Slide 1

Abdou Mekebri, Ph.D.

November 2, 2011

CALIFORNIA DEPT OF FISH AND GAMEOFFICE OF SPILL PREVENTION AND RESPONSE

FISH AND WILDLIFE WATER POLLUTION CONTROL LABORATORY

Recent Advances in the Analysis of Pyrethroid Insecticides in Surface Water and

Sediments

Slide 2

2009 Expert Tour

Outline

�Aquatic Toxicity of Pyrethroids

�GC/QQQ with Chemical

Ionization and Backflushing

�Results

�Conclusions

Slide 3

Why go to Lower Detection Limits?

�There is a direct (toxic) effect to aquatic and terrestrial

organisms from pyrethroid exposure and an indirect

effect because of the threat to their food supply.

�Synthetic pyrethroids are known endocrine disruptors

and many may be carcinogenic (USEPA).

�Synthetic pyrethroids are extremely toxic to aquatic

organisms with LC50 values < 1 ppb.

Slide 4

Toxicity in Water and Sediment

Pesticide Water Sediment

LC50

(ng/L)Organism LC50

(µg/kg)Organism

Bifenthrin 70 Hyalella azteca1 4-10 Hyalella azteca2

Cyfluthrin 140 Ceriodaphnia dubia1 4-10 Hyalella azteca2

Cypermethrin 1.5 Hyalella azteca3 3-6 Hyalella azteca3

Esfenvalerate 70 Ceriodaphnia lacustris1 4-10 Hyalella azteca2

λ-Cyhalothrin 4 Hyalella azteca4 4-10 Hyalella azteca2

Permethrin 30 Hyalella azteca1 4-10 Hyalella azteca2

1 From previous slide

2 Amwag et al., 2005, Environ. Toxicol. Chem., 24, 966-972.

3 Maund et al., 2002, Environ. Toxicol. Chem., 21, 9-15.

4 Maund et al, 1998, Pestic. Sci., 54, 408-417.

Slide 5

Detection Limits Needed

Optimal MDL use LC50/10

- Water: ~ 0.2 ng/L

- Sediment: ~ 0.3 ng/g

MDL equal to LC50

- Water: ~ 2 ng/L

- Sediment: ~ 3 ng/g

Slide 6

Improving MDLs

� Larger sample and/or injection volumes

� Use of MS/MS – lowers background noise

� Negative Chemical Ionization (nCI) – effective for

pyrethroids with Cl, F, or Br

Slide 7

PFE PFE

GPC GPC

Liq/Liq-SPE

SEDIMENT TISSUE WATER

PYRETHOID EXTRACTION METHODS

GC/ECDGC/MS/MS – EI - NCI

FLORISILFLORISILFLORISIL(if needed)

PFE: Pressurized Fluid ExtractionGPC: Gel Permeation Chromatography

DFG Sample Preparation/Analysis

Slide 8

Advantage Disadvantage

very sensitive (1-5 pg on column) dual column required for

confirmation, therefore dual data

processing

specific for halogenated compounds rely on retention times for

confirmation

excellent screening tool requires extensive clean-up

(sediment and tissue)

affordable extensive matrix interference

easy to operate cannot distinguish between

deltamethrin and tralomethrin

Analytical Instrument Comparison for Pyrethroids

Instrument: GC-ECD

Slide 9

sensitivity comparable with ECD

(2-10 pg)

narrow linearity range

MS spectra confirmation trap clean-up needed for tissue

no matrix interference requires experienced MS chemist

less data handling cannot distinguish between

deltamethrin and tralomethrin

short run time

Instrument: GC-MS Ion trap in MS-MS mode

Advantage Disadvantage

Slide 10

sensitivity comparable with

GC-MS-ion trap

not as sensitive as GC-MS-QqQ due

to polarity of compounds

best result will be obtained by APCI requires specially trained MS

chemist

distinguishes between deltamethrin and

tralomethrin

very expensive

excellent for thermo labile compounds

minimum sample clean-up required

excellent linearity

very promising for chiral separation

green chemistry

Instrument: LC-MS-MS

Advantage Disadvantage

Slide 11

extremely sensitive (0.1-2.0 pg on

column)

maintenance needed frequently

excellent for trace level in sediment

and tissue

very experienced MS chemist

needed

detects halogenated and

non-halogenated pyrethroids

expensive

best analytical tool for pyrethroids cannot distinguish between

deltamethrin and tralomethrin

Instrument: GC-MS Triple-quadrupole (QqQ)

Advantage Disadvantage

Slide 12

Instrumentation & Backflush Configuration

Purged Ultimate Union

Vent

Aux EPC

QQQ

MS

Purged UnionMM Inlet

Agilent 7890A/7000B GC/QQQWith Chemical Ionization

Two 15 m XLB

Slide 13

Vent

Aux EPC

QQQ

MS

Purged UnionInlet

Agilent 7890A 7000B

Two 15 m HP-5MS UI

Injection & analysis During Backflush

Mid-column Backflush

Vent

Aux EPC

QQQ

MS

Purged UnionInlet

Agilent 7890A 7000B

Two 15 m HP-5MS UI

Begin backflushing after last analyte passes purged union

or during post run

Slide 14

Bifenthrin, 1 ppb Standard: NCI-SIM and NCI-MRM (methane)

MRM transition: 386→205 (green)SIM ion: 386 (purple)

SIM

S/N = 55

MRM

S/N = 7158

Slide 15

Comparing Methane and Ammonia Reagent Gases for NCI GC/MS/MS Analysis of Pyrethroids

Fenvalerate I

Esfenvalerate

Ammonia

Methane

Slide 16

Pyrethroids-50fg (n=8)-NCI

Slide 17

Pyrethroids-20fg-NCI-large volume injection

Slide 18

Cypermethrin MRM by NCI Ammonia

0.020-10 µg/L

Slide 19

Deltamethrin MRM by NCI Methane

0.050-10 µg/L

Slide 20

Sediment-100fg spike

Slide 21

GC/ECD DB5 - Sediment Extracts

min10 15 20 25 30 35 40

Hz

500

1000

1500

2000

2500

ECD1 A, (L-200-10-SED_RR\L-200-10-SED_RR 2010-07-27 15-51-46\049F4901.D)

DB

OB

Bif

en

thri

n

DB

CE

Cy

h,L

-2

Pe

r-tr

an

s

Cy

f-1

Cy

f-2

Cy

f-4 C

yp

-1 C

yp

-2 C

yp

-3 C

yp

-4

Esf

-1

Es

f-2

De

ltam

eth

rin

Sediment - F4

ECD2 B, (L-200-10-SED_RR\L-200-10-SED_RR 2010-07-27 15-51-46\049F4901.D)

min10 15 20 25 30 35 40 45

Hz

250

500

750

1000

1250

1500

1750

2000

ECD1 A, (L-200-10-SED_RR\L-200-10-SED_RR 2010-07-27 15-51-46\026F2601.D)

DB

OB

Bife

nth

rin

Fe

np

rop

ath

rin

DB

CE

Cyh

,L-1

Cy

h,L

-2

Pe

r-c

is P

er-

tra

ns

Cy

f-3

Cyp

-2 C

yp-3

Es

f-1 D

elta

me

thri

n

Sediment - F2

Slide 22

Lambda-cyhalothrin MRM-NCI-MSMS

0.020 µg/L standard

methane

ammonia

Slide 23

Sediment Sample Extract by NCI - methane

deltamethrin

5.01 ng/g

es-fenvalerate

0.81 ng/g

Slide 24

22.5 25 27.5 30 32.5 35 37.5 40

Hz

0

2000

4000

6000

8000

10000

12000

14000

16000

ECD1 A, (L-333-11\L-333-11 2011-10-17 10-07-09\008F0801.D)

ECD - Round Robin Sediment Extract – 2011

Slide 25

NCI - Round Robin Sediment Extract – 2011

Slide 26

NCI - Round Robin Sediment Extract – 2011

Slide 27

MS/MS-EI-Signal to Noise and ELOD

Estimated Limit of Detection (ELOD)

Synthetic pyrethroid std conc µg/L MS/MS-EI-S/N Sediment ng/g

Allethrin 0.500 629 1.50

Parallethrin 0.500 325 0.80

Resmethrin 0.500 501 0.80

Bifenthrin 0.500 1408 0.30

Fenpropathrin 0.500 165 1.20

Tetramethrin 0.500 262 0.50

Phenothrin 0.500 686 0.50

Cyhalothrin 0.500 197 0.35

Permethrin cis 0.500 423 0.75

Permethrin trans 0.500 362 0.95

Cyfluthrin 0.500 24.5 0.35

Cypermethrin 0.500 40.0 0.45

Es-fenvalerate 0.500 57.4 0.25

Deltamethrin 0.500 30.9 0.50

Slide 28

MS/MS-NCI-Signal to Noise and ELOD

MS/MS-NCI-Signal to Noise S/N

Estimated Limit of Detection (ELOD)

Synthetic pyrethroid

std conc µg/L Methane Ammonia Water ng/L Sediment ng/g

Bifenthrin 0.050 973 2345 0.10 0.08

Cyhalothrin 0.050 683 1890 0.20 0.1

Permethrin 0.050 25 150 1.00 1.5

Cyfluthrin 0.050 277 1217 0.20 0.2

Cypermethrin 0.050 336 1410 0.20 0.18

Es-fenvalerate 0.050 2757 6450 0.05 0.05

Deltamethrin 0.050 190 463 0.15 0.3

Slide 29

Pyrethroid Method Detection and Reporting Limits for Water, Sediment and Tissue - ECD

Sample Matrix Water Sediment Tissue

Pyrethroid Pesticides

MDL RL MDL RL MDL RL

ppb (ug/L)

ppb (ug/L)

Dry wt ppb (ng/g)

Dry wt ppb (ng/g)

Fresh wt ppb (ng/g)

Fresh wtppb (ng/g)

Bifenthrin 0.001 0.002 0.50 1.00 0.65 2.00

Cyfluthrin 0.002 0.004 2.00 4.00 2.70 6.00

Cypermethrin 0.004 0.004 2.00 4.00 1.70 4.00

Deltamethrin 0.002 0.004 2.00 4.00 0.60 2.00

Es/Fenvalerate 0.001 0.002 1.00 2.00 1.70 4.00

Fenpropathrin 0.002 0.004 2.00 4.00 1.35 4.00

Lambda-cyhalothrin 0.001 0.002 1.00 2.00 1.50 4.00

Permethrin 0.003 0.005 4.00 8.00 2.20 6.00

Slide 30

Analysis of Deltamethrin(a) and Tralomethrin(b) using LC/MS

a

b

2x10

0

0 .05

0.1

0 .15

0.2

0 .25

0.3

0 .35

0.4

0 .45

0.5

0 .55

0.6

0 .65

0.7

0 .75

0.8

0 .85

0.9

0 .95

1

1 .05+ Sc an (0 .991-1 .333 min , 39 sc ans) W o rk listD a ta6 .d

523.00000100

1x10

0

0.1

0.2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1

1.1

1 .2

1 .3

1 .4

1 .5

1 .6

1 .7

1 .8

1 .9

+ Sc an (2 .415-2 .722 min , 41 sc ans) W o rk listD a ta7 .d

682 .78569100

703.7357450

104.993017 159.00000

5

Counts (%) v s. M ass-to -Cha rge (m/ z)

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760

b

a

Slide 31

Conclusions

� Analysis of pyrethroids by GC-MSMS-NCI yields low detection limits that are necessary for toxicity of these analytes

� GC-NCI-MSMS is at least 5 times more sensitive than GC-EI-MSMS but is not suitable for all pyrethroid pesticides

� Ammonia NCI is 5-10 times more sensitive than methane

� LC-MS is necessary to distinguish between deltamethrin and tralomethrin

Slide 32

Thank You

Any questions?