This document discusses pesticide residue analysis methods. It begins with an introduction to pesticide residue testing and maximum residue levels (MRLs). The main steps of pesticide analysis are then outlined, including extraction, clean-up, and analysis using techniques like gas chromatography-mass spectrometry (GC-MS). The document provides examples of extraction from vegetables and solid-phase extraction clean-up. It also discusses advances in analysis technology like triple quadrupole mass spectrometry.

1. Pesticide residue
analysis
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2. Outline
Introduction
Mrl analysis
Pesticide analysis steps
Extraction
Clean-up
Analysis of product
Advances in pesticide analysis
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3. Introduction
When crops are treated with pesticides very small amounts may
remain in or on the crop after harvesting or storage.
In order to determine any pesticide residues present in foodstuffs
analytical methods capable of accurately measuring extremely small
amounts of pesticides are required.
Gas chromatography is the most used equipment in pesticide
analysis others include High performance liquid chromatography
(HPLC) .
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4. Maximum residue levels (MRLS)
Mrl is the highest level of a pesticide residue that is legally tolerated in or on
food or feed when pesticides are applied correctly (Good Agricultural Practice).
Mrl covers the safety of all the consumers i.e children to adults
The maximum residue levels information is provided by the chemical produce
during chemical registration.
Any food or non-food should meet the levels of pesticide mrl of the different
pesticides
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5. Example of MRL analysis on grapes
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6. How Are Pesticide Residues on
Imported Produce Regulated
Imported produces tested for pesticide residues on random basis.
When a shipment is found to contain illegal residue all new
shipments after that required to be fully analyzed and certified by
approved laboratories for MRLS.
Countries whose produces are found to have excess pesticide
residual often have increased random inspection and testing beyond
the normal
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7. Example of MRL test carried out in USA
In 1986, a pesticide residue monitoring survey was conducted by
the California Department of Food and Agriculture (CDFA).
The survey of 5,500 food samples including fruits and vegetables
showed that nearly 85 percent had no detectable residues
whatsoever.
 In addition, 12 percent of the samples contained residues at less
than 50 percent of the established tolerance levels.
Less than 2 percent of the samples taken contained illegal residues.
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8. Extraction process
This is a fundamental process in pesticide residue analysis because no in-situ
technique has yet been invented.
There are several extraction approaches employed such as:
Pressurized liquid extraction(PLE)
Supercritical fluid extraction (SFE)
Aqueous extraction
Microwave-assisted extraction(MAE)
Solid-phase extraction (SPE)
Solid-phase microextraction (SPME)
Matrix solid-phase dispersion (MSPD)
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9. Pressurized liquid extraction(PLE)
PLE Is an accelerated solvent extraction.
Uses liquid for extraction.
Samples are loaded into vessels, which require a dispersant/drying agent to aid solvent flow.
Extraction occur at elevated temperature I.e room temp to 40-200 and pressure 1000-3000 psi
to improve recoveries.
Static extraction mode is used to equilibrate the sample to extraction conditions and dissolve
the analytes in liquid.
Vessel is flushed with solvent rapidly, rather than a controlled dynamic flow in SFE using a
restrictor thus making PLE faster than SFE.
Any liquid can be used provided it can be pumped through sample and its pH is between 2-12.
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10. PLE flow
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11. Supercritical fluid extraction (SFE)
SFE uses supercritical fluid as an extraction solvent.
CO2 is common solvent used in SFE, due to its safe nature,
uncreativeness, ready availability and inexpensiveness.
The rest of the process is similar to that of PLE.
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12. Solid-phase Extraction
Solid-phase extraction is a separation process by which compounds
that are dissolved or suspended in a liquid mixture are separated
from other compounds in the mixture according to their physical and
chemical properties.
SPE consists of four steps: column preparation (prewash), sample
loading (retention or sorption), column post wash, and sample
desorption (elution or desorption).
compounds of interest are retained on the sorbent while
interferences are washed away.
Analytes are recovered via an elution solvent.
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13. Example of pesticide extraction from vegetables
100 g of sample minced, 25g of homogenate macerated using 50ml of acetone
then decanted into 200ml of 2% sol of sodium sulphate in separating funnel.
50ml of hexane added into the separating funnel & mixed.
This proportion to an aqueous and non-aqueous layer(hexane layer).
Remove aqueous layer into beaker while the hexane layer is run through a
chromatographic column packed with anhydrous sodium sulphate.
The volume is evaporated using rotary evaporator to approximately 10ml then
made up to 25ml using hexane.
The extract is then analyzed for different pesticides.
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14. Clean-up
Clean-up is done to obtain uncontaminated products for analysis.
The clean-up process is vital especially in analysis of fatty foods.
Liquid-liquid and liquid-solid partitioning in which separation is
based on polarity is the primary mean of clean-up in residue
analysis.
Separations based on molecular weight, gel-permeation or size-
exclusion chromatography is common clean-up technique.
Other techniques used include precipitation, centrifugation,
ultrafiltration, dialysis and immunochemical.
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15. Gel permeation chromatography
Also known as size-exclusion chromatography
(SEC) separates compounds with large
molecular weight such as lipids and proteins
from smaller analytes such as pesticides using a
porous stationary phase material.
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16. Example of pesticide clean-up in vegetables samples
10g of florosil 60-100 mesh are packed using hexane into florosil
column.
1cm of anhydrous sodium sulphate is then added on top.
5ml of extract is then loaded and eluted with 200ml fractions of
6%,15% and 50% diethyl ether in hexane.
Elute mixture is collected in 500ml round-bottomed flask and
evaporated to 1ml
Extract then analyzed using gas-liquid chromatography using
electron capture detectors (ECD) for organochlorines and synthetic
pyrethroids and nitrogen phosphorous detectors (NPD) for OPS.
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17. Analysis
Capillary electrophoresis
Small amount of sample is typically sucked or forced into the capillary by pneumatic pressure,
then the voltage is applied.
Separation of ions occurs based on the mobility of ions, which is a function of size and charge.
Smaller cations in the buffer have higher mobility and force overall flow toward the anode.
Neutral molecules are carried with this flow in the electro-osmotic front while charged
molecules migrate faster or slower through the capillary based on their charge and mobility.
Plug flow occurs in CE because flow originates along the negatively charged capillary walls
unlike the laminar flow profile of chromatographic techniques.
This means peaks are sharper in CE and better separations can be achieved.
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18. Gas-liquid chromatography GC-MS
GC-MS is usually used in the analysis of mid- polar to non-polar
compounds whereas HPLC technique used for polar compounds.
Organophosphate and carbamate pesticides are mid polar in nature
thus gas chromatography is well suited for their analysis.
GC-MS systems are equipped with extensive mass spectral
“libraries”that can be extremely useful for identification and
characterization of unknown compounds.
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19. Gas chromatography flow
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20. Gas chromatography components
Carrier gas
The gas must be inert such as nitrogen, helium, argon, and carbon dioxide.
The type of gas is depended on type of detectors.
Sample injection port
Microsyringe are used to inject sample in small quantities.
It is introduced onto the column as a "plug" of vapour.
Sample port temperature is at 500c.
Sample size ranges from tenths of a microliter up to 20 microliters.
The sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised
solutes
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21. Columns
There are two general types of column, packed and capillary
Packed contain a finely divided, inert, solid support material coated with liquid
stationary phase.
Capillary -They can be one of two types; wall-coated open tubular (WCOT) or
support-coated open tubular (SCOT).
WCOT-Consist of a capillary tube whose walls are coated with liquid stationary
phase.
SCOT-The inner wall of the capillary is lined with a thin layer of support material
such as diatomaceous earth, onto which the stationary phase has been
adsorbed.
Temp of column is dependent on sample boiling temperature.
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22. Detectors
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23. LOD & LOQ
Limit of detection LOD- is usually defined as the lowest
quantity or concentration of a component that can be
reliably detected with a given analytical method e.g. GC.
Limit of quantification -is the lowest concentration at
which the analytes can not only be reliably detected but at
which some predefined goals for bias and imprecision are
met.
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24. Example of final GC data obtained
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Source: www.restek.com

25. Example of final GC data obtained
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Source : www.restek.com

26. Pesticides detected using GC
Pesticides analysed using GC
Organochlorines e.g. dieldrine, DDT, Lindane.
Organophosphorus e.g. diazinon, dichlorvos, malathion
Pyrethroids e.g. cypermethrin, deltamethrin
Aromatic fungicides eg chlorothalonil, quintozene
Dicarboximide fungicides eg folpet
Other pesticides also analyzed using GC but showing low or no response include:
Carbamates, imidazoles, benzoylureas, sulfonylureas
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27. Pesticides analysed using LC
Carbamates e.g. carbendazim
Conazoles e.g. fenamidone
Sulfonylureas e.g. triasulfuron
Triazines e.g. simazine
Phenylureas e.g. linuron
Strobilurins e.g. azoxystrobin
Neonicotinoids e.g. acetamiprid
All the above plus their metabolites can be analysed by Liquid chromatography
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28. Advances in pesticides residue analysis
Their has been a trend in advancement of analysis.
Chromatography was the 1st invention back in 1906.
Gas chromatography came into use in 1960 until late 1900.
High performance liquid chromatography was later used in 1970-
1990 era.
Gas chromatography mass spectrometry with advances came into
use from 2000-to date
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29. Mass spectrometry
Ideally suited for multiresidue analysis
Features of MS
Universal and selective
Screening and confirmation
Compatible with GC and LC
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30. Quadrupole (single MS)
It consists of four cylindrical rods, set parallel to each other.
Quadrupole mass spectrometry the quadrupole is the component of the instrument responsible for
filtering sample ions, based on their mass-to-charge ratio (m/z).
Features of quadrupole MS
Excellent quantitative capability.
Too low sensitivity in full scan mode for residue analysis
Selective if using at least three ions.
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Quadrupole MS (QMS)

31. Triple Quadrupole (QqQ)
Features of QqQ MS:
Excellent performances for quantitative multiresidue methods.
Monitoring of known and suspected residue only, unexpected compounds not detected.
 Usually, MRM acquisition in multiple acquisition groups.
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Triple Quadrupole QqQ MS

32. Thank you
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