analysis of diafenthiuron in tea strategies to overcome...

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Analysis of Diafenthiuron in Tea Strategies to Overcome the Degradation Problem Stefan Kittlaus, Astrid Dylda, Thomas Anspach Eurofins | Dr. Specht Laboratorien, Großmoorbogen 25, 21079 Hamburg Introduction Approaches to Prevent Degradation Diafenthiuron is a selective thiourea insecticide and acaricide, widely used in cotton, fruits, vegetables and tea. It is acting selectively on different groups of insects and mites. Currently, Japan, Australia and other countries set MRLs at the total value of 20 mg/kg for diafenthiuron and its two metabolites in tea. [1] In the European Union diafenthiuron is not approved. Thus, the default MRL is 0.01 mg/kg according to Art. 18(1)(b) Reg. 396/2005. The analytical determination of diafenthiuron often is complicated by its low stability in different matrices. In the European Union Proficiency Test for Screening Methods in 2015 (EUPT-SM07) a lack in the detection of diafenthiuron was observed for broccoli. Only 9% of the laboratories reported this compound. The authors of the PT report also supposed an oxidative degradation of diafenthiuron in this matrix. On their poster at RAFA 2015 they demonstrated that the presumed degradation is related to the presence of the matrix broccoli and cannot be observed in pure water. [2] [email protected] [1] L. Wang, P. Zhao, F. Zhang, F. Du, C. Pan; Ecotoxicology and Environmental Safety 79 (2012) 7579 [2] S. Uclés, A. Lonzano, S. Herrera López. A. Uclés, A.R. Fernández-Alba; Poster P13 on RAFA 2015, Prague Depending on the matrix diafenthiuron tends to rapid degradation. In this study this was demonstrated for black tea. Different approaches were tested to overcome this problem. The best results were obsorved after extraction with pure acetonitrile. For future studies it would be helpfull to also analyse the metabolites or to correct the results with the an isotopically labeled internal standard. But, to our knowlege both are not available until now. Conclusion 30% 40% 50% 60% 0 5 10 15 20 recovery time in h 0% 20% 40% 60% 80% 100% 0 20 40 60 80 100 120 recovery shaking time in minutes only acetonitrile (ACN) H 2 O / ACN + 200 mg Na 2 SO 3 H 2 O / ACN + 200 mg ascorbic acid H 2 O / ACN (classic QuEChERS extraction) iso-propanol / ACN EDTA / ACN addition of 10 mL acetonitrile shake spiking with diafenthiuron (0,5 mg/kg) addition of 10 mL LC-MS/MS 2 g black tea centrifuge H 2 O (classic QuEChERS) acetonitrile iso-propanol H 2 O + 200 mg ascorbic acid H 2 O + 200 mg Na 2 SO 3 EDTA-buffer 1 min 5 min 10 min 30 min 60 min 120 min 240 min QuEChERS Degradation Study in Tea diafenthiuron DP1 C 23 H 32 N 2 OS 385.2308 sulfomonoxide DP2 C 23 H 32 N 2 O 2 S 401.2257 carbodiimide DP3 C 23 H 30 N 2 O 351.2430 urea DP4 C 23 H 32 N 2 O 2 369.2536 Degradation of diafenthiuron according to [2] 0% 20% 40% 60% 80% 100% 58% 46% 13% recovery immediately after spiking 10 minutes after spiking 60 minutes after spiking sample preparation addition of 10 mL acetonitrile shake 5 min spiking with diafenthiuron (0,5 mg/kg) addition of 10 mL H 2 O LC-MS/MS 2 g black tea centrifuge 0 min 10 min 60 min wait Dependance of the recovery of diafenthiuron from the incubation time after spiking quantification and recovery calculation was done with solvent calibration Degradation in the final extract each point quantified with coresponding solvent standard to correct for instrument sensitivity variations Impact of different approaches on the degradation quantification and recovery calculation with solvent calibration The addition of iso-propanol and EDTA-buffer supported the degradation process. Significant improvements of compound recovery were observed after the addition of sodium sulfite. But, even after short extraction times recovery decreased dramatically. The addition of ascorbic acid had no impact. The best results were obtained with pure aceto- nitrile. But, also here a significant degradation of the diafenthiuron during extraction process was measured. To overcome the presumed degradation problem different approaches were checked. The impact of water addition and different solvents was tested by replacing water with acetonitrile or iso-propanol. The authors of the EUPT also proposed the use of antioxidants. [2] For this reason, we tested the addition of ascorbic acid and sodium sulfite. Finally, we also tried the addition of an EDTA/Cystein buffer (pH 11). The results demonstrated the high degradation rate of diafenthiuron. Increased waiting times lead to significant lower recoveries. Even in final extract (vial was placed in a cooled autosampler) a rapid degradation was detected. As opposed to that, this was not observed in pure solvent. In the past we also observed very low recoveries for diafenthiuron in spiking experiments. Not in all types of matrices but specifically in tea samples. One possible assumption is that some compounds of tea promote this degradation pathway. In the first experiment the correlation between different waiting times after spiking and the resulting recovery was determined. Moreover, the degradation in the final extract was observed.

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Page 1: Analysis of Diafenthiuron in Tea Strategies to Overcome ...eprw2016.com/wp-content/uploads/2016/06/PD-058.pdf · Analysis of Diafenthiuron in Tea – Strategies to Overcome the Degradation

Analysis of Diafenthiuron in Tea – Strategies to

Overcome the Degradation Problem

Stefan Kittlaus, Astrid Dylda, Thomas Anspach

Eurofins | Dr. Specht Laboratorien, Großmoorbogen 25, 21079 Hamburg

Introduction Approaches to Prevent Degradation

Diafenthiuron is a selective thiourea insecticide and acaricide,

widely used in cotton, fruits, vegetables and tea. It is acting

selectively on different groups of insects and mites. Currently,

Japan, Australia and other countries set MRLs at the total value of

20 mg/kg for diafenthiuron and its two metabolites in tea. [1]

In the European Union diafenthiuron is not approved. Thus, the

default MRL is 0.01 mg/kg according to Art. 18(1)(b) Reg. 396/2005.

The analytical determination of diafenthiuron often is complicated by

its low stability in different matrices. In the European Union

Proficiency Test for Screening Methods in 2015 (EUPT-SM07) a lack

in the detection of diafenthiuron was observed for broccoli. Only 9%

of the laboratories reported this compound. The authors of the PT

report also supposed an oxidative degradation of diafenthiuron in

this matrix. On their poster at RAFA 2015 they demonstrated that

the presumed degradation is related to the presence of the matrix

broccoli and cannot be observed in pure water. [2]

[email protected]

[1] L. Wang, P. Zhao, F. Zhang, F. Du, C. Pan; Ecotoxicology and Environmental Safety 79 (2012) 75–79

[2] S. Uclés, A. Lonzano, S. Herrera López. A. Uclés, A.R. Fernández-Alba; Poster P13 on RAFA 2015, Prague

Depending on the matrix diafenthiuron tends to rapid degradation. In

this study this was demonstrated for black tea. Different approaches

were tested to overcome this problem. The best results were obsorved

after extraction with pure acetonitrile.

For future studies it would be helpfull to also analyse the metabolites

or to correct the results with the an isotopically labeled internal

standard. But, to our knowlege both are not available until now.

Conclusion

30%

40%

50%

60%

0 5 10 15 20

rec

ove

ry

time in h

0%

20%

40%

60%

80%

100%

0 20 40 60 80 100 120

rec

ove

ry

shaking time in minutes

only acetonitrile (ACN)

H2O / ACN

+ 200 mg Na2SO3

H2O / ACN

+ 200 mg ascorbic acid

H2O / ACN

(classic QuEChERS extraction)

iso-propanol / ACN EDTA / ACN

addition of 10 mL

acetonitrile

shake

spiking with diafenthiuron

(0,5 mg/kg)

addition of 10 mL

LC-MS/MS

2 g black tea

centrifuge

H2O (classic QuEChERS)

acetonitrile

iso-propanol

H2O + 200 mg ascorbic acid

H2O + 200 mg Na2SO3

EDTA-buffer

1 min

5 min

10 min

30 min

60 min

120 min

240 min

QuEChERS Degradation Study in Tea diafenthiuron

DP1 C23H32N2OS

385.2308

sulfomonoxide

DP2 C23H32N2O2S

401.2257

carbodiimide

DP3 C23H30N2O

351.2430

urea

DP4 C23H32N2O2

369.2536

Degradation of

diafenthiuron

according to [2]

0%

20%

40%

60%

80%

100%

58%

46%

13%

rec

ove

ry

immediately

after spiking

10 minutes

after spiking

60 minutes

after spiking

sample preparation

addition of 10 mL

acetonitrile

shake 5 min

spiking with diafenthiuron

(0,5 mg/kg)

addition of 10 mL

H2O

LC-MS/MS

2 g black tea

centrifuge

0 min

10 min

60 min

wait

Dependance of the recovery of diafenthiuron from the incubation time after spiking

quantification and recovery calculation was done with solvent calibration

Degradation in the final extract

each point quantified with coresponding

solvent standard to correct for

instrument sensitivity variations

Impact of different approaches on the degradation

quantification and recovery calculation with solvent calibration

The addition of iso-propanol and EDTA-buffer

supported the degradation process. Significant

improvements of compound recovery were

observed after the addition of sodium sulfite. But,

even after short extraction times recovery

decreased dramatically. The addition of ascorbic

acid had no impact.

The best results were obtained with pure aceto-

nitrile. But, also here a significant degradation of

the diafenthiuron during extraction process was

measured.

To overcome the presumed

degradation problem different

approaches were checked. The

impact of water addition and

different solvents was tested by

replacing water with acetonitrile or

iso-propanol. The authors of the

EUPT also proposed the use of

antioxidants. [2] For this reason,

we tested the addition of ascorbic

acid and sodium sulfite. Finally,

we also tried the addition of an

EDTA/Cystein buffer (pH 11).

The results demonstrated the high

degradation rate of diafenthiuron.

Increased waiting times lead to

significant lower recoveries.

Even in final extract (vial was

placed in a cooled autosampler) a

rapid degradation was detected.

As opposed to that, this was not

observed in pure solvent.

In the past we also observed very low recoveries for diafenthiuron in

spiking experiments. Not in all types of matrices but specifically in

tea samples. One possible assumption is that some compounds of

tea promote this degradation pathway.

In the first experiment the correlation between different waiting times

after spiking and the resulting recovery was determined. Moreover,

the degradation in the final extract was observed.