Bull. Environm. Contam. Toxicol. 22,530-535 (1979)
Effect of Freezer Storage on Alkyl Phosphate Metabolites in Urine Gerald Ito, Wendell W. Kilgore, and John J. Seabury Department ci Environmental Toxicology, College of Agricultural and Environmental Sciences, University of Calffornia, Davis, Calif. 95616
For the past two years our laboratory has been involved in the monitoring of alkyl phosphate metabol i t e s of various organophosphorus insecticides in the urine of farm workers (KRAUS et al. 1977). Because of the large number of samples involved, the samples were stored frozen u n t i l analyzed. Recently, however, i t was reported that the parathion metabolites, O,O-diethylphosphoric acid (DEP) and O,O-diethyl phosphorothioate K+ (DETP, thionate isomer), are not stable in urine samples stored at -18~ (COMER et al. 1976). Thus, the purpose of this study wasoEo i--nvestigate these findings further to determine the v a l i d i t y of data previously obtained by ourselves and other investigators. Ail urine metabolites under investigation were studied over a twenty week period. EXPERIMENTAL PROCEDURE Standards. The alkyl phosphate standards, O,Odiethylphosp-h-oric acid and O,O-diethyl phosphorothioate K+, were supplied by American Cyanamid Corporation. Working solutions were prepared from stock solutions (l mg metabolite/mL acetone) by d i l u t i n g aliquots with acetone to provide final solutions containing O.Ol mg metabolite/mL acetone. Reagents. DOWEXl-X8 anion exchange resin, 50/I00 mesh, chloride form (BIO-RAD Laboratories); pesticide grade acetone; diazopentane as described by SHAFIK et al. (1973); and acetone containing I% formic acid. Equipment. A gas l i q u i d chromatograph (GLC) equipped with a flame photometric detector was used to separate and quantitate the metabolites. The glass column used was 1.8 m x 4 mm i . d . that had been silane-treated and packed with 4% OV-210 on acid washed, silane-treated 80/I00 mesh Gas Chrom Q (LEIBRAND and DUNHAM 1973). The GLC parameters were as follows: column oven temp., 155~ i n l e t temp., 200~ detector temp., 200~
0007-4861/79/0022-0530 $01.20 9 1979 Springer-Verlag New York Inc.
n i t r o g e n c a r r i e r gas f l o w r a t e , 85 mL/min. A Corning, Model 12, Research pH M e t e r , c a l i b r a t e d at pH 4.01 and pH 7 . 0 0 , was used f o r pH measurements. Method. Each of s i x male v o l u n t e e r s c o l l e c t e d u r i n e samples in o n e - q u a r t p l a s t i c b o t t l e s d u r i n g a normal w o r k i n g day. A I00 mL a l i q u o t of u r i n e from each s u b j e c t was f o r t i f i e d at 5 ppm O,Od i e t h y l p h o s p h o r i c acid and lO ppm O , O - d i e t h y l p h o s p h o r o t h i o a t e K+. The samples were mixed t h o r o u g h l y , d i v i d e d i n t o I0 mL a l i q u o t s (one I0 mL a l i q u o t f o r each a n a l y s i s ) , and then f r o z e n . Ail I0 mL a l i q u o t s were s t o r e d in g l a s s s c i n t i l l a t i o n v i a l s , covered w i t h P a r a f i l m , and s t o r e d at -25~ until analyzed. The samples were a l l o w e d to thaw at room t e m p e r a t u r e f o r 2-3 h b e f o r e a n a l y s i s . Each I0 mL a l i q u o t was s u f f i c i e n t to run d u p l i c a t e analyses. The f o r t i f i e d samples were a n a l y z e d at O, I , 2, 3, 4, 6, 8, 12, 16, and 20 weeks. Three I0 mL a l i q u o t s of u r i n e from each s u b j e c t were a n a l y z e d in d u p l i c a t e as c o n t r o l s at -2 days, 19 days, and 37 days a f t e r f r e e z i n g . Recovery s t u d i e s were a l s o performed in d u p l i c a t e from I0 mL a l i q u o t s of c o n t r o l u r i n e t h a t had been f r o z e n . The metabolites were analyzed as the d e r i v a t i v e s O,O-diethyl O-amyl phosphate (DEAmP) and O,O-diethyl S-amyl phosphorothiolate (DEAmPTH, t h i o l a t e isomer), which were prepared by a s l i g h t modification (WINTERLIN et al. 1975) of the method of LORES and BRADWAY (1977). Quantitation was accomplished by comparing peak heights to external standards. The s e n s i t i v i t y of the analysis was O.l ppm for a 2 mL urine sample when a 6-uL a l i q u o t from 6 mL of f i n a l solution was injected onto the GLC column (minimum detectable l i m i t of 200 pg of alkyl phosphate). The pH of the urines of each subject was also monitored.
RESULTS AND DISCUSSION The results of COMER et al. (1976) indicated considerable breakdown or d-Tsa-p-pearance of O,Odiethyl phosphorothioate K+ (DETP, thionate isomer) over a s i x week period. This was contrary to our f i n d ings in t h i s study. Under our conditions there was no apparent disappearance of e i t h e r DEP or DEPTh even a f t e r the urine samples were stored in the freezer for 20 weeks, as shown in Figures 1 and 2. Though the results of COMER et al. (1976) also indicated the disappearance
531
~2
n
6
o
0
0
L, I
I 2 I 3
I 4
Concentration Figure I. mean v a l u e s a r e p l o t t e d .
~ 3
Age of sample (Weeks)
I 8
I 12 o f DEP as a f u n c t i o n o f t h e age o f Standard deviation is indicated.
I 6
~'/l~.,T\!/t~!/"
-
?
the
sample.
I 16 Group
I 20
1~.
T
8
6
0
1
2
3
4
O E5 "' OE
~-
OE
E
9
10
!1
12
C
I 1
J 2
p~
I 3
! 4
I 6
AEe of sampie (W›
i,, 8
I 12
I 16
Concentration of DEPTh as a f u n c t i o n of the age of the samp!e. Figure 2. Standard d e v i a t i o n is i n d i c a t e d . Group mean values are p l o t t e d .
O
w
m
13 -
14 -
i 20
of O,O-diethyl phosphorothioate K+ over a range of approximately 4 ppm, we have found f l u c t u a t i o n s of up to 4 ppm in a sample analyzed one week apart. In t h e i r i n v e s t i g a t i o n LORES and BRADWAY (1977) found a s i g n i f i c a n t amount of v a r i a b i l i t y in t h e i r recoveries. Out r e s u l t s were also quite v a r i a b l e , as shown in Table l . TABLE 1 Percent Recovery of Alkyl Phosphate Metabolites in Urine
WEEK
DEP
DEPTh
0 l 2 3 4 6 8 12 16 20
43 38 47 44 46 53 49 39 37 38
46 61 35 37 33 94 77 73 68 27
Because the low recoveries did not give us as r e l i a b l e q u a n t i t a t i o n as desired, the a n a l y t i c a l method was used only as a s e m i - q u a n t i t a t i v e measurement. Also, the pH of the urine of each subject was monitored as a rough i n d i c a t o r of microbial growth and, thus, any possible breakdown. Although a considerable v a r i a t i o n existed between subjects, the pH did not change s i g n i f i c a n t l y during the storage period. In conclusion, i t is apparent from t h i s study that d i a l k y l phosphate metabolites do not break down or disappear in urine samples stored frozen for up to 20 weeks. I t is e v i d e n t , however, that the a n a l y t i c a l procedure used gives r i s e to highly variable data t h a t could be i n t e r p r e t e d as a loss or disappearance of the metabolites.
534
REFERENCES COMER, S.W., H.E. RUARK, and A.L. ROBBINS: Bull. Environ. Contam. Toxicol. 16, 618 (1976). KRAUS, J.F., D.M. RICHARDS, N.O. BORHANI, R.L. MULL, W.W. KILGORE, and W. WINTERLIN: Arch. Environ. Contam. Toxicol. 5, 471 (1977). LEIBRAND, R.J., and L.L. DUNHAM: 32 (1973). LORES, E.M., and D.E. BRADWAY: 25, 75 (1977).
Res. Dev. 24,
J. Agric. Food Chem.
SHAFIK, M.T., D.E. BRADWAY, H.F. ENOS, and A.R. YOBS: J. Agric. Food Chem. 21, 625 (1973). WINTERLIN, W., et a l . , Unpublished data, University of California, Davis. (1975).
535
Effect of Freezer Storage on Alkyl Phosphate Metabolites in Urine Gerald Ito, Wendell W. Kilgore, and John J. Seabury Department ci Environmental Toxicology, College of Agricultural and Environmental Sciences, University of Calffornia, Davis, Calif. 95616
For the past two years our laboratory has been involved in the monitoring of alkyl phosphate metabol i t e s of various organophosphorus insecticides in the urine of farm workers (KRAUS et al. 1977). Because of the large number of samples involved, the samples were stored frozen u n t i l analyzed. Recently, however, i t was reported that the parathion metabolites, O,O-diethylphosphoric acid (DEP) and O,O-diethyl phosphorothioate K+ (DETP, thionate isomer), are not stable in urine samples stored at -18~ (COMER et al. 1976). Thus, the purpose of this study wasoEo i--nvestigate these findings further to determine the v a l i d i t y of data previously obtained by ourselves and other investigators. Ail urine metabolites under investigation were studied over a twenty week period. EXPERIMENTAL PROCEDURE Standards. The alkyl phosphate standards, O,Odiethylphosp-h-oric acid and O,O-diethyl phosphorothioate K+, were supplied by American Cyanamid Corporation. Working solutions were prepared from stock solutions (l mg metabolite/mL acetone) by d i l u t i n g aliquots with acetone to provide final solutions containing O.Ol mg metabolite/mL acetone. Reagents. DOWEXl-X8 anion exchange resin, 50/I00 mesh, chloride form (BIO-RAD Laboratories); pesticide grade acetone; diazopentane as described by SHAFIK et al. (1973); and acetone containing I% formic acid. Equipment. A gas l i q u i d chromatograph (GLC) equipped with a flame photometric detector was used to separate and quantitate the metabolites. The glass column used was 1.8 m x 4 mm i . d . that had been silane-treated and packed with 4% OV-210 on acid washed, silane-treated 80/I00 mesh Gas Chrom Q (LEIBRAND and DUNHAM 1973). The GLC parameters were as follows: column oven temp., 155~ i n l e t temp., 200~ detector temp., 200~
0007-4861/79/0022-0530 $01.20 9 1979 Springer-Verlag New York Inc.
n i t r o g e n c a r r i e r gas f l o w r a t e , 85 mL/min. A Corning, Model 12, Research pH M e t e r , c a l i b r a t e d at pH 4.01 and pH 7 . 0 0 , was used f o r pH measurements. Method. Each of s i x male v o l u n t e e r s c o l l e c t e d u r i n e samples in o n e - q u a r t p l a s t i c b o t t l e s d u r i n g a normal w o r k i n g day. A I00 mL a l i q u o t of u r i n e from each s u b j e c t was f o r t i f i e d at 5 ppm O,Od i e t h y l p h o s p h o r i c acid and lO ppm O , O - d i e t h y l p h o s p h o r o t h i o a t e K+. The samples were mixed t h o r o u g h l y , d i v i d e d i n t o I0 mL a l i q u o t s (one I0 mL a l i q u o t f o r each a n a l y s i s ) , and then f r o z e n . Ail I0 mL a l i q u o t s were s t o r e d in g l a s s s c i n t i l l a t i o n v i a l s , covered w i t h P a r a f i l m , and s t o r e d at -25~ until analyzed. The samples were a l l o w e d to thaw at room t e m p e r a t u r e f o r 2-3 h b e f o r e a n a l y s i s . Each I0 mL a l i q u o t was s u f f i c i e n t to run d u p l i c a t e analyses. The f o r t i f i e d samples were a n a l y z e d at O, I , 2, 3, 4, 6, 8, 12, 16, and 20 weeks. Three I0 mL a l i q u o t s of u r i n e from each s u b j e c t were a n a l y z e d in d u p l i c a t e as c o n t r o l s at -2 days, 19 days, and 37 days a f t e r f r e e z i n g . Recovery s t u d i e s were a l s o performed in d u p l i c a t e from I0 mL a l i q u o t s of c o n t r o l u r i n e t h a t had been f r o z e n . The metabolites were analyzed as the d e r i v a t i v e s O,O-diethyl O-amyl phosphate (DEAmP) and O,O-diethyl S-amyl phosphorothiolate (DEAmPTH, t h i o l a t e isomer), which were prepared by a s l i g h t modification (WINTERLIN et al. 1975) of the method of LORES and BRADWAY (1977). Quantitation was accomplished by comparing peak heights to external standards. The s e n s i t i v i t y of the analysis was O.l ppm for a 2 mL urine sample when a 6-uL a l i q u o t from 6 mL of f i n a l solution was injected onto the GLC column (minimum detectable l i m i t of 200 pg of alkyl phosphate). The pH of the urines of each subject was also monitored.
RESULTS AND DISCUSSION The results of COMER et al. (1976) indicated considerable breakdown or d-Tsa-p-pearance of O,Odiethyl phosphorothioate K+ (DETP, thionate isomer) over a s i x week period. This was contrary to our f i n d ings in t h i s study. Under our conditions there was no apparent disappearance of e i t h e r DEP or DEPTh even a f t e r the urine samples were stored in the freezer for 20 weeks, as shown in Figures 1 and 2. Though the results of COMER et al. (1976) also indicated the disappearance
531
~2
n
6
o
0
0
L, I
I 2 I 3
I 4
Concentration Figure I. mean v a l u e s a r e p l o t t e d .
~ 3
Age of sample (Weeks)
I 8
I 12 o f DEP as a f u n c t i o n o f t h e age o f Standard deviation is indicated.
I 6
~'/l~.,T\!/t~!/"
-
?
the
sample.
I 16 Group
I 20
1~.
T
8
6
0
1
2
3
4
O E5 "' OE
~-
OE
E
9
10
!1
12
C
I 1
J 2
p~
I 3
! 4
I 6
AEe of sampie (W›
i,, 8
I 12
I 16
Concentration of DEPTh as a f u n c t i o n of the age of the samp!e. Figure 2. Standard d e v i a t i o n is i n d i c a t e d . Group mean values are p l o t t e d .
O
w
m
13 -
14 -
i 20
of O,O-diethyl phosphorothioate K+ over a range of approximately 4 ppm, we have found f l u c t u a t i o n s of up to 4 ppm in a sample analyzed one week apart. In t h e i r i n v e s t i g a t i o n LORES and BRADWAY (1977) found a s i g n i f i c a n t amount of v a r i a b i l i t y in t h e i r recoveries. Out r e s u l t s were also quite v a r i a b l e , as shown in Table l . TABLE 1 Percent Recovery of Alkyl Phosphate Metabolites in Urine
WEEK
DEP
DEPTh
0 l 2 3 4 6 8 12 16 20
43 38 47 44 46 53 49 39 37 38
46 61 35 37 33 94 77 73 68 27
Because the low recoveries did not give us as r e l i a b l e q u a n t i t a t i o n as desired, the a n a l y t i c a l method was used only as a s e m i - q u a n t i t a t i v e measurement. Also, the pH of the urine of each subject was monitored as a rough i n d i c a t o r of microbial growth and, thus, any possible breakdown. Although a considerable v a r i a t i o n existed between subjects, the pH did not change s i g n i f i c a n t l y during the storage period. In conclusion, i t is apparent from t h i s study that d i a l k y l phosphate metabolites do not break down or disappear in urine samples stored frozen for up to 20 weeks. I t is e v i d e n t , however, that the a n a l y t i c a l procedure used gives r i s e to highly variable data t h a t could be i n t e r p r e t e d as a loss or disappearance of the metabolites.
534
REFERENCES COMER, S.W., H.E. RUARK, and A.L. ROBBINS: Bull. Environ. Contam. Toxicol. 16, 618 (1976). KRAUS, J.F., D.M. RICHARDS, N.O. BORHANI, R.L. MULL, W.W. KILGORE, and W. WINTERLIN: Arch. Environ. Contam. Toxicol. 5, 471 (1977). LEIBRAND, R.J., and L.L. DUNHAM: 32 (1973). LORES, E.M., and D.E. BRADWAY: 25, 75 (1977).
Res. Dev. 24,
J. Agric. Food Chem.
SHAFIK, M.T., D.E. BRADWAY, H.F. ENOS, and A.R. YOBS: J. Agric. Food Chem. 21, 625 (1973). WINTERLIN, W., et a l . , Unpublished data, University of California, Davis. (1975).
535
