November 2008

Comprehensive Screening,
Confirmation, and Quantification of
Organic Pesticides in Foods by
GCMS and LCMS
The global focus on keeping food safe has intensified, resulting in major changes in the
number of pesticides that are being regulated and monitored, as well as the allowable
levels of those pesticides in food. These new regulations drive a need for a wide range
of comprehensive and complementary analytical methods and instrumentation that
possess the throughput, sensitivity, and breadth of pesticide compound coverage to
meet the demand of testing laboratories all over the world. This article provides an
overview of the instrument platforms, tools, and workflow for analyzing pesticides.
Philip Wylie, Jerry Zweigenbaum, Melissa Churley, Chin-Kai Meng, and Cao Zhe

ith the globalization of trade, food production

and distribution have become truly international
businesses. When we dine out, the fish might come
from Japan, the rice from Australia, the spices from China,
and the strawberries from Mexico. We take it for granted that
the food we eat is safe and free from contamination that could
make us seriously ill.
One of the 20th-century advancements that made this cornucopia possible was the development of pesticides. Crops
that were previously devastated by pest damage can now be
produced at very high yields to meet worldwide demand. However, all pesticide compounds are potentially harmful to
humans, and safeguards must be in place to assure that consumers are not exposed to dangerous levels of these pesticides.
Many countries have had regulations regarding the use of
pesticides, and their allowable levels in foods, for many years.

With the increasing globalization of the food industry, there

is greater scrutiny on food safety, resulting in major changes
in the number of pesticides that are being regulated and monitored, as well as the allowable levels of those pesticides in food.

A Global Effort to Keep Food Safe

The Food Quality Protection Act (FQPA) of 1996 updated federal regulation of pesticides in the United States to implement
a single health-based standard for all pesticides in foods. It provides special protection for infants and children, expedites
approval of safe pesticides, and requires periodic evaluation of
pesticide registrations and tolerances to ensure that the scientific data supporting registrations will remain up to date in the
future. Under this mandate, the US Food and Drug Administration (FDA) performs regulatory monitoring by sampling
individual lots of domestically produced and imported foods

their products to assure compliance with

FQPA, the EU regulation, and the Japan
Positive List, if they hope to sell their products in the U.S., Europe, and Japan, which
generate much of the worlds demand for
imported food. These new regulations
drive a global need for analytical methods that possess the throughput, sensitivity, and breadth of pesticide compound
coverage to meet the demand of testing
laboratories all over the world that must
ensure compliance.

A Broad Range of Complementary

Analytical Methods Is Needed

Figure 1: GCMS analysis of p,p-DDE in spinach. Data were generated using a J&W DB-5ms column,
a model 7890A GC system, and a model 5975C MSD mass spectrometer (Agilent Technologies).

and analyzes them for pesticide residues

to enforce established tolerances
(http://www.cfsan.fda.gov/~dms/pes0406.html). Domestic samples are collected
at the point of production, and import
samples are collected at the point of entry
into U.S. commerce. Products typically
are analyzed as the unwashed, whole, raw
commodity. The FDA also performs a
total diet study (TDS) on four regional
market baskets per year, each basket comprising about 300 foods that represent
the average U.S. consumers diet. The
foods are prepared as they would be consumed (table-ready) and tested for regulated pesticides. The FDA also performs
focused sampling that is generally used
to follow up on suspected problem areas
or acquire residue data on food commodities that are not usually covered during regulatory monitoring. There are
more than 1000 registered pesticides in
the U.S., and approximately 400 with tolerances established by the Environmental Protection Agency and enforced by
FDA.
The European Union (EU) has also
recently updated a harmonized regulation
of pesticide residues in food, with the new
standard taking effect on September 8,
2008 (http://ec.europa.eu/food/plant/protection/pesticides/index_en.htm). This
regulation sets maximum residue levels
(MRL) for food and animal feed. The

MRL is the highest level of a pesticide

residue that is legally tolerated. The regulation covers around 1100 pesticides currently or formerly used in agriculture in
or outside the EU. For pesticides that are
not specifically mentioned, a general
default MRL of 0.01 mg/kg (10 ppb)
applies. The EU designates the minimum
number of samples to be taken by each
member state, and EU reference laboratories coordinate, train staff, develop methods of analysis, and organize tests to evaluate the skills of the various national
control laboratories.
Japan also adopted a stricter pesticide regulation in 2006, referred to as
the Positive List system, which establishes MRLs for about 500 pesticides
(http://www.mhlw.go.jp/english/topics/foodsafety/positivelist060228/introduction.html). Produce with pesticide
residues exceeding these MRLs or the
default tolerance of the uniform limit of
0.01 ppm (10 ppb) for those pesticides for
which there is no Japanese MRL cannot
be marketed in Japan. The regulation also
lists 16 pesticides that must not be detected
in food. Japan tested more than 200,000
food samples in 2007.
The increased international focus on
pesticide contamination in food impacts
food producers in virtually any country,
since the food market is a global one. Producers in Thailand, for example, must test

Food testing laboratories need the ability

to detect and quantify hundreds of pesticides, in myriad foodstuffs, at very low
levels of contamination. They might be
screening for a set of hundreds of known
target compounds, using a defined test
method for each target in the set. Alternatively, a testing laboratory might wish
to look for the presence of any pesticide
(unknowns), without the need for
defining a method for each possible compound. They are often called on to do
both types of testing, and no single analytical approach can provide the flexibility required to meet this need. Wide variations in the chemical properties of
pesticide contaminants and the necessity
to detect a very large number of compounds require a range of chromatography and mass spectrometry (MS) systems.
For pesticides that can be vaporized
easily without degradation, gas chromatography (GC)MS (single quadrupole) is an ideal analytical tool, due to the
availability of large libraries of pesticide
spectra and deconvolution software. This
approach can be used to screen, confirm,
and quantify both target and unknown
compounds. Many foodstuffs, such as
garlic and ginger, are very complex, or
dirty, due to the presence of a very large
number of background compounds. GC
triple quadrupole MS (QQQ) is very useful for screening, confirming, and quantifying trace-level target compounds in
these complex matrices.
Pesticides that are quite polar, not easily vaporized, thermally labile, or not easily derivatized are best analyzed using liquid chromatography (LC) methods.
LCQQQ is particularly useful for analyzing sets of known target compounds.

Abundance
Abundance

m/z

Abundance

m/z

m/z

Figure 2: Confirmation of p,p-DDE in spinach using DRS and the Pesticide and Endocrine
Disruptor spectral library (Agilent Technologies).

A unique fragment ion for each pesticide

is routinely used for screening of each target compound a second precursor to
product ion transition can then be used
to confirm the identity of the compounds.
The transition with the greatest response
is typically used to quantify the pesticides.
Laboratories may be faced with the
challenge of determining all pesticides, if
any, present in a food sample. LCtime of
flight MS (TOF) screening methods provide extremely accurate molecular mass
information (typically < 2 ppm) that can
be searched against exact mass databases
of thousands of compounds. Compounds
found in this type of screen can be confirmed and quantified using LCMS-MS
analysis on either QQQ or quadrupole
time-of-flight (Q-TOF) instruments.

Analyzing for Target Pesticides

One of the most common activities in a
food-testing laboratory is analysis for a
set of target pesticides of specific interest. A finite set of pesticides might be
used for production of a particular food
crop, or a specific set might be especially

toxic to children, necessitating the routine analysis for those compounds.

GCMS (single quadrupole) utilizing
deconvolution software has broad applicability for this type of analysis. The
Automated Mass spectral Deconvolution
and Identification System (AMDIS) was
developed by the National Institute of
Standards and Technology, and is incorporated into deconvolution reporting
software (DRS) available from Agilent
Technologies. Deconvolution separates
unresolved peaks at any given retention
time into sets of cleaned component
spectra that can be matched to library
spectra. The analyst runs the method that
was used to generate the Pesticide and
Endocrine Disruptor Database Library
and locks the method, so that retention
times will match those stored in the database. DRS then uses deconvolution and
locked retention times to identify pesticides from the database.
This is particularly useful in the
analysis of extracts from foods such as
spinach (Figure 1). In this case, one of
the pesticides of interest is p,p-

dichlorodiphenyldichloroethylene (p,pDDE), a breakdown product of DDT. The

total ion chromatogram (TIC) does not
show a discernible peak at the p,p-DDE
retention time (24.07 min). Deconvolution analysis found 768 different components over the whole TIC. After a pesticide library search, 12 of these had a
match, including p,p-DDE. The black ions
in the bottom window of Figure 1 represent the raw scan, before deconvolution.
The white ions are a deconvoluted component that matches the known spectrum
of p,p-DDE. Plotting the extracted ion
chromatograms for this component (246,
248, 316, and 318) in the top window of
Figure 1 also helped to confirm the presence of the p,p-DDE peak in the TIC. Figure 2 illustrates the extraction of the p,pDDE spectrum from the raw spectrum,
and its match to the library spectrum,
resulting in a positive confirmation of the
presence of this pesticide in the spinach
sample. The use of powerful integrated
deconvolution software such as DRS, in
combination with specialized database
libraries such as the Japanese Positive
List Pesticide Library (1) or the more
comprehensive Pesticide and Endocrine
Disruptor Library (2) (Agilent Technologies, Palo Alto, California), can increase
the efficiency of a food testing laboratory greatly. These GCMS methods can
screen for hundreds of compounds and
quantify a subset of them in as little as
2 min after the run using DRS. With traditional GCMS data processing, an analyst would need as much as 15 min per
sample to review the data and confirm
pesticide hits.
LCMS methods are also very useful
for analysis of target pesticides that are
too polar or thermally labile for GCMS.
LCQQQ is especially suited to very
complex food matrices that can interfere with the analysis.

Dealing with Dirty Sample

Matrices
Sample preparation is a significant challenge with food analysis and makes up
a considerable portion of the total pesticide analysis time. Robust analysis
methods that can tolerate very complex
sample matrices without loss of sensitivity or accuracy are a must to reach
throughput goals.

y=208335.0516 x - 152717.0728
R2=0.99956076

Figure 3: Calibration curve for diazinon in pepper using a six-point curve from 0.1 to 100 ng/mL with a linear fit and no origin treatment. Data were
generated using a ZORBAX SB-C-18 1.8-mm LC column with a model G6410A QQQ system in the positive ESI mode (Agilent Technologies).

Area 1

1197

Area 2

1280

Area 3

1347

Area 4

1186

Area 5

1291

Mean

1260

RSD %

5.4

Multiple reaction monitoring (MRM)

with a QQQ system provides detection
of trace amounts of target compounds
in complex matrices, providing screening, confirmation, and quantification in
one analysis (although a replicate analysis is usually required in most laboratories for confirmation). The precursor ion
of interest generated by the source is
selected and isolated in the first (Q1)
quadrupole. A hexapole collision cell
(Q2) fragments the molecule, and the
product ions are selected in Q3. By choosing product ions from this transition that
are characteristic for the pesticides of

Figure 4: Precision of
GC-QQQ quantification of
500 fg of cyanophos
spiked in garlic. Data were
generated using an
HP5ms UI (Ultra Inert) 30
m 3 0.25 mm, 0.25-mm
film thickness GC column
and a model 7000A QQQ
MS system (Agilent
Technologies). The
pesticide cyanophos was
spiked into a garlic matrix
at 0.5 ppb and replicate
analyses performed at 500
fg on-column. The peaks
are superimposed for the
five runs, with the replicate
peak areas, mean peak
area, and RSD listed in the
table to the left.

interest, chemical noise is separated from

signal, providing very high sensitivity and
selectivity, even in very dirty matrices.
With careful selection of highly specific
product ions for each pesticide, it is possible to create an MRM assay that can be
used to identify and quantify hundreds
of pesticides in a sample simultaneously.
Efficient use of LCQQQ MRM
methods requires optimization of a few
parameters, including voltages on the
fragmentor and the collision cell. Parameters must be optimized to generate
the most intense product ions representative for each target compound, and

automated method optimization software simplifies this task enormously.

LCQQQ is an excellent platform for
screening, confirmation, and quantification of pesticides.A screening method uses
one transition per compound to look for
a large number of pesticides on the target
list. Confirmation of pesticides detected
in the screening is done using two transitions per compound. For example, 301
pesticides have been rapidly identified in
one analysis with most having a limit of
detection (LOD) of 0.01 mg/kg (10 ppb),
which is the MRL for baby food in the U.S.
and the general default MRL for foods in

the European Union. These levels were

reached in a single analysis using positive
ion electrospray with 99 transitions per
time segment, and a quantifying and qualifying ion for each compound (3). Two
types of vegetables (pepper and tomato)
were evaluated, and a linearity of response
over three orders of magnitude was shown
for quantification (r2 > 0.99; Figure 3).
GCQQQ also provides sensitive and

reproducible screening, confirmation, and

quantification of pesticides that are not
highly polar, particularly in very complex
matrices such as garlic and ginger. Despite
the high background of these matrices,
pesticide compounds can be detected at
levels as low as 0.5 ppb, with very high signal to noise and peak area precision as
good as 6% RSD (Figure 4). For certain
active pesticides such as acephate, the

Using oven backflush

it took an additional
33 min at 320 C to
remove those boilers

Run stopped at 42 min,

backflushed at 290 C for 7 min
Blank run after backflushed
shows the column was clean

Time (min)

use of chemical protectants can reduce

adsorption of analytes during the analysis, increasing sensitivity and improving
peak shape. The result can be a marked
improvement in accuracy of quantification in complex matrices (4).
Backflushing the GC column, rather
than using oven bakeout, also removes
many high-boiling interfering substances
in the sample (Figure 5) without altering retention times in subsequent runs
or shortening the life of the column (5).
Backflushing improves data quality while
shortening cycle time and increasing column life. It also eliminates ghost peaks
and reduces ion source contamination
by keeping excess column bleed and
heavy residues from being introduced
into the mass spectrometer.
One key to the successful rapid analysis of large numbers of pesticides is the
dwell time of the QQQ mass spectrometer the time spent to measure the signal of any one precursor to product ion
transition. The shorter the dwell time,
the more transitions that can be analyzed
per unit time, and the more pesticides
that can be quantified per unit time.

Figure 5: Backflushing the GC column for higher signal to noise and shorter cycle times. Comparison
of backflushing using capillary flow (red chromatogram) versus column bake-out (blue
chromatogram) utilizing a model 7890A GC system and an HP-5ms GC column (Agilent Technologies).

Figure 6: Pesticide screening for unknowns using LCQTOF. The top panel shows the full spectrum total ion chromatogram (TIC) using a model 1200 LC
system, a ZORBAX Eclipse XDB, 5-mm column, and a model 6510 LCMS QTOF system (Agilent Technologies). The Molecular Feature Extractor found 510
compounds in the TIC, 15 of which had hits from the EXACT MASS database search (bottom panel). The three highlighted compounds were further confirmed
by MSMS analysis.

Analyzing for Unknown Pesticides

Often, food-testing laboratories want to
determine the entire spectrum of pesticides that might be present in a sample,
rather than just a limited set of target compounds. While GCMS using deconvolution is an effective tool to screen for over
900 pesticides (2), LCTOF or LCQTOF
can be used to screen for any number of
LCMS-amenable compounds.
TOF and QTOF are extremely accurate
MS techniques, routinely achieving <2
ppm mass accuracy.Analysis software such
as the Molecular Feature Extractor (MFE)
from Agilent Technologies can find all ions
representing real compounds in the sample. Noise and other extraneous ions are
excluded. The resulting list of masses is
then searched against a database of theoretical exact masses of compounds based
on their molecular formulas and selected
adducts, to identify pesticide compounds
(Figure 6). This method has been used to
screen food extracts for 600 pesticides and
their degradates. The limit of detection
(LOD) was determined for 100 of the 600
pesticides, and varied from <0.01 mg/kg
(10 ppb) for 34% of the compounds, to
<0.5 mg/kg (500 ppb) for 95% of the compounds (6). Using the MFE algorithm
streamlines the data analysis for screening
of hundreds of compounds at sensitive levels to minutes, compared with hours or
even days using a manual approach. TOF
can be used to identify an unlimited number of ionizable compounds, and the sensitivity is not impacted by the number of
compounds screened.While LCTOF can
provide screening and quantification of
both unknown and targeted pesticides,
LCQTOF or another MS-MS approach
is required to confirm identification.

Conclusion
One need only look to recent events to
appreciate the importance of pesticide test-

ing in food.A series of poisonings in Japan

in December of 2007 and January of 2008
that sickened 10 people was linked to
methamidophos pesticide contamination
of gyoza dumplings imported from China.
The impact on Japanese food buying habits
was immediate, as importation of Chinese
vegetables fell 40% shortly after the source
of the poisoning was determined.
In another example, pet food vegetable
proteins contaminated with melamine
were imported into the United States from
China in early 2007, resulting in the deaths
of several cats and dogs. More recently, the
same contaminant has appeared in baby
formula in China.
Food-testing laboratories require a
broad range of complementary MS-based
analytical technologies and methods to
screen, confirm, and quantify the presence
of as many as 1000 pesticide compounds.
Screening methods can be used to detect
as many contaminants as possible in a single run, and these methods dont necessarily have to quantify all the compounds.
The screening method can be followed by
target compound analysis that is very selective, sensitive, and quantitative. For LCamenable pesticides, this means running
LCTOF or LCQTOF followed by
LCQQQ. For GC-amenable pesticides,
this means running GCMS with DRS for
screening followed by GCQQQ for target compound analysis at low levels. The
method used by any given laboratory is
determined by its specific pesticide analysis goals, existing instrumentation, the
budget for acquisition of new instrumentation, and required LODs.
When choosing new instrumentation,
laboratories doing pesticide testing also
should consider whether a particular manufacturer has a complete solution that
includes all of the above platforms, and
whether those platforms share common,
interchangeable ion sources, a unified soft-

Reprinted from Current Trends In Mass Spectrometry, a Spectroscopy Supplement, November 2008

ware platform, and transferable collision

voltages, thus providing a uniform workflow. The robustness of a manufacturers
systems is a key factor, including the tolerance of the ion source to contamination
and its required frequency of cleaning and
maintenance. Utility of the software and
its integration with comprehensive pesticide spectra libraries and exact mass databases are also keys to success. Finally, the
manufacturers reputation for excellence
in GC and LCMS instrumentation should
be considered.

References
(1) P.L. Wylie, Screening for pesticides in food
using the Japanese Positive List Pesticide
Method, Agilent Application Note 59897436 (2007).
(2) P.L. Wylie, Screening for 926 Pesticides
and Endocrine Disruptors by GC/MS with
Deconvolution Reporting Software and a
New Pesticide Library, Agilent Application
Note 5989-5076 (2006).
(3) J.A. Zweigenbaum, Multiresidue analysis
of 301 pesticides in food samples by
LC/Triple Quaduprole mass spectrometry,
Agilent Application Note 5989-8614
(2008).
(4) M. Anastassiades et al., J. Chromatogr., A
1015, 163184 (2003).
(5) C.-K. Meng, Improving Productivity and
Extending Column Life with Backflush,
Agilent Application Note 5989-6018
(2006).
(6) J.A. Zweigenbaum, Automated screening
of 600 pesticides in Food by LC/TOF MS
using a molecularfeature database
search, Agilent Application Note 59895496 (2006).

Philip Wylie, Jerry Zweigenbaum, Melissa Churley, ChinKai Meng, and Cao Zhe are Senior
Applications Chemists, Agilent Technologies,
Santa Clara, California. n
Printed in U.S.A.