at a carrier gas flow rate of 100 ml/min is shown in Figure 1A. The response was much more sensitive and more rapid than that obtained for the Delphi cell. The response times at various flow rates of some different Hersch cell systems are compared to the silver coated beads cathode cell system in Table I.
The usefulness of a working electrode of closely packed small spheres is demonstrated and it is believed that this concept should find future use in other galvanic analyzer systems, RECEIVED for review December 16,1970. Accepted February 18,1971.
Ion-Selective Electrode Procedure for Organophosphate Pesticide Analysis George Baum and Frank B. Ward Corning Glass Works, Research and Development Labs, Corning, N. Y . 14830
THE ECOLOGICAL PROBLEMS posed by the use of persistent pesticides have been well documented ( I , 2). As agriculturalists turn to increased use of organophosphates, the need for a reliable, sensitive, yet simple method for routine monitoring of these pesticides becomes evident. Although gas chromatographic procedures using highly sensitive detectors such as microwave (3), KC1 thermionic (4), or electron capture (5) have been successfully employed, the results may be of limited value. Organophosphates in the environment are known to undergo numerous, ill-defined conversions to intermediate products which may retain biological activity (6). A bioanalytical method based on cholinesterase in.hibition would have greater significance with respect to ingestion tolerance levels. This topic has been comprehensively reviewed by Gage (7). Numerous analytical procedures have been described for the determination of cholinesterase activity (8, 9). Most of these methods have been adapted to the study of cholinesterase inhibitors. We recently reported an electrometric method for the determination of cholinesterase activity (IO) based on the use of a liquid-membrane ion-selective electrode (11) which responds continuously to the concentration of acetylcholine remaining during an enzymic catalyzed hydrolysis. The present report describes our efforts to utilize the electrode for the determination of the anticholinesterase activity of organophosphate pesticides. (1) “Organic Pesticides in the Environment,” Aduan. Chem. Ser.. No. 60, American Chemical Society, Washington, D. C., 1966 (2) “Cleaning Our Environment,” American Chemical Society, Washington, D. C., 1969. (3) C. A. Bache and D. J. Lisk, ANAL.CHEM., 38, 1757 (1966). (4) H. B. Pionke, J. G. Conrad, G. Chesters, and D. E. Armstrong, Analyst, 93, 353 (1968). (5) W. L. Lamar, D. F. Goerlitz, and L. M. Law, Aduan. Chem. Ser., No 60, American Chemical Society, Washington, D. C., 1966, p 187. (6) R. D. O’Brien, ‘‘Toxic Phosphorous Esters,” Academic Press. New York, N. Y . , 1960. (7) J. G. Gage in “Advances in Pest Control Research,” R. L. Metcalf, Ed., Wiley, New York, N. Y . ,Vol. 4, 1961, p 183. (8) W. Pilz in “Methods of Enzymatic Analysis,” H-U. Fkrgmeyer, Ed., Weinheim Bergster, Verlag Chemie, 1965, p 765. (9) G. G. Guilbault, “Enzymatic Methods of Analysis,” Pergamon Press, Oxford, 1970, p 44 (10) G. Baum, Anal. Biochem., 39,65 (1971). (11) G. Baum, Ami. Lett., 3, 105 (1970).
EXPERIMENTAL
Solutions. The substrate solution was prepared from acetylcholine bromide (Eastman) dissolved in a buffer solution consisting of 0.10N NaCl, 0.005N KC1, 0.02N Na2HP04, 0.02M MgClz adjusted to p H 8.00 with 0.1N HC1. Acetylcholinesterase (3.1.1.7) was obtained from Worthington Biochemical Corporation. The specific activity reported was 110 I.U. A stock solution of 1.00 mg of enzyme in 10 ml of buffer was employed for all runs. Paraoxon, diethyl p-nitrophenylphosphate, was obtained from J. T. Baker, Tetram, 0,O’diethyl S-j3-diethylamminoethyl phosphorothioate hydrogen oxalate, was kindly furnished by R. D. O’Brien, Cornel1 University. Enzyme Activity Determination. An aliquot of 5 1.11 of the AChE solution was introduced to a stirred solution of 2.0 X 10-aM acetylcholine bromide in phosphate buffer. Fresh substrate solution was prepared daily. The potential developed across a Corning Model 476200 acetylcholine selective electrode with a Fisher cracked bead 13-639-57 calomel reference electrode was monitored by a Corning Model 101 digital electrometer which is equipped with a slope control and an analog concentration converter. The electrometer was calibrated to read out in concentration units directly. By means of a stopwatch, we noted the lapsed time for the substrate concentration to decrease from 1.95 X 10-3M to 1.85 X 10-3M. Inhibition Studies. Fresh inhibitor solutions were prepared daily. The enzyme and inhibitor were incubated, with shaking, at room temperature. At varying intervals an aliquot, usually 5-10 pl, was withdrawn and rapidly added to the substrate solution. Residual enzyme activity was determined in duplicate as described above. RESULTS AND DISCUSSION
The electrometric method yields cholinesterase activity values which are about 15% higher than the colorimetric procedure of Hestrin (12). No bias correction was applied to the data obtained in this study. The difference is probably due to a short induction period which was not determined in the colorimetric procedure. The reaction of cholinesterase with potent organophosphate inhibitors can be represented by Equation 1: E+ A
-B
k +1
z E...A
- B+
k+2
E -A
+B
(1)
! L1
(12) S. Hestrin, J . B i d . Chem., 180, 249 (1949). ANALYTICAL C H E M I S T R Y , VOL. 43, NO. 7 , J U N E 1971
947
At low inhibitor concentrations, the time-course of the inhibition follows the pseudo first order kinetic expression (13) of Equation 2 :
2.3 log 3 kg =
00 ~
It
The two organophosphates studied followed Equation 2 throughout the concentration range of interest. The data obtained at a series of concentrations can be replotted in the form of Equation 3 ut
log-
00
x
tki Z 100 = - + 2 2.3
(3)
Table I. Determination of Organophosphates Paraoxon, 2-minincubation, ng/ml Added Found % Diff. 132 170 378 576 756 945
133 162 376 588 745 943 Standard error in estimate of (I) = 12.0 ng/ml
+0.74 -4.69 -0.02 +3.42 -1.31 -0.38
Paraoxon, 30-min incubation, n g / d 10.0 27.2 50.1 71.2
9.05 27.1 54.7 67.6 Standard error in estimate of (I) = 4.3 ng/ml
-10.0 -0.4 +8.6 -5.4
to show the log of the per cent residual activity (P)after a fixed incubation time as a function of concentration. These preliminary measurements are required to determine the useful concentration range and incubation time for each inhibitor to be studied. Most reliable data are obtained within an inhibition level of 10% to 60% loss of initial cholinesterase activity. Organophosphate pesticides vary greatly in their rates of hydrolysis. It is therefore essential to limit incubation times to durations where a significant loss of anticholinesterase activity does not occur. After we established the linear inhibition regions, the analytical procedure followed for the determination of inhibitor concentration was to collect inhibition data at constant incubation times over a range of inhibitor concentrations. At an incubation time of two minutes, the data for five different concentrations, in duplicate, can be collected in a few hours. At an incubation time of two minutes, Paraoxon may be conveniently determined over a concentration range of about 100 to 1000 ng/ml. The standard error in estimate of Paraoxon in this concentration range is 12 ng/ml. Increased sensitivity can be obtained with an incubation time of 30 min. The useful inhibitor concentration range is 10-100
ng/ml; the observed standard error in estimate of Paraoxon was 4.3 ng/ml. Tetram was examined at an incubation time of two minutes only. The useful concentration range of inhibitor was 50 to 300 ng/ml; the observed standard error of estimate of Tetram was 19.2 ng/ml. Results are summarized in Table I. The sensitivity of a bioanalytical assay, such as described above, depends largely on the degree of anticholinesterase activity of the organophosphate pesticide or metabolite. Organocarbamates which also exhi bit anticholinesterase activity should be detectable by this procedure. For potent inhibitors, the sensitivity compares favorably with that obtained by GLC techniques.
(13) W. N. Aldridge, Biochem. J., 46,451 (1950).
RECEIVED for review December 21, 1970. Accepted February 24, 1971.
948
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, J U N E 1971
Tetrarn, 2-min incubation, ng/ml 66 132 192 265
74 127 194 270 Standard error in estimate of (I) = 19.2 ng/ml
$11
+6.9 +2.6 +1.9
