A Validated Method for the Determination of Neonicotinoid, Pyrethroid and Organochlorine

A Validated Method for the Determination of Neonicotinoid, Pyrethroid and Organochlorine Residues in Human Milk

Niharika Anand1, Abhishek Kundu2,3, Sujata Ray1*

SUPPLEMENTARY INFORMATION

1. Department of Earth Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, Nadia, West Bengal, India-741246

2. Department of Agricultural Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, India-741252

3. Department of Agricultural Chemistry and Soil Sciences, University of Calcutta, Kolkata, West Bengal, India - 700019

E mail addresses: *, ,

*Corresponding Author

Dr. Sujata Ray

Assistant Professor

Department of Earth Sciences

Indian Institute of Science Education and Research Kolkata

Mohanpur, Nadia,

West Bengal, India-741246

Contact No: +919836230611

Supplementary Table 1.1 3

Supplementary Table 1.2 5

Supplementary Table 1.3 6

Supplementary Table 2 7

Supplementary Table 3 9

Supplementary Table 4 10

Supplementary Table 5: 12

Supplementary Table 6 14

Supplementary Figure 1 15

Supplementary Figure 2 17

Supplementary Figure 3 19

Supplementary Table 1.1: Parameters of GC–MS/MS analyses of organochlorines

Compound / Retention time
(min) / Quantification transition(m/z)
[Collision energy/eV] / Confirmation Transition (m/z)
[Collision energy/eV] / Dwell
(ms) / Ion Ratio
HCB / 11.56 / 283.7→248.8 [20] / 283.7→214.0 [20] / 10 / 6.9
β-HCH / 11.96 / 216.9→181.0 [15] / 181.1→145.0 [5] / 10 / 48.4
γ-HCH / 12.10 / 216.9→181.0 [5] / 181.0→145.0 [15] / 10 / 97.4
δ-HCH / 12.68 / 181.1→145.1 [5] / 217.0→181.1 [15] / 10 / 93.5
Aldrin / 15.39 / 262.7→193.1 [20] / 262.7→227.8 [20] / 10 / 95.2
o,p- DDE / 17.69 / 246.0→176.2 [30] / 248.0→176.2[30] / 10 / 66.0
α-Endosulfan / 18.39 / 240.5→206.0 [10] / 194.9→159.0 [20] / 10 / 60.9
o,p-DDD / 19.73 / 235.0→165.2 [20] / 237.0→165.2 [20] / 10 / 63.8
β-Endosulfan / 20.58 / 194.6→159.0 [10] / 194.6→125.1 [30] / 10 / 11.6
p,p-DDD / 20.97 / 235.0→165.2 [20] / 237.0→165.2 [20] / 10 / 63.1
p,p-DDT / 22.25 / 235.0→165.2 [20] / 237.0→165.2 [20] / 10 / 61.8
Mirex / 24.96 / 272.0→237.0 [20] / 272.0→140.0 [40] / 10 / 0.6

Supplementary Table 1.2: Parameters of GC–MS/MS analyses of pyrethroids

Compound / Retention time
(min) / Quantification transition(m/z)
[Collision energy;eV] / Confirmation Transition (m/z)
[Collision energy;eV] / Dwell
(ms) / Ion Ratio
Allethrin / 17.37 / 123.0→81.0 [10] / 90.9→65.0 [15] / 10 / 25.4
Bifenthrin / 23.92 / 180.8→165.1 [30] / 180.8→166.1 [10] / 10 / 81.2
Fenpropathrin / 24.08 / 180.9→152.0 [20] / 207.9→181.0 [5] / 10 / 96.6
λ-Cyhalothrin / 25.32 / 180.9→152.0 [10] / 196.9→141.1 [30] / 10 / 97.2
Permethrin / 26.38 / 182.9→153.1 [15] / 182.9→77.1 [30] / 10 / 85.6
Cyfluthrin / 27.05 / 162.9→90.9 [15] / 162.9→127.0 [5] / 10 / 76.7
α-Cypermethrin / 27.54 / 162.7→91.2 [25] / 180.6→152.2 [20] / 10 / 56.1
Fenvalerate / 28.59 / 167.0→125.1 [5] / 208.9→141.1 [15] / 10 / 6.2
Deltamethrin / 29.79 / 252.9→93.0 [15] / 181.0→152.9 [25] / 10 / 53.3

Supplementary Table 1.3: Parameters of LC–MS/MS analyses of neonicotinoids

Compound / Retention time
(min) / Quantification transition (m/z)
[Collision energy;eV] / Confirmation Transition (m/z)
[Collision energy;eV] / Cone
(V) / Dwell
(ms) / Ion Ratio
Thiacloprid / 5.78 / 252.82→89.78 [41] / 252.82→98.65 [45] / 36 / 150 / 20.2
Imidacloprid / 5.10 / 255.63→174.83 [49] / 255.86→175.04 [17] / 22 / 150 / 87.3
Acetamiprid / 5.66 / 222.84→55.40 [13] / 222.84→89.78 [37] / 34 / 150 / 56.0
Thiamethoxam / 4.83 / 291.82→210.93 [15] / 291.82→181.00 [21] / 24 / 150 / 31.2

Supplementary Table 2: Comparison of Recovery (%) using all tested methods obtained in human milk spiked at 100 ng g-1

Compound / % Recovery (Mean±SD)
Selected Methoda / Original QuEChERSb / AOAC ACETATEc / QuEChERS with d-SPEd
HCB / 90±11 / 59±11 / 46±11 / 95±11
β-HCH / 103±6 / 122±9 / 87±7 / 105±11
γ-HCH / 94±8 / 115±10 / 75±9 / 95±10
δ-HCH / 98±7 / 122±11 / 83±8 / 100±11
Aldrin / 96±8 / 92±8 / 68±6 / 85±11
o,p-DDE / 97±89 / 119±10 / 85±10 / 109±6
α-Endosulfan / 92±8 / 100±9 / 79±6 / 82±11
o,p-DDD / 94±9 / 115±10 / 82±10 / 105±7
β-
Endosulfan / 99±6 / 124±9 / 82±11 / 84±10
p,p-DDD / 107±9 / 126±9 / 78±7 / 92±11
p,p-DDT / 91±4 / 106±11 / 80±10 / 102±6
Mirex / 97±7 / 86±11 / 65±7 / 102±9
Allethrin / 87±10 / 58±11 / 49±11 / 89±11
Bifenthrin / 92±9 / 78±9 / 67±7 / 100±11
Fenpropathrin / 90±7 / 89±10 / 71±9 / 81±10
λ-Cyhalothrin / 90±11 / 77±11 / 84±8 / 79±11
Permethrin / 93±11 / 92±8 / 79±6 / 82±11
Cyfluthrin / 84±10 / 72±9 / 77±5 / 82±11
α-Cypermethrin / 98±9 / 79±10 / 82±10 / 89±7
Fenvalerate / 91±11 / 68±9 / 78±11 / 84±10
Deltamethrin / 94±9 / 106±11 / 69±10 / 77±6
Imidacloprid / 90±9 / 86±11 / 63±7 / 71±9
Thiacloprid / 85±10 / 105±11 / 77±9 / 81±6
Acetamiprid / 95±9 / 89±9 / 65±10 / 69±9
Thiamethoxam / 93±7 / 87±8 / 75±9 / 78±7

a. The selected method includes extraction with ethyl acetate preceded by sulfuric acid treatment and cold centrifugation; b. Anastassiades et al., 2003; c. AOAC Official Method 2007.01 and d. Luzardo et al., 2013.

Supplementary Table 3: Schema of methods tested in human milk for optimization of clean-up

Variant of the method
Tested factors / Chemicals / Quantity / M1 / M2 / M3 / M4
Sorbent: / Basic / MgSO4 / 150 mg / + / + / + / +
Additional / PSA / 50 mg / + / + / + / +
C-18 / 25 mg / - / + / + / -
Florisil / 25 mg / - / - / + / +

Where, M1 = PSA+MgSO4; M2 = PSA+MgSO4+C-18; M3 = PSA+MgSO4 +C-18+ Florisil; M4 = PSA+MgSO4 +Florisil

Supplementary Table 4: Recovery (%) obtained from the clean-up experiments in human milk.

Compound / % Recovery (Mean±SD)
M1 / M2 / M3 / M4
HCB / 96±5 / 83±5 / 99±7 / 96±5
β-HCH / 97±10 / 104±7 / 95±5 / 104±6
γ-HCH / 115±7 / 75±6 / 94±5 / 95±5
δ-HCH / 86±5 / 86±8 / 99±9 / 88±2
Aldrin / 96±10 / 95±4 / 99±3 / 96±5
o,p DDE / 97±4 / 92±7 / 90±7 / 91±5
α-Endosulfan / 99±3 / 106±7 / 93±7 / 90±12
o,p-DDD / 96±4 / 92±7 / 91±7 / 93±6
β-Endosulfan / 100±4 / 94±6 / 87±8 / 90±9
p,p-DDD / 92±8 / 100±4 / 99±7 / 90±2
p,p-DDT / 95±7 / 96±3 / 99±8 / 92±19
Mirex / 91±15 / 100±2 / 99±8 / 91±7
Allethrin / 99±5 / 103±5 / 99±7 / 96±5
Bifenthrin / 99±10 / 104±7 / 95±5 / 104±6
Fenpropathrin / 111±7 / 75±6 / 92±2 / 95±5
λ-Cyhalothrin / 96±5 / 86±8 / 95±9 / 89±5
Permethrin / 96±4 / 95±8 / 91±3 / 96±7
Cyfluthrin / 99±4 / 106±7 / 93±5 / 91±7
α-Cypermethrin / 96±4 / 82±7 / 91±10 / 93±6
Fenvalerate / 106±4 / 95±6 / 87±9 / 90±8
Deltamethrin / 95±7 / 91±3 / 99±9 / 92±8
Imidacloprid / 91±10 / 100±8 / 99±7 / 91±8
Thiacloprid / 105±9 / 92±3 / 99±8 / 96±6

Where, M1 = PSA+MgSO4; M2 = PSA+MgSO4+C-18; M3 = PSA+MgSO4 +C-18+ Florisil; M4 = PSA+MgSO4 +Florisil

Supplementary Table 5: Calculation of Measurement uncertainty at level of 10 μg kg-1 (LVL) in human milk.

Measurement Uncertainty at the level of 10 μg kg-1 (LVL)
Compound name / Analyte Conc. / Rel. UCal / Rel. UIP / Rel. UR / UX / Expanded U
HCB / 9.01 / 0.02 / 0.02 / 0.01 / 0.25 / 0.49
β-HCH / 10.27 / 0.01 / 0.02 / 0.02 / 0.28 / 0.57
γ-HCH / 9.40 / 0.01 / 0.04 / 0.02 / 0.48 / 0.96
δ-HCH / 9.58 / 0.06 / 0.03 / 0.02 / 0.67 / 1.35
Aldrin / 9.28 / 0.02 / 0.03 / 0.02 / 0.35 / 0.69
Allethrin / 9.00 / 0.02 / 0.06 / 0.03 / 0.57 / 1.15
o,p-DDE / 9.04 / 0.01 / 0.04 / 0.03 / 0.47 / 0.95
α-Endosulfan / 9.20 / 0.02 / 0.03 / 0.02 / 0.41 / 0.83
o,p-DDD / 8.88 / 0.02 / 0.04 / 0.03 / 0.52 / 1.03
β-Endosulfan / 9.46 / 0.03 / 0.03 / 0.02 / 0.48 / 0.96
p,p-DDD / 9.20 / 0.02 / 0.03 / 0.03 / 0.43 / 0.85
p,p-DDT / 9.24 / 0.05 / 0.04 / 0.02 / 0.61 / 1.21
Bifenthrin / 9.12 / 0.00 / 0.03 / 0.02 / 0.28 / 0.57
Fenpropathrin / 9.05 / 0.03 / 0.01 / 0.01 / 0.31 / 0.62
Mirex / 9.27 / 0.01 / 0.03 / 0.02 / 0.36 / 0.71
λ-Cyhalothrin / 9.13 / 0.01 / 0.02 / 0.01 / 0.25 / 0.50
Permethrin / 9.35 / 0.00 / 0.03 / 0.02 / 0.36 / 0.71
Cyfluthrin / 8.77 / 0.06 / 0.02 / 0.03 / 0.65 / 1.30
α-Cypermethrin / 9.57 / 0.04 / 0.03 / 0.03 / 0.57 / 1.14
Fenvalerate / 8.93 / 0.03 / 0.05 / 0.03 / 0.59 / 1.19
Deltamethrin / 9.61 / 0.05 / 0.04 / 0.03 / 0.71 / 1.42
Thiacloprid / 8.83 / 0.07 / 0.04 / 0.02 / 0.74 / 1.48
Imidacloprid / 9.33 / 0.13 / 0.06 / 0.02 / 1.32 / 2.65
Acetamiprid / 9.13 / 0.02 / 0.05 / 0.04 / 0.82 / 1.51
Thiamethoxam / 9.51 / 0.05 / 0.04 / 0.03 / 0.72 / 1.45

Where, Rel. Ucal refers relative uncertainty for calibration curve; Rel. UIP refers relative uncertainty associated with intermediate precision; Rel. UR refers relative uncertainty associated with repeatability and Uxrefers to combined uncertainity.

Supplementary Table 6: Demographic characteristic of donor mothers and day of collection of human milk

Sample ID / Day of collection / Birth wt. / Mother's age / Parity
NA1 / 3 / 3.0 / 19 / 1
NA2 / 2 / 2.5 / 19 / 1
NA3 / 3 / 2.8 / 24 / 2
NA4 / 2 / 3.5 / 32 / 3
NA5 / 2 / 2.6 / 19 / 1
NA6 / 3 / 2.3 / 22 / 1
NA7 / 2 / 3.3 / 22 / 1
NA8 / 3 / 3.2 / 30 / 2
NA9 / 3 / 2.5 / 20 / 2
NA10 / 2 / 2.4 / 22 / 1

Supplementary Figure 1: Extracted chromatograms of small peaks of Allethrin, β-Endosulfan and p,p-DDT along with Total Ion Chromatogram (TIC).

Supplementary Figure 2: LC-MS/MS chromatograms of neonicotinoids compound (A. Imidacloprid, B. Thiacloprid, C. Thiamethoxam, D. Acetamiprid).

Supplementary Figure 3: Showing calibration curve for selected pesticides.

A Validated Method for the Determination of Neonicotinoid, Pyrethroid and Organochlorine

Contents