Anal. Chem. 1987, 59, 2123-2127 2123

Electrocatalytic Reduction of Ethylene Dibromide by Vitamin B,, in a Surfactant-Stabilized Emulsion

James F. Rusling,* Thomas F. Connors,’ and Azita Owlia

Department of Chemistry (U-601, Uniuersity of Connecticut, Storm, Connecticut 06268

Alkyl vlclnal dlbromldes were reduced at glassy carbon electrodes In Isooctane/water emulslons stablllzed by Aero- SOCOT (AOT) and tetraethylammonlum perchlorate (TEAP) by udng an electrocatalytk cyde InvoMng vttamln B12. Catatysls lowers the overpotentlal by about 0.8 V compared to dlrect reductlon In a homogeneous medlum. The heterogeneous rate constant (0.0056 cm s-l) for the key Co( I I)/Co( I ) re- ductlon step of the catalyst was about 3-fold smaller than In pH 2.3 aqueous acetonltrlle. Apparent rate constants on the order of IO‘ L mol-‘ s-’ for the rate-Umlthg chemlcal reactions of 1,ldlbromoethane and 1,Pdlbromobutane wlth Co(1) were consklerably smaller than In aqueous acetonltrlle because of the partltionlng of substrate into the hydrocarbon phase. Desplte these klnetlc Ilmltatlons, square-wave voltammetry could be used wlth the Catalytic cycle to estlmate total ethylene dlhalldes In unleaded and leaded gasollne In AOT/ TEAP emulsions prepared wlth gasoline.

Ethylene dibromide (EDB), or l,pdibromoethane, is a suspected human carcinogen (1) which has been used as an agricultural biocide and as a scavenger of lead in leaded gasoline (2). These and other sources have caused serious contamination of groundwaters in the northeastern United States (1,3,4). Cross-contamination of storage tanks has led to trace quantities of EDB in unleaded gasoline.

Vicinal dibromides can be reduced directly at mercury electrodes to yield alkenes and bromide ions (5-8). Direct reduction by dc or pulse polarography was recently proposed as a method of determining EDB (8). Although good precision and accuracy were obtained, direct reduction required po- tentials of about -1.6 V vs. SCE. With such a high reducing power a t the electrode, other species in some real sample matrices might be reduced at potentials close to -1.6 V and interfere in the determination of EDB. Also, use of mercury as an electrode can complicate the electrode process by chemical reaction with alkyl halides or reduction intermediates (7, 9).

We recently found (10) that vicinal dibromides are reduced in an efficient electrocatalytic cycle involving vitamin BIza, a naturally occurring cobalt(II1) corrin complex (Figure 1). In this reaction, the Co(1) form of vitamin BI2 is produced at a cathode in aqueous acetonitrile at about -0.75 V, and reduces vicinal dibromides to alkenes and bromide ions with a savings in overpotential of 0.84 V compared to direct reduction. High water solubility and poor solubility in nonpolar organic sol- vents creates a problem in the choice of medium when vitamin B12 is used to catalyze reductions of nonpolar substrates. For this reason, we used a mixed acetonitrile-water solvent in previous studies (IO). However, even in this system the solubility of 1,2-dibromobutane, for example, is limited to 35 mM. An alternative is to use a two-phase system containing

Present address: Connecticut State Department of Health, Laboratory Division, Hartford, CT.

0003-2700/87/0359-2 12380 1.50/0

a large proportion of hydrocarbon to solubilize the nonpolar substrate and water to dissolve the vitamin. Such mixtures can be emulsified by suitable surfactants (11). For example, acetonitrile/water mixtures containing cationic surfactant have been suggested for electroanalysis of oxidizable organics (12). Oxygen was determined in an oil/water emulsion of per- fluorotributylamine by coulometric and polarographic re- duction (13). Mass transport to electrodes in a dodecane/ water emulsion stabilized by nonionic surfactants was studied with ferrocene as an electroactive probe (14). Thus, electro- catalytic reactions in emulsions seemed viable.

Fendler and co-workers (15) found that vitamin BIZ pro- motes formation of large inverted micelles of bis(2-ethylhexyl) sulfosuccinate (Aerosol-OT, or AOT) in clear surfactant- hydrocarbon-water microemulsions. When [ water] / [AOT] was <20, these authors found large enhancements in rates of certain ligand exchange reactions. The vitamin was shown to reside in water pools enclosed by surfactant aggregates surrounded by bulk hydrocarbon. Unfortunately, micro- emulsions containing such small amounts of water and no additional electrolyte are too resistive for electrochemical measurements with conventional sized voltammetric elec- trodes. However, relatively stable emulsions with good con- ductivities can be prepared as media for electrocatalytic re- ductions. In this paper, we describe an extension of the catalytic reduction of alkyl vicinal dibromides with electro- chemically generated vitamin BIz8 [Co(I)] to an emulsion of isooctane, water, tetraethylammonium perchlorate, and Aerosol-OT. This reaction was used to estimate ethylene dihalides in leaded and unleaded gasoline.

EXPERIMENTAL SECTION Chemicals. Vitamin B1% was obtained as hydroxocobalamine

hydrochloride (crystalline, 99%) from Sigma Chemical Co. and used as received. Purity was confirmed by UV-vis spectroscopy. l,2-Dibromoethane and 1,2-dibromobutane were from Aldrich Chemical Co. and they were used as received. Isooctane was Baker ‘Resi-analyzed” grade, Aerosol-OT as bis(2-ethylhexyl) sulfo- succinate sodium salt was from Fluka, and tetraethylammonium perchlorate (TEAP) was from Eastman Kodak Co. Distilled water was further purified with a Barnstead Nanopure system and had a specific resistance 115 MQ cm. All other chemicals were reagent grade.

Solutions. Stock microemulsions containing 0.1 M AOT, 2.0 M water, and 0.1 M TEAP were prepared in isooctane and stirred overnight at room temperature. Emulsions were prepared by vigorously mixing the appropriate amounts of stock microemulsion and water, and by adding additional solid TEAP to saturate the system. The latter was necessary to stabilize the emulsion, which could be used for several hours without phase separation. Typical compositions of the emulsion for analytical work were 3% AOT, 6% TEAP (saturated), 33-50% water, and the remainder iso- octane. For the gasoline-based emulsions, 49% water was used with the other components in the same proportions.

A Bioanalytical Systems BAS-100 electrochemical analyzer was used for cyclic voltammetry (CV) and Osteryoung-type (16) square-wave voltammetry (SWV). A three-electrode, amber glass cell, with a planar glassy carbon working electrode (A = 0.088 or 0.071 cm2), a platinum-wire counter electrode, and an aqueous saturated calomel reference

Apparatus and Procedures.

0 1987 American Chemlcal Society

2124 ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987

Flgure 1. Structure of vitamin B12a in the base-on form.

(SCE) were used. The SCE was connected to the sample com- partment by a salt bridge containing saturated KCl and termi- nating in a medium porosity glass frit covered with an Agar/KCl plug. The glassy carbon electrode was metallographically polished as described previously (17) before each voltammetric scan. In addition, for analytical determinations only, the polished electrode was electrochemically treated by holding the potential at -1.5 V (vs. SCE) for 60 s, then at 0.4 V for 60 s . Cell resistance was externally compensated by the BAS-100. All solutions for elec- trochemistry were purged for 10-15 min with purified nitrogen prior to the experiment and prior to addition of vicinal dibromides followed by a 0.5-min period of purging. Nitrogen passed through an emulsion of similar composition before entering the cell. This procedure with the emulsions minimized the rate of escape of the dibromides from the analyte medium.

However, in stirred emulsions, 0.7 mM concentrations of EDB were removed in less than 10 min. Thus, for the analytical de- terminations, the required amounts of water, vitamin B12 (0.48 mM), and TEAP were mixed and purged with nitrogen for 20 min. A recently prepared gasoline microemulsion made with purged water, containing EDB standard additions where appropriate, was then added. A separate emulsion was prepared for each standard addition. Vitamin B12 samples prepared in this way in isooctane emulsion free of EDB showed no catalytic peak, confirming the absence of significant levels of oxygen. For quantitation in SWV, the catalytic peak current for EDB was found by subtracting the estimated current from the overlapping diffusion peak from the net peak current. All experiments were done at ambient tem- perature (23 f 2 "C).

Conducitivity measurements were made with a Yellow Springs Instruments Model 31 conductivity bridge and Model 3400 con- ductivity cell calibrated against standard KCl solutions.

Gas chromatography was done on gasoline samples diluted 1/1000 in pure pentane. Standards were EDB and 1,2-di- chloroethane in pentane. A Perkin-Elmer Sigma 300 gas chro- matograph equipped with an electron-capture detector was used. Gas chromatographic conditions were as follows: columns, 6-ft X 2- or 4-mm i.d. with 10% squalane on 80/100 mesh Chromosorb W; oven temperature, 68 O C ; detector temperature, 300 "C; injector temperature, 150 OC; nitrogen carrier gas pressure, 60 psi at flow rates of 65 cm min-' (4-mm i.d.) and 24 cm min-' (2-mm id.). Results were essentially the same on both columns.

Table I. Specific Resistance of Selected Micro- and Macroemulsions

proportions of components specific AOTlwaterli-octlTEAP [water]/ resistance,

(Bi,/DBB) [AOTI fl cm

5.4/9.9/84.7/0 (O/O) 45 1 700 000 5.3/9.6/82.3/satd (010) 45 50 000 3.5/42.0/54.3/0 (0.021/0) 298 80 000

3.4/41.3/53.5/satd (0.021/0.003) 298 510 3.4/41.3/53.5/satd (0.020/0.033) 298 700

3.4/41.3/53.3/satd (0.021/0) 298 435

T to& 1

EIUOLTI Flgure 2. Cyclic voltammogram at 0.5 V s-l of 1.3 mM vitamin BlZa in emulsion of AOT/water/isooctane/TEAP (3.414 1.3/53.5/saturated).

RESULTS AND DISCUSSION Choice of the Medium. As noted above, the stock mi-

croemulsion was much too resistive for electrochemical measurements with our glassy carbon electrodes. Addition of water to give concentrations between 33% and 50% and saturation with TEAP gave a macroemulsion with a specific resistance suitable for voltammetric measurements (Table I). Saturation with TEAP was necessary to obtain a reasonable conductivity and also to physically stabilize the emulsion. Although the isooctane emulsions with good conductivities were turbid, they remained free from phase separation for several hours. Addition of dibromobutane to the emulsions had a small but measurable influence on conductivity. The use of heptane in place of isooctane also gave acceptable conductivities.

It had been reported that addition of an alcohol could increase conductivity in AOT systems (II), but addition of propanol to the macroemulsion caused immediate phase separation. A similar result occurred when attempts were made to control the pH of the aqueous phase by addition of mineral acid or phosphate buffer.

Electrochemistry of Vitamin B12* in the Emulsion. Cyclic voltammograms of vitamin B12a (aquocob(II1)alamine) revealed electron-transfer reactions of intermediate rate for the Co(III)/Co(II) and Co(IIj/Co(I) redox couples (Figure 2). Such quasireversible electrode reactions are characterized (18) by increasing anodic-cathodic peak separations, negative shifts in cathodic peak potential, and decreasing current functions ( i P d l 2 ) as scan rate (u) increased. All of these characteristic trends are evident in the CV data (Table 11) for the two redox couples. The apparent diffusion coefficient (D ') estimated from current functions for the Co(I1) peak at u C 0.3 V s-l by using the Randles-Sevcik equation was 2 X lo4 cm2 s-l. This value is between that (10) of 2.7 X low6 cm2 s-l in water/ acetonitrile (l:l, viscosity (19) 0.9 cP) and that of 1.4 X lo4 cm2 s-' in purely aqueous media (21). Since the bulk viscosity of the macroemulsion is considerably greater than that of water (20), the value of D'suggests that vitamin BlZr travels predominantly in the aqueous phase and is probably not bound to surfactant aggregates.

Apparent heterogeneous rate constants ( k o ) for the redox couples were estimated from the anodic-cathodic peak sep-

ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987 2125

Table 11. Voltammetric Characteristics of Vitamin BIZ, in AOT Emulsion"

scan rate, -E,,, V &u-' /~ , pA v s-1 vs. SCE sl'* LIZ,, mV i,,/i,,

Co(III)/Co(II)

0.05 0.081 36.8 93 1.07 0.10 0.090 43.1 110 1.07 0.30 0.112 38.9 158 0.99 0.50 0.116 31.6 161 0.97 1.00 0.106 21.8 146 1.06 5.12 0.158 16.3 244 0.98

CO(II)/CO(I)

0.05 0.943 44.1 81 1.06 0.10 0.945 51.0 82 0.97 0.30 0.954 44.7 100 1.16 0.50 0.957 32.8 99 1.12 1.00 0.961 24.1 119 1.05 5.12 0.950 21.1 118 0.96

AOT/water/isooctane by weight: 3.4/41.3/53.2, saturated in TEAP. Concentration of vitamin BIZ was 1.3 mM.

T

A -1 .o

E LUOLT I Flgure 3. Cyclic voltammogram at 0.5 V s-' of 1.3 mM vitamin B,, and 1.3 mM 1,2dibromobutane in emulsion of AOTlwaterlisooctaneI TEAP (3.4/41.3/53.5/saturated).

arations by the method of Nicholson (22), assuming an elec- trochemical transfer coefficient of 0.5. The value of 2.5 f 0.7 X cm s-l for the Co(III)/Co(II) couple was similar to that at polished glassy carbon in aqueous acetonitrile (10). How- ever, k" of 5.6 f 0.9 X cm s-' for the Co(II)/Co(I) reaction was about 3-fold smaller than in pH 2.3 aqueous acetonitrile. The slower Co(II)/Co(I) reaction in the emulsion is probably a consequence of the uncontrolled pH in the water phase. Comparison of the potential of the Co(I1) reduction peak in the emulsion with the pH dependence of this peak potential in water (21) suggests a pH > 4.7 for the water phase in the emulsion. At this pH, vitamin BlZr exists in the base-on form (Figure l), to which electron transfer is slower (21) than to the base-off form predominating in solution a t pH 2.3.

Electrocatalytic Reduction of Vicinal Dibromides. Since our work on catalytic reduction of vicinal dihalides in aqueous acetonitrile focused on 1,2-dibromobutane, we studied this compound f i t in the emulsion. When 1,2-dibromobutane (DBB) was added in equimolar amounts to a vitamin BlZe containing emulsion, the current of the Co(I1) reduction peak increased about 2-fold at 0.5 V s-l, and the anodic peak for oxidation of Co(1) disappeared (Figure 3). This behavior is characteristic for homogeneous electrocatalysis (23). A peak is not observed for Co(1) because it reacts with substrate to recycle Co(I1) a t the electrode, thus increasing the cathodic peak current, which is directly related to the rate of the ho- mogeneous catalytic reaction (23). As expected, a larger in- crease in the Co(I1) peak current (Table 111) occurs at larger ratios of y = [substrate] / [catalyst]. Similar results were obtained when EDB was the substrate, and also in emulsions prepared with heptane rather than isooctane.

The pathway for reduction of vicinal dibromides by vitamin Blzs in aqueous acetonitrile was shown by voltammetric, bulk

Table 111. Apparent Rate Constants for Reduction2 Vicinal Dihalides by Vitamin BIzs in AOT Emulsion"

scan rate, V s-l ic/2?'id log k,, log (L mo1-ls-l)

1,2-Dibromobutane, y = I

0.05 1.00 2.78 0.10 0.90 2.92 0.50 1.25 4.27 1.00 1.15 4.22

1,2-Dibromobutane, y = 10

0.05 0.50 3.42 0.10 0.42 3.60 0.50 0.46 4.36 1.00 0.39 4.58

3.77 f O.6gb

1,2-Dibromoethane, y = 10

0.05 0.99 3.95 0.10 0.73 3.98 0.50 0.71 4.66

4.20 f 0.40b

" Composition as in Table 11. Mean value.

electrolytic, and spectroelectrochemical studies to follow Scheme I. Voltammetric data in aqueous acetonitrile were

Scheme I

Co(I1) + e- = Co(1) k" kl

Co(1) + RBr, - [BrCo(III)RBr] - Co(I1) + RBr' + Br- (2)

Co(1) + RBr' - Co(I1) + RBr- fast (3)

RBr- - alkene + Br- fast (4) successfully modeled (10) by expanding grid digital simulation (24) based on this scheme. The model assumed semiinfinite planar diffusion, a ratio of diffusion coefficients of substrate to catalyst of 10, and experimentally measured D and ko values. Assuming that Scheme I holds in the emulsions, catalytic efficiencies (ic/2yid, where ic and id are peak catalytic and diffusion currents) can be used to estimate the rate constant kl for reaction of the Co(1) species with vicinal di- bromide. This was done by comparing experimental catalytic efficiencies with a plot of values computed by digital simu- lation vs. the kinetic parameter X = (RT/Fu)C*kl , where C* is the concentration of catalyst (23), and the other symbols have their usual electrochemical meanings. Since factors such as partition of reactants not included in the model may in- fluence k, , the values obtained are apparent rate constants.

Values of I t , show reasonably good agreement at different scan rates (Table 111), but reflect a smaller reaction rate than does the k , of 6 X lo6 L mol-' s-l found in pH 2.3 aqueous acetonitrile (IO). Although a quantitative exploration of such kinetic effects is beyond the scope of the present work, it is likely that the decrease in rate is related to partition of reactants between phases. The highly water soluble vitamin BlZs resides entirely in the aqueous phase (15). However, by analogy with other alkyl bromides (W), vicinal dihalides should be substantially partitioned into the hydrocarbon phase. Thus, the true rate constant in the aqueous phase is attenu- ated in Itl by partition of the substrate (26).

Estimation of EDB in Gasoline. This application was explored to demonstrate practical utility of the catalytic re- action in emulsions. We used square-wave voltammetry (SWV) for electroanalysis because of its superior sensitivity and resolution over CV. Forward and reverse SWV currents

2126 ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987

f =f

I , . , , I . . . . l . . J 40.300 M.0 -0.5 -1 .o -1 .mO

ElUOLfl Flgure 4. Square-wave voltammogram of 0.5 mM vitamin B,, and 0.001 mM EDB in emulsh of AOT/water/isooctaneTAP (as in Figure 2). SWV conditions: f = 15 Hz, amplitude = 50 mV, and step = 2 mV.

30 -

20

6 -

4 -

[EDBI , pM [ E D B I added, p M

F b w 5. Influence of concentration of ED6 on SWV catalytic current h emuldons: (a) emulsion of AOT/water/ie/TEAP (as in Figure 2) containing 1.0 mM vitamin B,,, and (b) emulsion containing 0.48 mM vitamin B,, prepared with gasoline. SWV conditions as in Figure 4.

(16) of vitamin BIZ in the macroemulsion confirmed the quasireversible nature of the Co(II)/Co(I) reduction. Addition of EDB in less than equimolar amounts to the emulsion containing vitamin BIZ resulted in a catalytic peak at potentials slightly more positive than that of the Co(I1) peak (Figure 4). Analogous double peaks are found in CV (23) for very fast catalytic reactions, where only a small amount of catalyst is needed to drive the reaction. Such behavior has been observed in electrocatalysis with vitamin BI2 in aqueous acetonitrile (10). Thus, the first peak corresponds to total consumption of substrate present at a low concentration in the reaction layer close to the electrode. When the potential of the electrode approaches the Eo' of Co(II)/Co(I), the unreacted Co(I1) is reduced in the second, diffusion-controlled peak. No change in the Co(III)/Co(II) peaks was observed upon addition of substrate.

Under conditions chosen to encompass the range of con- centrations found in unleaded gasoline, peak catalytic currents were linear with increasing concentrations of EDB up to 100 p M (Figure 5a). Linear regression gave a slope of the cali- bration m e of 0.293 f 0.026 AIM, a coefficient of correlation ( r ) of 0.992, and an intercept of 0.95 f 1.34 @A, the latter showing that the line passed through zero. The limit of de- tection, estimated as the concentration at which the catalytic peak can first be distinguished from the Co(II) peak, was about 30 p M under the present conditions. This limit can be im- proved by lowering the catalyst concentration. The catalytic peak was explored for estimating EDB in unleaded gasoline by preparing the emulsion with gasoline instead of isooctane. This required more water (see Experimental Section) with gasoline than with isooctane to keep resistance low. Phase separation in the gasoline emulsions occurred in minutes

Table IV. Estimated Vicinal Dihalide Content in Regular Gasolines

dihalides found, ppm

gasoline ECAT" GCb [EDB] reported ref

unleaded 1 1.18 h 0.03 1.11 0.1-5.7" 27

unleaded 2 2.24 * 0.31 2.36 reg leaded 65.0d 69-371' 27

av 171 24-120e 28

"By electrocatalytic SWV reported as [EDB] with standard er- rors estimated from standard deviations of slope and intercept from linear regression on standard addition data. *By gas chro- matography as sum of EDB and EDC; relative precision about &IO%. Gasolines from 9 major manufacturers, EDB determined by gas chromatography. Estimated by comparison of catalytic CV peak with calibration curve. eLimits set by US EPA.

av 1.9

rather than hours, so that stirring was required immediately before the voltammetric scan. SWV of vitamin B12r in the gasoline emulsions revealed a new peak at -0.6 V vs. SCE. Since this peak was a possible interference with the catalytic peak for the reduction of EDB at about -0.85 V, we used standard addition experiments for the EDB determinations. Only the peak at -0.85 V increased in height as standard was added, and good linearity (Figure 5b) and coefficient of cor- relation were obtained ( r = 0.9995).

Gas chromatography of the unleaded gasoline samples re- vealed the presence of another vicinal dihalide, ethylene di- chloride (EDC), at comparable levels to EDB. Electrochemical studies showed that the catalytic cycle was also active for EDC. Thus, the electrocatalytic method in gasoline yields both EDB and EDC, estimated as EDB. Results for this quantity by GC and by electrocatalysis show good agreement for unleaded gasoline, and are similar to previous estimates in the literature (Table IV).

An alternative approach to electrocatalytic determination of EDB in gasoline emulsions would be to use a concentration of catalyst much lower than that expected for EDB. This would provide a single catalytic peak for the analysis; i.e. under those conditions no double peak is observed (IO). However, if the impurity peak in the gasoline is present, it might create a large error if its current exceeded the catalytic current. By using a larger concentration of catalyst, the second-order rate-limiting reaction (eq 2, Scheme I) is made faster and a larger current is observed relative to the impurity. However, overlap of the catalytic peak with the Co(I1) peak still needs to be addressed. Also, great care must be taken to exclude oxygen from the medium and to avoid loss of vicinal dihalides by volatilization.

In summary, this work demonstrates the usefulness of surfactant-stabilized emulsions in electrocatalytic reductions as a basis for quantitative estimation of vicinal dihalides. Voltammetric results for the reduction of vicinal dibromides catalyzed by vitamin Blz are similar to those found in ho- mogeneous media, with rates limited by the properties of the surfactant-stabilized medium. For the estimation of ethylene dihalides in gasoline, the sample becomes a component of the emulsion, which simplifies handling procedures. We are presently exploring other organized surfactant-containing media for applications in analysis, synthesis, and modeling of biological redox events.

ACKNOWLEDGMENT The authors thank Dermot Jones and Ana Pita of the

Hydrocarbon Section, Laboratory Division of the Connecticut State Department of Health, for the GC analyses of gasoline.

Registry No. EDB, 106-93-4; l,Z-dibrornobutane, 533-98-2; vitamin B,,, 68-19-9.

Anal. Chem. 1987, 59, 2127-2130 2127

LITERATURE CITED (1) Van Bladeren, P. J. J. Am. Coll. Toxlcol. 1983, 2 , 73-83. (2) Borman, S. Anal. Chem. 1984, 56, 573A. (3) US EPA Report, Fed. Reg. 1983. 48(No. 197), 46229-46230. (4) Isaacson, P. J.; Hankln, L.; Frlnk, C. R. Sdence 1984, 225. 672. (5) Casanova, J.; Rogers, H. R. J . Org. Chem. 1974, 39, 2408-2410. (6) Hawley, M. D. I n Encyclopedic of Electrochemistry of the Elemsnts;

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(8) Tokoro, R.; Bllewicz, R.; Osteryoung, J. Anal. Chem. 1986, 58, . - 1964- 1969. HIII, H. A. 0.; hatt, J. M.; O'Riordan, M. P.; Williams, F. R.; Williams, R. J. P. J. Chem. SOC. A 1971, 1859-1862. Connors, T. F.; Arena, J. A,; Rusllng, J. F.. unpublished results, 1987. Sharma, M. K.; Shah, D. 0. Introduction to Macro and Mlcroemul- slons: Shah, D. 0. Ed.; ACS Symp. Ser. No. 272; American Chemical Society, Washington, DC, 1985; pp 1-18. Franklin, T. C.; Iwunze, M. Anal. Chem. 1980, 52, 973-976. Berthod, A.; Georges, J. Anal. Chim. Acta 1983, 147, 41-51. Georges, J.; Berthod, A. J. Electroanal. Chem. Interfaclel Electro- chem. 1984, 175, 143-152. Fendler, J. H.; Nome, F.; Van Woert, H. C. J. Am. Chem. SOC. 1974,

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RECEIVED for review January 5,1987. Accepted May 26,1987. This work was supported by US PHS Grant ES03154 awarded by the National Institute of Environmental Health Sciences and, partly, by the donors of the Petroleum Research Fund, administered by the American Chemical Society. This paper is part 4 in the series Electrocatalytic Reactions in Organized Assemblies.

113, 19-40.

4608-461 1.

1988, 58, 2366-2372.

Anodic Behavior of the Antineoplastic Agent Amethopterin at a Mercury Electrode and Its Determination in Body Fluids by Liquid Chromatography with Indirect Anodic Polarographic Detection

Antonio Guerrieri and Francesco Palmisano*

Laboratorio di Chimica Analitica, Dipartimento di Chimica dell' Universitd, Via G . Amendola, 173, 70126 Bari, I taly

Amethopterln has been found to be anodlcaHy electroactlve on mercury due to a depolarlratlon effect arlslng from the formation of a sparingly soluble mercury complex. Hlgh- petformame HquM chromatography wlth anodlc pdarographk (sampled dc) detectlon at +0.19 V vs. Ag/AgCI has been employed for the determlnatlon of the drug In body flulds. Detector response was found to be Unear In the range 5-1000 ng on column. A detectlon llmlt of 1.5 FM amethopterln in serum was achieved. The wlthlnday and day-today coeffl- clent of varlatlon at the 10 bg/ml level were 1.5 and 3.7% respectively.

Amethopterin (Methotrexate, MTX or L-(+)-N-(~-[ ((2,4- diamino-6-pteridinyl)methyl)methylamino] benzoy1)glutamic acid) is a competitive inhibitor of dihydrofolate reductase currently used in the treatment of several human cancers including acute lymphocytic leukemia, osteosarcoma, non- Hodgkin's lymphoma, breast carcinoma, and choriocarcinoma. The effectiveness of MTX increases with high dosage regimens but the risk of haematologic and renal toxicity also increases. Citrovorum factor (Leucovorin) rescue is used to protect patients from overdosage toxicity effects associated with high concentrations of MTX in plasma and/or with a delayed MTX elimination. However, a 6% incidence of drug-related

deaths has been reported (I). Such a noticeable mortality rate accounts for the stringent need of clinical and pharmacokinetic monitoring of high-dose MTX treatments. Several protocols (2) are presently used for monitoring high-dose MTX therapy, all of them requiring measurement of serum or plasma drug concentration. Fluorometry (3), radioimmunoassay (4-6), enzyme immunoassay (7), and high-performance liquid chromatography (HPLC) with UV detection (8-12) are the most often used techniques.

A significant amount of work in the field of liquid chro- matography/electrochemical detection (LC/EC) methodology for the determination of pteridine derivatives, other than MTX, has been accomplished by Lunte and Kissinger (13, 14), Picomole amounts of several oxidized and reduced pteridines were simultaneously determined in biological samples by LC/EC with a dual electrode (glassy carbon) am- perometric detector. Recently (15) some preliminary data concerning the anodic electroadivity of MTX on glassy carbon have been reported, as well as a LC/EC method for MTX determination in body fluids.

In this paper the peculiar anodic behavior of MTX at a mercury electrode is described. An anodic wave caused by the formation of an insoluble film has been observed. Al- though the ability of purine and pyrimidine derivatives to yield anodic polarographic currents arising from formation of sparingly soluble mercury compounds has been extensively

0003-2700/87/0359-2127$01.50/0 0 1987 American Chemical Society