An Overview
of Multi-residue
Pesticide Testing
CONTENTS
Introduction .......................................................................................................................................................................................3
Drivers for Pesticide Testing ............................................................................................................................................4
Regulatory Limits ......................................................................................................................................................................5
Multi-residue Pesticide Methods .................................................................................................................................6
Sample Preparation .................................................................................................................................................................... 7
Sample Extraction .................................................................................................................................................................... 7
QuEChERS Extraction ...........................................................................................................................................................8
Dispersive Solid Phase Extraction (dSPE) .............................................................................................................9
Solid Phase Extraction (SPE) .........................................................................................................................................10
Gaining Efficiencies in Liquid Handling ................................................................................................................. 11
Sample Analysis ...........................................................................................................................................................................12
Key Technologies for the Determination of Multi-residues ....................................................................12
Gas Chromatography Tandem Quadrupole Mass Spectrometry (GC-MS/MS) .....................13
Liquid Chromatography Tandem Quadrupole Mass Spectrometry (LC-MS/MS) ................14
Retention .......................................................................................................................................................................................15
Sample Injection .....................................................................................................................................................................16
Matrix Effects ..............................................................................................................................................................................17
Data Processing ......................................................................................................................................................................18
Why Waters? ...................................................................................................................................................................................19
Enabling Technologies and Services from Waters .......................................................................................19
Introduction
Plant protection products, more commonly known as pesticides,
are used to control pests, weeds, and diseases. They may include
the following:
■
Herbicides to control weeds before and during growth
■
Insecticides to protect seeds and plants from damage by insects
■
Nematicides and molluscicides to control attack on growing plants
by worms and slugs
■
Rodenticides to prevent damage and contamination by small
mammals such as mice and rats during growth and storage
■
Fungicides to prevent mold forming on plants in the field and in store
Pesticide residues resulting from the use of plant protection products on
crops or food products may pose a risk factor for public health or hinder
trade. This eBook aims to provide a short background on multi-residue
pesticide analysis, discussing elements of sample preparation and
sample analysis.
3
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
DRIVERS FOR PESTICIDE TESTING
WHY TEST FOR PESTICIDES?
Many individual governments or groups of nations
monitor pesticide residues in domestic and imported
agricultural produce and other foods each year.
This monitoring verifies that:
■
No unexpected residues are occurring in crops in
support of the national statutory approvals process
for pesticides
■
Residues do not exceed the statutory maximum
residue limits (MRLs)
■
Human dietary intakes of residues in foods are within
acceptable levels
In food safety, the phrase “due diligence” refers to being
able to prove that a business has taken reasonable steps
to prevent food safety breaches.
Food businesses are responsible for ensuring that the
food they produce, or import is compliant with current,
relevant legislation, including MRLs. This may cover
more pesticides at concentrations lower than legal limits,
analysis of finished products as well as ingredients.
A second reason for food businesses to conduct testing
is to protect their brand.
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An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
REGULATORY LIMITS
Maximum residue levels (MRLs) are established in raw
food commodities and animal tissues. MRLs are set at
the highest level of pesticide that the relevant regulatory
body would expect to find in that crop when it has been
treated in line with critical Good Agricultural Practice.
WHAT DOES AN MRL DO?
The main function of an MRL is to act as a control
mechanism to ensure the product has been correctly
used according to its label. The value assigned to
a selected pesticide will vary depending upon the
commodity for which the MRL was set.
RESIDUE DEFINITIONS
In some cases, the compound applied as the plant
protection product is transformed by the time samples
are taken for analysis. These changes are considered as
part of the approval process when the residue definition
is created. For example, in the EU, the residue definition
for aldicarb is the sum of aldicarb, its sulfoxide, and its
sulfone, expressed as aldicarb. Residue definitions vary
between country and commodity.
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An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
MULTI-RESIDUE PESTICIDE METHODS
WHY MULTI-RESIDUE ANALYSIS?
A primary goal for all laboratories involved
with the analysis of pesticide residues in food
is accurately determining as many compounds
as possible or compounds in a prescribed list
in the most cost-effective manner.
Laboratories must address capacity and
efficiency issues constantly to meet sample
throughput requirements.
By implementing multi-residue methods,
many labs have significantly extended their
scope of analyses and achieved effective
and efficient implementation using generic
extraction, matrix clean-up, and determination
with gas and liquid chromatography coupled
with mass spectrometry.
Waters LC-MS/MS and APGC™-MS/MS systems in the NofaLab laboratory.
Read this case study to learn how NofaLab meets the growing
needs of its customers by improving method development for
rapid food contaminant detection.
6
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
Sample Preparation
There are several key aspects to consider prior to sample extraction.
PREPARING SAMPLES FOR ANALYSIS
Samples received at the laboratory may need work to prepare a test
sample for analysis by removal of parts (soil, stones, bones, etc.) not
to be analyzed. Representative samples should be homogenized
to provide uniform particle size, uniform residue distribution, and
to increase the surface area available for extraction. To minimize
degradation of labile compounds, samples of fruit and vegetables are
comminuted (cut and homogenized) at low temperature (e.g., frozen
and in the presence of “dry ice” or liquid nitrogen) to transform the
sample into a fine, homogenous powder which is frozen for storage
until required for analysis.
IMPROVING EXTRACTION EFFICIENCY
Test portions should be extracted frozen or while in the process of
thawing (except dry samples with water content <20%). To improve the
extraction efficiency of low moisture commodities (cereals, spices, and
dried fruits), addition of water to the milled samples prior to extraction
is recommended. When looking at the influence of various factors
on the extraction yields, the use of samples with incurred pesticide
residues is advised.
SAMPLE EXTRACTION
CHALLENGES WITH LEGACY METHODS
Legacy multi-residue methods relied on liquid-liquid extraction
and clean-up using gel permeation chromatography (GPC) or
solid phase extraction (SPE). These methods were typically
labor-intensive, required specialist equipment, and involved
the use of a lot of glassware and large volumes of organic
solvents, including chlorinated solvents for the extraction, with
associated disposal costs.
MODERN APPROACHES
There are now several generic extraction protocols in common
use for multi-residue methods:
■
Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS)
remains the most popular approach for pesticide residue
analysis in fruit, vegetables, cereals and sometimes products
of animal origin1
■
Dutch mini-Luke (“NL-”) method using extraction with
acetone followed by partition with dichloromethane/
petroleum ether (1:1 v/v)2
■
Swedish ethyl acetate (“SweEt”) method3.
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An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
QuEChERS EXTRACTION
WHAT IS QuEChERS?
QuEChERS is a versatile, streamlined approach using a rapid solvent-
based extraction in a centrifuge tube, often followed by dispersive
solid-phase extraction (dSPE) for clean-up.
WHY QuEChERS?
QuEChERS requires a small sample size (10 – 15 g) and low solvent
volume (10–15 mL) so waste disposal costs are minimized (no
chlorinated solvents). There is no requirement for glassware so no
need for washing or storage. It speeds up sample throughput and
simultaneously generates extracts for both GC-MS(/MS) and LC-MS/
MS. Ready-to-use extraction and clean-up tubes are commercially
available, which contain pre-weighed salts and sorbents.
The use of acetonitrile (MeCN) provides extraction of a broad range of
the compounds, from polar to nonpolar. The addition of buffering salts
into a mixture of water and MeCN causes the formation of a two-phase
system and improves recoveries of pH-dependent analytes.
>> Click here to learn more about our commercially available products.
Analyst transferring QuEChERS extract to dSPE tube.
Watch this video on Simplifying your QuEChERS
Extractions using DisQuE™ Sample Preparation Products.
Extraction
Transfer 10 g sample into 50 mL tube, add internal standards (optional) and 10 mL MeCN; shake vigorously (1 min)
Partition
Add salts (e.g. 4 g MgSO
4, 1 g NaCl, 0.5 g C6H9NaO8 and 1 g Na3C6H5O7); immediately shake vigorously (1 min)
Centrifugation
5 min at > 3000 g (RCF) and store extracts from fat or wax containing samples in freezer overnight
Clean-up
LC-MS/MS
GC-MS/MS
QuEChERS CEN method workflow.
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An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
DISPERSIVE SOLID PHASE EXTRACTION (dSPE)
WHAT IS dSPE?
Dispersive solid phase extraction (dSPE) is a rapid, simple, and
straightforward technique suitable for the clean-up of extracts from a
wide variety of food and agricultural commodities.
An aliquot of acetonitrile supernatant from a QuEChERS extraction is
added to a disposable tube containing sorbent(s). The tube is shaken,
centrifuged and the supernatant is collected for further analysis. Unlike
in conventional SPE, it is the co-extractives that are retained on the
sorbent whilst the analytes remain in the solution. Varying amounts
of different sorbents are added depending on the commodity and
pesticides targeted.
The most popular commercially available kits were developed in
accordance with the official standardized version of QuEChERS and
typically include magnesium sulfate to remove any remaining water
and one or more of three sorbents for the dSPE step:
■
Graphitized carbon black (GCB; for pigments)
■
Primary-secondary amine (PSA; for sugars, fatty acids, organic acids)
■
Octadecyl-bonded silica (C
18; for long chain hydrocarbons,
lipids, waxes)
dSPE sorbent suggestions for different commodities.
Commodity Type
Cleanup Provided
Size
AOAC Method 2007.01
CEN Method 15662
General Fruits and
Vegetables
(Celery, Head Lettuce, Melon)
Removes polar
organic acids, some
sugars and lipids
2 mL Tubes
15 mL Tubes
50 mg PSA, 150 mg MgSO
4 Part #186004572
400 mg PSA, 1200 mg MgSO
4 Part #186008072
25 mg PSA, 150 mg MgSO
4 Part #186004831
150 mg PSA, 900 mg MgSO
4 Part #186004833
Fruits and Vegetables with
Fats and Waxes (Cereals,
Nuts, Dairy, Avocado)
Removes polar
organic acids, some
sugars, more lipids
and sterols
2 mL Tubes
15 mL Tubes
50 mg PSA, 50 mg C
18, 150 mg MgSO4 Part
#186004830
400 mg PSA, 400 mg C
18, 1200 mg MgSO4 Part #
186008073
25 mg PSA, 25 mg C
18, 150 mg MgSO4 Part
#186004832
150 mg PSA, 150 mg C
18, 900 mg MgSO4 Part
#186004834
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An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
SOLID PHASE EXTRACTION (SPE)
LIMITATIONS OF dSPE
The use of dSPE is not without problems, especially when dealing with
certain commodities. The dSPE sorbents can decrease the recovery of
some compounds and may not provide sufficient clean-up of the extract,
increasing the possibility of faster contamination of the instrumentation.
ALTERNATIVES TO dSPE
Laboratories may turn to conventional trap and elute SPE, which can be
more effective for removal of matrix co-extractives but increases the risk
of poor recovery of certain pesticides.
Using SPE in pass-through mode, the extract is passed straight through
the cartridge and the matrix co-extractives remain on the sorbent and
analytes are collected in the eluant.
One advantage over conventional SPE is the omission of the extra
load, rinse and subsequent elution steps typically involved with SPE.
SPE using Oasis™ PRiME HLB can be used as an alternative to dSPE,
especially where removal of lipids is important or to avoid use of PSA
or GCB sorbents in dSPE.
Oasis™ PRiME HLB Plus Cartridge, Pass-Through SPE
Discover the benefits a simple pass through offers for the
removal of chlorophyll from highly pigmented samples in
this
application brief.
10
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
GAINING EFFICIENCIES IN LIQUID HANDLING
LIQUID HANDLING: FROM PAINS TO GAINS
A common and significant step in multi-residue pesticide
testing workflows is the preparation of matrix matched
standards. The accuracy, repeatability and traceability
of this step are essential for the reliable quantitation
of pesticides.
Automation of routine liquid handling steps such
as this can provide several benefits, including:
■
Minimized pipetting errors
■
Reduced waste
■
Increased traceability
■
Reduced risks of repetitive injury
■
Freeing analysts to be deployed for other
high value tasks
Read this application note: Automating
Preparation of Matrix-Matched Standards
for Pesticide Residue Analysis Using the
Andrew+™ Pipetting Robot.
11
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
Sample Analysis
KEY TECHNOLOGIES FOR THE DETERMINATION OF
MULTI-RESIDUES
Mass spectrometry coupled with both gas chromatography (GC)
and liquid chromatography (LC) is needed to provide comprehensive
analysis of the wide range of pesticide residues. Tandem quadrupole
mass spectrometry (MS/MS) has become the most widely adopted
technique due to its high selectivity in multiple reaction monitoring
(MRM) mode and sensitivity to meet global MRL regulations.
Like the evolution of sample preparation techniques, MS/MS has
continued to develop to meet users’ growing needs, where:
■
Sensitivity to exceed MRL requirements has allowed for sample
preparation to be streamlined and reduce matrix loaded
■
Acquisition speeds yield sufficient data points to reliably quantify
even the most well-focused and overlapping peaks
■
The reduced instrument footprint offers laboratories improved
performance per square meter of coveted bench space
Read this application note on the determination of
multi-residue pesticides in fruits and vegetables by
UPLC and APGC coupled with the Xevo™ TQ-S micro.
12
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
GAS CHROMATOGRAPHY TANDEM QUADRUPOLE MASS
SPECTROMETRY (GC-MS/MS)
Originally gas chromatography coupled to mass spectrometry
(GC-MS) methods were mostly based on selected ion monitoring
(SIM) or full scan modes, using either single quadrupole or ion
trap mass analyzers. However, the determination of GC-amenable
pesticides in food by using MS/MS has grown considerably over the
last decade as this provides higher selectivity and sensitivity that
minimizes most chromatographic interferences.
SOFTER IONIZATION FOR ENHANCED SENSITIVITY AND SELECTIVITY
GC-MS/MS typically uses EI at 70 eV, extensive fragmentation is
often observed so the selection of the precursor ion for MS/MS is
often a compromise between sensitivity and selectivity. APGC™ is
a soft ionization technique which generates higher abundance of
molecular ions, with less fragmentation than conventional EI. As
the molecular ion (or protonated molecular ion) is highly abundant
under the soft ionization occurring in APCI, selectivity and sensitivity
are notably enhanced when it is used as the precursor ion for MRM
methods. Moreover, APGC operates at atmospheric pressure which
removes the restriction imposed by pumps, allowing a much wider
range of flow rates for GC separations and use of alternative gases
to helium, such as nitrogen.
Read this white paper on Atmospheric Pressure Gas
Chromatography (APGC) to learn more.
Waters APGC System, the Xevo TQ-XS.
13
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
LIQUID CHROMATOGRAPHY TANDEM QUADRUPOLE
MASS SPECTROMETRY (LC-MS/MS)
With the introduction of UltraPerformance Liquid
Chromatography™ (UPLC™), laboratories reported significant
method efficiency benefits over traditional LC. The true
benefits of this technology was further realized with the
advancement of MS/MS technology almost a decade
later, offering:
■
Improved system sensitivity, allowing for simple dilution
of crude QuEChERS extracts, avoiding timely and labor-
intensive extract cleanup
■
Increased MS/MS scanning capabilities enabling the
analytical scope in a single injection to be extended,
without impacting data quality
■
Reduced analytical runtimes, allowing twice the samples
to be analyzed by UPLC-MS/MS vs traditional LC-MS/MS
■
Less solvent usage and associated disposal costs
ACQUITY™ UPLC Premier and Xevo TQ Absolute system
Read this application note: Multi-residue Method for
the Quantification of Pesticides in Fruits, Vegetables,
Cereals and Black Tea using UPLC-MS/MS.
14
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
RETENTION
WHAT IS RETENTION TIME?
Retention time (RT) is the time between the start of an injection to the
emergence of the peak maximum of the analyte(s).
WHY IS RT IMPORTANT?
If this interaction is too short, then little to no chromatography has taken
place, separations will be less stable, and there will be a high chance
of ion suppression when LC-MS is used. The column void volume (v)
is a measure of the internal volume inside the column packed with the
stationary phase particles and can be estimated from a column’s length
(L) and internal diameter (ID).
Analytical quality control, performance and method validation guidelines
such as SANTE/11312/2021 state “the minimum acceptable retention
time for the analyte(s) should be at least twice the retention time
corresponding to the void volume of the column.”4
Methamidophos retention time in different samples.
Spinach
Strawberry
Soybean
Wheat flour
Methamidophos
Acephate
Methomyl
Black tea
Visit the Column Coach to Assist in Column Selection
for your Analysis.
15
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
SAMPLE INJECTION
Pesticides, such as methamidophos and acephate, are polar and elute
early in the chromatogram. Injecting samples containing moderate
amounts of organic solvent (≥25%) on a reversed-phase column often
results in fronting and/or split peaks for the early eluting compounds.
Reducing the organic content used in the sample diluent prior to transfer
onto the column may help to improve the peak shape of early eluting
analytes. However, maintaining a portion of organic in the extract is
essential for analyte stability.
ALTERNATIVES TO MANUAL DILUTION
Post injector mixing can allow the injection of typical QuEChERS extracts
into high aqueous mobile phase without compromising peak shape.
An extension loop is placed in between the injector port and column.
Before the injection is made, the extension loop is filled with the high
aqueous mobile phase, which provides more volume to aid dispersion
of the sample into the aqueous solvent prior to transfer onto the column.
Methamidophos in a QuEChERS extract with different acetontrile and water ratios.
Read this application note: Multi-residue Method for
the Quantification of Pesticides in Fruits, Vegetables,
Cereals and Black Tea using UPLC-MS/MS.
Increasing
aqueous
content
100% MeCN QuEChERS extract
of a vegetable
25% MeCN QuEChERS extract
of a vegetable
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An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
MATRIX EFFECTS
The hyphenation of chromatographic and mass spectrometry
technologies has revolutionized food contaminant testing laboratories.
However, one major drawback is the potential for the phenomenon of
matrix effects. The influence of matrix on the reliability of your method
should be determined when implementing new methodologies,
commodities or analytes into your laboratory’s analytical scope.
COMPENSATING FOR MATRIX EFFECTS
Matrix effects are known to occur frequently in LC-MS/MS methods
and should be assessed during method validation. Ionization efficiency
in the source is impacted, resulting in ion suppression or enhancement.
These effects are caused by the co-elution of matrix co-extractives
with analytes. Significant variations in the magnitude of matrix effects
have been observed between different commodities. The use of matrix-
matched calibration is recommended to mitigate for matrix effects.
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
0
5
10
15
20
25
Peak response
Concentration (ng/ml)
Matrix
Solvent
Matrix effects causing ion suppression of a matrix matched calibration curve.
Watch this bite-size webinar on compensating for matrix
effects in complex samples to learn more.
17
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
DATA PROCESSING
Whilst quantitation itself is a routine
experiment, the analysis of hundreds of
pesticides, each with two MRM transitions
creates a significant amount of data for review.
Along with peak integration, retention time,
ion ratios, signal to noise, calibration curve
data and quality control standard performance
must be assessed. As the scope of analytes
continues to increase, the data review can create
bottlenecks for routine testing laboratories.
Data processing software with Exception
Focused Review (XFR) functionality
allows customized rule-sets based on the
laboratory’s standard operating procedures
(SOPs) to be created and enables analysts to:
■
Review data more efficiently
and consistently
■
Quickly identify injections that fall outside
the laboratory’s analytical quality system
■
Visually and quickly identify samples
with suspected incurred residues
■
Submit the sample batch for approval
with confidence
The MS Quan Software Dashboard makes it easy to visualize your data.
Read this white paper on the benefits of waters_connect™
MRM Processing Application, MS Quan.
18
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
Why Waters?
ENABLING TECHNOLOGIES AND
SERVICES FROM WATERS
The use of plant protection products
helps to secure global food supplies
but can leave pesticide residues in our
food chain. With an extensive regulatory
framework in place across the globe and
varying physicochemical characteristics
of pesticide classes, food manufacturers
and testing services need flexible, fast and
reliable testing solutions. Whether meeting
food safety regulations, quality control
stipulations or undertaking metabolite
discovery, efficiency is paramount to
keep pace with demands.
With an extensive portfolio of instruments,
services and support we provide quality,
knowledge and confidence for optimum
productivity in your laboratory. We partner
with you to ensure a successful purchase
outcome, employing our global team of
application experts to assist in instrument
setup and user training.
COLUMN
CHEMISTRIES
APGC
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SOFTWARE
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AWARD
WINNING
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Enabling technologies across
the analytical workflow.
>>Click each icon to learn more.
19
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
Resources
1. Anastassiades M et al. (2003). Fast and easy multi-residue method
2. Lozano A et al. (2016). Miniaturisation and optimisation of the Dutch
3. Pihlström et al. (2007). Analysis of pesticide residues in fruit and
4. SANTE 11312/2021. Analytical Quality Control and Method Validation
Procedures for Pesticide Residues Analysis in Food and Feed.
20
An Overview of Multi-residue Pesticide Testing
Introduction
Sample Preparation
Sample Analysis
Why Waters?
Waters Corporation
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F: 1 508 872 1990
waters.com
Waters, The Science of What’s Possible, Oasis, Andrew+, Xevo, ACQUITY, UltraPerformance
Liquid Chromatography, UPLC, waters_connect, DisQuE are trademarks of Waters
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©2022 Waters Corporation. Produced in the U.S.A. May 2022 720007619EN GJ-PDF
