1. ISTITUTO ZOOPROFILATTICO SPERIMENTALE
DELL’UMBRIA E DELLE MARCHE-PERUGIA
Comparison of three preparation procedures for determination of Organochlorine pesticides in food of animal origin (fat)
Angela Paoloni, Sabrina Alunni, Alessandro Pelliccia, Ivan Pecorelli
Laboratorio Contaminanti Ambientali - Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, via Salvemini 1, Perugia Italy.
Introduction
A number of sample preparation techniques and methods of analyses have been developed for the determination of pesticide residues in a wide range of food of animal origin. Analyses of pesticides from food of animal origin were generally performed with an accelerated solvent extraction (ASE) step by a solvent mixture of n-hexane and acetone with a suitable ratio (example 3:2 v/v), fat separation was then carried out with gel permeation chromatography (GPC) and a clean-up step by SPE silica columns was finally performed. Most of these procedures are time and solvent consuming with consequent high cost of analyses. An alternative method may be QuEChERS, developed for the purification of pesticide residues in food of plant origin, is also now being used for other matrices such as milk, eggs or food of animal origin in general.
The aim of this work was to evaluate the application of QuEChERS method, coupled with GC-MS/MS (QqQ) detection in Multiple Reaction Monitoring (MRM) for the determination of Organochlorine pesticide residues in fatty animal matrices, alternatively, to classic preparation. The performance of two different types of dispersive solid phase extraction methods (d-SPE) with different concentration of MgSO4 (900 mg and 1200 mg); PSA (150 mg and 400 mg) and C18 (150 mg and 400 mg) respectively, with the presence of ceramic homogenizer in the second type of d-SPE, valuated and compared to validation data obtained by classic ASE/GPC method.
GC-MS/MS conditions
Agilent 7890 A GC-MS/MS equipped with PTV injector
Analytes were separated by a Restek GC column Rtx-Pesticides2 (30 m x 0,25 mm, 0,20 µm) with in-line Siltek filter (5 m x 0,25 mm ID).
Total Run Time: 40 min
Temperature Program (see Table 1);
Carrier gas: Helium 5.5
Collision gas: Nitrogen 6.0
Injection volume: 2 μL
Table 1:GC oven parameter
Sample preparation
Add
4 g of anidrous Magnesium Sulfate (MgSO4)
1 g of Sodium Chloride (NaCl)
1 g of Sodium Citrate Tribasic Dihydrate (C6H5Na3O72H2O)
0.5 g of Sodium Hydrogencitrate Sesquihydrate (C6H6Na2O71.5H2O).
1 g of fat
Extraction with (ACN/H2O)
The mixture is shaken by vortex for 1 min and then centrifuged at 3000 rpm for 15 min.
The supernatant is transferred into a specific d-SPE for purification.
Extract is evaporated to dryness and the residue is re-dissolved in 200 μL of isooctane and injected into GC-MS/MS.
Figure 1: Cromatogram of matrix matched calibration standard at 0.10 µg/mL.
Time (min.)
Temp. (°C)
Flow
(°C/min) 80 20
160 5
300
3 (Post run)
Method Comparison
The method performances were evaluated in terms of linearity in solvent and in matrix, sensitivity, mean recovery (as measure of trueness or bias) and precision by analysis of 5 replicates, at mg/Kg (LOQ) and mg/Kg (10xLOQ) in fat. In table the data for the LOQ (0,010 mg/Kg) level for the three methods are reported: ASE+GPC, QuEChERS with classic d-SPE (150 mg) and with high concentration (400 mg) of C18.
Table 2: Validation data at LOQ for ASE + GPC procedure Table 3: Validation data at LOQ for QuEChERS method (150 mg C18) Table 4: Validation data at LOQ for QuEChERS method (400 mg C18)
ASE +GPC
Pesticide
spike 1
spike 2
spike 3
spike 4
spike 5
Average R% SD
RSDr
alpha-HCH 94 76 80
102 81 87 11 13
Aldrin 95 82
105
101 92 12 14
alpha-Endosulfan 77 66 88 75 8 10
beta-Endosulfan
117 86 85 96 91 16 17
beta-HCH
109
106 83 90 98
cis-Chlordane
100 84 99
Chlorpyrifos(-ethyl) 97
120
107
Chlorpirifos-methyl
119 19
Dieldrin
118
103
Endosulfan sulfate 89
112
115
Endrin 67 20
cis-Heptachlorepoxid 79 9
trans-Heptachlorepoxid
Heptachlor 29 72 73 26 36
gamma-HCH (Lindane) 5 6
Hexachlorobenzene (HCB) 64 63 54 15
4,4'-Methoxychlor 65 -- 18
o,p'-DDD
161
116 30
o,p'-DDE
o,p'-DDT
p,p'-DDD
114
174 35 31
p,p'-DDE 71 93
p,p'-DDT
trans-Chlordane
108 57 42 74
D-SPE 150 mg C18
Pesticide
spike 1
spike 2
spike 3
spike 4
spike 5
Average R% DS
RDSr
alpha-HCH 75
100 89
126 80 94 21 22
Aldrin 69 88 65
107 53 76 28
alpha-Endosulfan 74
117 93
129 77 98 24 25
beta-Endosulfan 51 84 81 20
beta-HCH 78
110
102
115 16
cis-Chlordane 72 85 97 91 12 13
Chlorpyrifos(-ethyl)
127 95 18 19
Chlorpirifos-methyl
105 96 15
Dieldrin 79 86
108 87
Endosulfan sulfate
121
113 17
Endrin
103 10 11
cis-Heptachlorepoxid
256
101 60
118 67
trans-Heptachlorepoxid 64 27 68 32 46
Heptachlor -- 90
gamma-HCH (Lindane) 92
120
Hexachlorobenzene (HCB) 47 62 43 40
4,4'-Methoxychlor 83
109
o,p'-DDD
112 70
o,p'-DDE 42 29
o,p'-DDT 66
p,p'-DDD
114
p,p'-DDE 59 99 23
p,p'-DDT
trans-Chlordane 71
D-SPE 400 mg C18
Pesticide
spike 1
spike 2
spike 3
spike 4
spike 5
Average R% DS
RDSr
alpha-HCH 86 85 95 94 89 5 6
Aldrin 66 70 65 73 68 3
alpha-Endosulfan 75 82 78 92 88 83 7 9
beta-Endosulfan 81 80 84 97 87 8
beta-HCH 96 93
101
cis-Chlordane 77 71
Chlorpyrifos(-ethyl) 90
100 91
Chlorpirifos-methyl
102
Dieldrin 72
Endosulfan sulfate
Endrin
cis-Heptachlorepoxid
trans-Heptachlorepoxid
Heptachlor 10
gamma-HCH (Lindane)
Hexachlorobenzene (HCB) 53 57 62 59 11
4,4'-Methoxychlor
116
105
103
o,p'-DDD
o,p'-DDE 76 69
o,p'-DDT 74 4
p,p'-DDD 98
p,p'-DDE
p,p'-DDT
trans-Chlordane
Conclusions
In accordance with performance parameters described in point G6 of Document N° SANTE/11945/2015, high C18 (400 mg) QuEChERS method validation results, obtained from 5 replicates, showed mean recovery in the range of % and particularly good RSDs values below 12% for all the 24 molecules analyzed. Only Hexachlorobenzene (HCB) and Aldrin has a mean recovery value below 70% (with good precision). The presence of high PSA and C18 sorbent with ceramic homogenizers seem to be essential to achieve good performances and more clear purified extracts .The proposed method is now applied in our laboratory for routine samples and will be tested in the next EUPT-AO on pig lard.
Reference: [1] Document N° SANTE/11945/2015.