.482

| ||||||

12.303

11.70 11.90 12.10 12.30 12.50 12.700

10000

20000

30000

40000

50000 12.482|

|

|

|

|

| ||||||12.303

Zoom in A factor of 150x !


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

References• Christopher P. Sandy, “A Blind Study of Pesticide Residues in Spiked and Unspiked Fruit

Extracts Using Deconvolution Reporting Software”Agilent Technologies publication, 5989-1654EN, October 2006

• M. Anastassiades, S. J. Lehotay, D. Stajnbaher, and F. J. Schenck, “Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and ‘Dispersive Solid-Phase 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.


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