These Slides show the commonly used MRM method for pesticides. It also guides you to select the right way to build a new one.

1. Common Multi Pesticide
Residue Extraction Methods
Dr. Sherif M. Taha

2. The more information on pesticides and its related matrices, the easier you can
get the appropriate residue analysis method for these pesticides.
Main physical and chemical properties for analyzed pesticides include;
chemical structure, pKow, pKa, and stability.
Main characterization for the desired matrix (sample content) include; water/
oil content, pH, the presence of specific compounds that may be similar to
analyzed pesticides or may largely effect on selected
technique for pesticide analysis.
What to know before looking for a pesticide extraction method
https://www.bbcgoodfood.com/howto/guide/health-benefits-avocado
https://www.organicfacts.net/health-benefits/fruit/health-benefits-of-lemon.html

3. Chemical structure, solubility, and pKa
3
From the chemical structure of a pesticide, It can be easily classified into a polar, moderately
polar, and non polar.
pKow : 6.5 4.7 0.75 -0.44
the proper analysis techniques can be suggested from the chemical structure, see lecturers on mass
spectrometry for pesticides residue analyses

4. Chemical structure, solubility, and pKa
4

5. Chemical structure, solubility, and pKa
5

6. Chemical structure, solubility, and pKa
6

7. Luke method
QuEChERS
EtOAc QuEChERS
Swedish method

8. MRM for pesticides residue analysis
8
 Nowadays, there are a huge number of used pesticides worldwide. Agricultural products
are susceptible to contain high residue levels from such pesticides.
 The probability of finding many different pesticide residues in the same sample is high.
This situation is twofold in countries that have uncontrolled pesticides practices.
 Pesticides control not only enhance citizen's health but also will increase the export and
import of safe food.
 Methods that can extract a higher number of pesticides (of widely different physical and
chemical properties) are highly recommended. These methods are known as Pesticide
multi residue methods (MRMs), which are capable of simultaneously determining
hundred of pesticide residue in a single analysis.

9. Luke MRM Method
9
Luke, M.A., et al. (1975) J. Assoc. Off. Anal. Chem. 58, 1020-1026
 At 1975, Luke MRM method was introduced for pesticide residue analysis. It has the
capability to analyze both nonpolar pesticides (organochlorines) and polar ones
(organophosphorus) reverse to other previously reported methods before 1978.
 Luke method: pesticides extraction was carried out using polar solvent (acetone) which
followed by a liquid- liquid partitioning step using nonpolar solvents (petroleum ether
and dichloro methane).
 A brief on the main steps .of Luke method will be discussed in the next slide

10. Luke MRM Method
10Luke, M.A., et al. (1975) J. Assoc. Off. Anal. Chem. 58, 1020-1026
50 g
Homogenized sample
+
200 ml acetone
blind for 2 min
40 filtrate +50 ml petroleum ether
50 ml dichloromethane
Collect the previous lower layer+
2* 50 ml dichloromethane
Collect the upper layer over sod
sulfate
Collect the lower layer over sod
sulfate

11. QuEChERS- Original
 QuEChERS MRM method is the most commonly used MRM method for pesticides residues
analysis nowadays. This method enables extraction of hundreds of pesticide residues.
 QuEChERS are the abbreviations for the following words: Quick, Easy, Cheap, Effective,
Rugged, and Safe.
 This method employs a single-phase solvent extraction (MeCN/Water), then partitioning
using salts.
 Although MeCN does not extract lipophilic matrices as EtAC and Acetone, It is not suitable
for direct GC analysis. Since it has a high expansion volume during evaporation in GC
analysis.
11MICHELANGELO ANASTASSIADES, STEVEN J. LEHOTAY, Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning an
“Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce, AOAC (86) 2003.

12. QuEChERS- Original
12
10 g
Homogenized sample
+
10 ml MeCN
& shack for 1 min
4 g MgSO4 + 1g NaCl
& shack for 1 min
Centrifuge for 5 min/5000 rpm
Use 25 mg PSA and 150 mg
MgSO4 for 1 ml MeCN extract.
& centrifuge for 1 min/6000 rpm

13. Advantages of QuEChERS Vs Luke
 Using metal surfaces for sample bending during pesticide extraction was avoided.
Therefore, losing time and solvents during cleaning the used blinder was also avoided.
 A single tube is used for each sample, which prevents cross-contamination.
 Shaking can be carried out by hand or using shaking instruments for many tubes in one
time.
 Finally, QuEChERS can extract a wider number of pesticide residue with a lower
consumption of organic solvents, and without using chlorinated ones.
13

14. QuEChERS- Original / MeCN
14MICHELANGELO ANASTASSIADES, STEVEN J. LEHOTAY, Fast and Easy Multiresidue Method Employing Acetonitrile
Extraction/Partitioning an “Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce, AOAC (86) 2003.
 MeCN gives the lowest amount of co-extracted matrix
components, from a mixture of fruit and vegetables, even
after using primary secondary amine bonded silica (PSA)
as a dispersive solid phase extraction (dSPE).
 The presence of water in MeCN layer enables it to recover
even basic pesticides like imazlil at lower pH conditions,
reverse to the case of using EtAC at this acidic condition.

15. QuEChERS- Original / MgSO4
15
MICHELANGELO ANASTASSIADES, STEVEN J. LEHOTAY, Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning an
“Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce, AOAC (86) 2003.
 MgSO4 induce the highest partitioning efficiency for MeCN/ water by introducing a
high amount of a well separated MeCN upper layer.
 MeCN layer that contains water residue has a higher ability (through increasing its
polarity) for recovering highly polar pesticides, like methamidophos.
 Note, the used MgSO4 must be first activated at 500 C for 5 h before application. To
remove phthalate and give a good partitioning for MeCN/ Water.

16. QuEChERS- Original / NaCl
16MICHELANGELO ANASTASSIADES, STEVEN J. LEHOTAY, Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning an
“Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce, AOAC (86) 2003.
 the polarity of MeCN can be controlled using NaCl with MgSO4, where the higher
amount of NaCl the lower amount of water molecules in MeCN layer, leading to
lowering its ability to the recovery of both polar matrices and highly polar pesticides,
and vice versa.
 It was reported that using a mixture of 4 g MgSO4 and 1 g NaCl is sufficient for the
recovery of a wide range of pesticides of different polarities with a lower amount of co-
extracted matrices components in the MeCN layer

17. QuEChERS- Original / pH
17MICHELANGELO ANASTASSIADES, STEVEN J. LEHOTAY, Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning an
“Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce, AOAC (86) 2003.
 Generally, most pesticides are stable at lower pH.
 Original QuEChERS,
commodities of basic pH were adjusted to be below 4. At strong pH conditions, amount of
co-extracted Matrices (acidic ones like Fatty acid) increased.
 Also, recovery of basic pesticides was lower at low pH (orange, lemon samples), will be
discussed in the next slides.

18. Some problematic pesticides
18
Pesticide pKa KOW logP Stability
pymetrozine / - 0.18 Low at acidic condition
dioxacarb 0.67 Low at acidic and alkaline cond.
dichlofluanid / 3.70 Low at alkaline cond.
chlorothalonil / 2.92 Low at alkaline cond.
tolylfluanid / 3.90 Low at alkaline cond.
dicofol / 4.30 Low at alkaline cond.
imazalil 6.53 wb 3.82 Stable in dilute acid/ base cond.
thiabendazole 4.73 & 12.00 2.39 Hydrolytically stable
carbendazim 4.2 wb 1.51 Low in alkaline cond.
captan / 2.8 Low at alkaline cond.
folpet / 3.11 Low at alkaline cond.

19. Partitioning of imazalil, pKa = 6.53
19
Cl
CH
Cl
OCH2CH=CH2
CH2
N
N
Cl
CH
Cl
OCH2CH=CH2
CH2
N
N
At pH <its pKa , in lemon .Imazalil
will be protonated , it will be in lower
aq. layerNCeM reppu naht rehtar
At pH > its pKa .Imazalil will be
neutral , it will be in MeCN layer
naht rehtarlower aq. one
H+

20. QuEChERS / Acetate buffer
20
15 g (fresh sample)+ 15 ml MeCN (containing1 % HOAC)
6 g MgSO4 + 1.5 g NaOAC
50 mg PSA + 50 C50+ 150 mg MgSO4
/ mL of extract
AOAC Official Method 2007.01
Shake
/cfg
Shake
for 1 m
Shake
for 1 m

21. QuEChERS / Citrate buffer
21
10 g (fresh sample)+ 10 ml MeCN
4 g MgSO4 + 1g NaCl+
1 g Na3Citrate: di H2O + 0.5 g Na2H-Citrate: di H2O
25 mg PSA + 7.5 mg GCB + 150 mg MgSO4
/ mL of extract
Shake
/cfg
Shake
for 1 m
European Committee for Standardization (CEN) Standard Method EN 15662
Shake
for 1 m

22. 22
Which QuEChERS
you can use

23. Which QuEChERS
23
*Steven J. Lehotay; Kyung Ae Son; Hyeyoung Kwon; Urairat Koesukwiwat; Wusheng Fu; Katerina Mastovska; Eunha Hoh; Natchanun Leepipatpiboon, Comparison of
QuEChERS sample preparation methods for the analysis of pesticide residues in fruits and vegetables, Journal of Chromatography A, 1217 (2010) 2548–2560.
*C. Christia, E. Bizani , C. Christophoridis, K. Fytianos, Pesticide residues in fruit samples: comparison of different QuEChERS methods using liquid
chromatography–tandem mass spectrometry, DOI 10.1007/s11356-015-4456-0.
 Both acetate and citrate buffered QuEChERS cover a wider range of pesticides than the
original unbuffered QuEChERS.
 However, it was reported that acetate buffered QuEChERS is more suitable for the
analysis of some problematic pesticides like pymetrozine and thiabendazole .
 In addition, chlorothalonil and folpet can be better screened by the acetate buffer
QuEChERS. It was also reported that analysis of pesticides using acetate QuEChERS for
most fruits samples (except peach) is better than using citrate one, based on recoveries
results.

24. EtOAc QuEChERS Methods
24*Steven J. Lehotay; Kyung Ae Son; Hyeyoung Kwon; Urairat Koesukwiwat; Wusheng Fu; Katerina Mastovska; Eunha Hoh; Natchanun Leepipatpiboon, Comparison
of QuEChERS sample preparation methods for the analysis of pesticide residues in fruits and vegetables, Journal of Chromatography A, 1217 (2010) 2548–2560.
 MeCN can't directly be injected to GC instruments as it has a high expansion volume.
 MeCN extract is evaporated and reconstituted with suitable GC solvents like EtOAc,
Hexane, Acetone, and its mixtures.
 Some researchers studied using EtOAc instead of MeCN, especially after the shortage of
MeCN in some periods of times.
 Using EtOAC instead of MeCN has been studied in the acetate QuEChERS method, but it
gives a high amount of co-extracted components with low recoveries for highly polar
pesticides.

25. EtOAc QuEChERS Methods
25
10 g (fresh)+ 10 ml EtOAC
8 g MgSO4 + 1,5 g NaCl
Shake
3S
Shake
25 m
 EtOAc has been used for GC MS/ MS
analysis of 210 pesticides in fruits and
vegetables.
 To use this method for analysis more polar
compounds by LC MS/MS, a buffer
system may be required.
P. Taylor, S. Uclés, N. Belmonte, M. Mezcua, A.B. Martínez, M.J. Martinez-, M. Gamón, Journal of Environmental Science and Health , Part B : Validation of a multiclass multiresidue method
and monitoring results for 210 pesticides in fruits and vegetables by gas chromatography-triple quadrupole mass spectrometry Validation of a multiclass multiresidue
method and monitoring results for 210 pesticides in fruits and vegetables by gas chromatography-triple quadrupole mass spectrometry, (2014) 37–41. doi:10.1080/03601234.2014.911566.

26. Sweden MRM EtOAC method/ SweEt
26
10 g (fresh)+ 3 g NaHCO3
+ 20 ml EtOAC
10 g Na2SO4
Shake
1 m
European Committee for Standardization (CEN) Standard Method EN 15662
Shake
for
15 m
 The final extract of this method is
directly injected to both
LC Ms/MS and GC MS/MS.
 This method have been validated
for more than 300 pesticides using
LC MS/MS and for more than 140
using GC MS/MS.

27. Pesticide residue analysis in herbal plants
27
 Pesticide residue analysis of herbal plants can be accomplished using
QuEChERS or Sweden method after adding appropriate water volume for
the dried sample.
 However, using Sweden method result in cleaner herbal extract. New proof
that herbal plants (chamomile, marjoram,..) have more polar components
that co-extracted when using more polar solvents like MeCN in QuChERS
method.

28. Pesticide residue analysis in herbal plants
28
▰TICs for chamomile extracts
using
A. QuEChERS (MeCN)
B. EtAC
C. EtAC/ Hexane (6/4)
D. EtAC/ Hexane (6/4) with CP
Talanta 174 (2017) 767–779

29. 29
Talanta 174 (2017) 767–779

30. 30
Thank You
Sherif2taha@gmail.com