CIPAC/4683/R

Determination of deltamethrin and/or piperonyl

butoxide in Long-Lasting (incorporated into
polyethylene) Insecticidal Mosquito Nets

Validation and performance verification

Centre wallon de Recherches agronomiques

of the analytical method by GC-FID

REPORT TO CIPAC

by Olivier PIGEON

Walloon Agricultural Research Centre – (CRA-W)

Pesticides Research Department

WHO Collaborating Centre

for the Quality Control of Pesticides

Centre wallon de Recherches agronomiques

Département Phytopharmacie
Rue du Bordia, 11
B - 5030 GEMBLOUX - BELGIQUE
Tél : ++ 32 (0) 81 62 52 62
Fax : ++ 32 (0) 81 62 52 72
phytopharmacie@cra.wallonie.be
http://cra.wallonie.be

1
 CIPAC/4683/R

1. Validation of the analytical method

The validation of the analytical method for determination of deltamethrin and/or piperonyl
butoxide in Long-Lasting (incorporated into polyethylene) Insecticidal Mosquito Nets
(incorporated LNs) was performed on April 2008 in the framework of the CRA-W study
21678.

1.1 Specificity
The analysis of reagent blanks (method without sample) and untreated net samples in
comparison with the analysis of standard solutions, spiked untreated net samples and
treated net samples showed the absence of compound interfering with the determination of
deltamethrin, deltamethrin R-isomer and piperonyl butoxide.

Representative chromatograms
FID1 A, (RE21898A\001F0201.D)
5.788 - Standard Interne

pA 8.438 - Piperonil Butoxide

15.771 - Deltamethrin
90

80

70

60

50

40

30

20

10

0
2 4 6 8 10 12 14 16 min

Deltamethrin and piperonyl butoxide calibration solution (C5)

2
 CIPAC/4683/R

FID1 A, (RE21898A\003F0802.D)

5.789 - Standard Interne

8.421 - Piperonil Butoxide

pA

90

80

70

15.730 - Deltamethrin
60

50

40

30

20

10

0
2 4 6 8 10 12 14 16 min

Deltamethrin and piperonyl butoxide calibration solution (C3)

FID1 A, (RE21898A\005F1402.D)
5.791 - Standard Interne

8.396 - Piperonil Butoxide

pA

90

80

70

60

50
15.692 - Deltamethrin

40

30

20

10

0
2 4 6 8 10 12 14 16 min

Deltamethrin and piperonyl butoxide calibration solution (C1)

3
 CIPAC/4683/R

FID1 A, (RE21898A\006F5301.D)
pA

90

80

70

60

50

15.117 - Deltamethrin R-Isomer

40

30

20

10

0
2 4 6 8 10 12 14 16 min

Deltamethrin R-isomer calibration solution

FID1 A, (RE21898A\031F3901.D)
5.787 - Standard Interne

pA

90

80

15.725 - Deltamethrin
70
8.388 - Piperonil Butoxide

60

50
15.111 - Deltamethrin R-Isomer

40

30

20

10

0
2 4 6 8 10 12 14 16 min

PermaNet 3.0® side sample solution (Re 16029/71)

4
 CIPAC/4683/R

FID1 A, (RE21898A\033F4201.D)

5.790 - Standard Interne

8.448 - Piperonil Butoxide

pA

15.750 - Deltamethrin
90

80

70

60

15.131 - Deltamethrin R-Isomer

50

40

30

20

10

0
2 4 6 8 10 12 14 16 min

PermaNet 3.0® roof sample solution (Re 16029/73)

5
 CIPAC/4683/R

FID1 B, (RE21672A\007F0402.D)

5.513 - dipropyl phthalate

14.898 - deltamethrin
pA

45

14.332 - deltamethrin R-isomer

40

35

5.423
30

25

15.112
20

15

10

0
2 4 6 8 10 12 14 16 m in

NetProtect® sample solution (134 blue)

FID1 B, (RE21672A\018F2102.D)
5.515 - dipropyl phthalate

14.882 - deltamethrin
pA

45

14.320 - deltamethrin R-isomer

40

35

30
5.442

25
5.476
5.666
5.605

20

15

10

0
2 4 6 8 10 12 14 16 m in

NetProtect® sample solution (158 white)

6
 CIPAC/4683/R

1.2 Linearity of the detector response

Deltamethrin
The linearity was demonstrated over a range of concentrations from 5 µg/mL to 50 µg/mL
expressed as deltamethrin by measuring detector response factor (deltamethrin to internal
standard area ratio) versus deltamethrin concentration for a series of 5 standard solutions
of deltamethrin of 5, 12, 24, 36 and 50 µg/mL). Linear regression (least squares method)
was calculated on the mean of minimum 2 injections of each standard solution.

Calibration curve of deltamethrin by GC-FID

(linear regression)
1.8

1.6
y = 0.0330x - 0.0030
1.4
R² = 0.9999
1.2
Response factor

0.8

0.6

0.4

0.2

0
0 10 20 30 40 50 60
Concentration of deltamethrin (µg/mL)

The response factor (deltamethrin to internal standard area ratio) is linear in the range from
5 µg/mL to 50 µg/mL (R² > 0.99).

7
 CIPAC/4683/R

Piperonyl butoxide
The linearity was demonstrated over a range of concentrations from 24 µg/mL to 240
µg/mL expressed as piperonyl butoxide by measuring detector response factor (piperonyl
butoxide to internal standard area ratio) versus piperonyl butoxide concentration for a
series of 5 standard solutions of piperonyl butoxide of 24, 60, 120, 180 and 240 µg/mL).
Linear regression (least squares method) was calculated on the mean of minimum 2
injections of each standard solution.

Calibration curve of piperonyl butoxide by GC-FID

(linear regression)
12

10
y = 0.0434x + 0.0397
R² = 1.0000
8
Response factor

0
0 50 100 150 200 250

Concentration of piperonyl butoxide (µg/mL)

The response factor (piperonyl butoxide to internal standard area ratio) is linear in the
range from 24 µg/mL to 240 µg/mL (R² > 0.99).

8
 CIPAC/4683/R

1.3 Repeatability

The repeatability of the method was assessed by performing 5 or 6 replicate determinations

on 2 NetProtect samples.

Weightin Deltamethrin Deltamethrin R-isomer

Sample Sample
g content content
identification number weight (g) (g/kg) (g/kg)
134 blue 1 0.2994 1.55 0.248
134 blue 2 0.2998 1.59 0.265
134 blue 3 0.2997 1.56 0.259
134 blue 4 0.2996 1.50 0.260
134 blue 5 0.2997 1.54 0.265
Mean 1.55 0.259
SD 0.03 0.007
RSD 1.9% 2.7%
158 white 1 0.2995 1.51 0.358
158 white 2 0.3004 1.48 0.361
158 white 3 0.2999 1.52 0.362
158 white 4 0.2995 1.50 0.361
158 white 5 0.3000 1.50 0.367
158 white 6 0.2994 1.52 0.374
Mean 1.51 0.364
SD 0.01 0.006
RSD 0.9% 1.6%

The repeatability of the method is very good (RSD < 2% for deltamethrin content and <
3% for deltamethrin R-isomer content).

9
 CIPAC/4683/R

1.4 Accuracy and repeatability (recoveries)

Recovery values were calculated by fortifying untreated net samples with known amounts
of deltamethrin and piperonyl butoxide. The untreated net sample was analysed prior to
spiking and showed the absence of compound interfering with determination of
deltamethrin and piperonyl butoxide (< 30 % LOQ).

Deltamethrin

Sample Fortification level Extraction Injection Recovery (*)

(g/kg as deltamethrin) date date (%)
Mosquito net 0.398 April 2008 April 2008 85
0.399 April 2008 April 2008 92
0.399 April 2008 April 2008 95
0.403 April 2008 April 2008 94
0.403 April 2008 April 2008 98
Mean 93
Standard deviation 5
Relative standard deviation 5.2 %
Confidence interval (**) 93 ± 6
1.99 April 2008 April 2008 92
1.99 April 2008 April 2008 97
1.99 April 2008 April 2008 96
2.01 April 2008 April 2008 95
2.00 April 2008 April 2008 98
Mean 96
Standard deviation 2
Relative standard deviation 2.4 %
Confidence interval (**) 96 ± 3
3.97 April 2008 April 2008 99
3.98 April 2008 April 2008 97
3.97 April 2008 April 2008 98
4.01 April 2008 April 2008 92
4.02 April 2008 April 2008 98
Mean 97
Standard deviation 3
Relative standard deviation 2.9 %
Confidence interval (**) 97 ± 3
Mean 95
Standard deviation 4
Relative standard deviation 3.9 %
Confidence interval (**) 95 ± 2
(*) Each result is the mean of 2 chromatographic injections.
(**) Student t-test with a probability of 95 %.

The average recovery for deltamethrin on mosquito net for each fortification level is in the
range 90-110 % with a RSD < 5 %.

10
 CIPAC/4683/R

Piperonyl butoxide

Sample Fortification level Extraction Injection Recovery (*)

(g/kg as piperonyl date date (%)
butoxide)
Mosquito net 1.98 April 2008 April 2008 88
1.98 April 2008 April 2008 93
1.98 April 2008 April 2008 94
1.91 April 2008 April 2008 99
1.91 April 2008 April 2008 98
Mean 94
Standard deviation 4
Relative standard deviation 4.7 %
Confidence interval (**) 94 ± 6
9.89 April 2008 April 2008 98
9.86 April 2008 April 2008 99
9.89 April 2008 April 2008 98
9.56 April 2008 April 2008 99
9.51 April 2008 April 2008 97
Mean 98
Standard deviation 1
Relative standard deviation 0.9 %
Confidence interval (**) 98 ± 1
19.7 April 2008 April 2008 99
19.8 April 2008 April 2008 98
19.7 April 2008 April 2008 98
19.1 April 2008 April 2008 97
19.8 April 2008 April 2008 97
Mean 98
Standard deviation 1
Relative standard deviation 0.9 %
Confidence interval (**) 98 ± 1
Mean 97
Standard deviation 3
Relative standard deviation 3.1 %
Confidence interval (**) 97 ± 2
(*) Each result is the mean of 2 chromatographic injections.
(**) Student t-test with a probability of 95 %.

The average recovery for piperonil butoxide on mosquito net for each fortification level is
in the range 90-110 % with a RSD < 5 %.

11
 CIPAC/4683/R

1.5 Estimated limit of quantification (LOQ)

The limit of quantification (LOQ) was appraised as the concentration of deltamethrin and
piperonyl butoxide yielding a peak signal of at least 5 times the baseline noise.

Actine substance expressed as LOQ (g/kg)

deltamethrin 0.01

piperonyl butoxide 0.1

2. Performance verification of the analytical method

2.1 Accuracy and reproducibility (recoveries)
Between April 2008 and March 2009, the performance of the analytical method was
controlled in the framework of several (WHOPES) Phase I and II studies by performing
recoveries concurrently with the analysis of more than 830 samples of PermaNet® 3.0, 2.5
and 2.0. Recovery values were calculated by fortifying untreated net samples with known
amounts of deltamethrin and piperonyl butoxide. The untreated net samples were analysed
prior to spiking and showed the absence of compound interfering with determination of
deltamethrin ad piperonyl butoxide (< 30 % LOQ).

Recoveries for deltamethrin at 0.4 g/kg

N = 20
Mean recovery = 98.2%
RSD = 8.3%
150
140
130
120
110
Recovery %

100
90
80
70
60
50
0 5 10 15 20 25

12
 CIPAC/4683/R

Recoveries for deltamethrin at 2 g/kg

N = 20
Mean recovery = 95.6%
RSD = 3.5%
150
140
130
120
110
Recovery %

100
90
80
70
60
50
0 5 10 15 20 25

Recoveries for deltamethrin at 4 g/kg

N = 20
Mean recovery = 97.1%
RSD = 3.2%
150

140
130

120

110
Recovery %

100

90

80
70

60

50
0 5 10 15 20 25

13
 CIPAC/4683/R

Recoveries for piperonyl butoxide at 2 g/kg

N = 20
Mean recovery = 96.2%
RSD = 4.8%
150
140
130
120
110
Recovery %

100
90
80
70
60
50
0 5 10 15 20 25

Recoveries for piperonyl butoxide at 10 g/kg

N = 20
Mean recovery = 98.7%
RSD = 2.0%
150
140
130
120
110
Recovery %

100
90
80
70
60
50
0 5 10 15 20 25

14
 CIPAC/4683/R

Recoveries for piperonyl butoxide at 20 g/kg

N = 20
Mean recovery = 98.4%
RSD = 1.9%
150
140
130
120
110
Recovery %

100
90
80
70
60
50
0 5 10 15 20 25

The control of the performance of the analytical method performed during analysis of more
than 830 samples show mean recoveries of 95-99 % with RSD ranging from 1.9% to 8.3%
depending on the concentration level. Very few recoveries are outside the warning limit
(green line) and all the recoveries are within the action limit of the quality control chart.
Recoveries performed concurrently with the analysis of samples show therefore a good
accuracy and within laboratory reproducibility of the analytical method.

15
 CIPAC/4683/R

2.2 Comparison with another laboratory

The analytical method from CRA-W was compared with the analytical method from
Vestergaard Frandsen for deltamethrin content in 90 samples of PermaNet 3.0 and 2.0.
The analytical method from Vestergaard Frandsen involved extraction by heating under
relux with toluene, solvent exchange from toluene to mixture isooctane / 1,4 dioxane and
chromatographic determination by High Performance Liquid Chromatography with UV
Detection (HPLC-UV). The following graphs show comparable results (R² > 0.99).

5.00

4.50 y = 0.9849x + 0.0141
R² = 0.9919
4.00

3.50
Deltamethrin VF (g/kg)

3.00

2.50

2.00

1.50

1.00

0.50

0.00
0.00 1.00 2.00 3.00 4.00 5.00

3. Conclusion
The analytical method was successfully validated. The control of the performance of the
analytical method performed concurrently with the analysis of more than 830 samples
showed also a good accuracy and a good within laboratory reproducibility. The proposed
method is considered appropriate for the determination of deltamethrin and/or piperonyl
butoxide in Long-Lasting (incorporated into polyethylene) Insecticidal Mosquito Nets.

Acknowledgements
The author wish to gratefully thank the WHO Pesticides Evaluation Scheme, the LN
manufacturers Vestergaard Frandsen and Intelligent Insect Control, the IRD/CREC, the
London School of Tropical Hygiene & Medicine, The Institute of Tropical Medicine and
the personnel (LN Team) of the Pesticides Research Department of the Walloon
Agricultural Research Centre (CRA-W).
16