Detection and Estimation of Organophosphorus Compounds with Hydroxamic Acids Using a Chemical Analog of the Cholinesterase Inhibition Method Joseph Epstein and M. M. Demek Research Laboratories, Edgewood Arsenal, Edgewood Arsenal, Md. 21010 A chemical analog to the enzyme inhibition technique for determination of organophosphate anticholinesterase esters is described. Hexanehydroxamic acid is used in lieu of an esterase. In this method, the organophosphate ester is allowed to react with an excess of the hydroxamic acid at pH 9. The unreacted hydroxamic acid then catalyzes the hydrolysis of a pale yellow substrate to a highly red-colored species. After a given time, the hydrolytic reaction is stopped by the addition of acid and the initial inhibitor concentration is calculated from the measured intensity of color. While the sensitivity attained in the determination of the organophosphorus esters is not so high using the described technique as is attainable using enzymatic techniques, the advantages in the use of stable chemicals make this procedure worthy of recommendation.
dure potentially useful, especially where sensitivity is not of major concern. Moreover, this work is of interest as a guide to future research in studies of chemical analogs of enzyme inhibition techniques. Organophosphorus compounds such as isopropyl methylphosphonofluoridate (GB) o r diisopropylphosphorofluoridate (DFP) react rapidly with hydroxamic acids in dilute aqueous solution to produce fluoride ion and an unstable phosphorylated intermediate (I), which spontaneously undergoes a Lossen rearrangement (8). The final products of the reaction are fluoride ion, the corresponding phosphonic or phosphoric acid, and a product (11) resulting from the reaction of a n alkyl isocyanate [one of the products of rearrangement of (I)] with excess hydroxamic acid. 0
BECAUSE OF THE HIGH SENSITIVITY and selectivity attainable, greater use is being made of enzymes in the fields of analysis and detection ( I ) . A particularly useful application is in the detection and analysis of various organophosphate acids (after conversion to a mixed anhydride) ( 2 ) and esters which phosphorylate the enzyme cholinesterase, thus preventing it from catalyzing the hydrolysis of a suitable carboxylic acid ester substrate. The rate of production of the carbon ester hydrolysis products is related to the concentration of the inhibitor, the rate decreasing with increasing inhibitor concentration. The types of carbon esters used in the above scheme have been many, ranging from simple esters which produce acid products upon hydrolysis (3), t o esters which hydrolyze t o form colored species from colorless or differently colored ones (4, 5), to electrochemically inactive esters which form active moieties (6) upon hydrolysis, t o nonfluorescent esters which hydrolyze t o fluorescent species (7). While the use of enzymes has the advantages of potential sensitivity and selectivity, it has been recognized that several of the properties inherent in enzymes limit their usefulness ( I ) . The use of a chemical system qualitatively similar to that of the system involving a n esterase is described in this paper. Although the sensitivity using the chemical system is not so high as that attainable with some enzymatic procedures, the advantages in the use of organic chemicals (such as the stability and reproducibility of reagent solutions, etc) make this proce-
(1) G. G. Guilbault, ANAL.CHEM., 35, 527R-36R (1966). (2) H. W. Yurow, D. H. Rosenblatt, and J. Epstein, Talanta, 5, 199 (1960). (3) P. A. Giang and S. A. Hall, ANAL.CHEM., 23, 1830 (1951). (4) D. N. Kramer and R. M. Gamson, Zbid.,30, 251 (1958). (5) J. Epstein, M. M. Demek, and V. Wolff, Zbid., 29, 1050 (1957). (6) G. G. Guilbault, D. N. Kramer, and P. L. Cannon, Jr., Zbid., 34, 1437 (1962). (7) G. G. Guilbault and D. N. Kramer, Zbid., 37, 120 (1965). 1 136
ANALYTICAL CHEMISTRY
0
’/
RC-NHOH
//
+ FP-(0R)a
+
0
I * RNCO
//
+ HOP-(OR)2
0
’ /
RNCO
+ RC-NHOH
+
RCONH-OCONHR’ (11)
For a given hydroxamic acid, the facility with which the Lossen rearrangement will occur, is related to the ionization constant of the acid formed (9), the rate increasing with increasing acid strength. Because in the above cases, the acids (phosphonic and phosphoric) are relatively strong (pKa-l), the rearrangement is extremely rapid. In fact, the rate controlling step in the overall reaction between G B and a hydroxamic acid is the one producing fluoride ion (10). If acylating agents possessing groups which ultimately form acids of weak character are used, the intermediate acylated hydroamic acids d o not undergo a Lossen rearrangement but hydrolyze instead to form the acids of the acylating agent and regenerate the hydroxamic acid. The hydroxamic acid, in effect, catalyzes the hydrolysis of the acylating agent ( I I ) . (8) B. E. Hackley, Jr., R. Plapinger, M. A. Stolberg, and T. Wagner-Jauregg, J . Am. Cliem. SOC.,77, 3651 (1955). (9) H. L. Yale, Chem. Reo., 33, 209 (1943). (10) R. Swidler and G. M. Steinberg, J . Am. Cliem. SOC.,1956, p.
3594. (11) M. M. Demek, G. F. Endres, and J. Epstein, U. S. Patent, 3,068,072, Dec. 11, 1962.
0
0
//
RC--X
0
//
+ R'C-NHOH
/ / / /
RC-NHOC-R'
RC--X
+ HX
0
0
//
//
+ HzO OH; R'C-NHOH + RC-OH
0
//
//
RC-NHOC-R'
0
0
0
//
0
+ HzO
// R'C--"oH+
0
//
RC-OH
+ HX
These reactions have been made the basis for a n analytical method for GB. The phosphorylating material is allowed to react with an excess of a hydroxamic acid; the concentration of the unreacted hydroxamic acid is determined from the rate of splitting of an acylating agent by the hydroxamic acid; the initial concentration of the phosphorylating material is determined from the concentration of the unreacted hydroxamic acid. The acylating agent used in this study was the pale yellow 2-azobenzene-l -naphthyl acetate, which, upon hydrolysis, produces the intensely colored cherry-red naphthol (5). Thus, simple colorimetric measurements are feasible. The excess hydroxamic acid can also be determined colorimetrically via its reaction with ferric ion. EXPERIMENTAL
Apparatus. A constant temperature water bath set at 25 =t0.5" C was used in these experiments. All measurements were made in a Beckman Spectrophotometer, Model DU at 540 mp. Reagents. HEXANEHYDROXAMIC ACID. Sodium metal, 1.10 grams, was dissolved in 24 ml of absolute ethanol in a round-bottom flask fitted with a reflux condenser and a drying tube. An ethanolic solution of hydroxylamine was made up by dissolving 1.74 grams (ca. 0.024 mole) of hydroxylamine hydrochloride (Coleman and Bell, Reagent grade, minimum assay 96%) in 36 ml of absolute ethanol, and treating this solution dropwise with about one half of the sodium ethoxide solution. The flask was cooled in ice during the neutralization, which was carried on until a drop of the solution was alkaline to phenolphthalein. The precipitated sodium chloride was then filtered off using suction, and the filter washed with a small amount of absolute ethanol. The hydroxylamine solution was added to a solution of 3.2 grams (0.022 mole) of ethyl caproate in 10 ml of absolute ethanol and permitted to stand at room temperature for 4 hours before addition of the remaining sodium ethoxide. Then an equal volume of absolute ether was added. Only a small amount of solid separated after standing in the refrigerator for several days, but concentration of the solution and further addition of absolute ether produced several crops of the salt. This was dissolved in the minimum amount of water (ca. 40 ml) and the pH adjusted to 7.4 by the addition of glacial acetic acid. After chilling in ice, the white solid precipitate was filtered off, washed with a little ice cold water, and dried. Recrystallization from benzene produced 1.3 grams (47 % yield) of mica-like plates melting at 61.5-63.5" C and giving a n intense red-violet coloration with ferric chloride. The melting point reported in the literature is 63.5-64.0" C. Aqueous solutions in the range 10-5 to lO-3M were prepared for use. ESTERSUBSTRATE SOLUTION.The substrate is 2.5 X 10-3M solution of 2-azobenzene-I-naphthyl acetate in acetone. The substrate can be prepared as follows. To 5.5 grams of 1,2naphthoquinone (0.035 mole) in 75 ml of glacial acetic acid is added 5.8 grams of phenylhydrazine hydrochloride (0.04 mole) in 175 ml of water. The dark red precipitate which forms almost immediately is filtered, washed, and recrystallized from absolute alcohol. The naphthol forms dark red needles melting at 137 to 139" C (uncorrected).
To 0.5 gram of the recrystallized naphthol in 10 ml of anhydrous pyridine is added 6 ml of acetic anhydride. The mixture is refluxed for 5 min, cooled to room temperature, and poured into a beaker containing 75 grams of ice. The orange precipitate is filtered, washed with 100 ml of 10% hydrochloric acid solution, and then washed with 100 ml of water. The air-dried 2-azobenzene-1-naphthyl acetate is recrystallized from 95% alcohol; its melting point is 120" C. BUFFERSOLUTION.The buffer solution is 0.001M in sodium tetraborate, adjusted to pH 9.0 with 0.05M HCI. ACETONE.ACS grade acetone is used. HYDROCHLORIC ACID. 0.05M is used. GB SOLUTION.Stock solutions of G R (98% purity, furnished by the Organic Chemistry Department, Research Laboratories, Edgewood Arsenal) are made up by diluting accurately weighed quantities of GB with distilled water. Procedure. Optimum experimental conditions will vary with the sensitivity and speed of analysis desired, and the nature of the phosphorus compound under investigation (See Results and Discussion). As an example, for analysis of isopropyl methylphosphonofluoridate in concentrations ranging from 10-5 to 10-4M, with an analysis time of approximately 20 minutes, the following procedure is satisfactory: T o 4 ml of the GB solution, add 1 ml of the hydroxamic acid solution, 2 x lO-3M, and 1 ml of borate buffer solution; mix well and let stand at 25" C for 10 minutes. Add 4 ml of ester substrate solution; mix well and let stand for 5 minutes. Add 0.5 ml of 0.05N HCl and read in a Beckman spectrophotometer at 540 mp after 2 minutes. RESULTS
Spectral absorbance curves of the ester substrate and its hydrolysis product are shown in reference 5. Various ancillary studies of relevance to its use in this work have also been reported therein and the reader is urged to consult the original publication. In brief, at a wavelength of 500 mp, the wavelength of maximum absorbance of the hydrolyzed productLe., the naphthol-there is no appreciable absorbance by the ester substrate even at twice the concentration of the naphthol. Hydrolysis of only 5 of the acetate may be expected to produce a solution with an absorbance of approximately 0.5 optical density unit greater than the unhydrolyzed acetate at 500 mp. In this study, because of the high concentration of the ester substrate used and the relatively low concentrations of the naphthol formed under the experimental conditions prescribed, it was advantageous to make the measurements at 540 mp. At 540 mp, there was a negligible loss in sensitivity and much reduced blank values. The rate of appearance of color due to 2-azobenzene-lnaphthol was measured in a solution in which the solvent was a 25% solution of acetone in water buffered at pH 8.0. The solution was 5 X 10-4M with respect to both 2-azobenzene-lnaphthol acetate and hexanehydroxarnic acid and the temperature was 25" C. It was established that the reaction was first order with respect to the acetate with a rate constant of 5 x 10-3 min-1. Following almost complete destruction of the acetate, additional acetate was added which decomposed at the same rate, demonstrating the catalytic nature of the hexanehydroxamic acid. As in the case of the reaction between hydroxamic acids and phosphorylating agents, the rate of the reaction between hydroxarnic acid and acylating agents is dependent upon the concentration of the hydroxamate anion. Thus, the reaction rates are very low in acid solution and addition of 0.5 rnl of 0.05N HCI brings the reaction to a halt for all intents and purposes, when the concentration of the hydroxamic acid used is of the order of lOWM; with higher concentrations of hydroxVOL. 39, NO. 10, AUGUST 1967
1137
amic acid, it becomes necessary, as is specified in the general procedure, to make the optical density readings as close to 2 minutes after the addition of acid as possible. The concentrations of 2-azobenzene-1-naphthol formed when the ester substrate is allowed to react with different concentrations of the hydroxamic acid for 5 and 50 minutes are shown in Tables I, 11, and 111. These data show clearly that there is a linear relation between concentration of the naphthol
Table I. Formation of 2-Azobenzene-1-Naphthol from 2-Azobenzene-1-Naphthyl Acetate" (Ten determinations at each concentration) Net O.D. Net readings, x lo-' Av readings, [HAY O.D. units M x 104 O.D. units [HAl0 0 0.0950 f 0.0004 0.40 0.1150 =t 0.0004 0.020 5.0 0.80 0.1352 f 0.0008 0.0402 5.0 1.20 0.1549 + 0.0009 0.0599 5.0 0.0800 5.0 1.60 0.1750 =t 0.002 2.00 0.195 f 0.000 0.1000 5.0 Reaction conditions: 5 ml HA s o h ; 1 ml buffer; 4 ml2-azobenzene-1-naphthyl acetate solution; let stand for 5 minutes at 25' i 0.5" C; add 0.5 ml 0.05N HCI, read at 540 mp. 5 Concentration shown is that of hydroxamic acid in the reaction mixture (10 ml). 0
Table 11. Formation of 2-Azobenzene-1-Naphthol from 2-Azobenzene-1-Naphthyl Acetate" (Ten determinations at each concentration) Net O.D. X Av readings, Net readings, M x 108 O.D. units O.D. units [HA], 0.0 0.200 f 0.002 0.4 0.220 f 0.001 0.020 5.0 0,041 5.1 0.8 0.241 f 0.002 1.2 0.260 3z 0.001 0.060 5.0 1.6 0.283 & 0.002 0.083 5.2 2.0 0.301 f 0.002 0.101 5.0 a Reaction conditions: 5 ml HA soln; 1 ml buffer, 4 ml 2-azobenzene-1-naphthyl acetate solution; let stand for 50 minutes at 25' & 0.5" C; add 0.5 ml 0.05M HC1, read at 540 mp. b Concentration shown is that of hydroxamic acid in the reaction mixture (10 ml).
-
Table 111. Formation of 2-Azobenzene-1-Naphthol from 2-Azobenzene-1-Naphthyl Acetate" (Ten determinations at each concentration) Net O.D. [HAhb Av readings, Net readings, x M x 104 O.D. units O.D. units [HA], 0 0.206 f 0.0008 0.40 0.4062 f 0.0004 0.2002 5.0 0.80 0.6008 & 0.0026 0,3948 4.9 1.20 0.7966 f 0.0027 0.5906 4.9 1.60 1.OOO z!= 0.009 0.7944 5.0 1.204 & 0.0028 0.998 5.0 2.00 Reaction conditions: 5 ml HA soln; 1 ml buffer, 4 ml 2-azobenzene-1-naphthyl acetate solution; let stand for 50 minutes at 25 f 0.5" C; add 0.5 ml 0.05M HCI, read at 540 mp. * Concentration shown is that of hydroxamic acid in the reaction mixture (10 ml).
1138
ANALYTICAL CHEMISTRY
found and the concentration of the hydroxamic acid used; and although the kinetics of formation of the naphthol are pseudo first order, under the conditions of the experiments described herein, where only a small fraction of the total ester is hydrolyzed, one gets apparent zero order kinetics. Hence, for the same concentration of hydroxamic acid, there is found an almost direct dependence of the naphthol concentration on the reaction time (of Tables I and 111). Tables IV, V, and VI give the results of complete experiments. Tables IV and V give the results of identical experiments using two different sources of GB. Standard curves are constructed by plotting the G B concentration against the average colorimetric readings. In agreement with theory, two moles of hydroxamic acid are destroyed per mole of G B reacting. In Table IV and V, the concentration of the G B (column l), is calculated for a 10-ml volume. In actual test, the total of the G B would be added in 4 ml. Hence the lowest concentration of G B used in these experiments was 3.73 X 10PM (ca. 5 ppm) rather than 1.49 X lO-5M. Similarly in the experiments shown in Table VI, the lowest concentration of G B is ca. 0.5 PPm. It should be pointed out also, that, although not critical to the use of the procedure (as one uses a calibration curve for analysis) the hydroxamic acid concentrations recovered in two sets of experiments (Tables IV and V) are considerably higher than the added hydroxamic acid concentrations. The discrepancies may have been due to the blank value which was taken from other experiments. DISCUSSION Choice of Reaction Conditions. The usefulness of detection and estimation methods for highly toxic and rapidacting chemicals such as G B depends to a large extent upon their rapidity, simplicity, and sensitivity. The concentrations of reactants and p H of the reaction mixture chosen for these experiments were calculated from available data on the reaction between G B and hydroxamic acids (12a) and G B and hydroxyl ion (126) to meet the requirements of speed and sensitivity. In connection with the latter requirements, it is desirable, as this is an analysis by difference, that a minimum quantity of the hydroxamic acid, (consistent with the requirements of rapid and complete reaction with the phosphorylating agent) be used. (Actually, the reactivities of G B to the anions of hydroxamic acid and to hydroxyl ion were determined at two different temperatures. Therefore the numerical figures given in subsequent equations in which both reactions are involved are only approximately correct.) It had been established that the rate of the reaction between G B and a hydroxamic acid is proportional to the concentration of the hydroxamic acid anion and that, for a series of hydroxamic acids of varying ionization constants, the bimolecular rate constants are related to the strengths of the acids according to the equation k2 = GBKBB where GB and /3 are constants for a given reaction, solvent, temperature, and series of similar reactants. KB is the base dissociation constant of the hydroxamic acid anion, related to the ionization constant of the conjugate acid (KA)by the equation KB = Krp/KA,where KT
