FIuorometric Determination of Pesticides George G. Guilbault and M. H. S a d a r Chemistry Department, Louisiana State University in New Orleans, New Orleans, La. 70122
THEOBJECT of this study is t o develop sensitive procedures for the determination of chlorinated insecticides, carbamate insecticides, and herbicides, based on the use of enzyme systems. Fluorescence methods were used, because whenever tried such techniques have been shown to allow the determination of much lower concentration of enzymic inhibitors (for lower enzymic activities can be measured). The enzyme system screened for inhibition was lipase, which has been reported to be inhibited by small amounts (0.1 pg) of organophosphorus compounds ( I ) . A highly sensitive fluorometric method (2, 3) was used to monitor the lipase activity. The nonfluorescent substrate, 4-methyl umbelliferone heptanoate was used, which is cleaved by as little as 10-5 unit of lipase t o 4-methyl umbelliferone. The chlorinated insecticides aldrin, heptachlor, lindane, and DDT, the carbamate Sevin and the herbicide 2,4-D were found to inhibit lipolytic activity, and could be determined in varying concentrations. EXPERIMENTAL
Reagents. 4-Methyl Umbelliferone Heptanoate was prepared as described by Jacks and Kirchner (3). A stock 10-3M solution was prepared in methyl cellosolve. A 0.1 mg/ml solution of lipase was prepared by dissolving the enzyme (porcine pancreas, Sigma, activity 2.1 Wilson units per mg. A unit represents 0.05 meq of titratable fatty acid formed from the action of enzyme powder on 1.0 ml of neutral olive oil in 30 minutes at 37 "C, as assayed by the procedure of LazoWasem ( 4 ) in triply distilled water. Pesticides. Stock solutions of all pesticides were prepared in methyl cellosolve. The pesticides were all of the highest available purity, and were obtained as follows: Sevin (1naphthyl N-methyl carbamate), Union Carbide; 2,4-D (2,4-dichlorophenoxy acetic acid), Eastman Organics; aldrin, lindane (1,2,3,4,5-hexachlorocyclohexane),heptachlor (3,4,5,6,7,8,9-heptachlorodicyclopentadiene), and DDT [2,2-bis(p-chlorophenyl) 1,l trichloroethane] were obtained from Polyscience Corp., Evanston, 111. Apparatus. An Aminco Bowman spectrophotofluorometer (SPF), equipped with a thermoelectric cooler t o maintain a constant temperature of 25 "C and a linear Beckman recorder to automatically record rates, was used in all studies described. A A, of 330 mp and a A, of 450 mp were used to monitor the 4-methyl umbelliferone formed in enzymic cleavage of the heptanoate ester. Procedures. DETERMINATION OF LINDANE.To 3.0 ml of citrate buffer, 0.1M pH 7.0, and 0.1 ml of 10-3M 4-methyl umbelliferone heptanoate is added 0.1 ml of a solution of lindane or heptachlor to be analyzed. The fluorescence is adjusted to zero, and 0.1 ml of a 0.1 mg/ml solution of lipase is added. The rate of change in fluorescence with time, AF/min, is recorded. A blank (uninhibited) rate is recorded with no pesticide present, but with 0.1 ml of the solvent for lindane or heptachlor added. The per cent inhibition is calculated as below:
0
20
40
60
80
100
120
140
160
180
220
200
ALDRIN, p g / m l
Figure 1. Effect of enzyme concentration on the sensitivity of detection of aldrin. No preincubation. A-0.006 unit/ml of lipase B-0.060 unit/ml of lipase
Inhibition
=
(AF/min)Blnnk - (AF/rninh (AF/min)B l a n k
loo
The concentration of pesticide is determined from a calibration plot of per cent inhibition us. concentration of lindane. DETERMINATION OF SEVIN, HEPTACHLOR, OR ALDRIN. The procedure as described above is followed, except that the enzyme is preincubated with the pesticide (10 minutes for Sevin and 30 minutes for aldrin and heptachlor) in the presence of buffer (3 ml) but with no substrate added. After incubation the fluorescence of the solution is again adjusted t o zero, 0.1 ml of the substrate is added, and the rate of change in fluorescence with time is recorded. A blank is run with 0.1 ml of solvent incubated with enzyme and buffer with no pesticide present, The per cent inhibition is calculated, and the concentration of Sevin, heptachlor, or aldrin determined from a calibration plot of per cent inhibition rs. concentration. DISCUSSION AND RESULTS
Effect of Substrate and Enzyme. 4-Methyl umbelliferone heptanoate has been found to be the best substrate for the assay of lipase ( 2 ) from considerations of stability, blank rate, rate of enzymic hydrolysis, and lowest detectable enzyme concentrations. The conditions for assay (substrate contration, pH, etc.) were the same as previously found optimal R - C - O
u, cH3 (Noniluorescent)
Highly Fluorescent
(1) G. G. Guilbault and D. N. Kramer, ANAL.CHEM., 36, 409
(1964). (2) G . G. Guilbault and M. H. Sadar, A m l . Letr., 1,460 (1968). (3) T. J. Jacks and H. W. Kirchner, Anal. Biockem., 21,279 (1967). (4) E. A. Lazo-Wasem, J . Pliurrn. Si., 50,999 (1961). 366
ANALYTICAL C H E M I S T R Y
( 2 ) . The rate of production of the highly fluorescent 4-methyl umbelliferone from the nonflourescent heptanoate ester, as modified by the pesticide present, is measured.
The effect of enzyme concentration on the sensitivity of detection of the pesticide is shown in Figure 1. In general, greater sensitivity was achieved with decreasing enzyme concentration, a 0.006-unit/ml concentration beng more sensitive then a 0.06-unit/ml. In fact one of the chief advantages of a fluorescence monitoring system lies in its ability to monitor lower enzyme concentration, which in turn allows the assay of lower pesticide concentrations. Although a further decrease in lipase concentration caused a further increase in assay sensitivity, a decrease in reproducibility and precision resulted, as might be expected. Therefore, an enzyme concentration of 0.006 unit/ml was used in the assay of all pesticides. Effect of Solvent. It has been shown ( I ) that methyl cellosolve (ethylene glycol monomethyl ether), the solvent used for preparing stock solutions of the pesticide, is inhibitory to the enzyme in concentrations above 3-5z.For this reason only 0.1 ml of the pesticide solution is added to the reaction mixture of 3.3 ml (overall 373, and the blank uninhibited rate is always run with 0.1 ml of methyl cellosolve added. Likewise in the preincubation procedure the pesticide is mixed with enzyme only after buffer has been added, in a total volume of 3.2 ml. Effect of Incubation. The chlorinated insecticides, aldrin and lindane, and the carbamate insecticide, Sevin, were found to be potent inhibitors of lipase activity (Table I), even without incubation. Heptachlor and DDT, two other chlorinated pesticides, and the herbicide (2,4-D) were less potent, and large concentrations were required for 50z inhibition of the enzyme (Iso). Preincubation of enzyme and inhibitor before addition of substrate has a pronounced effect on aldrin, Sevin, lindane, and heptachlor, but little effect on 2,4-D. Smaller concentrations of D D T effect a 50% inhibition (276 pg/ml compared to a maximum inhibition of 4 1 z with 500 pg with no preincubation), but DDT still remains a poor inhibitor. Optimum preincubation times are indicated in Table I, being found by studying the % inhibition with a constant pesticide concentration as a function of time of incubation. Although preincubation had some effect on lindane, the
9
r
I
-
-
-
-
A
-
d
-
i
>
PESTICIDE,
yg/ml
Figure 2. Plot of per cent inhibition us. concentration for a number of inhibitors of lipase. A-Sevin, 10 minutes preincubation B-Aldrin, 30 minutes preincubation C-Heptachlor, 30 minutes preincubation D-Lindane, 30 minutes preincubation E-DDT, 20 minutes preincubation
Table I. Comparison of 150 for Various Pesticides Both with and without Preincubation Lipase = 0.006 unit/ml 150, M a
Inhibitor Aldrin Sevin Lindane 2,4-D Heptachlor DDT
No incubation 1.64 X 4.0 X 1 . 2 x 10-4 1.0
x
Preincubationb 1.93 X 10-6 1 . 7 X 10-6
6.18
10-3"
Optimum incubation time, min 30 10 30
x 10-6
...
...
4.24 x 10-6
1.46 x IO-*
. . .d
7.8
x
30 20
10-4
Concentration of pesticide giving 50% inhibition of enzymic activity. At optimum preincubation time. c Preincubation has little effect. Reaches a maximum of 41 inhibition at 500 pg/ml, then decreases. Never reaches 50 inhibition,
z
z
Table 11. Comparison of 160 for Sevin with Various Enzymes
(I
Enzyme Cholinesterase, horse serum Cholinesterase, bovine erythrocyte Cholinesterase, human plasma Cholinesterase, fly head Lipase, porcine pancreas Data from reference (5).
Lo, 1.8
4.0 5.0
0.04a 3.4
effect was not as pronounced as that observed with other pesticides. Furthermore, better results were obtained with no incubation with lindane than with preincubation. Without incubation 3-100 pg/ml of lindane can be analyzed with a relative error of lt2.5%, whereas with incubation 0.8-40 pg/ml were analyzed with a relative error of f3.5 %. Plots of per cent inhibition us. concentration of inhibitor were found to be linear (Figure 2), over the following range of inhibitors: 0.1-2 pg/ml of Sevin and heptachlor, 0.1-5 pg/ml of aldrin, 0.8-10 pg/ml of lindane, and 10-1000 pg/ml of DDT and 2,4-D. The pesticides were added to Mississippi River water samples, and the amount of pesticide present was calculated from a standard calibration plot. Average error and precision of about 2-3% was obtained in all analyses in the concentration ranges indicated above, which is quite acceptable for the low concentrations of pesticides analyzed. The results reported indicate that the method of lipase inhibition is the most sensitive enzymic method yet reported for heptachlor, aldrin, lindane, and 2,4-D. It was found in this study that lipase is more sensitively inhibited by Sevin than are bovine erythrocyte and human plasma cholinesterase, although some insect cholinesterases are inhibited by smaller concentrations of Sevin (5) (Table 11). The proposed inhibition scheme is likewise a good one for DDT. Although carbonic anhydrase is inhibited by lower concentrations of D D T (1-10 pg) (6),the lipase procedure described is an easier, more convenient one to carry out. Instead of an elaborate gaseous C02monitoring system, only a recording fluorometer is needed. The effect of the pesticides is believed to be a direct inhibition of the enzyme lipase. These substances were found to (5) T. E. Archer and G . Zweig, J . Agr. Food Chem., 6,910 (1958). (6) H. Keller, Naturwissenschaften, 39, 109 (1952). VOL. 41,NO. 2, FEBRUARY 1969
367
have no effect on the fluorescence of 4-methyl umbelliferone over the time of assay (3-5 minutes). Because the lipase assay system used with 4-methyl umbelliferone heptanoate as substitute is a completely homogeneous, soluble one the effect of the inhibitor is not believed to be on the physical state of the substrate. This is in contrast to other lipase systems involving insoluble (emulsified) substrates in which the inhibitor does affect the physical state of the substrate (7). Interferences. Organophosphorus compounds such as Sarin and Systox inhibit lipase (I),and would thus interfere in any procedure for the determination of the chlorinated pesticides. In order to eliminate possible interference from inorganic ions, the pesticide can be extracted from the sample with methyl cellosolve prior to analysis. Mixtures of pesticides can be separated by thin layer chromatography and each (7) P. Desnuelle, Adcan. Enzymol. 23, 130 (1961).
inhibitor determined separately. Thus the specific analysis of mixtures of pesticides in samples of urine, crop materials, animal tissue, milk, etc. is possible. Current research in these laboratories is directed toward the use of different enzymes with simple separation techniques for the assay of complex mixtures of pesticides. Results of this study will be forthcoming. ACKNOWLEDGMENT
The authors thank H. Beckman, Agricultural Toxicology Labs, for supplying a sample of the Sevin used in this study.
RECEIVED for review August 12, 1968. Accepted October 14, 1968. The financial assistance of the Office of Saline Water, U. S. Department of the Interior (Grant No. 14-010001-1337) and the United States Public Health Service (Contract PH 21-2016 to Louisiana State University School of Medicine) is gratefully acknowledged.
Application of Interrupted-Elution to Combustion Radio Gas Chromatography Fulvio Cacace and Giorgio Perez Laboratorio di Chimica Nucleare del C.N.R.-Istituto di Chimica Farmaceutica e Tossicologica, Unicersity of Rome, Rome, Italy
THECOMBUSTION of the effluents, followed by the reduction of the resulting water to molecular hydrogen, is a practice widely employed in the radio gas chromatography of tritiated compounds (1-10). Such a technique allows, in fact, the radioactivity detector to be operated at room temperature, outside the oven of the chromatograph, a feature which is most desirable in many cases. In addition, the combustion of the effluents largely eliminates the variations of the detector efficiency and background caused by the elution of certain types of organic substances (7, I / ) ,because these effluents are converted to inorganic gases, such as Hz, CO, N2, etc., and affect to a much smaller extent the operation of the radioactivity detector. With methods based on the continuous combustion of the effluents, the residence time of a given peak in the combustion furnace depends entirely on the chromatographic separation being carried out, and the result may be inadequate for a quantitative conversion. While in a properly constructed and operated unit, ocerall conversions of 97.8 to 99.5% have been measured (12) even under difficult conditions (high flow rates, (1) F. Cacace, R. Cipollini, and G. Perez, Science, 132,1253 (1960). (2) F. Cacace, Nucleonics, 19, 5, 45 (1961), (3) A. T,James and E. A. Piper, J . Chromatogr., 5,265 (1961). (4) J, W. Winkelman and A. Karmen, ANAL. CHEM.,34, 1067 (1962). (5) A . Karmen, I. McCaffrey, and R. L. Bowman, J . Lipid Res., 3, 372 (1962). (6) A. T . James and E. A . Piper, ANAL. CHEM., 35, 515 (1963). (7) A. Karmen, I. McCaffrey, J. W. Winkelman, and R. L. Bowman, ibid., p 537. (8) F. Cacace, R. Cipollini, and G. Perez, ibid., p 1348. (9) A. Karmen, J. Assoc. Ofic.Agr. Chemists, 47, 15 (1964). (10) A. Karmen, J. Gas Chromatogr., 5,502 (1967). (11) J. K. Lee, E. K. C. Lee, B. Musgrave, Y.N. Tang, J. W. Root, and F. S. Rowland, ANAL. CHEM.,34, 741 (1962). (12) F. Cacace, R. CipoIlini, G. Perez, and E. Possagno, Gazr. Chim. Ztal. 91, 804 (1961). 368
ANALYTICAL CHEMISTRY
relatively large samples, etc.), the possibility of isotopic fractionation during the combustion (13) makes it very desirable to achieve in all cases, and especially in the assay of tritiated compounds, a complete conversion. The difficulties which are encountered in the continuous combustion approach could be largely overcome by the application of the principle of interrupted elution (14), which allows sufficient time to ensure the complete conversion of any eluted peak, and permits an effective removal of any activity left in the combustion train before the conversion of the next peak is undertaken. The advantages of the static activity analysis allowed by the interrupted-elution radio gas chromatography in applications not requiring the combustion of the effluents have been discussed elsewhere (15). The present paper describes the application of the interruptedelution principle to the combustion radio gas chromatography of tritiated compounds. EXPERIMENTAL
Apparatus. The apparatus used is a Model B Fractovap of Carlo Erba Co. (Milan, Italy), modified as shown in Figure 1. The carrier gas (He, A, Nz etc.) flows from the pressure regulator, 1, through the injection port, 2, and the column, 3, housed in the oven, 4. A two-way stainless steel stopcock, 5 , is inserted between the injection port and the column, and a three-way stainless steel stopcock, 6, is inserted between the column and the thermal conductivity detector, 7, whose outlet is connected
(13) R. F. Glascock, “Isotopic Analysis for Biochemists,” Academic Press, New York 1954, p 87. (14) R. P. W. Scott, I. A . Fowlis, D. Welti, and T. Wilkins, “Gas Chromatography 1966,” A. B. Littlewood, Ed., the Institute of Petroleum, London 1967, p 318. (15) F. Cacace and G. Perez, ANAL. CHEM.39, 1863 (1967).
