Quantitative Determination of Haloxyfop in Human Urine by Gas

Chapter 20

by

Quantitative Determination of Haloxyfop in Human Urine Gas Chromatography—Mass Spectrometry

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 17, 2016 | http://pubs.acs.org Publication Date: December 23, 1988 | doi: 10.1021/bk-1988-0382.ch020

R. A. Campbell, P. E. Kastl, B. E. Kropscott, and M . J. Bartels Analytical and Environmental Chemistry, Health and Environmental Sciences, Dow Chemical Company, Midland, MI 48674

An analytical method has been developed for the quantitative determination, at low to sub ppb levels, of haloxyfop in human urine. This method could be used to determine potential worker exposure to VERDICT herbicide during typical product use. Urine samples containing haloxyfop were fortified with isotopically labeled haloxyfop, extracted using benzene, derivatized, cleaned-up via HPLC and analyzed by gas chromatography-mass spectrometry. The mean relative recovery (±S.D.) of haloxyfop from urine (test material vs. internal standard) for the 7 concentrations ranging from 100 ng/mL to 0.2 ng/mL, was 107±7%. The coefficient of variation for relative recovery from urine was 3% at the highest (100 ng/mL) concentration and 58% at the lowest (0.2 ng/mL) concentration. A stability evaluation showed haloxyfop to be stable in urine samples stored at room temperature for at least two weeks. H a l o x y f o p m e t h y l e s t e r ( I ) , [methyl 2-(4-((3-chlorq-5(trifluoromethyl)-2-pyridinyl)oxy)phenoxy)propionate], t h e a c t i v e i n g r e d i e n t i n VERDICT h e r b i c i d e f o r m u l a t i o n s , i s b e i n g e v a l u a t e d f o r r e g i s t r a t i o n as a h e r b i c i d e f o r e r a d i c a t i o n o f a n n u a l and p e r e n n i a l grasses. The method d e s c r i b e d i n t h i s r e p o r t was d e v e l o p e d u s i n g a r a c e m i c m i x t u r e o f h a l o x y f o p and does n o t d i f f e r e n t i a t e between t h e S and R f o r m s . 0097-6156/89/0382-0251$06.00/0 • 1989 American Chemical Society

Wang et al.; Biological Monitoring for Pesticide Exposure ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

252

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE


U n p u b l i s h e d d a t a show h a l o x y f o p m e t h y l e s t e r i s r a p i d l y h y d r o l y z e d t o t h e a c i d i n man. An a n a l y t i c a l method was r e q u i r e d t o d e t e r m i n e low l e v e l c o n c e n t r a t i o n s o f h a l o x y f o p i n human u r i n e . A method u s i n g an i s o t o p i c a l l y l a b e l e d (D/j-haloxyfop) i n t e r n a l s t a n d a r d and gas chromatography-mass s p e c t r o m e t r y w i t h m u l t i p l e i o n d e t e c t i o n (GC/MS MID) was d e v e l o p e d t o q u a n t i t a t i v e l y d e t e r m i n e h a l o x y f o p i n u r i n e as low as 0 . 2 ng/mL. T h i s p r o c e d u r e i n c l u d e s an a u t o m a t e d h i g h r e s o l u t i o n sample c l e a n u p u s i n g r e v e r s e d - p h a s e HPLC.

(I) MATERIALS AND

HALOXYFOP

METHODS

MATERIALS. H a l o x y f o p was s u p p l i e d by A g r i c u l t u r a l P r o d u c t s Department, The Dow C h e m i c a l Company. The chemical p u r i t y of the t e s t m a t e r i a l , a racemic m i x t u r e , was d e t e r m i n e d t o be ± 9 9 % by l i q u i d chromatography. I s o t o p i c a l l y labeled haloxyfop, (99.5% pure) f o r use as an i n t e r n a l s t a n d a r d was s y n t h e s i z e d u s i n g a deuterated intermediate [prepared by r e f l u x i n g w i t h D 2 S O 4 a t 9 5 ° C ] ( B a r t e l s , M. J . ; G a t l i n g , S. C. J . L a b e l l e d Compounds Radiopharm., i n p r e s s .) T h i s procedure r e s u l t e d i n a n e a r l y complete exchange o f d e u t e r i u m f o r t h e f o u r h y d r o g e n s on t h e phenyl (center) r i n g . D i s t i l l e d - i n - g l a s s benzene, m e t h a n o l , UV s p e c t r o p h o t o m e t r y g r a d e hexane, a c e t o n i t r i l e and c o n c e n t r a t e d h y d r o c h l o r i c a c i d were o b t a i n e d from F i s h e r S c i e n t i f i c ( F a i r Lawn, N J ) . WISP L i m i t e d Volume I n s e r t s f o r HPLC a n a l y s i s were o b t a i n e d from Waters D i v . ( M i l l i p o r e , M i l f o r d , MA). Conical GC/MS a u t o s a m p l e r v i a l s were o b t a i n from Wheaton S c i e n t i f i c ( M i l l v i l l e , NJ). Human u r i n e f o r method v a l i d a t i o n was o b t a i n e d from v o l u n t e e r s w i t h i n t h e T o x i c o l o g y Lab, The Dow C h e m i c a l Company. STANDARDS. S t o c k s o l u t i o n s A and B were p r e p a r e d by d i s s o l v i n g a c c u r a t e l y weighed amounts o f h a l o x y f o p and D 4 ~ h a l o x y f o p , r e s p e c t i v e l y , i n methanol. A l i q u o t s of e a c h o f t h e s e s t o c k s o l u t i o n s were combined and d i l u t e d i n methanol t o prepare a t h i r d s t o c k s o l u t i o n (C). E x t e r n a l s t a n d a r d s o l u t i o n s f o r t h e GC/MS were p r e p a r e d by m e t h y l a t i n g an a l i q u o t o f s t o c k s o l u t i o n C u s i n g diazomethane, e v a p o r a t i n g t o dryness under n i t r o g e n and t h e n d i l u t i n g w i t h hexane t o a c h i e v e t h e


20. CAMPBELL ET AL.

Haloxyfop in Human Urine


desired concentration [i.e. ( 2 4 9 ng/mL h a l o x y f o p / 5 0 3 ng/mL i n t . s t d . ) t o ( 4 . 9 8 ng/mL h a l o x y f o p / 1 0 . 1 ng/mL int. std.)]. S o l u t i o n s f o r s p i k i n g u r i n e were p r e p a r e d by d i l u t i n g an a l i q u o t o f s t o c k s o l u t i o n C with methanol to y i e l d s o l u t i o n s of [ 2 . 4 9 JLlg/mL h a l o x y f o p / 5 . 0 3 Jlg/mL i n t . std.) to ( 0 . 2 4 9 jlg/mL h a l o x y f o p / 0 . 5 0 3 Jlg/mL i n t . s t d . ) . For recovery d e t e r m i n a t i o n s u r i n e s a m p l e s were s p i k e d w i t h 10-125 mL o f t h e a p p r o p r i a t e s p i k i n g s o l u t i o n p r i o r to extraction. E X T R A C T I O N METHOD AND H P L C C L E A N - U P . The appropriate amount o f h a l o x y f o p a n d i n t e r n a l s t a n d a r d were s p i k e d i n t o 30 mL o f u r i n e . C o n c e n t r a t e d HC1 (1 mL) was a d d e d t o t h e u r i n e a n d t h e s a m p l e was h e a t e d a t 80°C f o r 1 hour. A f t e r a l l o w i n g t o c o o l , 5 mL o f b e n z e n e was a d d e d t o t h e s a m p l e w h i c h was a g i t a t e d f o r 30 s e c o n d s on a v o r t e x m i x e r f o l l o w e d by centrifugation at 1500 rpm f o r 5 m i n u t e s . The b e n z e n e e x t r a c t was r e m o v e d b y p i p e t a n d a n a d d i t i o n a l 5 mL o f b e n z e n e was a d d e d t o t h e s a m p l e w h i c h was a g i t a t e d a n d c e n t r i f u g e d again. T h e s e c o n d b e n z e n e e x t r a c t was c o m b i n e d w i t h the f i r s t extract. An e t h e r e a l s o l u t i o n of d i a z o m e t h a n e was g e n e r a t e d u s i n g D i a z a l d (Aldrich C h e m i c a l Company, Inc., Milwaukee), and approximately 1 mL a d d e d t o e a c h s a m p l e . A f t e r complete methylation (-10 m i n . ) t h e s o l u t i o n s were e v a p o r a t e d u n d e r a gentle stream of nitrogen. T h e r e s i d u e was t h e n r e c o n s t i t u t e d i n d i e t h y l e t h e r and quantitatively t r a n s f e r r e d t o a WISP L i m i t e d V o l u m e I n s e r t . The e t h e r was e v a p o r a t e d u n d e r n i t r o g e n a n d t h e s a m p l e was r e c o n s t i t u t e d i n 125 |1L o f m e t h a n o l . For automated c l e a n - u p of the d e r i v a t i z e d urine extracts, a 100 JUL a l i q u o t was i n j e c t e d o n a r e v e r s e d p h a s e HPLC s y s t e m c o l l e c t i n g t h e f r a c t i o n (typically 3.75 t o 5.2 minutes) c o n t a i n i n g t h e h a l o x y f o p m e t h y l e s t e r and the c o - e l u t i n g i n t e r n a l s t a n d a r d peak. The c l e a n - u p was p e r f o r m e d u s i n g a W a t e r s R a d i a l C o m p r e s s i o n M o d u l e (RCM-100) a n d a NOVA-PAK c o l u m n . A g u a r d c o l u m n ( 2 . 5 4 cm x 3 . 9 mm) c o n t a i n i n g j l B o n d a p a k Cl8 ( 3 7 - 5 0 Jim) was u s e d f o r t h e p r o t e c t i o n o f t h e a n a l y t i c a l column. The HPLC c o n d i t i o n s were a s follows: autosampler: W a t e r s WISP ( M o d e l 5 1 0 B ) w i t h L i m i t e d Volume i n s e r t s ; D e t e c t o r : W a t e r s 4 8 1 (X= 227 nm)with s e n s i t i v i t y a t 0 . 0 1 A U F S ; pump: Waters 590; column: N O V A - P A K CIQ (10 cm x 5 mm i d ) ; f l o w r a t e : 1.0 mL/min; pressure: 1700 p s i ; i n j e c t i o n volume: 100 JUL; mobile phase: 70/30 a c e t o n i t r i l e / d i s t i l l e d water. A f r a c t i o n c o l l e c t o r f i t t e d w i t h an a i r a c t u a t e d v a l v e t h a t a l t e r n a t e d the column e f f l u e n t b e t w e e n w a s t e a n d a new c o l l e c t i o n v i a l was u s e d . The t i m i n g o f t h e v a l v e s w i t c h i n g was c o n t r o l l e d b y t h e W a t e r s 590 p u m p .


253


254

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

T h e UV d e t e c t o r was u s e d o n l y f o r c h e c k i n g t h e r e t e n t i o n window o f s t a n d a r d s . Samples c o n t a i n e d h i g h l e v e l s o f UV a b s o r b i n g c o m p o n e n t s , t h u s f r a c t i o n s w e r e c o l l e c t e d b a s e d on r e t e n t i o n times. The 3 . 7 5 t o 5.2 m i n u t e f r a c t i o n o f t h e effluent f r o m t h e UV d e t e c t o r was c o l l e c t e d i n a 1 d r a m v i a l . 500 |IL o f h e x a n e was a d d e d t o t h e v i a l w h i c h was a g i t a t e d f o r 15 s e c o n d s o n a v o r t e x m i x e r a n d c e n t r i f u g e d a t 3000 rpm f o r 1 m i n u t e . The hexane (top) l a y e r was r e m o v e d a n d p l a c e d i n a 5 0 0 |1L conical vial. T h e s a m p l e was b l o w n t o d r y n e s s under n i t r o g e n a n d r e c o n s t i t u t e d i n 1 0 0 |1L o f h e x a n e prior t o i n j e c t i o n on t h e GC/MS. T h e 10 a n d 1 0 0 ng/|lL s p i k e d u r i n e e x t r a c t s were r e c o n s t i t u t e d i n 1.0 and 10.0 ml hexane, respectively. GAS CHROMATOGRAPHY-MASS S P E C T R O M E T R Y . A F i n n i g a n 4600 GC/MS e q u i p p e d w i t h a S u p e r l n c o s d a t a s y s t e m (Finnigan MAT, S a n J o s e , C A ) , a H e w l e t t P a c k a r d 7 6 7 3 a u t o s a m p l e r and o p e r a t e d i n t h e MID m o d e , was u s e d f o r quantitative analysis of f i n a l extracts. S e p a r a t i o n o f the h a l o x y f o p as i t s m e t h y l e s t e r f r o m i n t e r f e r i n g s p e c i e s i n t h e u r i n e was a c h i e v e d w i t h a 15 m x 0 . 3 2 mm i . d . D X - 3 c a p i l l a r y c o l u m n , 0 . 2 5 Jim f i l m t h i c k n e s s (J&W S c i e n t i f i c , I n c . F o l s o m , CA) . H e l i u m a t 11 p s i g was u s e d a s t h e c a r r i e r g a s . During t h e a n a l y s i s , t h e c o l u m n was m a i n t a i n e d a t a temperature of 220°C, the i n j e c t i o n port and i n t e r f a c e o v e n t e m p e r a t u r e s were 275 a n d 2 7 0 ° C , respectively. T h e i n j e c t i o n v o l u m e was 3 . 0 j l L . A modified fixedr a t i o s p l i t t i n g s y s t e m (1) was u s e d . A 10 cm x 0 . 1 mm f u s e d s i l i c a r e s t r i c t o r t u b e was s e a l e d i n t h e i n j e c t o r s p l i t v e n t on t h e f r o n t i n s t r u m e n t p a n e l . The s p l i t v a l v e was f u l l y o p e n e d , a l l o w i n g f l o w c o n t r o l b y the r e s t r i c t o r tube. T h e e f f l u e n t was v e n t e d t o a f u m e h o o d t h r o u g h 1/8 i n c h t e f l o n t u b i n g . T h e GC/MS was a l s o e q u i p p e d w i t h a r e d u c e d - p r e s s u r e interface (2). O t h e r MS o p e r a t i n g p a r a m e t e r s a r e a s follows: preamp: l O ^ amps/volt; e l e c t r o n energy: 70 e V ; emission current: 0 . 4 mA; m u l t i p l i e r v o l t a g e : 1.4 kV; source temperature: 150°C.; i n d i c a t e d p r e s s u r e : 5 x 10~6 t o r r . H a l o x y f o p was q u a n t i f i e d u s i n g internal s t a n d a r d t e c h n i q u e s by m o n i t o r i n g t h e m o l e c u l a r i o n s , m/z 3 7 5 f o r t h e m e t h y l e s t e r o f h a l o x y f o p a n d m/z 3 7 9 for the deuterated haloxyfop i n t e r n a l standard methyl ester. -

CALCULATIONS I n t e r n a l s t a n d a r d c a l c u l a t i o n s were u s e d i n t h i s analysis. The r e s p o n s e f a c t o r (Rf) f o r a s t a n d a r d c a l c u l a t e d from the f o l l o w i n g equation:


was

Rf

where

HAL

x

A

I S

A

HAL

X

C

I S

(1)

CJJ^L

standard

and in

Cj5

ng/mL

are of

the

compounds The

GC/MS p e a k in

the

the

from

A

the

A

and

of

the

D4-internal

and A J S

are

the

obtained

for

these

L

of

the

recovery

of

spiked

urine

following

standard

haloxyfop

two

solutions.

vs. D 4 -

samples

was

calculated

equation: Rf

% Recovery

H

areas

analysis

relative

haloxyfop

concentrations

haloxyfop

respectively.

respective


C

=

compounds

from

255

Haloxyfop in Human Urine

20. CAMPBELL ET AL.

x

(A

-BKG)

H A L

x

Ng

I

S

=

x A

IS

x

N

Quantitative Determination of Haloxyfop in Human Urine by Gas

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