Transform Analytical Chemistry With Micro Solid Phase Extraction
eBook
Published: January 17, 2025

Credit: PAL System
Traditional solid phase extraction (SPE) revolutionized sample preparation, but it often involves high solvent consumption and complex procedures.
Micro solid phase extraction (μSPE) offers a groundbreaking alternative, minimizing solvent use, simplifying workflows and enhancing precision for trace-level analysis in complex matrices.
This eBook explores the advantages of μSPE over traditional methods and its broad applicability across analytical platforms such as gas and liquid chromatography mass spectrometry (GC-MS and LC-MS).
Download this eBook to learn:
- How µSPE improves efficiency and sustainability compared to traditional SPE methods
- Key applications of µSPE for trace-level analysis
- The advantages of simplified workflows and reduced solvent use for modern labs
Transforming Analytical
Chemistry: The Power of
Micro Solid Phase Extraction2
Contents
Foreword 3
Enhanced Analytical Chemistry With
Micro-Solid Phase Extraction 4
Clean-up for GC/MS and LC/MS
Analysis of Pesticides and Environmental
Contaminants in QuEChERS Extracts of Foods 7
Enhancing Efficiency With Automated Micro-SPE 8
Unlocking the Power of Miniaturization in SPE:
Insights From an Expert 9
LC/MS Application Note: Analysis
of Highly Polar Pesticides 12
Time and Money Savings by the Implementation
of Automated Micro-SPE for Cleanup of
QuEChERS Extracts of Veterinary Drugs 21
Streamlining Lab Workflows: Practical
Insight Into How Micro-SPE Transforms
Pesticide Analysis 22
Transitioning Workflows: From QuEChERS
Extraction to µSPE Clean-up 24
Fully Automated QuEChERS for Organochlorine
and Organophosphate Pesticides in Tomato
Juice and Red Wine 25
GC/MS Application Note: Fully Automated
QuEChERS Extraction and Clean-up for
Homogenous Matrices 26
GC/MS Application Note: Routine Pesticide
Analysis Using Micro-SPE 31
Additional Resources 403
One of the most important advances in the field
of analytical chemistry is Solid Phase Extraction
(SPE), a technique that has laid the foundation for
sample separation and purification. Traditional SPE
emerged as a powerful alternative to liquid–liquid
extraction, simplifying analyte purification and
improving sensitivity. However, challenges such
as high solvent consumption, complex multi-step
procedures and environmental impacts persist.
In an attempt to overcome traditional limitations,
Micro-SPE (μSPE) methods have been developed
and are rapidly changing the landscape of
analytical chemistry. Adopting μSPE methods
allows laboratories to achieve greater efficiency,
precision and sustainability in their workflows.
By significantly reducing solvent volumes and
streamlining sample processing, μSPE meets the
growing demand for high-throughput and ecofriendly practices in modern laboratories.
Beyond simplifying workflows, μSPE enhances
sensitivity and selectivity, which is for applications
requiring trace-level analysis in complex matrices.
The value of this technique is also emphasized by
the technique’s adaptability to a range of analytical
platforms, including gas chromatography-mass
spectrometry and liquid chromatography-mass
spectrometry (GC/MS and LC/MS, respectively).
This eBook explores the potential of μSPE to
transform analytical workflows. It highlights the
key benefits of μSPE, compares it with traditional
techniques and examines its application across
various industries. This resource provides a
comprehensive guide for scientists and lab
managers looking to optimize their sample
preparation processes, achieve higher precision
and embrace sustainable practices in analytical
chemistry.
Foreword4
limitations, particularly concerning its capacity. In
scenarios involving heavy matrices, smaller loading
volumes must be carefully considered to ensure
optimal performance.
This eBook will explore the analytical capabilities
of µSPE, provide an overview of the technique and
demonstrate key applications.
The evolution of SPE
Traditional SPE has been a common technique in
sample preparation for five decades.3 It involves
using solid adsorbent materials to selectively
isolate and concentrate target analytes from liquid
samples, simplifying the process of purification
and analysis.1,4 In comparison, dispersive Solid
Phase Extraction (dSPE), a variant of SPE, involves
dispersing the solid adsorbent directly into the
sample matrix, improving contact with the analytes
and enhancing extraction efficiency. Dispersive
SPE has found broad applications in fields where
the accurate detection of chemicals, contaminants
or biological markers is critical, such as food
safety, environmental testing, forensic science and
biomedical research.5
Despite its advantages, dSPE comes with
limitations. The multi-step processes, including
conditioning, loading, washing and eluting the
Introduction
Solid Phase Extraction (SPE), which separates
analytes based on their physical and chemical
properties, has become a vital technique in modern
analytical chemistry. SPE was initially developed as
an alternative to traditional liquid–liquid extraction
(LLE), a technique that relied on large volumes of
organic solvents, extended processing times and
a high risk of error.1 By simplifying the sample
preparation process, SPE provides a more efficient
means of purifying and concentrating analytes. This
improvement significantly enhances sensitivity and
precision across multiple applications, including
environmental monitoring, pharmaceuticals and
clinical diagnostics.
Micro-SPE (µSPE) is a more recent evolution of
the SPE technique, designed to meet the growing
need for high-efficiency analysis while minimizing
resource use.2 µSPE builds upon the principles of
traditional SPE but employs much smaller volumes
of samples and solvents. Despite its downsized
format, µSPE maintains the same high-performance
levels, offering improved selectivity, sensitivity
and sustainability. µSPE represents another
leap forward in analytical chemistry, making it
an attractive option for a range of applications,
particularly when analyzing trace-level compounds
in complex matrices. However, µSPE does have
Enhanced Analytical Chemistry With
Micro Solid Phase Extraction5
to diverse compounds and complex sample types,
making it highly versatile. For labs that deal with a
wide range of applications, this level of adaptability
is essential and can be used to optimize workflows
and maintain efficiency.6 Furthermore, the high
reproducibility and consistency of µSPE results are
essential for ensuring regulatory compliance and
maintaining quality control.
Analytically, µSPE offers enhanced sensitivity and
selectivity over traditional SPE methods. With µSPE,
labs can achieve lower detection limits and higher
recovery rates. This is particularly important for
applications requiring trace-level analysis, such as
detecting pollutants in environmental samples or
identifying biomarkers in clinical research.6,7,8
Overall, µSPE overcomes many of the limitations
associated with traditional SPE by combining
analytical power with a simplified workflow.
Moving towards efficient and green
analytical chemistry
Beyond its analytical advantages and flexibility,
µSPE offers significant logistical and financial
benefits, making it an ideal solution for modern
laboratories. The reduced consumption of
solvents and adsorbent materials lowers the
cost per sample, creating immediate short-term
savings. Over the long term, µSPE’s ability to
minimize solvent and sorbent waste translates
into continuous savings on waste disposal and
improved efficiency for high-volume labs. Industries
that rely on extensive sample cleanups, such as
pharmaceuticals or environmental monitoring,
can particularly benefit from these cost savings
alongside the improved labor productivity that µSPE
provides.
µSPE is also compatible with a wide range
of analytical techniques, including gas
chromatography–mass spectrometry (GC/MS) and
liquid chromatography–mass spectrometry (LC/MS).
Its seamless integration with these workflows allows
for high-throughput analysis, further improving the
efficiency of a lab’s operations. Automation enhances
this integration, making it easier to process large
batches of samples with minimal intervention while
still ensuring high precision.
Another key advantage of µSPE is its reduced
environmental footprint. Traditional SPE methods
rely on large volumes of solvents and sorbents,
generate considerable waste and consume
significant amounts of energy. µSPE directly
addresses these concerns by reducing solvent and
waste production. As more laboratories prioritize
sustainability and seek green alternatives in their
workflows, µSPE emerges as a key solution for
reducing the environmental burden of analytical
processes.
sample, are time-consuming and labor-intensive.
Likewise, the use of large volumes of organic
solvents simultaneously increases costs and
generates significant amounts of waste. For
laboratories dealing with large sample volumes,
these factors lead to high operational costs and a
large environmental footprint.
In recognition of these challenges, exploration of
cutting-edge methods has led to the development
of µSPE.1 µSPE relies on smaller volumes of
samples and solvents while retaining the analytical
capabilities of dSPE. This miniaturization has
opened new possibilities for laboratories aiming to
improve accuracy and efficiency while addressing
environmental and cost concerns (Figure 1).6,7,8
μSPE provides advanced analytical
capabilities
One of the primary benefits of µSPE is the ability
to streamline sample preparation by combining
analyte concentration and cleanup into a single,
simplified step. In traditional SPE methods,
analytes are often diluted during extraction; this
process requires additional concentration steps,
such as solvent evaporation, to achieve the
desired sensitivity. In contrast, µSPE eliminates
this multistep process while maintaining the initial
analyte concentration, enabling higher recovery
rates. This improvement shortens processing
times while ensuring greater consistency in the
results.6,7,8
In addition, µSPE demonstrates remarkable
flexibility when handling various analytes and
sample matrices. From environmental water
samples to biological fluids, the technique adapts
Figure 1. Summary of the key limitations of traditional
dSPE techniques.6
Conclusions and future prospects
Advances in SPE, particularly the development
of µSPE, are revolutionizing laboratory sample
preparation and analysis. µSPE offers significant
improvements in efficiency, precision and
sustainability, transforming how laboratories
handle complex samples and trace-level analytes.
By reducing solvent usage, minimizing waste and
improving sensitivity, µSPE provides laboratories
with a more cost-effective and environmentally
friendly solution, without sacrificing analytical power.
In summary, µSPE offers numerous benefits to
modern analytical workflows, including enhanced
sensitivity, higher recovery rates, reduced costs and
a smaller environmental footprint. As laboratories
evolve towards more sustainable practices, µSPE
will play a vital role in achieving these goals.
For lab managers, scientists and analysts, the
incorporation of µSPE into daily operations offers
a pathway to more efficient, accurate and green
analytical processes.
References
1. Badawy MEI, El-Nouby MAM, Kimani PK, Lim LW, Rabea EI. A
review of the modern principles and applications of solid-phase
extraction techniques in chromatographic analysis. Anal Sci.
2022;38(12):1457–1487. doi: 10.1007/s44211-022-00190-8
2. Naing NN, Tan SC, Lee HK. Micro-solid-phase extraction. In:
Poole CF, eds. Solid-Phase Extraction. Elsevier, 2020:443–471.
doi: 10.1016/b978-0-12-816906-3.00016-9
3. Liška I. Fifty years of solid-phase extraction in water analysis
– historical development and overview. J Chromatogr A.
2000;885(1–2):3–16. doi: 10.1016/S0021-9673(99)01144-9
4. Płotka-Wasylka J, Szczepańska N, de la Guardia M,
Namieśnik J. Miniaturized solid-phase extraction techniques.
Trends Analyt Chem. 2015;73:19–38. doi: 10.1016/j.
trac.2015.04.026
5. Zwir-Ferenc A, Biziuk M. Solid phase extraction technique
– Trends, opportunities and applications. Pol J Environ
Stud. 2006;15(5):677–690. https://www.pjoes.com/SolidPhase-Extraction-Technique-Trends-Opportunities-andApplications,87920,0,2.html. Accessed November 11, 2024.
6. Chong CM, Hubschmann HJ. Fully Automated QuEChERS
Extraction and Cleanup of Organophosphate Pesticides in
Orange Juice. LC GC N Am. 2021;39(s1):12–16. https://www.
chromatographyonline.com/view/fully-automated-quechersextraction-and-cleanup-of-organophosphate-pesticides-inorange-juice. Accessed November 11, 2024.
7. Hakme E, Poulsen ME. Evaluation of the automated microsolid phase extraction clean-up system for the analysis of
pesticide residues in cereals by gas chromatography-Orbitrap
mass spectrometry. J Chromatogr A. 2021;1652:462384. doi:
10.1016/j.chroma.2021.462384
8. Michlig N, Lehotay SJ. Evaluation of a septumless minicartridge for automated solid-phase extraction cleanup
in gas chromatographic analysis of >250 pesticides and
environmental contaminants in fatty and nonfatty foods.
J Chromatogr A. 2022;1685:463596. doi: 10.1016/j.
chroma.2022.463596
Click here to learn more about the
analytical power of μSPE7
Clean-up for GC/MS and LC/MS Analysis
of Pesticides and Environmental Contaminants
in QuEChERS Extracts of Foods
Dispersive SPE µSPE
• Limited selectivity, limited clean-up
◊ High sample and solvent volumes
◊ Requires evaporation with N2
◊ End volume > 0.5 ml in vial
• High selectivity
◊ Sharp elution peak profile, no concentration
◊ Final volume < 100–200 µl (or online), no
sample concentration by evaporation
• Good clean-up compared to LC separation
• Manual operation
◊ Time consuming
◊ Low sample throughput
◊ Batch processing
• Walk away automation
◊ Fast with < 10 min
◊ High productivity
◊ Prep on chromatographic timescale
• Not traceable
◊ Manual operation
◊ No activity log
• Traceable
◊ Processing well documented
◊ 21 CFR part 11 compatible
μSPE offers significant advancements
in automated sample preparation by
eliminating analyte dilution and the
need for solvent evaporation or cartridge
drying. This streamlined approach is
particularly well-suited for applications
such as QuEChERS pesticide analysis,
veterinary drug testing, filtration of life
science samples and more.
By replacing traditional manual SPE
workflows that require large solvent
volumes, µSPE represents a step forward
in green analytical chemistry by reducing
waste and lowering costs. The µSPE
cartridge functions like a compact liquid
chromatography (LC) column, utilizing
only micro-volumes of sample and
elution solvents. With a syringe serving
as the solvent pump, consistent flow
and pressure ensure high repeatability
in automated procedures, enhancing
efficiency and reliability in the laboratory.µSPE lets researchers
achieve their
goals faster.
Pesticide analysis plays a key role in ensuring food and environmental safety. By ensuring
compliance with regulatory standards, it helps minimize the risk of exposure to potentially
harmful contaminants.
QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction is commonly
used to clean samples during pesticide analysis. Initial extraction uses solvents, such
as acetonitrile, and partitioning salts to separate the sample from unwanted matrix
compounds. Following this, additional extraction often relies on manual dispersive solidphase extraction (d-SPE) to further purify samples. However, this conventional approach
creates challenges in terms of time, labor and cost efficiency and requires large quantities
of solvent during analysis. In contrast, the latest technological advances in micro-solid
phase extraction (μSPE) overcome many of these limitations and offer the ability
to automate sample clean-up.
This infographic explores how μSPE provides a streamlined solution for pesticide analysis.
Streamlined Pesticide Analysis:
Enhancing Efficiency With Automated Micro-SPE
The traditional dispersive approach
The analytical power of μSPE
Multi-step dispersive cleanup
Fully automated µSPE cleanup
Achieve cutting-edge sample
cleanup and analysis.
Analyse
+250
pesticides across different matrices.
Manual dispersive workflows use a series of labor-intensive processes to extract samples of
interest. Each additional step in this process prolongs the analytical workflow, creating more
opportunities for inaccuracies and increased processing costs.
By automating repetitive tasks and minimizing manual intervention, μSPE accelerates
throughput without compromising accuracy. μSPE streamlines the sample preparation
process, significantly reducing labour time and solvent usage, allowing scientists to spend
more time on valuable data analysis.
Understanding the limitations of traditional workflows allows laboratories to make informed
decisions about which clean-up approach suits their needs. The adoption of cutting-edge
techniques, such as μSPE, can unlock efficient workflows, accurate analysis and provides
cost-effective approaches to pesticide cleanup and analysis.1-3
μSPE offers the opportunity to improve a range of workflows through cost savings, efficiency
and advanced analytical capabilities.
By unlocking efficient and streamlined workflows, μSPE empowers laboratories to achieve
operational excellence and meet regulatory requirements with confidence. μSPE cartridges
pair with the PAL System to provide the analytical power needed across a range
of applications, from food safety to environmental monitoring.
Depending on batch size,
manual sample transfer can
add hours of intensive labor
to the workflow.
A streamlined workflow
reduces the number of
manual sample transfer
steps and allows analysis and
sample preparation to be
carried out in parallel.
Throughout this workflow,
samples need to be
manually tracked to maintain
data integrity and regulatory
compliance.
Automated sample tracking
eliminates human error
and variability.
Less solvent is used
during processing without
compromising performance.
The μSPE workflow allows for
a significant reduction (factor 2)
in solvent consumption
compared to dispersive
solid phase extraction.
Requires significant
quantities of solvents,
driving up consumable costs
and generating excess waste.
Reduce labor requirements
by more than 30%, freeing
up hours of valuable time
for data analysis and high
impact activities.
Relies on time-consuming
manual operations,
with low sample throughput.
Overlap sample cleanup
and analysis, drastically
increasing throughput.
Extended workflows demand
time and reduce throughput.
Sample transfer
with adsorbent
Load cartridge
Inject ready
for GC/MS
Elute extract
Sample mixing
Sample transfer
ready for
GC/MS analysis
Centrifugation
Excessive
solvent usage
Timeconsuming
Inaccurate
data
Costly
!
!
Manual sample
tracking and
labelling
throughout
workflow
Documented
sample processing
Lower solvent
requirements
Reduced
solvent usage
High solvent usage
Low-labor input
Reduce solvent usage
in line with green
chemistry principles.
High throughput
workflow
Increase operational
efficiency to minimize
labor input and reduce
overall costs.
Fully automated
clean-up
Enhance sample
throughput using
automated sampling
and analysis.
Increased
productivity
Eliminate sources of
error to ensure reliable
and precise results.
Reliable and
sensitive analysis
Labor-intensive processes
Walk away
automation
Sequential sampling
and analysis
Reduce
labor hours
High
selectivity
Minimize
costs
! ! ! !
! ! !
Choosing the right workflow
Improved workflow through μSPE
Explore the capabilities of μSPE
Unlock the power of μSPE cartridges
References
1. Michlig N, Lehotay SJ. Evaluation of a septumless mini-cartridge for automated solid-phase
extraction cleanup in gas chromatographic analysis of >250 pesticides and environmental
contaminants in fatty and nonfatty foods. Journal of chromatography A/Journal of
chromatography. 2022;1685:463596-463596. doi: 10.1016/j.chroma.2022.463596
2. Hakme E, Poulsen ME. Evaluation of the automated micro-solid phase extraction clean-up
system for the analysis of pesticide residues in cereals by gas chromatography-Orbitrap
mass spectrometry. Journal of Chromatography A. 2021;1652:462384.
doi: 10.1016/j.chroma.2021.462384
3. Lorena Manzano Sánchez, Jesús F, Ferrer C, M. Mar Gómez-Ramos, Amadeo
Fernández-Alba. Evaluation of automated clean-up for large scope pesticide
multiresidue analysis by liquid chromatography coupled to mass spectrometry.
Journal of chromatography A/Journal of chromatography. 2023;1694:463906-463906.
doi: 10.1016/j.chroma.2023.4639069
Micro solid phase extraction (µSPE) represents
a significant leap forward in the evolution of
sample preparation techniques, offering enhanced
efficiency, precision and sustainability. In this
interview, Hans-Joachim Hübschmann, a seasoned
expert with decades of experience in food safety,
environmental analysis and chromatography, shares
his insights on the benefits of µSPE. Hans discusses
the key motivation behind the miniaturization of
SPE, the role µSPE plays in modern laboratories
and its potential to revolutionize various analytical
workflows. With a rich background in developing
automated sample preparation solutions, Hans
provides valuable perspectives on how µSPE
can enhance both laboratory performance and
environmental sustainability.
Q: How and why has SPE become
miniaturized over time, and what are
the advantages of performing µSPE?
Hans-Joachim Huebschmann (HJH): Traditional
solid phase extraction, using milliliter cartridges,
celebrates its 50th anniversary this year. This
milestone highlights the pivotal publication by
Reginald Adams, Thomas Good and Michael
Telepchak that replaced the large separation
funnels that required liters of solvents. Over
these last 50 years, advancements in analytical
instruments have brought generations of progress.
The performance of today’s techniques, such as gas
chromatography–mass spectrometry (GC/MS) and
liquid chromatography–mass spectrometry (LC/MS),
is incomparable to those from the 1970s or ‘80s.
Miniaturization has largely been driven by the
incredible sensitivity of these modern instruments,
which can detect analytes in femtogram ranges.
We no longer need to extract and concentrate
from large samples; a few grams of homogenized
material serve today’s needs. Likewise, we don’t
inject large extract volumes anymore; a few
microliters of a cleaned extract work well for both
GC/MS and LC/MS.
Unlocking the Power of Miniaturization in SPE:
Insights From an Expert
Hans-Joachim
Huebschmann,
PhD10
mode. The matrix is kept on the sorbent and the
pesticides elute in a sharp band, like from an LC
column.
Additionally, the µSPE workflow supports the
traditional “enrichment” method – also known as
the load-wash-and-elute mode – commonly used
for applications like drinking water. Any type of
sorbent material can be used in µSPE cartridges,
depending on the application. The different types
of µSPE cartridges available are continuously
expanding, based on collaboration with
customers and sorbent material manufacturers.
In some cases, µSPE cartridges are prepared by
manufacturers using proprietary sorbents. Some
examples include:
C18 materials: These have the widest application
outside of QuEChERS, where they are used for
veterinary drug analysis by LC/MS.
Strong anion exchange material (SAX): This
is the standard for very polar pesticides, extracted
using the quick polar pesticide (QuPPe) extraction
for compounds like glyphosate, glufosinate or
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA).
Weak anion exchange material (WAX): This is
the solution for per- and polyfluoroalkyl substances
(PFAS) analysis.
Q: What are the advantages and
limitations of using µSPE in these
applications?
HJH: Routine laboratories benefit from automated
processing using XYZ robotic samplers, which
are already widely installed in most labs and
are industry-standard, but upgrades to µSPE
are always possible. In addition to the abovementioned cost savings and analytical benefits,
automated µSPE workflows reduce handling errors,
deliver the necessary results faster and allow
overnight and unattended weekend runs. It is also
worth mentioning that these workflows are well
documented in preparation for potential audits.
As life sciences applications mostly use LC or LC/
MS already, the design of µSPE cartridges and the
functionality of the PAL XYZ robot allows the direct
elution of a cleaned extract into the sample loop of
the HPLC injection valve. No additional glassware
or vials are needed, avoiding absorption and
contamination in additional handling steps.
Currently there isn’t a standard approach, but inline
processing with GC/MS or LC/MS and standalone
robotic samplers to handle µSPE tasks for multiple
systems are both viable options depending on the
lab’s needs.
So the question becomes, why stay at the level
of traditional SPE? Why prepare milliliters of
cleaned extract if we only inject a few microliters?
One reason for the continued use of traditional
SPE methods is that established protocols have
become entrenched, hindering progress toward
more innovative approaches. µSPE, as the name
suggests, steps down from the milliliter scale to
microliters. It needs less sorbent material and
much less solvent, which offers a big cost saving
for laboratories. As a result of this scale-down,
µSPE is faster, avoids evaporation and can be more
selective for the analytes of interest.
Q: What is driving the need for
miniaturization?
HJH: Green analytical chemistry is a big drive
for miniaturization. However, it is more than a
trend: it’s a responsibility for analysts in food,
environment and life sciences. Our role is to reduce
hazardous solvent waste, minimize plastic usage,
save energy and protect colleagues from harmful
reagents.
From a commercial perspective, cost savings and
faster report generation are major advantages of
using µSPE. Also, as µSPE cleanup is often done
with hyphenated techniques (e.g., GC/MS and LC/
MS), companies can utilize MS instrumentation
in which they already have significant financial
investment.
The µSPE clean-up can also be performed in
parallel using automated inline processes while a
previous extract is being analyzed. We call this the
“prep ahead” mode. There’s no longer a waiting
period for the last manually prepared extract
before injection. Likewise, µSPE handles all of the
samples identically, an approach which improves
reproducibility.
On the practical side, µSPE facilitates workload and
logistics in a laboratory. µSPE is fully automated
and gives researchers the time they need for other
essential tasks. For instance, during QuEChERS
clean-up for pesticide extracts, µSPE facilitates
the often cumbersome choice of sorbent material
compared to dispersive SPE, where there are too
many choices and recommendations.
Q: Outside of the conventional
QuEChERS workflow, where else can
µSPE cartridges be used?
HJH: For the cleanup of QuEChERS extracts, a
specific type of µSPE cartridge has proven to be
highly effective, even when analyzing a large
variety of pesticides using GC/MS and LC/MS. For
the QuEChERS extract we use the “scavenging”11
materials. Currently, µSPE is being adopted for
routine SPE applications without altering the
underlying chemistry, but it offers advantages in
terms of speed, efficiency and scalability for routine
analysis. Future developments of sorbent materials
will continue to enhance µSPE. For special matrices
and samples targeted to low-recovery compounds,
dedicated types of µSPE cartridges may be
developed.
Q: Is there any ongoing research to
expand the use of µSPE in unexplored
fields or for new applications?
HJH: As a developer of µSPE cartridges and
automated workflows, we closely follow the
innovation of new sorbent materials, both
in research and manufacturing. Advanced
sorbents (e.g., molecular-imprinted materials,
immunosorbents or nanostructured materials),
despite being more expensive, will benefit from
µSPE’s low material requirements.
Other applications of µSPE cartridges can be
observed when filtration is required, which offer
a range filter materials and different technical
approaches. In its simplest form, this could involve
automated, inline filtration of extracts directly into
the HPLC injection valve.
Q: Can you tell us about WAX SPE
cartridges? What applications are
they used for, and do they have any
advantages in these fields?
HJH: The Environmental Protection Agency (EPA)
Method 533 uses WAX SPE cartridges for PFAS
analysis, particularly in drinking water. These
cartridges are ideal for retaining short-chain acidic
compounds, allowing for detection at the required
low parts-per-trillion (ppt) levels. This approach
is particularly important in PFAS analysis, which
requires a system free from background noise. We
find PFAS compounds in almost all components
of the analytical flow path. All suspected parts
need to be replaced by PFAS-free materials. This
also applies to the WAX sorbent material and the
cartridge itself. The µSPE cartridge material is
the same type used for pipette tips – free from
leachables and, crucially, PFAS.
For the PAL System used in PFAS µSPE analysis, a
PFAS-free upgrade kit with PFAS-free tubing and
syringe is available, as are PFAS-free consumables.
Results presented at a recent conference on
Halogenated Persistent Organic Pollutants in
Singapore confirmed the excellent PFAS-free
background and performance of the dedicated PAL
System.
Q: Where do you see µSPE being used
in the future?
HJH: 50 years of analytical SPE has shown a
great degree of versatility across a wide range of
applications, thanks to newly developed sorbent12
LC/MS Application Note
www.palsystem.com
Analysis of Highly Polar Pesticides13
2
Analysis of Highly Polar Pesticides
Automated Micro-SPE Clean-up of Complex Food Matrices for LC-MS/MS
Florencia Jesús1 , Amadeo Rodríguez Fernández-Alba1, Hans-Joachim Hübschmann2
1) Department of Chemistry and Physics, Agrifood Campus of International Excellence (ceiA3), University of Almeria, Spain
2) CTC Analytics AG, Zwingen, Switzerland.
Introduction
Micro-SPE (μSPE) emerged as an automated green
micromethod for sample preparation and clean-up in
food safety, proteomics, forensic, environmental and
analysis since more than ten years. Applications are wideranging and cover drugs, environmental contaminants,
and, in particular, the extract clean-up in multiresidue
pesticide analysis. The automation of the μSPE sample
preparation steps led to the desired standardization of
the applied sorbent materials for extract clean-up for
the large variety of food commodities, an increase in
sample throughput, and the welcome potential for offline
and online hyphenation with GC-MS, LC-MS or even IC
analysis.
Highly Polar Pesticides Analysis
The application of “highly polar pesticides” (HPPs) in
agricultural production is increasing due to their low cost
and high efficiency, with glyphosate as the well-known
active ingredient in the popular herbicide Roundup™.
The EFSA reports compounds like fosetyl-Al, etephon,
chlormequat and glyphosate within the top ranking of
frequent maximum residue limit exceedances (MRL) in
Europe1,2.
Such highly polar pesticides are not amenable for
extraction and clean-up by the widely used QuEChERS
protocol. This group of pesticides is mainly characterized
by its high water solubility. Glyphosate as the best-known
pesticide in this group serves well as a lead substance
for method development. Due to the high polarity and
dissociation in aqueous media LC-MS/MS is the method
of choice for high-throughput multi-residue analysis and
simultaneous quantification.
In this application report the analysis of eleven HPPs
including glyphosate, glufosinate, ethephon, fosetylaluminium, and their related metabolites is described
for analysis from complex food matrices such as honey
and pollen. For the first time the micro-SPE clean-up
is introduced also for HPPs to overcome the known
analytical limitations by removal of polar co-extracts with
automated clean-up processing of the sample extracts3.
Offline Sample Pretreatment: Extraction of
raw extract from different matrices
The extraction of the HPPs from a representative test
portion of fruit and vegetables is described with acidified
methanol in the Quick Polar Pesticides Method QuPPe4.
After centrifugation a filtered aliquot is transferred to
autosampler vials for direct LC-MS/MS analysis. For the
described analysis of honey and pollen samples the
applied extraction method is modified as follows:
Honey: To 5 g of homogenized sample 9 mL of water
is added and spiked with 100 μL of a mixed solution of
isotopically labelled glyphosate-13C2,15N and AMPA-13C,15N
at 1 mg/L. After vortexing 10 mL of MeOH is added,
and the tubes are shaken at 1000 rpm for 10 min and
centrifuged at 3220 g for 5 min. The sample concentration
in the extract is 0.25 g/mL.
Pollen: To 2 g of homogenized sample 10 mL of water
is added and spiked with 100 μL of a mixed solution of
isotopically labelled glyphosate-13C2,15N and AMPA-13C,15N
at 1 mg/L, and 100 μL of a solution of fosetyl-Al-D15 at
1 mg/L. After vortexing the tubes are centrifuged at
3220 g for 30 min. A 5 mL aliquot of the supernatant is
then treated with 250 mg of C18 material and 5 mL of
acetonitrile, vortexed, and again centrifuged at 3220 g for
5 min. The sample concentration in the extract is 0.1 g/mL.
For both types of sample the supernatant is then applied
for a manual SAX SPE to compare with the automated μSPE
clean-up on the PAL System.14
3
Automated SAX μSPE clean‑up
For the micro-solid-phase extraction (μSPE) clean-up, a PAL RTC System was used as a benchtop device for automated
sample preparation. The benchtop installation allows the distribution of the cleaned extracts to different analysis
systems like LC-MS or IC.
μSPE uses SPE cartridges in the dimensions of 35 mm
hight and 8.5 mm OD. These novel PAL μSPE cartridges
contain for this application a layer of SAX sorbent material,
shown in Figure 1.
A syringe is used to load the raw extract, wash the sorbent
bed and elute the analytes applying a precise extract and
solvent volume with a predefined and constant flow of
5 μL/s, as illustrated in Figure 3. The syringe is also used
for the cartridge transport between the conditioning
rack, elution rack and to the waste container. The needle
transport works with the needle solidly sticking in the
cartridge needle seal. The executed clean-up steps are
highly reproducible using the automated workflow.
The μSPE configuration uses a trayholder with three
dedicated racks, as shown in Figure 2.
The vials with raw extracts are placed into position 1, the
one closest to the rail. The elution rack is positioned in
the center of the trayholder in position 2 with empty vials
under the shown aluminium vial cover. The cartridge tray
in position 3 is used also for the cartridge conditioning. A
solvent station with three glass bottles of 100 mL each provides different solvents employed for cartridge conditioning,
elution, and sample dilution. MeOH:H2O (1:1 v/v), and MEOH p.a. for a fast and thorough syringe washing are
provided by a Fast Wash Station with an integrated solvent pump connected to external solvent reservoirs.
Fully automated raw extract clean-up from complex matrix samples using PAL System μSPE
A general clean-up of extracts other than dilution is not part of the above mentioned QuPPe method, except a
dispersive SPE with C18 sorbent material in case of a necessary removal of protein and lipids from cereals, nuts, seeds
or oily fruits. Though, with complex matrix samples, such as honey, pollen or even with coffee or tea, the co-extraction
of polar matrix components was observed. These matrix compounds co-elute with the analytes of interest and
interfere the peak detection and integration even in LC-MS/MS analysis, hence limiting the identification and detection
sensitivity.
Solid-phase extraction (SPE) and micro solid-phase extraction (μSPE) employing a strong anion exchange (SAX)
sorbent material were implemented for a clean-up of the primary extracts. The automation and miniaturization of the
SAX clean-up for HPP compounds is demonstrated in this application for the first time.
Manual SAX SPE clean‑up
Honey: An aliquot of 2.4 mL extract is diluted to a final volume of 10 mL with MeOH. The sample is then loaded on
an SPE cartridge (HyperSep™ SAX 500 mg/6 cc, previously conditioned with 10 mL of MeOH), then washed with 6 mL
of MeOH, and the water content removed by a vacuum pump for 3 min. The analytes are then eluted with 3 mL of
MeOH:HCl 1M (9:1). Sample concentration in the extract is 0.2 g/mL.
Pollen: An aliquot of 6 mL extract is diluted to a final volume of 10 mL with MeOH. The sample is then loaded on an
SPE cartridge (HyperSep™ SAX 500 mg/6 cc, conditioned with 10 mL of MeOH), then washed with 6 mL of MeOH, and
dried from water by a vacuum pump for 3 min. The analytes are then eluted with 3 mL of MeOH:HCl 1M (9:1). Sample
concentration in the extract is 0.2 g/mL.
Figure 1: The novel PAL μSPE cartridge (cross section).15
4
Figure 3: Principle of the PAL μSPE operation
Figure 2: μSPE rack on a PAL RTC system: raw extract vials (Pos.1),
the eluate rack, gets the cleaned extract (Pos.2), and the conditioning
rack connected to waste (Pos.3), μSPE tool with 1000 μL prep syringe
A dedicated μSPE tool with a 1000 μL smart syringe
with flat-tipped 22 gauge needle is used for the sample
preparation and clean-up workflow. The principle of
the automated μSPE operation on the PAL System is
illustrated with Figure 3.
For the automated μSPE clean-up the novel septumless μSPE
cartridges are containing 50 mg of strong ion exchange (SAX)
sorbent material.
For the automated SAX μSPE clean-up 360 μL of honey or
900 μL of the pollen extracts was diluted to a final volume
of 1500 μL with MeOH. The diluted extracts are placed in
2 mL PP vials on the dedicated PAL μSPE clean-up tray in
rack position 1 (Figure 2).16
5
Activity Description Parameter Flow Source Target
Select syringe Liquid tool 1000 μL Tool park station
Wash syringe Volume 1000 μL Solvent station Waste
Cycles 3 Methanol Waste
Conditions cartridge Liquid tool 1000 μL 5 μL/s Methanol Conditioning rack
Load sample Liquid tool 1000 μL 5 μL/s Raw extract Conditioning rack
Wash syringe Volume 1000 μL Solvent station Waste
Cycles 3 Methanol Waste
Wash cartridge Liquid tool 600 μL 5 μL/s Methanol Conditioning rack
Wash syringe Volume 660 μL Solvent station Waste
Cycles 3 Methanol Waste
Take elution solent Liquid tool 400 μL Solvent station
Move cartridge Liquid tool Conditioning rack Elution rack
Elute analytes Liquid tool 400 μL 5 μL/s Elution rack
Move cartridge Liquid tool Elution rack Waste container
Wash cartridge Volume 440 μL Solvent statio Waste
Cycles 3 Methanol Waste
Table 1: SAX μSPE clean-up workflow parameter
The Automated μSPE Workflow
The automated μSPE clean-up workflow uses the
classical enrichment mode, shown in the sequence
of steps in Figure 4. The prepared raw extracts,
empty collection vials for the cleaned extract, and the
appropriate number of cartridges are loaded to the
dedicated racks on the PAL μSPE trayholder. In a first
step the prep syringe is selected and thoroughly cleaned
with methanol from the solvent reservoirs. For every
raw extract vial a cartridge and an empty collection vial
are held ready in their respective racks in Pos. 2 and
3 (Figure 2). The automated workflow processes the
prepared number of samples fully unattended. Using a
benchtop installation the cleaned extracts are then ready
for analysis by LC-MS or IC. In online installations the
PAL RTC System can change the syringe to an injection
syringe for applying the cleaned extract via a 6-port
injection valve to LC-MS analysis.
For the detailed parameters used in this workflow see
Table 1.
Figure 4: Sequence of the automated PAL workflow for benchtop extract clean-up using SAX μSPE cartridges (*manual steps)17
6
a Detected as fosetyl
b Detected as fosetyl-D
Table 2: Compounds analysed with optimised SRM transitions and collision energy.
LC/MS Analysis
For the LC analysis, a Thermo Scientific™ Transcend™ DUO LX-2 (Thermo Scientific™, Germering, Germany) was used.
Chromatographic separations were performed on an anionic polar pesticide column (APP column) from Waters™ (Milford,
MA, USA) at a constant temperature of 50 °C. Mobile phase A was water containing 1.2% formic acid, mobile phase B was
acetonitrile containing 0.5% formic acid. The mobile phase flow was set at 0.5 mL/min throughout the analysis. A gradient
elution started at 10% A and held for 0.5 min, then ramped up to 80% A at 1.5 min, and to 90% A at 3 min. The final
mobile phase composition was maintained until 16.5 min. After completed analysis the column was re-equilibrated again
to 10% A to for the next injection. The injection volume was 10 μL.
A TSQ Altis LC-MS/MS system (Thermo Scientific™, San Jose, USA) equipped with an OptaMax™ NG (H-ESI II) ion source
operated in negative ionisation mode was used. The source parameters were set as follows: ion spray voltage: 2500 V,
sheath gas: 60 (arbitrary units), aux gas: 15 (arbitrary units), sweep gas: 0 (arbitrary units), ion transfer tube temperature:
325 °C; vaporiser temperature: 350 °C. The triple quadrupole was operated in the SRM mode for the target compounds
listed in Table 2. The cycle time of the MS was set to 0.8 s. Resolution of Q1 was 0.7 u, Q3 was set to 1.2 u. As CID gas
argon was used at a constant pressure of 1.5 mTorr.
Compound SRM 1 CE SRM 2 CE
(m/z > m/z) (V) (m/z > m/z) (V)
AMPA 110 > 63 20 110 > 79 28
AMPA-13C,15N 112 > 63 20 112 > 107 28
Ethephon 143 > 107 7 145 > 79 7
Fosetyl-Ala 109 > 81 12 109 > 63 29
Fosetyl-Al-D15b 114 > 82 13 114 > 63 30
Glufosinate 180 > 63 40 180 > 95 17
Glyphosate 168 > 150 10 168 > 63 22
Glyphosate-13C2,15N 171 > 153 10 171 > 63 22
HEPA 125 > 79 21 125 > 95 14
MPPA 151 > 133 13 151 > 63 32
N-Acetyl-AMPA 152 > 110 13 152 > 63 23
N-Acetyl-glufosinate 222 > 79 22 222 > 180 17
N-Acetyl-glyphosate 110 > 136 13 210 > 148 16
Phosphonic acid 81 > 79 15 81 > 63 2718
7
Figure 5: Extracted LC-MS/MS ion chromatograms of standards of AMPA, glufosinate, and glyphosate at 0.010 mg/kg spiked to honey and
pollen matrix without (left) and after SAX clean-up (right).
Results and discussion
The effect of the SAX clean-up of honey and pollen extracts on the removal of matrix components is observed by the
extracted ion chromatograms of analytes such as AMPA, glufosinate, and glyphosate as it is shown in Figure 5. After the
automated SAX μSPE clean-up the compounds are detected with improved selectivity and sensitivity for a more reliable
identification and peak integration, a higher signal-to-noise ratio for the analytes was achieved. The reduction of the signal
suppression by the matrix, particularly for AMPA and glufosinate, is seen with the significantly reduced baseline and a signal
enhancement on their peak heights.
Compared to the previously used and above described manual extract clean-up procedure significant advantages are
achieved with the automated PAL System μSPE clean-up:
• Instead of 500 mg SAX material only 50 mg sorbent
material is applied leading to reduced cost and faster
processing.
• Also, the solvent and extract volumes and the time
required for conditioning, sample loading, and washing are reduced by a factor of 10.
• The time-consuming drying step of the manual SPE
method before and after the elution using a vacuum
pump can be omitted.
• The recovery results obtained with the automated
SAX μSPE clean-up are comparable or slightly higher
to those obtained for the manual clean-up
(see Table 3).
• In particular, the recoveries from honey for AMPA
were between 98 and 113% (manual 70%). MPPA
showed improved recoveries of 74–86%, both with
acceptable RSDs.
• Overall recoveries for the HPP compounds investigated are between 70 and 120%, with RSDs below 20%,
and LODs from 0.005 to 0.020 mg/kg. A linearity is
achieved from 0.002 to 0.200 mg/kg.
• The considerably reduced manual labour allows the
increased sample throughput for routine work.
• The automated workflow prevents human bias and
leads to improved reproducibility of the results.8 19
Table 3: Recoveries, relative standard deviations (RSDs), limits of quantitation (LOQs), linear ranges, and matrix effects (MEs) obtained for honey with
manual and automated SAX SPE cleanup.
For the quantitative calibration the matrix-matched calibration points are also submitted to the automated clean-up. This
calibration approach, called “semi-procedural standard calibration” (because the spiking was done just prior to clean-up, and
not prior to the whole extraction method), was previously employed and recommended by Hakme and Poulsen5 and by
Manzano et al.6 for the accurate multiresidue analysis of more than 200 pesticides in tomato, orange, rice, avocado, and black
tea using automated μSPE clean-up for LC-MS/MS to compensate for potential analyte losses from the clean-up step.20
9
References
1 P. Medina-Pastor and G. Triacchini. 2020. “The 2018 European union report on pesticide residues in food,”
EFSA J. 18(4), 1–103. https://doi.org/10.2903/j.efsa.2020.6057.
2 L. Carrasco Cabrera and P. Medina Pastor. 2022. “The 2020 European Union report on pesticide residues in food,”
EFSA J. 20(3). https://doi.org/10.2903/j.efsa.2022.7215.
3 Jesus, Florencia, Adrián Rosa García, Tommaso Stecconi, Víctor Cutillas, and Amadeo Rodríguez Fernández-Alba. 2023.
“Determination of Highly Polar Anionic Pesticides in Beehive Products by Hydrophilic Interaction Liquid Chromatography
Coupled to Mass Spectrometry.” Analytical and Bioanalytical Chemistry. https://doi.org/10.1007/s00216-023-04946-7.
4 M. Anastassiades, A.-K. Wachtler; et al. 2023. “Quick Method for the Analysis of Highly Polar Pesticides in Food Involving
Extraction with Acidified Methanol and LC- or IC-MS/MS Measurement, I. Food of Plant Origin (QuPPe-PO-Method),
Version 12.1 (17.03.2023)”. https://www.quppe.eu/quppe_doc.asp
5 Elena Hakme, Mette Erecius Poulsen. “Evaluation of the automated micro-solid phase extraction clean-up system for the
analysis of pesticide residues in cereals by gas chromatography-Orbitrap mass spectrometry”. J. Chrom. A 1652 (2021)
462384. https://doi.org/10.1016/j.chroma.2021.462384.
6 Manzano Sanchez, Lorena, Florencia Jesus, Carmen Ferrer, M. Mar Gomez-Ramos, and Amadeo Fernandez-Alba. 2023.
“Evaluation of Automated Clean-up for Large Scope Pesticide Multiresidue Analysis by Liquid Chromatography Coupled to
Mass Spectrometry.” J. Chrom. A, 0–23. https://doi.org/10.1016/j.chroma.2023.463906.
Imprint
Date of print: 12.2023
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Conclusion
Automated µSPE is a viable alternative for cleanup of
QuEChERS extracts of veterinary drugs.
▪ For a batch of 15 samples, 80 minutes of time is saved, a
26% time savings.
▪ Each sample is analyzed immediately after cleanup, reducing
degradation time by sitting in an autosampler.
▪ Automated µSPE delivers consistent flow rates for each
sample.
▪ Recovery will be compared to manually extracted dSPE
samples in future work, rather than to spiked bovine matrix
samples.
References
1. Lehotay, S. J., Han, L., Sapozhnikova, Y. (2016). Automated Mini-Column
Solid-Phase Extraction Cleanup for High-Throughput Analysis of Chemical
Contaminants in Foods by Low-Pressure Gas Chromatography—Tandem Mass
Spectrometry. Chromatographia, DOI 10.1007/s10337-016-3116-y
Overview
Purpose: Demonstrate the use of automated µSPE cleanup Vs.
manual cleanup of animal tissue extracts to save time and money in
a veterinary drug research laboratory.
Methods: Automated µSPE and manual dispersive SPE are
compared via LC-MS/MS analysis.
Results: Short synopsis of the results.
Introduction
In this poster presentation, we compare two workflows for the
cleanup of multi-class veterinary drugs in animal tissue matrices,
based on the Thermo Scientific™ VetDrugs Explorer Collection
(P/N VDX-TSQ02-10001, Thermo Fisher Scientific, Waltham MA,
USA). A manual sample cleanup using a QuEChERS (Quick, Easy,
Cheap, Effective, Rugged, and Safe) extraction is performed. The
final cleanup steps and dilution with mobile phase prior to injection
into an LC-MS/MS are amenable to automation. By automating
these steps, we save time and labor, solvent volumes, and
introduce a more consistent and reproducible cleanup and dilution,
thus improving the analytical results. The amount of time, labor, and
solvents saved are quantitated for a typical lab and presented in
this poster.
Methods
Sample Preparation
Samples were prepared following the protocol included with the
VetDrugs Explorer Collection. Tissue samples were prepared by
weighing 5g of bovine muscle and placing it in a 50mL centrifuge
tube. 2mL of acetonitrile was added as well as 0.5mL of 0.2M
ammonium oxalate and 0.1M disodium EDTA solution. An additional
13mL of acetonitrile is added and the sample is shaken to
homogenize the mixture. Next, 5g of anhydrous sodium sulfate is
added and shaken. After a 30-minute wait, samples are centrifuged
for 10 minutes (4500 rpm), then the supernatant is decanted. At this
point, samples for automated µSPE are placed on a robot for
automated cleanup, while the samples for manual cleanup are
mixed with a dispersive SPE material, filtered, and diluted.
Manual SPE Cleanup
The decanted supernatant is placed in a centrifuge tube and 500mg
of CEC18 (Carbon End Capped C18) dispersive SPE (dSPE) is
added to the supernatant. The tube is shaken a minimum of 4 times
at intervals over at least 15 minutes. The tube is then centrifuged,
(4500 rpm at 20°C) for 5 minutes. 3mL of the extract is added to
1mL of Mobile Phase A (see below for composition) and mixed.
Finally, the final extract is filtered with a 0.45 µm PTFE syringe filter
into and amber 2mL autosampler vial for analysis.
Figure 1. TriPlus RSH autosampler. From left to right, Tool
Park Station, Peltier Stack, µSPE tray holder, Solvent Module,
Valve Drive Module, and Wash Station.
Money Savings From Automated µSPE
The monetary savings gained from µSPE are largely due to labor
costs, and smaller solvent volumes that can be employed. Labor
costs of 80 minutes per 15 sample batch. While the initial 8 steps of
the sample preparation are identical in this study, further studies will
investigate smaller initial sample sizes to scale down the
homogenization, which uses substantial amounts of costly
acetonitrile. If the sample could be scaled down by a factor of 10,
the subsequent solvent usage would correspondingly be reduced
by the same factor.
Time Savings From Automated µSPE
The TriPlus RSH Autosampler takes care of extraction steps 9-13
listed in Table 1. This results in a time savings of 80 minutes for
every 15 samples prepared. These 80 minutes are converted into a
single 8.5 minute extraction, a nearly 10x time savings. The very
first sample of a given sequence starts with the first extraction,
taking 8.5 minutes prior to LC-MS/MS injection, however, every
subsequent sample in the sequence is prepared during the LC
runtime of the previous sample through “look ahead” or overlapping
sample preparation. By utilizing look ahead sample prep, the next
sample is ready for injection as the previous sample’s LC-MS/MS
run is completed. This saves 8.5 minutes per sample in our
automated process. As sample batch sizes are increased, this same
time savings is realized for additional samples. Additionally, a more
consistent extraction is achieved due to the constant 2 µL/min flow
rate through the µSPE cartridge. Finally, each extract is analyzed in
near “real-time,” immediately after cleanup. This helps with
compounds that are susceptible to degradation.
Time and Money Savings by the Implementation of Automated µSPE for Cleanup of QuEChERS Extracts of Veterinary Drugs
Jonathan Beck1, Tom Flug1, Laura Burns2, Dwayne Schrunk2, Dipankar Ghosh3, Ed George3
1CTC Analytics AG, Lake Elmo, MN, USA, 2Iowa State University Veterinary Diagnostics Laboratory, Ames, IA, USA, 3Thermo Fisher Scientific, San Jose, CA, USA
Registered names, trademarks, etc. used, even when not marked as such, are not to be considered unprotected by law.
Jonathan R. Beck
Automated µSPE Cleanup
The automated µSPE cleanup and sample injection is carried out
using the Thermo Scientific™ TriPlus™ RSH autosampler. The
decanted supernatant is placed in a 2mL amber autosampler vial,
and placed in the cooled Peltier autosampler rack at 8°C. The
autosampler aspirates 300µL of supernatant using the µSPE tool,
selects a µSPE cartridge packed with 15 mg of CEC18 from the
cartridge tray, and elutes the extract through the cartridge at a flow
rate of 2 µL/sec. This loading amount and flow rate was based on
the work of Lehotay et al. (2016) [1]. The extract is collected at the
elution tray in a 2nd 2 mL autosampler vial with a 350 µL glass
insert. After the extract is collected, the µSPE cartridge is returned
to the cartridge tray. The autosampler then transfers 100 µL of the
extract to a 3rd vial with insert in the cooled stack, then adds 33 µL
of Mobile Phase A from the solvent module to the 3rd vial and mixes
the solution. The autosampler exchanges the µSPE tool for the
LCMS injection tool. The autosampler aspirates 6 µL of the final
diluted extract and injects into a 2 µL sample loop on the injection
valve. The whole process takes 8.5 minutes to complete. A
photograph of the autosampler with its modules is shown in Figure
1, while a detail of the extraction is shown in Figure 2.
LC Separation
HPLC Thermo Scientific Ultimate™ 3000 HLPC pump,
column oven, and degasser unit.
Column Thermo Accucore™ VDX column (100 mm x 2.1 mm
x 2.6 µm, P/N VDX-102130).
Mobile Phase A Water with 0.05 % Formic Acid
Mobile Phase B 47.5% Methanol, 47.5% Acetonitrile, 5%
Water, 0.05% Formic Acid
HPLC Gradient
Mass Spectrometry
Thermo Scientific TSQ Fortis™ triple quadrupole mass
spectrometer operating in SRM mode to monitor ~130 Veterinary
Drug compounds. A total ion chromatogram is shown in Figure 3.
Data Analysis
Samples were acquired and processed with Thermo Scientific™
TraceFinder 4.1 Software.
Time
(min)
Flow
(mL/min)
%B
0 0.3 2
2 0.3 2
3 0.3 20
11 0.3 100
13 0.4 100
14.4 0.4 100
14.5 0.35 2
16 0.3 2
17 0.3 2
Tool park
• LCMS Tool
• μSPE Tool (D8/57)
• Additional D7/57 syringe
Tool for internal standard
addition
Peltier Stack 6DW
• Higher sample capacity
• Sample temperature control
Dedicated μSPE handling tray holder
• 2x VT54 rack
• Elution/elute tray cover
• μSPE cartridge tray
Solvent Module
3x 100 mL Bottles
Fast Wash
Station
Valve Drive module
• 6-port Injection valve
• includes 2 μL loop
PAL terminal
Step Step Description Time
(min)
1 Prep and Label Tubes 40
2 Weigh Samples 45
3 Spike Samples 25
4 Add Ammonium Oxalate/EDTA 6
5 Add Acetonitrile 10
6 Multi Tube Vortex Homogenization 15
7 Add NaSO4 30
8 NaSO4 Wait, then Centrifuge 50
9* Decant to Falcon Tube, add dSPE 20
10* Multi Tube Vortex with dSPE 15
11* Centrifuge 15
12* Dilute 3:1 Extract:Mobile Phase A 15
13* Transfer to Autosampler Vial 15
Table 1. Extraction steps and timing for each step. *Steps 9-13 are
automated in the automated µSPE experiment.
Results
Timing of 15 Sample Extracts
A batch size of 15 samples was timed to compare manual to
automated SPE cleanup. The time required for each step is listed in
Table 1.
1 Load and elute sample onto μSPE cartridge
Cleaned
extract
Elution Tray
Eluate Tray
2 Get cleaned extract, dilute.
Figure 2. Side profile of automated sample loading onto µSPE
cartridge, elution, and transfer of cleaned extract to dilution vial
(not pictured) prior to dilution and injection.
Results, continued
Recovery
Extraction recovery was calculated for 13 compounds, shown in
Table 2. Extraction recovery was determined by comparing the
quantitated amount for triplicate injections at a concentration of 5
ng/g cleaned up using µSPE compared to a direct injection of the
same extract. The intended veterinary use for each compound is
also listed. For the 5 ng/g concentration, recoveries ranged from
49.2% for Clopidol, to 144.5% for Ketoprofen. Recoveries are
skewed high because analyzing raw extracts that were not
subjected to SPE cleanup of any kind resulted in ion suppression
compared to the samples that were cleaned up via µSPE.
Table 2. Extraction recoveries for a range of veterinary drug
compounds calculated at the 5 ng/g concentration level.
Veterinary
Drug
Usage Recovery
%
Acepromazine Sedative 88
Albendazole Anthelmintic 120
Azaperone Sedative 92
Brilliant Green Dye 123
Chlorpromazine Anti-nausea/Muscle relaxant 114
Clopidol Coccidiostat 84
Derquantel Anthelmintic 69
Difloxacin Antibiotic 75
Ipronidazole Antiprotozoal 95
Ketoprofen NSAID 128
Thiabendazole Anthelmintic 175
Victoria blue bo Dye 175
Xylazine Adrenegic agonist 117
Figure 3. Total Ion Chromatogram for the µSPE cleaned sample at
a concentration of 100 ng/g.22
In today’s fast-paced analytical laboratories,
efficiency and precision are key to meeting the
growing demands of modern science. For Daniela
Rechsteiner and her team, adopting Micro Solid
Phase Extraction (µSPE) technology brought
significant improvements to their pesticide analysis
workflow. In this interview, Daniela shares her
insights on what led her lab to embrace µSPE, the
challenges they overcame during the transition
and how the technology has not only streamlined
their processes but also opened doors for future
applications in food safety testing.
Q: What initially led you to consider
adopting µSPE technology in your lab?
Daniela Rechsteiner (DR): We wanted to reduce
manual work in our processes and improve the
precision of our cleanup procedures. Additionally,
the fact that National Reference Laboratories
(specialist laboratories responsible for maintaining
standards for routine testing of feed, food and
animal health) are already using µSPE was another
significant factor in our decision to adopt it.
Q: What were the pain points in your
previous workflow and how does
µSPE improve your current workflow?
DR: One of the main issues we faced was the
manual preparation of cleanup materials like
primary secondary amine (PSA) and magnesium
sulfate mixtures. We used to purchase raw
materials and mix them ourselves, which was quite
labor-intensive. Our technicians had to prepare
these mixtures and then manually transfer the
cleanup to high-performance liquid chromatography
(HPLC) vials, which added a lot of steps to the
process. This step was repetitive and required
precision. µSPE significantly reduces this manual
labor, making the process more efficient by
Streamlining Lab Workflows: Practical Insight Into How
Micro-SPE Transforms Pesticide Analysis
Daniela
Rechsteiner, PhD23
Q: Has µSPE affected the sustainability
of your lab practices, or changed your
lab’s environmental footprint?
DR: Honestly, not much. The cartridges we use
come with a lot of packaging material, and we still
require 10 millilitres of acetonitrile for extraction,
so we haven’t been able to reduce the solvent
volume yet. That said, we do use less PSA and
magnesium sulfate because those cleanup materials
are no longer required with µSPE.
Q: How do you see the role of µSPE
evolving in your lab’s future work?
Are you planning any upcoming
projects where you anticipate its use?
DR: We have some interesting projects coming
up. For example, we are exploring the possibility
of using µSPE for cleaning up polycyclic aromatic
hydrocarbons (PAHs) in chocolate. Currently, we use
gel permeation chromatography (GPC) to remove
fats, but we’re also investigating if µSPE can help
with this. Likewise, we analyze veterinary drugs, and
I see the potential for µSPE in that area too.
Q: What advice would you give to labs
considering adopting µSPE?
DR: Pay close attention to the details. Make sure
you have the right syringes, vials and vial caps,
and that your workflow is meticulously planned.
It’s the small details that can slow down adoption.
In our case, it took us a while to adjust to all the
specifics of the method. Finally, it is also worth
mentioning that the support we received from
CTC was invaluable throughout the process. They
were always available to assist, whether it was for
programming or troubleshooting, and they were
very responsive to our needs.
eliminating the need for manual filling and reducing
the overall workload.
Q: How was the transition from
traditional SPE methods to µSPE?
Were there any challenges when
adopting the new method?
DR: Our transition had some unique challenges
because we wanted to use µSPE offline, specifically
for GC/MS analysis, rather than injecting directly.
We had to program our instruments with CTC
Analytics to handle µSPE according to our needs.
One of the issues we faced was handling the small
sample volumes – splitting the cleaned extract
for different instruments was tricky. Validation
was another challenge, as performance varies for
different pesticides across both µSPE and traditional
methods. We had to figure out how to conduct the
method comparison and decide when performance
differences were acceptable from a validation
perspective.
Q: What types of samples and
analyses do you primarily use µSPE
for? How many samples do you
analyze per day?
DR: We use µSPE to analyze pesticides, specifically
in a multi-method for 400 pesticides. Our samples
include fruits, vegetables, dried fruits, nuts and
spices. On average, we process about 50 samples
per week.
Q: What are the most common causes
of errors in an analytical workflow?
What problems do they cause?
DR: Common errors in our workflow often
occurred during the manual extraction, cleanup
and transfer to HPLC vials. Technicians might
mix up samples, perform the steps in the wrong
sequence or forget to add important elements like
the internal standard or matrix protectants. We also
encountered weighing errors. Another issue with
µSPE was the potential to use the wrong cartridge
or have it in the wrong position, such as using a
fruit-specific cartridge for a nut sample, which could
affect results.24
The following collection of content highlights
workflows that are essential for achieving
consistent and efficient sample preparation in
modern analytical laboratories. As emphasized
by Daniela Rechsteiner in the previous interview,
a critical starting point in many workflows is the
loading of the QuEChERS extract onto the cartridge
for µSPE clean-up. This step ensures optimal
removal of matrix interferences and prepares the
sample for accurate analysis.
For some workflows, such as those detailed in
“Fully Automated QuEChERS Extraction and
Clean-Up for Homogeneous Matrices”, the process
begins even earlier, with the QuEChERS extraction
itself being automated on the PAL System. This
integration streamlines sample preparation,
saving time and reducing manual handling errors.
Both approaches underscore the flexibility and
robustness of the PAL System in accommodating
diverse application needs while maintaining high
analytical standards.
The following content offers a wealth of insights
into these innovative techniques and their practical
applications.
Transitioning Workflows:
From QuEChERS Extraction
to µSPE CleanupConclusion
A fully automated QuEChERS based on the PAL
RTC system is stable and reliable in the analysis
of organochlorine and organophosphate pesticides
from tomato juice and red wine.
Overview
Using PAL Robotic Tool Change (RTC) system, a fully
automated QuEChERS was developed for the extraction and
clean-up of organochlorine and organophosphate pesticides
from homogeneous samples. The automated QuEChERS
workflow includes extraction with acetonitrile, salting out with
saturated sodium chloride solution and the clean-up with PAL
µSPE prior to injection into the GC-MS/MS for analysis.
Method validations were achieved by using the automated
matrix matches calibration spiked from 1 ng/mL to 100 ng/mL
of organochlorine and organophosphate pesticide standards
into the PAL µSPE-cleaned tomato juice and red wine samples.
The matrix match calibration curve linearities were at R2 0.995
or better. By spiking 10 ng/mL of pesticide standards into a raw
tomato juice and red wine, the recoveries were obtained in the
range of 70% - 130%, with n = 6 samples to determine the
automated workflow precisions.
Introduction
QuEChERS is the quick, easy, cheap effective, rugged and
safe sample preparation method developed by M.
Anastassiades and S.J. Lehotay in 2003. Since then, this
technique has become the widely used sample preparation
approach in pesticide residue analyses. According to the
QuEChERS website, about 45 min are needed to manually
prepare 8 samples in the laboratory [1]. In the QuEChERS
method, acetonitrile is used as extraction solvent, followed by
adding NaCl and buffer salts. After shaking and centrifugation
traditionally dispersive solid phase extraction (dSPE) is used
for extract clean-up before analysis by GC-MS or LC-MS. High
matrix load in GC-MS, the matrix effects in LC-MS, enhancing
or suppressing analyte signals, varying extraction recoveries
were major challenges that were approached to overcome by
many matrix specific modified clean-up agents and procedures.
Sample Evaluation
The organochlorine and organophosphate
pesticide recoveries were evaluated based on preand post-spike of pesticide standards into the
tomato juice and red wine samples, respectively.
Matrix match calibration curves were generated by
using the PAL RTC System to spike seven
different concentrations ranging from 1 ng/mL to
100 ng/mL automatically into the cleaned tomato
juice and red wine samples (prepared by the
automated QuEChERS).
A series of five to six samples, spiked with
10 ng/mL organochlorine and organophosphate
pesticide standards, were processed the full
automated QuEChERS workflow to determine the
recovery and precision based on the matrix match
calibration curve. The detailed results of matrix
match calibration curve linearity, precision and
recovery of this fully automated QuEChERS
sample preparation of tomato juice and red wine
are listed in Table 1.
Fully Automated QuEChERS for Organochlorine and
Organophosphate Pesticides in Tomato Juice and Red Wine
Chiew Mei Chong, Gwen Lim Sin Yee, Hans-Joachim Huebschmann
CTC Analytics Asia Ple. Ltd., 117610 Singapore
Contact: GLIM@CTC.CH
Analytical Strategy
A cartridge based miniaturized solid phase extraction, known
as PAL µSPE, was fully automated by using the PAL RTC
system to perform the fully automated QuEChERS preparation
workflow.
Using PAL µSPE in Matrix Clean-Up
Figure 1. Principle of the PAL µSPE clean-up
Vortex
Mixer
Solvent
Module
Fast Wash
Module Tray Holder
Tool
Park Station
Figure 3. PAL RTC system configuration for the fully automated QuEChERS analysis
Automated QuEChERS Workflow in Analyzing Tomato Juice and Wine Samples
Instrument Setup
The fully automated workflow, including sample blank, post
spike with calibration and internal standards was carried out
with the instrument setup as shown in Figure 3. The
automated QuEChERS was established by the PAL RTC
system with a vortexing module, solvent module to store up to
100 mL of acetonitrile and saturated sodium chloride solution,
a fast wash module with acetonitrile and water for active
syringe wash. In the Tray Holder, as shown in Figure 3, rack 1
was used to place the tomato juice and red wine samples in 2
mL vials. The clean up cartridges were placed on the
dedicated cartridge holder in rack 3. Empty vials with slitted
septa were placed at the center rack 2 underneath of the
aluminium vial cover to receive cleaned extract.
Results
Figure 5. QuEChERS in tomato juice
Figure 7. Total Ion Chromatograms (TICs) of 100ng/mL organochlorine and organophosphate pesticide standards by the GC-MS/MS.
Table 1. Linearity, precision, and recovery of the organochlorine and organophosphate pesticides in
tomato juice and wine based on the fully automated QuEChERS sample preparation workflow and
GC-MS/MS analysis.
Tomato and wine samples
Cleaned extract
PAL µSPE cartridges
For the full report please request from GLIM@CTC.CH.
References
[1] QuEChERS Home Page https://www.quechers.com/index.php
(accessed Sep 7, 2020)
An optimized sorbent bed for GC QuEChERS analysis,
containing a mixture of 45 mg of MgSO4, PSA, C18EC and
CarbonX was filled by CTC Analytics into proprietary PAL µSPE
cartridges. This PAL µSPE cartridges work in scavenging mode
to retain the sample matrix inside the sorbent bed. A clean
extract delivering the pesticide compounds is eluted into empty
vials beneath the cartridges for online injection to GC-MS.
Pos1: Acetonitrile
Pos2: NaCl(sat.)
Pos3: Not use
Tomato juice Tomato juice
+
Acetonitrile
Tomato juice
+
Acetonitrile
+
NaCl (sat.)
Figure 6. QuEChERS in wine
Compounds
(based on elution
order)
Tomato Juice Red Wine
Linearity Precision
%RSD (n=5) Recovery Linearity %RSD (n=6) Precision Recovery
Methacrifos 0.9995 8% 129% 0.9984 24% 103%
Sulfotep 0.9990 7% 94% 0.9982 16% 87%
alpha-BHC 0.9977 13% 104% 0.9924 20% 101%
beta-BHC 0.9956 6% 115% 0.9958 12% 115%
delta-BHC 0.9966 7% 118% 0.9956 11% 115%
Terbufos 0.9994 8% 108% 0.9953 14% 81%
gamma-BHC 0.9991 9% 87% 0.9979 13% 113%
Tolclofos-methyl 0.9998 5% 107% 0.9998 10% 114%
Heptachlor 0.9972 5% 99% 0.9969 20% 84%
Ronnel 0.9990 4% 105% 0.9970 10% 112%
Aldrin 0.9989 15% 85% 0.9973 21% 72%
Malathion 0.9972 9% 86% 0.9960 13% 115%
Fenthion 0.9964 10% 112% 0.9986 11% 106%
Bromophos 0.9990 8% 104% 0.9994 9% 109%
Heptachlor epoxide 0.9961 13% 84% 0.9973 13% 86%
Clofenvinfos 0.9608 12% 89% 0.9971 13% 111%
Bromophos-ethyl 0.9977 3% 86% 0.9995 8% 68%
Endosulfan I 0.9959 5% 79% 0.9997 16% 77%
tChlordane 0.9970 4% 115% 0.9985 17% 81%
Bromfenvinphos 0.9761 13% 80% 0.9989 28% 103%
Iodofenphos 0.9966 18% 91% 0.9993 16% 82%
Prothiofos 0.9989 10% 79% 0.9998 14% 69%
4,4-DDE 0.9982 15% 83% 0.9997 19% 86%
Profenofos 0.9865 10% 86% 0.9995 11% 94%
Dieldrin 0.9965 6% 112% 0.9985 18% 75%
Endrin 0.9968 12% 84% 0.9993 14% 101%
Endosulfan II 0.9984 6% 94% 0.9981 14% 107%
Chlorthiophos III 0.9982 11% 76% 0.9988 9% 64%
4,4'-DDD 0.9988 6% 102% 0.9985 15% 88%
Ethion 0.9980 6% 111% 0.9993 14% 83%
Endrin aldehyde 0.9988 3% 55% 0.9712 30% 91%
Chlorthiophos 0.9956 11% 81% 0.9992 13% 61%
Sulprofos 0.9990 8% 94% 0.9990 17% 76%
Endosulfan sulfate 0.9986 6% 115% 0.9996 13% 109%
Carbophenothion 0.9963 6% 92% 0.9990 11% 74%
4,4-DDT 0.9987 2% 147% 0.9990 19% 94%
Endrin ketone 0.9962 12% 95% 0.9985 11% 83%
Methoxychlor 0.9966 15% 136% 0.9979 18% 99%
Leptophos 0.9953 19% 59% 0.9967 6% 57%
Prior to analyzing the tomato juice and red wine samples, a Shimadzu GCMS-TQ8040, equipped with Rxi-5Sil MS (30 m x 0.25 mm x
0.25 µm) capillary column was optimized to achieve the detection limit to at least 1 ng/mL. A full MRM total ion chromtogram (TIC) of
the 100 ng/mL organochlorine and organophosphate standards is shown as Figure 7. Upon completed QuEChERS extraction and
clean-up, the tomato juice and wine samples were injected at 3 µL into this optimized GC-MS/MS system for analysis.
The only manual step in the automated QuEChERS workflow was a pipetting of
500 µL of sample into 2 mL autosampler vials. The subsequent steps such as
adding Acetonitrile, salt solution and clean-up by PAL µSPE were carried out
automatically by the PAL RTC system based on the workflow shown in Figure 4.
The organic and aqueous phases were well separated after adding in the
saturated salt solution. No centrifugation was required. The PAL µSPE cartridge
with MgSO4, PSA, C18EC and CarbonX sorbents provided a high clean-up
efficiency, as shown in Figure 5 for tomato juice and Figure 6 for red wine
samples.
Add 500 µL sample
into sample vial
Add 750 µL Acetonitrile
into sample vial
Vortex sample vial
at 1000 rpm
Add 250 µL saturated
NaCl solution
into sample vial
Vortex sample vial
at 2000 rpm
Wait 90 s for
phase separation
Take 300 µL of
organic layer (upper)
Clean-up through
PAL µSPE
Inject cleaned extract online
into GC-MS/MS for analysis
Tomato juice
(after PAL µSPE cleanup)
Wine Wine
+
Acetonitrile
Wine
+
Acetonitrile
+
NaCl (sat.)
Wine
(after PAL µSPE cleanup)
Figure 4. Automated QuEChERS workflow by PAL RTC System
Figure 2. Red wine sample, before and after the PAL µSPE clean-up
Before After
Methacrifos
Sulfotep
alpha-BHC
beta-BHC
delta-BHC
Terbufos
gamma-BHC
Tolclofos-methyl
Heptachlor
Ronnel
Aldrin
Malathion
Fenthion
Bromophos
Heptachlor epoxide
Clofenvinfos
Bromophos-ethyl
Endosulfan I
Chlordane
Bromfenvinphos
Iodofenphos
Prothiofos
Profenofos
4,4’-DDE
Dieldrin
Endrin
Endosulfan II
Chlorthiophos II
4,4’-DDD
Ethion
Endrin aldehyde & Chlorthiophos
Sulprofos
Endosulfan sulfate & Carbophenothion
Edifenphos
4,4’-DDT
Triphenyl Phosphate (ISTD)
Endrin Ketone
Methoxychlor
Leptophos
Presented at the analytica Vietnam 2023,
Ho Chi Minh City, Vietnam
2526
GC/MS Application Note
Fully Automated QuEChERS Extraction and
Clean-up for homogeneous matrices
www.palsystem.com27
2 IngeniousNews 02/2021 IngeniousNews 02/2021
Fully Automated QuEChERS Extraction and Clean-up for
homogeneous matrices
Chiew Mei Chong, Hans-Joachim Hübschmann, CTC Analytics Pte. Ltd., Singapore
A fully automated, true green analytical method, for high
sensitivity and precision pesticide analysis. This report describes
for the first time a fully automated QuEChERS extraction and
clean-up workflow for homogeneous matrices like fruit juices,
demonstrated for orange juice.
Introduction
A fully automated QuEChERS extraction and extract cleanup method for GC-MS and LC-MS analysis is presented by
using a PAL RTC robotic sampling system. QuEChERS is the
well-established quick, easy, cheap effective, rugged, and safe
pesticide extraction procedure, developed by M. Anastassiades
and S.J. Lehotay in 2003 1 and has become a widely used
sample preparation approach in pesticides residue analyses.
The steps for pesticide residue analysis start with the
representative sampling and comminution pre-treatment, the
necessary manual steps to provide a homogeneous subsample
for processing. Vegetable and fruit juices are considered
homogenous after thorough shaking the commercial
packaging, the bottles or carton packages, before transferring
an aliquot to analysis vials.
Only 0.5 mL of homogenized juice are transferred into a regular
2 mL autosampler vial for the automated extraction, cleanup and online analysis by LC-MS or GC-MS. The raw extract
clean-up and removal of the high matrix load are achieved by
using micro-SPE cartridges (µSPE). The advantage of µSPE is
the straightforward separation of the pesticide fraction from
the matrix by elution of the pesticide fraction through a small
sorbent bed keeping the matrix behind. Solvent evaporation
in this micro method is completely avoided, keeping the initial
concentration level of the pesticides, providing high recoveries.
The automated extraction and clean-up process takes only a
few minutes and is well compatible with chromatographic
runtimes. A prep-ahead mode allows the processing already
of a next sample during the chromatographic run.
PAL System Configuration
A PAL RTC System with automated tool change was employed.
The system configuration as shown in Figure 1 further
comprises a vortex mixer, solvent and wash modules as well
as trayholder with the vial racks for the sample and extract
vials, and the micro-SPE clean-up cartridges.
For the automated QuEChERS extraction, a saturated NaCl
solution is provided in the solvent module. The acetonitrile
for extraction is provided with a fast wash module from an
external reservoir.
The workflow includes the addition of internal standards and
optional analyte protectants (APs) as well as the automated
preparation of the pesticide calibration from reference
standards as well. The same vial rack carries the sample vials,
empty vials to collect the cleaned extract, as well the necessary
µSPE cartridges for the clean-up, shown in Figure 2.
Automated Workflow
The automated analysis of pesticides from juices comprises
five stages:
• Preparation of the calibration standards
• Standards addition
• Extraction with acetonitrile
• Extract clean-up
• GC-MS and/or LC-MS injection and analyses
The first part with a fresh preparation of the calibration curve
can be used optionally, as well as the addition of internal
standards to the sample. Typically, commercial multi-residue
pesticide standards are applied. The dilution of standards is
achieved in routine by entering the desired dilution factors.
The automated extraction and clean-up workflow up to a GCMS injection is illustrated in Figure 3. The QuEChERS extraction
step is performed by intense vortexing using the original high
NaCl salting-out conditions. A pH adjustment as of AOAC
2007.01 or EN 15662 methods can be achieved by providing
the required buffer salts in accordingly prepared vials before
adding a juice sample.
The extract clean-up is achieved by applying the raw extract
after phase separation to µSPE cartridges. Here the syringe
1 3
2
4
5
7
6
1 Tool park station for 3 syringes
2 Vortex Mixer module
3 Solvent module
4 Head of robotic sampler with tool
5 Fast Wash module
6 Tray Holder for vial racks and
μSPE cartridges
7 Handheld terminal
Figure 1. Configuration of the PAL RTC System for the automated QuEChERS extraction and clean-up of juice samples.
Rack #1 Rack #2 Rack #3
Calibration standards and internal standards
Juice samples
Cleaned extract
μSPE cartridges
Figure 2. Trayholder top view showing the rack placement of standards, samples, cleaned extracts and the µSPE cartridge reservoir.4 28 IngeniousNews 02/2021 IngeniousNews 02/2021 5
works like an LC pump and pushes the extract in constant slow
flow of 2 µL/s through the cartridge (see the CTC Newsletter
IngeniousNews 02/2015). The pesticides fraction elutes first
leaving the sample matrix behind on the cartridge. The cleaned
extract is collected in empty vials on the same trayholder.
The sorbent material mix of the clean-up cartridge is optimized
for GC-MS and LC-MS analysis 2 . A big benefit of the optimized
sorbent material mix for laboratory logistics is the wide
versatility of the cartridges for any kind of food samples. This
also includes high fat content and spice containing samples
making any further modification of the sorbent material mix
for different kind of sample matrices unnecessary 3, 4 .
In the online configuration to GC-MS and LC-MS, every sample
is processed on an identical time axis within 5 to 7 minutes.
A so-called ‘prep-ahead’ mode allows the processing of the
next same during analysis of the previous one, as shown in
Figure 3. The ‘prep-ahead’ mode increases sample throughput
significantly and maximizes the duty cycle of the connected
analysis system. The described workflow integrates into the
chromatography data systems of the leading instrument
manufacturer for GC-MS and LC-MS.
Experimental
The only manual step in the project was transferring orange
juice from the well-shaken bottle into 2 mL sample vials.
The subsequent QuEChERS extraction steps, such as adding
acetonitrile, adding saturated sodium chloride salt, clean-up
and injection into the GC-MS/MS are all carried by the PAL RTC
System with the aid of the PAL Method Composer software
(PMC) to build the automation workflow.
Analysis Parameter
PAL RTC Robotic Sampler
Sample volume 400 µL
Standard volumes 50 µL each, for calibration and ISTD
MeCN volume 3x 200 µL (extraction solvent)
Salting-out 200 µL (NaCl sat.)
Vortexing speed, time 1500 rpm, 60 s
Extract clean-up 250 µL raw extract (applied to µSPE)
Clean-up flow 2 µL/s
GC-MS System
(Triple quadupole GC-MS System Shimadzu TQ8040)
GC Conditions
Inlet Temperature 250°C
Inlet Mode Splitless
Injection Volume 3 µL
Column SH-Rxi-5Sil MS, 30 m x 0.25 µm x 0.25 mm
Oven Temperature 50°C (2 min),
30°C /min to 75°C (1 min),
4°C/min to 250°C (1 min),
20°C/min to 300°C (0.92 min)
MS Parameter
Ion source Temp. 250°C
Detection MRM mode, as of manufacturer pesticide
database
Results and Discussion
For the automated extraction the sample size of the
homogeneous juice sample is scaled down from the usual
sample amount of 10 g to only 400 to 500 µL of juice.
Figure 4 shows the orange juice sample in undergoing
QuEChERS sample preparation and clean-up steps. After adding
the saturated sodium chloride, two liquid layers are formed, in
which, after vortexing and sedimentation, the upper layer is the
raw extract of acetonitrile. An aliquot of this extract is transferred
to the µSPE cartridge for clean-up. The clean-up effect can
visually be very well noticed by the removed colourants.
A group of organophosphate pesticides was evaluated based
on pre-spike and post-spike of pesticide standards into the
juice samples. By using the PAL Method Composer software,
the pre-spike and post-spike steps can be integrated optionally
into the automation workflow.
Chromatograms and Calibration Curves
A total ion chromatogram of the extracted orange juice after the
automated QuEChERS extraction and clean-up using the postspike of standards with 100 ng/mL of the organophosphate
pesticide compounds is shown in Figure 5.
The calibration was prepared in a concentration range from
1.0 to 100.0 ng/mL with the standards post-spiked into a blank
and µSPE cleaned orange juice extract. A very good linearity
with correlation coefficients better than 0.995 for all the
investigated organophosphorus pesticides was achieved. The
calibration curves of the late eluting compounds piperonyl
butoxide, leptophos and coumaphos are shown representative
for the group of compounds in Figure 6.
Pre- and post-spiked data from seven consecutive sample
runs were used to calculate the method recovery values and
method detection limits (MDL) listed in Table 1. The resulting
data show a high recovery between 71% and 114% for all
pesticides investigated. The MDLs confirm a very good and
regulation-compliant sensitivity of the described method.
Selected real-life mass chromatograms of the lower recovery
and late eluting compounds at the 10 ng/mL decision level are
shown in Figure 7.
Figure 3. Automated workflow for juice extraction, extract clean-up and GC
injection
Figure 4. Workflow steps visualized in the 2 mL vials of the automated juice extraction and clean-up
(a) (b) (c) (d)
a Orange juice from juice box c Orange juice + acetonitrile + NaCl sat. phase separation
b Orange juice + acetonitrile, vortexed d Cleaned extract after µSPE step, injected6 IngeniousNews 02/2021 IngeniousNews 02/202129 7
Compound Name Retention Time
(min)
Linearity
1 ng/mL - 100 ng/mL
Pre-Spike at 10 ng/mL
%RSD
(n=7)
Recovery
Post-Spike at 10 ng/mL
%RSD
(n=6)
MDL
(ng/mL)
Methacrifos 20.167 0.9985 8.7% 114 % 7.8% 3.1
Sulfotep 25.200 0.9989 9.7% 106 % 8.2% 3.2
Phorate 25.581 0.9988 10.9% 115 % 8.8% 4.0
Terbufos 27.816 0.9972 7.0% 91 % 6.9% 2.0
Fonofos 27.884 0.9979 8.4% 115 % 10.7% 3.1
Disulfoton 28.662 0.9980 4.8% 110 % 11.6% 1.7
Tolclofos-methyl 30.770 0.9982 5.9% 91 % 6.3% 1.7
Fenchlorphos (Ronnel) 31.293 0.9966 7.2% 95 % 6.0% 2.1
Malathion 32.620 0.9960 12% 108 % 11% 4.1
Fenthion 33.014 0.9962 6.3% 91 % 5.6% 1.8
Parathion 33.168 0.9974 10% 99 % 8.1% 3.1
Bromophos methyl 33.789 0.9977 7.0% 90 % 5.9% 2.0
Bromfenvinfos-methyl 34.888 0.9974 8.3% 82 % 7.7% 2.1
Chlorfenvinphos 34.952 0.9977 7.8% 91 % 2.9% 2.2
Bromophos-ethyl 35.847 0.9976 7.6% 81 % 2.1% 1.9
Tetrachlorvinphos 36.167 0.9985 7.7% 86 % 9.7% 2.1
Bromfenvinphos 36.814 0.9990 9.1% 88 % 4.7% 2.5
Iodofenphos 36.938 0.9971 9.4% 76 % 8.4% 2.3
Fenamiphos 36.951 0.9976 7.8% 85 % 10% 2.1
Prothiofos 37.168 0.9962 9.2% 74 % 5.8% 2.1
Profenofos 37.366 0.9989 10% 87 % 6.7% 2.6
Ethion 39.571 0.9957 7.5% 76 % 3.9% 1.8
Chlorthiophos 39.694 0.9950 7.9% 80 % 1.7% 2.0
Triazophos 40.227 0.9982 7.9% 88 % 8.4% 2.2
Sulprofos 40.252 0.9968 8.5% 84 % 2.3% 2.2
Carbophenothion 40.675 0.9984 8.2% 74 % 6.4% 2.0
Edifenphos 40.729 0.9960 9.9% 80 % 8.8% 2.5
Piperonyl butoxide 42.393 0.9993 8.5% 86 % 6.9% 2.3
Leptophos 44.947 0.9982 9.1% 71 % 6.9% 2.0
Coumaphos 47.854 0.9947 9.7% 80 % 8.3% 2.5
Conclusion
The fully automated QuEChERS extraction and clean-up
procedure frees up resources in the routine laboratory. The
industry standard PAL RTC x, y, z-sampling system provides
a reliable method for pesticides analysis of homogeneous
samples as shown for organophosphates pesticides analysis
from orange juice. The typical high amount of solvents,
glassware and consumables required for pesticides analysis is
significantly reduced providing a true green analytical method.
The automated method avoids solvent evaporation steps,
uses only one cartridge type for all matrices, and is fast to be
executed online during a chromatographic run in ‘prep-ahead’
mode optimizing the sample throughput of the MS detection
system in use.
The analytical data show an excellent sensitivity for the
investigated organophosphates pesticides with MDLs in the
Figure 5. Total ion chromatogram of post-spiked orange juice (100 ng/mL) after the automated QuEChERS extraction and µSPE clean-up.
Figure 6. Linear calibration curves post-spiked into µSPE cleaned orange juice extracts of the late eluting compounds piperonyl butoxide, leptophos and coumaphos.
Figure 7. Real-life mass chromatograms (3 MRM transitions each) at the 10 ng/mL decision level, (a) spike 10 ng/mL, (b) blank run.
Table 1. Linearity, precision, recovery and method detection limits (MDL) of the organophosphates pesticides investigated automatically extracted from orange juice.30
Imprint
Date of print: 05.2021
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range of 3 to 4 ng/mL. The quantitative calibration is linear
in the range of 1 to 100 ng/L. The method precision at the
decision level is excellent with less than 10% RSD for all
compounds, making this automated method a well suitable
solution for the pesticide analysis of homogeneous juice
samples. The described automated extraction and clean-up
workflow can be applied for unattended online GC-MS and
LC-MS analysis.
The original full text publication5 can be downloaded
from the LC-GC website at
https://www.chromatographyonline.com/view/fullyautomated-quechers-extraction-and-cleanup-oforganophosphate-pesticides-in-orange-juice
1 Anastassiades, M., S.J. Lehotay, D. Stajnbaher and F.J. Schenck. 2003.
“Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/
Partitioning and ‘Dispersive Solid-Phase Extraction’ for the Determination of
Pesticide Residues in Produce”. JAOAC Int 86(2) 412-31.
2 Bruce D. Morris and Richard B. Schriner, J. Agric. Food Chem. 2015, 63,
5107−5119.
3 Steven J. Lehotay, Lijun Han, Yelena Sapozhnikova, Automated Mini-Column Solid-Phase Extraction Cleanup for High-Throughput Analysis of Chemical Contaminants in Foods by Low-Pressure Gas Chromatography-Tandem Mass Spectrometry, Chromatographia, 13.06.2016, DOI 10.1007/
s10337-016-3116-y.
4 Goon A., Shinde R., et al. 2019. “Application of Automated Mini–Solid-Phase
Extraction Cleanup for the Analysis of Pesticides in Complex Spice Matrixes
by GC-MS/MS”. J. AOAC Int. 103(1) 40–45. DOI https://doi.org/10.5740/
jaoacint.19-0202
5 Chong, C.M., Hubschmann, H.-J. (2021). Fully Automated QuEChERS Extraction and Cleanup of Organophosphate Pesticides in Orange Juice. LCGC
Special Issue 04-01-2021 (39) 12–16.31
GC/MS Application Note
www.palsystem.com
Routine Pesticide Analysis using
Micro-SPE2 32
Routine Pesticide Analysis using Micro-SPE
Andreas Schürmann1, Claudio Crüzer1, Veronika Duss1, Thomi Preiswerk2, Hans-Joachim Huebschmann2
1) Cantonal Laboratory Zürich, Official Food Control Authority of the Canton of Zürich, Department Pesticide Analysis, Zürich, Switzerland. 2) CTC Analytics AG,
Zwingen, Switzerland.
Introduction
The department of pesticides analysis of the Official Food Control Authority of the Canton of Zürich in Switzerland examines
goods from production and trade and analyses food for pesticide residues. Starting from Steve Lehotay’s publication1 and
presentation at the 5th Latin American Pesticide Residue Workshop (LAPRW), the department of pesticide analysis introduced
the automated clean-up of extracts in their routine lab as early as 2020. The methodology of generic extraction with ethyl acetate
is well established for many years already, but clean-up procedures turned out to be a major obstacle for the steadily increasing
sample throughput due to the additional manual workload addressing separately the different kind of food commodities of a
governmental laboratory. Two years of experience led to a thorough understanding of the new automated micro-SPE (µSPE)
clean-up workflow and its successful implementation into the laboratory routine procedure for pesticides analysis. This report
presents for the first time the application of µSPE for the clean-up of the raw extracts using ethyl acetate as extraction solvent for
pesticides from different also more complex food commodities.
Keywords
food, pesticides, multi-compound method, ethyl acetate extraction, automated clean-up, micro-SPE, high lipid content, avocado, liver, spices, cereals, captan, folpet, PTV, GC-MSMS
Project Goal
Generic extraction methods for food have found their solid place for the multi-compound pesticides analysis in private,
industrial, and governmental laboratories. Ethyl acetate and acetonitrile extraction methods have been developed as fast and
easy to handle standard multi-compound methods. An up to recently unsolved bottleneck became the clean-up of the raw
extracts as the direct injection to GC is impaired by the high matrix content. Also, the LC analyses are affected by matrix effects
and frequent maintenance requirements. A suitable and also easy to handle clean-up procedure was missing to complement
the very capable extraction using acetonitrile (aka QuEChERS method ) or ethyl acetate (aka SweEt method2,3) as solvents.
Using the suggested dispersive SPE (dSPE), it turned out in practice that different food commodities required different adequate sorbent mixes to handle the many diverse matrix components (like chlorophyl, carbohydrates or lipids) of varying food
commodities optimally without losses of the target pesticides. It is reported that ethyl acetate achieves high recoveries also for
polar pesticides but also extracts a large amount of non-polar co-extractives, such as lipids and wax materials, which must be
removed before the chromatographic determination. Typically, an additional gel permeation chromatography (GPC) is applied
for ethyl acetate extracts as a clean-up method, in particular for fat containing samples4.
1 Anastassiades, M., Lehotay, S.J., et al. “Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and ‘Dispersive Solid-Phase Extraction’ for the
Determination of Pesticide Residues in Produce.” Journal of AOAC International, 86(2) 2003, 412–31.
2 Ekroth, S. „Simplified Analysis of Pesticide Residues in Food Using the Swedish Ethyl Acetate Method (SweEt)“. National Food Administration (NFA) Sweden, 2011.
http://www.laprw2011.fq.edu.uy/pdf/Lunes/Susanne Ekroth.pdf.
3 Ekroth, S. „The SweEt Method: An Efficient Alternative to Analyze Pesticide Residues in Food“. National Food Administration (NFA) Sweden, presented at the LAPRW
Conference, San Jose, Costa Rica, 2017.
4 Barceló, D. “Food Contaminants and Residue Analysis.” N.A., edited by Y. Picó, In: Comprehensive Analytical Chemistry, Vol. 51, Elsevier B.V., 2008.333
The goal of the project was to establish in the pesticides laboratory of the Official Food Control Authority of the Canton of
Zürich a generic clean-up procedure for the extracts of the applied ethyl acetate extraction method which is useful for all
incoming food types without requirement for a food type dedicated clean-up procedure. An automated extract clean-up
was envisioned for an improved sample throughput on the three already installed GC-MSMS systems providing less manual
variability and more reproducible pesticides recoveries. Based on the early publications by Bruce Morris and Richard Schriner
from Hill Laboratories, Hamilton, New Zealand5, as well as by Steve Lehotay, US Department of Agriculture, Wyndmoor, PA,
USA6, the application of the reported µSPE clean-up promised to be available as a viable solution.
Instrumentation
Three triple quadrupole GC-MSMS systems are in operation (TSQ 8000 Evo and TSQ 9000, Thermo Fisher Scientific, Austin,
TX, USA) equipped with TriPlus RSH robotic systems (Thermo Fisher Scientific, Austin, TX, USA) for online µSPE extract cleanup and injection (Figure 1). One unit carries in addition a headspace tool and agitator module for the determination of dithiocarbamate pesticides after acid cleavage and CS2 detection. Thanks to the automated tool change (ATC) both methods can
be executed without user interaction, the TriPlus RSH robot automatically selects the appropriate tool for online µSPE extract
clean-up or the acid cleavage for CS2 detection. The TraceFinder software (Thermo Fisher Scientific) is used for the execution
of the automated sample preparation workflow and the GC-MS instrument control, data acquisition and reporting.
5 Morris, B.D., and R.B. Schriner. “Development of an Automated Column Solid-Phase Extraction Cleanup of QuEChERS Extracts, Using a Zirconia-Based Sorbent, for
Pesticide Residue Analyses by LC-MS/MS.” J. Agric. Food Chem., 63 (2015) 5107−5119. doi:10.1021/jf505539e.
6 Lehotay, S.J., Han, L., et al. “Automated Mini-Column Solid-Phase Extraction Cleanup for High Throughput Analysis of Chemical Contaminants in Foods by Low- Pressure Gas Chromatography-Tandem Mass Spectrometry.” Chromatographia, 79 (2016) 1113–30. doi:10.1007/s10337-016-3116-y.
Figure 1. TriPlus RSH System for automated online µSPE extract clean-up
All GCs are equipped with a temperature programmable injector (PTV) allowing the injection at a low temperature of
55 °C for performance improvements of the lower volatile components. Excess solvent vapor of a 3 µL injection is vented by
applying 3 s of split flow, followed by a splitless completion of the vaporization and transfer to the column to minimize loss
of the higher volatile compounds. A DB-5ms Ultra Inert GC column (Agilent Technologies Inc.) is used with only 15 m length,
0.25 mm ID, and 0.25 µm film thickness. A standard baffled inlet liner without glass wool is used as displayed in Figure 2
(Restek Corporation, Bellefonte, PA, USA).
Each GC-MS system runs about 100 sample injections per week, in addition to the calibration and system suitability checks.4 34
Sorbent Bedmass Units %
PSA 12 mg 27
C18EC 12 mg 27
CarbonX 1 mg 2
MgSO4 20 mg 44
Total 45 mg 100
Figure 2. PTV inlet liner after more than 100 analyses during the weekly liner change.
As a result of the applied online µSPE raw extract clean-up, a liner exchange is performed only once a week, reducing system
downtime significantly. Even at the time of change after about 100 sample runs, the liner still appears to be clean without
visible residues, as shown in Figure 2.
The low matrix burden after the online µSPE raw extract clean-up also shows up with the extended lifetime of the GC column
in use. The column gets clipped about half a meter only after six months of use and more than 2600 sample analyses run on
the system. An MS ion source maintenance is performed, when deemed necessary, approximately once a month. Thanks to
the Thermo Fisher ‘Never Vent’® technology the ion source exchange is a maintenance procedure that only takes about two
hours until the system is ready again to process the next samples.
Workflow
Ethyl Acetate Extraction
The processing of food samples starts manually from a bulk sample by cryomilling to achieve a representative test portion.
From the homogenized test portion about 10 g are weighed into a regular 50 mL centrifuge extraction tube-containing 6 g
MgSO4 and 1.5 g sodium acetate. 10 mL of ethyl acetate are added, and the tube is shaken mechanically for 5 minutes. After
centrifugation 1 mL of the supernatant is transferred to 2 mL autosampler vials.
7 2 g for dry spices, 5 g for grain, dry samples wetted with water (10 mL) prior to extraction
Table 1. µSPE cartridge sorbent material composition355
Extract Clean-up
The automated procedure using µSPE cartridges (60101-45 GC Thermo Scientific GC SPE Cartridge) was established as
extract clean-up. The cartridges in use for the purification of the GC-MS samples contain 45 mg of a mixture of PSA, C18EC,
CarbonX and MgSO4 sorbent materials5, as specified in Table 1.
The configuration of the TriPlus RSH robotic system with the dedicated µSPE trayholder is shown in Figure 3. The vials with
the ethyl acetate raw extract are placed into slot 1 of the µSPE tray holder of the TriPlus RSH robot. Slot 3 in the front holds
the µSPE cartridges. The eluted and cleaned extracts are collected in empty 2 mL vials in slot 2 in the center of the tray holder.
The processing of the sample is executed serially including the online injection of the cleaned extract to GC-MS.
Figure 3. µSPE tray holder configuration with standards and solvents on the TriPlus RSH robotic system.
Raw extracts get processed on a self-controlled time axis of the TriPlus RSH robot so that the extract is ready for injection
when the GC Ready signal is expected. Extract purification is prepared in-time and avoids compound degradation by different
and increasingly long wait times.
The automated clean-up workflow starts with the conditioning of the cartridges held ready in slot 1 with 300 µL ethyl acetate.
After the cartridge conditioning with elution solvent from the reservoir the syringe loads 200 µL of the raw extract in ethyl
acetate from a sample vial in slot 1 and moves to the cartridge tray to pick a conditioned cartridge by inserting the needle.
The cartridge is moved by the syringe to the elution tray and inserted into an empty vial (held ready below the cover) at
slot 2.
Figure 4. Automated µSPE clean-up workflow, here shown for online GC-MS injection.6 36
The raw extract is then pushed through the sorbent bed of the cartridge with a constant speed of 2 µL/s by the syringe. The
extracted matrix is retained on the cartridge, the cleaned extract elutes and gets collected in the vial below. Additionally, a
blow-out step using the syringe can be added. The automated µSPE clean-up workflow is graphically illustrated in Figure 4.
After the clean-up procedure the TriPlus RSH robot cleans the preparation syringe with polar and less polar solvents and finally
changes to the injection tool with a regular 10 µL GC injection syringe. After dilution and a mixing step, 3 µL of the cleaned
extract are injected. The PTV injector is kept at 55 °C during injection with a 3 s split open time, before starting the injector
and GC oven heating ramp, see Table 2. The described automated clean-up procedure takes 15 min of the total GC-MSMS
analysis time of 45 min, see the analysis parameters used for the MS in Table 3.
Carrier gas: Helium
Carrier gas mode: Constant pressure
Carrier gas pressure: 70 kPa (depending on column length)
GC Column: DB5-ms UI / 15 m x 0.25 mm x 0.25 µm
Injection mode: PTV
Injection volume: 3 µL
Rate [°C/s] Temp [°C] Time [min] Split [mL/min]
0 55 0.1 30
2.5 330 12 0
PTV injector temperature program:
Table 2. GC parameters
Temperature program:
Ionisation mode: EI pos
MS transfer line temp.: 290 °C
Ion source temp.: 220 °C
Scan mode: Timed SRM
Total scan time: 0.3 s
Total compounds: 209 (418 MRM)
Table 3. Parameters of the MSMS system
It is important to note at this point that the extracts get injected online right after the clean-up step. Waiting times, in particular
different waiting times after contact with the sorbent material, are avoided so that all samples are treated on the identical
timeline to avoid uncontrolled decomposition thus improving reproducibility of the recovery of the target analytes.
# Rate [°C/min] Temperatur [°C] Hold Time [min]
Initial 55.0 2.00
1 20.0 165.0 0.00
2 3.0 205.0 0.00
3 10.0 290.0 0.00
4 10.0 310.0 3.00377
Workflow Preparation
The TriPlus RSH µSPE clean-up workflow is created using the TriPlus Method Composer software (Thermo Fisher Scientific,
Austin, TX, USA) and optimized for the parallel execution (‘prep-ahead’ mode) with the ongoing GC separation of a previous
analysis. The graphical user interface is shown in Figure 5. The tools and modules of the used TriPlus RSH configuration are
shown on the right side. The available workflow activities for this configuration are offered in the box on the left. The required
tasks are pulled by ‘drag & drop’ into the centre of the screen to build the workflow sequence and customized by adaptation
of the default parameters. The saved workflow is selected within the TraceFinder sequence table of the GC-MS systems for
execution within the planned sample sequence.
With the typical ‘prep-ahead’ mode of the TriPlus RSH system the maximum sample throughput for each of the employed
GC-MS systems is achieved.
There is no wait time for the GC-MS system. A next analysis run starts right away after the Ready signal of the GC.
The clean-up method is optimized to match the GC-runtime. The sample preparation of the next sample will start in order to
align the readiness of the consecutive sample with the Ready signal for injection to the GC. In case of a faster GC cycle time
the TriPlus RSH Method Composer can also be used to further optimize the method to match a faster sample to sample cycle.
Figure 5. PAL Method Composer Software including the µSPE clean-up workflow steps shown in the center.
Experience with selected pesticides and critical matrices
It could be shown that the recovery of the pesticides folpet and captan benefit from the GC injection volume of 3 µL as
demonstrated in Figure 6. Smaller injection volumes of less diluted extracts are contra-productive for these two difficult to
analyse fungicides. Nevertheless, it is also crucial for the analysis of captan and folpet that the liner and the analytical column
are in good condition as it can be achieved in routine with the µSPE clean-up.
Figure 6. Pak-Choi spiked with 100 µg/kg captan and folpet, measured on a TSQ 8000 EVO8 38
The current experience and setup allow the clean-up of samples with a lipid content of up to 15 % which for instance is the
approximate fat content of avocados. Also, liver samples can be run without the need of a separate freeze-out of fats.
Critical matrices like spices with a high content of essential oils (e.g. chilly, paprika, etc.) are cleaned-up online as well. 2 g
of spices are treated with 10 mL of water before extraction with ethyl acetate. The raw extract is then cleaned as described
above.
A pre-treatment is also required for grains and cereals. 5 g of sample material is soaked in 10 mL of water before extraction,
then the raw extract is automatically cleaned-up as described.
New matrices are treated using the described workflow without any additional dilution.
Quantitation
A screening procedure is used to identify potential non-compliant samples. The non-compliant residues in the selected
samples are quantified by using the standard addition method with the identified pesticide taking into account any potential
matrix effect. Three data points are prepared in an automated workflow with online GC-MS injections. A processing
(procedural) standard is added at the beginning to correct for possible losses of pesticides during extraction and clean-up by
µSPE.
Conclusion
The comparison of the earlier manual method using the optimized dSPE clean-up sorbent mix for a particular food
commodity with the automated µSPE workflow showed very good compliance within the normal and accepted error range in
pesticide analysis.
Folpet and captan, two typical but difficult GC-analytes, were successfully analysed with the automated µSPE workflow.
The PAL Method Composer is a versatile tool for the adaption of the µSPE workflow to the specific needs as published by
Steve Lehotay. After a short learning phase of less than a day, the described µSPE workflow could subsequently be developed
in less than 3 days without the need of any programming knowledge.
The described µSPE workflow has been in routine operation for two years now and showed high reliability also applied for
unattended overnight runs, releasing time from earlier manual workload to be used for other duties such as data evaluation
and quantitation.
Future work
As the utilized cartridges showed excellent potential for lipid removal, the scope of samples using the described workflow
will be extend to cheese and liver samples to avoid the time-consuming manual freeze-out process of the contained fat,
after extraction. Also under investigation is the applicability of the clean-up procedure for the analysis of PCBs and PAH
contaminations of the food samples.39
Imprint
Date of print: 09.2022
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CH-4222 Zwingen
Switzerland
T +41 61 765 81 00
Contact: info@ctc.ch
Legal Statements
CTC Analytics AG reserves the right to make improvements and/or changes to the
product(s) described in this document at any time without prior notice.
CTC Analytics AG makes no warranty of any kind pertaining to this product, including
but not limited to implied warranties of merchantability and suitability for a particular
purpose.
Under no circumstances shall CTC Analytics AG be held liable for any coincidental
damage or damages arising as a consequence of or from the use of this document.
© 2022 CTC Analytics AG. All rights reserved. Neither this publication nor any part
hereof may be copied, photocopied, reproduced, translated, distributed or reduced
to electronic medium or machine readable form without the prior written permission
from CTC Analytics AG, except as permitted under copyright laws.
CTC Analytics AG acknowledges all trade names and trademarks used as the property
of their respective owners.
PAL is a registered trademark of CTC Analytics AG | Switzerland
www.palsystem.com
Visit our homepage for more information.40
Additional Resources
If you would like to learn more about µSPE, use the resources below.
1. Manzano Sánchez L, Jesús F, Ferrer C, Mar Gómez-Ramos M, Fernández-Alba A. Evaluation of automated
clean-up for large scope pesticide multiresidue analysis by liquid chromatography coupled to mass
spectrometry. J Chromatogr A. 2023;1694:463906. doi:10.1016/j.chroma.2023.463906
2. Hakme E, Poulsen ME. Evaluation of the automated micro-solid phase extraction clean-up system for
the analysis of pesticide residues in cereals by gas chromatography-Orbitrap mass spectrometry. J
Chromatogr A. 2021;1652:462384. doi: 10.1016/j.chroma.2021.462384
3. Chong CM, Hubschmann HJ. Fully Automated QuEChERS Extraction and Cleanup of Organophosphate
Pesticides in Orange Juice. LC GC N Am. 2021;39(s1):12–16. chromatographyonline.com/view/fullyautomated-quechers-extraction-and-cleanup-of-organophosphate-pesticides-in-orange-juice. Accessed
November 11, 2024.
4. Michlig N, Lehotay SJ. Evaluation of a septumless mini-cartridge for automated solid-phase extraction
cleanup in gas chromatographic analysis of >250 pesticides and environmental contaminants in fatty
and nonfatty foods. J Chromatogr A. 2022;1685:463596. doi: 10.1016/j.chroma.2022.463596
5. Performance Qualification Test. palsystem.com/fileadmin/user_upload/Files/Micro-SPE_Scripts/PQ_
Tests_uSPE-GCQuE1-45-V.pdf. Accessed December 4, 2024
6. Performance Qualification Tests for ΜSPE-LCQuE1-30-T Cartridges Analysis Protocol. palsystem.com/
fileadmin/user_upload/Files/Micro-SPE_Scripts/PQ_Tests_uSPE-LCQuE1-30-T.pdf. Accessed December 4,
2024
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