Q-TOF LC/MS Screening andConfirming of Non-TargetedPesticides in a Strawberry Extract

Abstract

A process for rapid screening and confirming of non-target pesticides is presented

using a fruit-extract example. A strawberry extract was rapidly screened for pesticide

content using the Molecular Feature Extraction (MFE) algorithm of the Agilent

MassHunter software, a 600-pesticide database, and accurate mass spectra using

liquid-chromatography, electrospray, quadrupole-time-of-flight mass spectrometry

(Q-TOF LC/MS). Accurate masses of the ions detected and identified by MFE were

compared to the exact masses of compounds in the pesticide database. Positive com-

pounds were then confirmed using the MS/MS (Q-TOF LC/MS) mode.

Authors

Chin-Kai Meng and Jerry Zweigenbaum

Agilent Technologies Inc.

2850 Centerville Road

Wilmington, Delaware 19808

USA

Peter Fürst and Eva Blanke

Chemisches Landes- und Staatliches

Veterinäruntersuchungsamt

Joseph-König-Straße 40, D-48147

Münster, Germany

Application Note

Food Safety

2

Introduction

As the food industry has globalized in recent years, govern-

ment regulations to enforce food safety standards have

increased. The United States has more than 1000 registered

pesticides. Of these, approximately 400 have EPA-established

and FDA-enforced tolerance levels in foods. Over 1100 pesti-

cides are monitored in food and animal feed in Europe as a

result of the European Union's strict food pesticide regula-

tions. Over 500 pesticides have established maximum residue

levels (MRLs) in Japan [1]. Consumer concern along with

these types of regulations has led to an increased demand for

data on pesticide levels in foods. This includes both target

pesticides, which the analyst investigates, as well as, nontar-

get pesticides, which the analyst might not anticipate.

LC/MS/MS technology using LC in tandem with a triple

quadrupole mass spectrometer (LC/QQQ) has become the

approach of choice for target analyses of LC-amenable ana-

lytes in food. This highly selective and sensitive methodology

has proven remarkably rugged and rapid for analyzing target-

ed pesticides at trace levels in complex matrices. Now, with

the advent of Q-TOF LC/MS as a complementary form of

LC/MS/MS, rapid screening for non-target pesticides at trace

levels in foods becomes feasible.

LC/QQQ uses the Multiple Reaction Monitoring (MRM)

process to essentially remove chemical background from the

sample matrix. However, LC single and triple quadrupole

instruments cannot readily be used for nontarget identifica-

tions for two reasons. Full scan mode is required to acquire

the full spectra needed to identify unknowns, but in scanning,

the instrument sensitivity is significantly reduced. In this

mode, LC single and triple quadrupole instruments do not

have the selectivity gained by the MRM approach that

focuses on ion transitions. Without good selectivity, charac-

teristic ions of an analyte are sometimes overwhelmed by

chemical noise. Plus, common LC-MS spectral libraries are

unavailable due to difficulties in standardizing and reproduc-

ing fragmentation energies among instruments from different

vendors. In contrast, the Q-TOF LC/MS application acquires

accurate-mass spectra providing superior selectivity while

maintaining high sensitivity.

LC/Q-TOF, first introduced commercially in the mid-nineties, is

similar to LC/QQQ except the third quadrupole is replaced by

an analyzer known for delivering accurate mass, the TOF. This

configuration gives excellent selectivity from high mass reso-

lution and high mass accuracy with full spectra [2, 3, 4]. The

Agilent 6510 Q-TOF LC/MS system used for this work has

demonstrated mass accuracy values of < 2 ppm for small mol-

ecules. With this extraordinary accuracy comes the ability to

distinguish between compounds of very similar molecular

mass. Since the exact or theoretical mass is uniquely deter-

mined by the molecular formula, obtaining accurate experi-

mental masses allows an analyst to quickly narrow a search

to a limited number of possible molecular formulas. In addi-

tion, using the accurate mass and relative abundance of iso-

topes further limits the possible molecular formulas. Unlike

fragmentation patterns, molecular formulas by exact mass are

reliably catalogued in databases and can be easily and quickly

searched using computerized algorithms.

For confirmation of identity, compounds can be further ana-

lyzed using the collision induced dissociation (CID) in Q-TOF

MS/MS mode. Comparison of the structure of the proposed

compound with the fragments obtained can confirm the iden-

tity. Accurate mass data and isotopic distributions for the pre-

cursor and product ions can be compared to spectral data of

reference compounds, if available, obtained under identical

conditions for final confirmation.

Several unique features allow the Agilent 6510 Q-TOF LC/MS

system to achieve the 2 ppm level of mass accuracy. One fea-

ture is automatic internal referencing. The Agilent 6510

adjusts for variation in conditions by using internal reference

standards. In this technique, two compounds of known mass

are introduced continuously in the Q-TOF ion source. The soft-

ware automatically calibrates the mass axis of every spec-

trum.

A second feature includes a shift from a time-to-digital con-

verter (TDC) to analog-to-digital converter (ADC). Older TDC

technology only registers an ion's arrival if its signal is above

a certain intensity and gives the same response whether the

signal is the result of one or many ions. As a result, the TDC

approach cannot maintain mass accuracy over a wide dynam-

ic range. ADC technology creates a continuous digital repre-

sentation of the detector's signal and can provide up to three

decades of dynamic range, thereby allowing components to

be found in the presence of higher-abundance components

[5].

Another benefit of the Agilent 6510 Q-TOF LC/MS system is

temperature stability. The flight tube in this model is con-

structed from a metal alloy with an extremely low coefficient

of thermal expansion. This effectively insulates the system

from temperature fluctuations such that 1 to 2 ppm errors in

mass accuracy can be readily attained [5].

While excellent MS and MS/MS mass-accuracy enables con-

fident, simultaneous screening for thousands of compounds

using confirmatory MS/MS data [6], the quantity and com-

plexity of data produced is significant. Powerful software

tools are needed to efficiently process the data. Agilent's

Molecular Feature Extraction (MFE) algorithm reduces data

3

To confirm the identity, a "Targeted MS/MS" method is devel-

oped for the pesticides that were found in the MFE and data-

base search. In the method, the retention time and precursor

ion for each target are entered. A second injection using the

targeted MS/MS method is made. In the second analysis, the

experimental accurate-mass MS/MS information is used to

calculate formulas with the molecular formula generation

(MFG) tool. This confirmation step compares not only exact

mass and retention times but also fragment patterns and iso-

tope ratios. A diagram depicting this "screen and confirm"

workflow is shown in Figure 1.

Experimental

LC/MS

• Agilent 1200 Series Rapid Resolution LC system with:

• Agilent 1200 Series binary pump SL and degasser

• Agilent 1200 Series high performance autosampler

SL (ALS SL)

• Agilent 1200 Series thermostatted column com-

partment (TCC)

• Agilent 1200 Series diode-array detector SL (DAD

SL) - for method development and troubleshooting

processing time by automatically finding all sample compo-

nents down to the lowest-level abundance and extracting all

relevant spectral and chromatographic information [6, 7]. In

addition, the proprietary, Molecular Formula Generator (MFG)

software provides high-confidence identification of

unknowns, using multiple dimensions of information to gener-

ate and score lists of possible molecular formulas. The MFG

reduces the number of plausible formulas by using the accu-

rate-mass of adduct ions and their isotopes [6].

Screening and Confirming WorkflowThis application note describes a "screen and confirm" work-

flow for the identification of nontargeted pesticides in a

strawberry extract. The process starts with an injection of an

aliquot of the sample extract into the Q-TOF LC/MS. The ana-

lytes from the chromatographic column are ionized and

passed through the first quadrupole and into the TOF without

CID. The resulting data file is cleaned of extraneous back-

ground noise and unrelated ions by the Molecular Feature

Extraction (MFE) tool. The MFE then creates a listing of all

possible components as represented by the full TOF mass

spectral data. An exact mass database is then searched for

hits to identify the pesticides in the data file.

QuEChERSextraction

1. Screen

Backgroundnoise andunrelated ions

MolecularFeatureExtractor(MFE)

Match vs.database ofpesticides

aliquotLC Q-TOF

Full Single MassSpectra Data File

List ofcomponents

Identification ofpesticide

2. Confirm, if necessary

LC

aliquot

Pesticideconfirmed by

formula

Confirmation

MolecularFormulaGenerator(MFG)

Analyst reviewsion fragments toconfirm identity

TargetedMS/MS Data

File

Q-TOF

Figure 1. Screen and Confirm - LC/Q-TOF analysis and software workflow.

4

• Agilent 6510 Accurate-Mass Q-TOF LC/MS

• Column: 2.1 mm × 100 mm, 1.8 µm

ZORBAX Eclipse Plus C18, RRHT, 600 bar (p/n, 959764-902)

QuEChERS Sample Preparation Method QuEChERS is the acronym for the sample preparation method,

which stands for quick, easy, cheap, effective, rugged, and

safe. It is a method that is widely receiving acceptance for

rapid extraction of pesticides in food (8, 9).

Extraction: • Chop samples into small pieces and freeze in a bag

overnight before grinding. Dry ice should be added during

grinding.

• Weigh 10 g of an homogenized sample into a

50-mL Teflon centrifuge tube.

• Add 10 mL of acetonitrile (and ISTD solution, if used).

• Add 4 g of anhydrous magnesium sulphate, 1 g of sodium

chloride, 1 g of trisodium citrate dehydrate, and 0.5 g of

disodium hydrogen citrate sesquihydrate to the tube.

• Adjust the pH to 5-5.5 using 5 N NaOH.

• Shake the sample vigorously for 1 min by using a vortex

mixer at maximum speed or by hand shaking.

• Centrifuge for 5 minutes at 3000 rpm.

Cleanup: • Transfer 6 mL of supernatant into a 12-mL polypropolyene

centrifuge tube which contains 150 mg of primary-secondary

amine (PSA) adsorbent and 900 mg of MgSO4.

• Shake for 30 s.

• Centrifuge for 5 min at 3000 rpm.

• Adjust the pH of the cleaned extract to 5.0 for analysis, if

necessary.

Results and Discussion

A sample of strawberries was extracted with acetonitrile

using the QuEChERS protocol and analyzed as described in

the screening workflow section. The raw TOF (MS1 Mode)

Full Spectrum TIC for the sample is shown in Figure 2. The

TOF data was processed using the Molecular Feature

Extraction (MFE) algorithm of the MassHunter Workstation

software to find likely compounds. This algorithm is designed

to find all ions in the data file that represent real compounds

[7]. Figure 3 shows the molecular feature method editor

menu and specific search criteria for the strawberry extract.

LC (1200) and MS (6510 QTOF) MethodParameters

The HPLC and MS were operated under the following conditions:Flow Rate 0.3 mL/min

Injection Volume 10 µL

Solvent A 0.1% formic acid in water

Solvent B 100% acetonitrile

Gradient Time (min) Solvent B

0 10%

20 95%

25 95%

Ion Source ESI

Drying Gas Temperature 325 °C

Drying Gas Flow 10 L/min

Nebulizer 50 psi

VCap 4000 V

Fragmentor 175 V

Reference Masses 121.050873 and 922.009798

Acquisition Mode MS1

Min Range 100

Max Range 1000

Scan Rate 1

Acquisition Mode Targeted MS/MS

MS Min Range 100 MS/MS Min Range 100

MS Max Range 1000 MS/MS Max Range 1000

MS Scan Rate 1.4 MS/MS Scan Rate 0.7

Max Time Between MS 10

Varied Collision Energy with Mass

Slope 5

Offset 5

Extraction Peak filter: Use peaks with height £ 1000

Ion species Positive Ions: H, Na, K, NH4

Charge state Peak spacing tolerance: 0.0025 m/z, plus 7.0 ppm

Limit assigned charge states to a maximum of 1

Compound filters Relative height £ 0.2 %

Mass filters None

Mass defect Filtering not used

Results Delete previous compounds

Highlight all compounds

5

Figure 2. The raw TOF (MS1 Mode) Full Spectrum TIC for an acetonitrile extract of a sample of strawberries.

Figure 3 . MFE Method Editor Menu and settings for pesticide screening of the strawberry extract.

6

The MFE produced 822 potential compounds. Figure 4 shows

the TIC, the hyperlinked extracted compound chromatogram

(ECC), and the mass spectrum for one of these compounds.

Since ECCs are created when one of the MFE “Find

Compounds” algorithms is run, the ECC consists of all the

related ions and no chemical noise.

The accurate mass of each of these compounds was subse-

quently searched against a working exact mass database of

600 pesticides1. The criteria used in this search are shown in

Figure 5. Twenty-six of the 822 compounds had mass matches

(3 ppm tolerance) with pesticides in the database. Three plau-

sible exact-mass match compounds, cyprodinil, azoxystrobin,

and boscalid were then selected for further confirmation

using MS/MS (Q-TOF) analysis with the same instrument.

The ECC and mass spectrum for each of the three are provid-

ed in Figure 6 along with the database search results showing

a difference of less than 1 ppm in experimental and database

masses. The precursor ion (M + H)+ masses chosen for the

MS/MS analysis of the strawberry extract were exact masses

from the database: 226.13395, 404.12410, and 343.03995 for

cyprodinil, azoxystrobin, and boscalid, respectively. Criteria for

finding compounds in the resulting MS/MS chromatogram

are listed in Figure 7. Using the accurate MS/MS masses for

the fragment ions, formulas were generated for each com-

pound found in this step.

Figure 4. The ECC and mass spectrum are shown for one of 822 compounds found using the MFE software along with the TIC.

Search criteria Match Mass only with 3.00 ppm tolerance

Database Your Exact Mass compound database

Peak limits 10

Positive Ions H, Na, K, NH4

Charge state range 1-2

No neutral losses

Search results Limit to the best 5 hits

Figure 5. Exact mass database search menu and settings.

1 A 1600-compound Mass Hunter Personal Pesticide Database (G6854AA) is available for Exact Mass Searching.

7

Figure 6. The hyperlinked ECC and mass spectrum for three positive pesticides, cyprodinil, azoxystrobin, and boscalid, found in the

strawberry extract, are shown along with the exact mass database search results (a portion of the results is shown).

IntegratorMS/MS Integrator

ProcessingMaximum chromatogram peak width 0.25 min

Cpd TIC Peak FiltersFilter on peak area

Limit (by height) to the largest 10 peaks

Peak SpectrumSpectra to include average scans > 10% of peak height

Exclude TOF spectra anywhere if above 40.0% of saturation

MS/MS peak spectrum background: None

ResultsDelete previous compounds

Highlight all compounds

Extract MS/MS chromatogram

Extract MS/MS spectrum

Figure 7. Software settings for “Find Compounds by Targeted MS/MS”.

8

The confirmation process for azoxystrobin will be further dis-

cussed as an example. In Figure 8, the best-fit (with mass

accuracy of 0.26 ppm and isotopes) formula generated from

the Targeted MS/MS analysis for one of the compounds was

C22H17N3O5, the formula for azoxystrobin. The two associated

fragment masses for this peak had less than 1 ppm difference

in mass (0.31 and 0.2 ppm) when compared to the database

masses for fragments expected from the C22H17N3O5 parent

formula. In addition, the three isotope masses for the molecu-

lar ion all differed by less than 1 ppm. The table outlined in

Figure 8 shows that the experimental isotope abundances of

the three isotopes match well with the calculated (theoreti-

cal) abundances. The boxes in Figure 9 surrounding the iso-

topes represent the theoretical isotope abundances.

Final confirmation of the structure is obtained by comparing

the experimental fragment masses to likely theoretical frag-

ment ion masses. Analysis of the structural formula, as seen

in Figure 10, shows two likely fragments, with masses of

344.10351 and 372.09843 that match closely in mass to the

Figure 8. Results from the formula analysis of MS and MS/MS data of a compound (azoxystrobin) in the strawberry

extract.

9

two fragment ions found in the MS/MS data: 344.10286 and

372.09781 amu. The difference between experimental and cal-

culated is 0.31 and 0.20 ppm, respectively. The accuracy of

this comparison, along with the MS data identification and

MS/MS formula generation results all strongly suggest the

presence of azoxystrobin in the analyzed strawberry extract.

Conclusion

This application note demonstrates that the high degree of

mass accuracy (and selectivity) now available in combination

with powerful database searching tools can be used to suc-

cessfully identify nontargeted pesticides in food. The mea-

sured mass is used to generate a few potential molecular for-

mulas and thereby frees the analyst from labor-intensive man-

ual comparison of fragmentation patterns. These formulas,

when combined with one or two fragment ions of accurate

mass, can be used to quickly and confidently identify nontar-

geted pesticides in food samples.

Figure 9. The experimental isotope abundances of the three isotopes match well with the theoretical abundances (outlined in boxes).

O

O

O

NN

O

N

OH3C CH3

344.10351

372.09843

Figure 10. Structural analysis for Azoxystrobin fragments.

www.agilent.com/chem

Agilent shall not be liable for errors contained herein or

for incidental or consequential damages in connection

with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc., 2009

Printed in the USA

May 5, 2009

5990-3935EN

References

1. P. Wylie, J. Zweigenbaum, M. Churley, C.-K. Meng, and C. Zhe, "Comprehensive

Screening, Confirmation, and Quantification of Organic Pesticides in Foods by

GC-MS and LC-MS," (2008) Current Trends in Mass Spectrometry, November.

2 I. V. Chernushevich, A. V. Loboda, and B. A.Thomson, "An Introduction to

Quadrupole-Time-of-Flight Mass Spectrometry," (2001) J. Mass Spectrom.

36: 849-865.

3. H. R. Morris, T. Paxton, A. Dell, J. Langhorne, M. Berg, R. S. Bordoli, J. Hoyes,

and R. H. Bateman, "High Sensitivity Collisionally-activated Decomposition

Tandem Mass Spectrometry on a Novel Quadrupole/Orthogonal-acceleration

Time-of-flight Mass Spectrometer," (1996) Rapid Commun. Mass Spectrom.

10: 889.

4. A.A. Shevchenko, I.V. Chernushevich, W. Ens, K.G.Standing, B, Thomson,

M.Wilm, and M. Mann, "Rapid ‘de novo’ peptide sequencing by a combination

of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass

spectrometer," (1997) Rapid Commun. Mass Spectrom. 11: 1015.

5. K. Imatani, "Advances in Accurate-Mass TOF and Q-TOF LC-MS Systems,"

(2008) American Laboratory, 40:12-16.

6. "Achieving Unmatched Speed and Confidence for Complex Sample Analyses:

Agilent True High-Definition TOF Technology," Agilent Technologies, publication

5990-3168EN.

7 E. M. Thurman, I. Ferrer, and J. A. Zweigenbaum, "Automated Screening of 600

Pesticides in Food by LC/TOF MS Using a Molecular-Feature Database Search,"

Agilent Technologies, publication 5989-5496EN.

8. 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) Journal of AOAC International, 86:412-431.

9. 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) Journal of AOAC

International, 88:615-629.

For More Information

For more information on our products and services, visit our Web site at

www.agilent.com/chem.