Anal. Chem. 1986, 58, 135-139 (28) Amrnann, D.; Anker, P.; Metzger, E.; Oesch, U.; Simon, W. I n “Ion Measurement in Physiology and Medicine”; Kessler, M.; Hoper, J.; Harrison, D. K., Eds.; Springer-Veriag: Berlin, Heidelberg, New York, and Tokyo, 1985. (27) Gullbauit, G. 0.; Durst, R. A.; Frant, M. S.; Freiser, H.; Hansen, E. H.; Light, T. s.; PUngOr, E.; Rechnitz, G.; Rice, N. M.; Rohm, T. J.; Simon. W.; Thomas, J. D. R. Pure Appl. Chem. 1978, 48, 127. (28) Oesch, U.; Dinten, 0.; Ammann, D.; Simon, W. I n “Ion Measurement
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in Physiology and Medicine”; Kessier, M.; Hoper, J.; Harrison, D. K., Eds.; Springer-Veriag: Berlin, Heidelberg, New York, and Tokyo, 1985.
RECEIVED for review July 11,1985. Accepted September 11, 1985. This work was partlysupported by E. 1. du pant de Nemours & CO., InC.
Competitive Heterogeneous Enzyme Immunoassay for Digoxin with Electrochemical Detection Kenneth R. Wehmeyer, H. Brian Halsall,* and William R. Heineman*
Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221 Charles P. Volle and I-Wen Chen
T h e Radiobiology Department, E. L. Saenger Radioisotope Laboratory, University of Cincinnati Medical Center, Cincinnati, Ohio 45267
A competltlve heterogeneous enzyme Immunoassay utlllzlng electrochemlcal detectlon has been developed for the determlnatlon of dlgoxln In human plasma. Alkallne phosphatase conjugated to dlgoxln Is the labeled hapten, and enzyme actlvlty Is measured by using phenyl phosphate as the enzyme substrate. The enzyme-generated phenol Is detected amperometrlcally In a thin-layer cell at a carbon paste electrode after separatlon on a 5-cm octyldecylsllane column. The procedure allows the determlnatlon of dlgoxln throughout Its therapeutlc range with a detection llmit of 50 pg/mL. A good correlation between thls method and radlolmmunoassaywas obtained for the determlnatlon of digoxin In patient samples. The feaslblllty and llmltatlons of employing flow lnjectlon analysis with electrochemlcal detection for quantltatlon of the enzyme product were also demonstrated.
Immunoassay is an extremely important tool in the low-level (pg/mL-ng/mL) determination of clinically important compounds in a variety of biological matrices (I). Radioisotopes have been the label of choice in such assays for the past 20 years owing to their sensitive detection by scintillation counting techniques. However, the drawbacks associated with the use of radioisotope labels have prompted the development of immunoassays based on nonisotopic labeling schemes (2, 3). A number of alternatives have been investigated including radical (4,5), fluorescent (6, 7), electroactive (&IO), and enzyme labels (11,12). Enzyme labels have been the most successful, and both homogeneous and heterogeneous assays are commercially available. Both of these assay formats are based on the inherent signal amplification capabilities of an enzyme label, with quantitation being achieved by measuring the conversion of substrate to product. An advantage of the heterogeneous assay is the removal of potentially interfering sample matrix components prior to the determination of enzyme-generated product. The application of electrochemical techniques to immunoassay methodology has been investigated by a number of grbups (ref 13 and references therein). The combination of
electrochemical detection with noncompetitive heterogeneous enzyme immunoassay results in extremely sensitive assays for macromolecules (14).The objective of the present research was to demonstrate the feasibility of electrochemical detection for competitive heterogeneous enzyme immunoassays for small molecular weight compounds. Digoxin was chosen as a model compound and alkaline phosphatase (EC 3.1.3.1) as the labeling enzyme. Digoxin, a steroidal cardiac glycoside, has a relatively narrow therapeutic range from 0.5 to 2.0 ng/mL (15). Alkaline phosphatase catalyzes the conversion of phenyl phosphate to phenol, which can be detected electrochemically by oxidation (13,14,16). The general assay protocol is shown in Figure 1. Digoxin in the sample or standard and digoxin labeled with alkaline phosphatase compete for solid-phase antibody coated on the walls of reagent tubes. Unbound digoxin and labeled digoxin are then rinsed from the tubes. The labeled digoxin bound to the solid-phase antibody is determined by incubation with the enzyme substrate solution. The phenol produced by the enzyme reaction is quantitated by oxidative hydrodynamic amperometry in a thin layer cell using either flow injection analysis/electrochemistry (FIAEC) or liquid chromatography/electrochemistry (LCEC). FIAEC involves the direct injection of the sample into a thin-layer electrochemical cell, while in the LCEC approach the phenol is retarded by a 5-cm C-18 column. The peak currents obtained for plasma solutions containing known amounts of digoxin are used to construct a standard curve from which the values of digoxin levels in patient samples can be determined.
EXPERIMENTAL SECTION Apparatus. The instrumentation and procedure for the determination of phenol by LCEC has been reported (14,16). The same instrumentation was used for FIAEC, but with no columns. Analyses by FIAEC were done with 0.05 M carbonate buffer mobile phase, a flow rate of 0.6-1.6 mL/min, and an applied potential of +870 mV vs. Ag/AgCl. Reagents. Digoxin was obtained from Sigma Chemical Co., St. Louis, MO. Digoxin-alkaline phosphatase conjugate was purchased from Immunotech Corp., Cambridge, MA. Digoxin antibody in goat serum was a gift from the Centers for Disease Control, Atlanta, GA. The immunoglobulin G fraction of the goat serum was isolated by ammonium sulfate precipitation, followed
0003-2700/88/0358-0135$01.50/00 1985 American Chemical Society
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Figure 1. General heterogeneous enzyme immunoassay protocol: (1) walls of polystyrene cuvettes coated with Ab and Tween 20 (0); (2) enzyme-labeled digoxin (D-E) and digoxln (D), samples or standards, added to cuvettes for competitive equllibratlon with Ab; (3) solution aspirated from cuvettes; (4) substrate (s,phenyl phosphate) added to cuvettes; and (5) product (p, phenol) detected after fixed reaction time by LCEC or FIAEC. by DEAE cellulose chromatography (17,18). Sources for phenyl phosphate, Tween 20, and the polystyrene cuvettes and the composition of the phosphate (pH 7.0, 0.1 M and pH 7.4 with Tween 20 and saline) and carbonate (pH 9.6,0.05 M) buffers have been reported (14). Pooled human plasma was prepared by combining 3 or 4 units of human plasma obtained at University Hospital in Cincinnati. Antibody coating solutions of 10,5.0,and 0.5 pg/mL were prepared by dissolving the appropriate amount of digoxin-specificantibody in carbonate buffer containing 0.02% sodium azide. Digoxinalkaline phosphatase conjugate dilutions of 1/125, 1/250, and 1/500 were prepared from a stock solution by dilution with an appropriate amount of PBS-Tween. Standard solutions of digoxin (0-5.0 ng/mL) were prepared in human plasma. The enzyme substrate solution was 1.0 X low3M phenyl phosphate and 1.5 X M MgC12.6H20in carbonate buffer. Immunoassay Procedure. The general assay protocol is outlined in Figure 1. Polystyrene cuvettes were coated with digoxin-specific antibody by passive adsorption according to a reported procedure (14). A 375-pL portion of the digoxin standard or sample and 25 p L of a digoxin-alkaline phosphatase conjugate dilution were added at the same time to the antibody-coated cuvettes. The assay solutions were incubated at room temperature. The contents of the cuvettes were aspirated and the cuvettes washed consecutively, twice with PBS-Tween, once with PBSTween ( 5 min), and twice with carbonate buffer. Following the washing step, 300 pL of the enzyme substrate solution was added to each cuvette and incubated at room temperature for a timed interval. For the FIAEC measurement of phenol, 20 pL of sample solution from the cuvettes was injected without further treatment. For LCEC, the enzyme reaction was first stopped by the addition of 25 pL of 5.5 M NaOH, and phenol was then determined as previously described (14). Optimization of Immunoassay Conditions. The time required for the antigen/antibody reaction to reach equilibrium was evaluated by using 375 pL of blank pooled plasma (0 ng/mL), 25 pL of a 1/125 dilution of the digoxin-alkaline phosphatase conjugate,and cuvettes coated with either a 0.5,5.0, or a 10 pg/mL antibody solution. Antigen/antibody incubation times ranging from 1 to 300 min were employed. Antibody coating solutions of 10, 5 , and 0.5 pg/mL and digoxin-alkaline phosphatase conjugate dilutions of 1/125,1/250, and 1/500 were investigated to find optimal values for the assay. Comparison of Electrochemical Enzyme Immunoassay with Radioimmunoassay (RIA). Samples from patients receiving digoxin therapy were obtained from University Hospital in Cincinnati. The samples were analyzed at University Hospital by an RIA (ARIA-I1System, Becton Dickinson) procedure (19).
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Figure 2. FlAEC traces for 20-pL injection of 0.05 M sodium carboM nate buffer containing 9.2 X lo-' M phenyl phosphate, 1.5 X magnesium chloride and (A) 0, (B) 1.5 X and (C)2.9 X M phenol. For the LCEC enzyme immunoassay, digoxin standards in pooled human plasma and patient samples were diluted 5/1 with pooled human plasma. For the FIAEC enzyme immunoassay patient samples and standards were filtered by ultracentrifugation and then diluted 5/1 with pooled human plasma. For both assays the 5/1 plasma dilution of standards and patient samples was required to eliminate an observed matrix effect. The specific conditions for both types of assays were 10pg/mL coating solution, 1/125 digoxin-alkaline phosphatase conjugate dilution, antigen/ antibody incubation time of 4 h, and substrate reaction time of 40 min. Duplicate analyses were performed for each digoxin standard. Phenol was determined by LCEC or FIAEC.
RESULTS AND DISCUSSION Detection of Phenol by FIAEC a n d LCEC. Cyclic voltammetry of phenol in carbonate buffer shows an irreversible oxidation wave (Epa= +670 mV vs. Ag/AgCl) at a carbon paste electrode (16). Since phenyl phosphate is electroinactive over the potential range of +lo00 to -200 mV vs. Ag/AgCl in this supporting electrolyte, phenol is detectable without interference from the substrate phenyl phosphate that would be present in an assay solution. In FIAEC a small volume of sample is injected into a mobile phase that carries the sample plug directly into the thin-layer electrochemical cell for detection of phenol by oxidation. A current-time trace for the repetitive injection of blank substrate solution and substrate solutions containing two concentrations of phenol that are near the detection limit is shown in Figure 2. Even though the components of the substrate solution are electroinactive, injection of the solution gives rise to a capacitive current, as shown by the peaks labeled A. This is caused by the slight difference in the matrix of the substrate solution and the carbonate mobile phase, which contains no phenyl phosphate or MgC12. FIAEC requires careful matching of the mobile phase and sample matrices in order to minimize this blank signal. The ipof the blank signal showed a variation of 2-15% on various days. Since FIAEC affords no mechanism for the separation of phenol from the substrate components, the faradaic current signal for the oxidation of phenol is superimposed on the capacitive current blank signal. Consequently, the detection of phenol was limited by the presence of the blank signal to a concentration of 1.5 X M, which gave a signal twice that of the blank as shown by the peaks labeled B. The maximum i, for the oxidation of phenol was reached at a potential of +870 mV, which was used for the immunoassays. FIAEC detection of phenol a t this potential had a linear dynamic range from 1.5 X to 2.8 M (r = 0.999). There was no indication of electrode x fouling after repeated injections of phenol solutions with concentrations as high as 1.0 X M. Approximately 25 s
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Flgure 3. Heterogeneous enzyme immunoassay with LCEC detection for a series of digoxin standards in plasma solutions. Concentration of digoxin in plasma samples was (A) 5.0, (B) 2.0, (C) 1.0, (D) 0.5, and (E) 0.0 ng/mL. A s s a y conditions were as follows: 10 pglmL antibody coating concentration, 11125 digoxin-alkaline phosphatase conjugate dilution, 4 4 antigenlantibody incubation interval, and 40-min substrate reaction time.
was required for sample analysis. The LCEC detection of phenol has been described previously (13,14,16).Retardation of phenol on a 5 c m precolumn slurry packed with 10-pm octyldecylsilane particles separates the phenol peak from other assay components that contribute to the interfering capacitance spike, as shown by the chromatograms in Figure 3 for the repetitive injection of phenol samples for an immunoassay. The small, constant peak is the capacitance spike. The separation enabled phenol to be detected to a much lower limit by LCEC than was possible by FIAEC. The LCEC determination of phenol had a detection limit of 5 x lo4 M and a linear range of 3 orders of magnitude. Approximately 2.5 min was required for the analysis of each sample by this technique. Due to its lower detection limit, LCEC was used for the investigation of the optimum immunoassay condtiions. The FIAEC and LCEC methods offer different advantages. LCEC has a ca. 100-fold better detection limit than FIAEC, since the interfering capacitance spike is separated chromatographically. However, this advantage is gained a t the expense of a longer analysis time per sample (ca. 2.5 rnin for LCEC compared to 25 s for FIAEC). The analysis time is determined by the efficiency of the separation. In this case, phenol is just base-line resolved from the capacitance peak, as shown in Figure 3, which requires a total time of 2.5 min for this chromatographic column. FIAEC, conversely, has a much shorter analysis time at the expense of a poorer detection limit. In this mobile phase, the capacitance peak associated with the blank limited the detection limit to 1.5 X IO-' M. The magnitude of the capacitance peak is highly dependent on the composition of the mobile phase and the components in the sample. Consequently, different detection limits may be obtained for other electrodes, mobile phases, and samples.
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Optimization of Immunoassay Parameters. In the development of an immunoassay method it is critical to ascertain the optimum conditions for each stage of the procedure. The heterogeneous enzyme assay required the evaluation of the antigen/antibody incubation interval, the antibody coating concentration, and the enzyme-conjugate dilution. For best sensitivity a competitive assay should be allowed to proceed to equilibrium (20). The time required for the digoxin/antibody reaction to reach equilibrium was determined by incubating the 0 ng/mL standard and labeled digoxin with the antibody-coated cuvettes for various periods of time. The amount of bound labeled digoxin was then determined by reaction with the substrate solution. As shown in Figure 4, equilibrium for a 5.0 pg/mL antibody coating concentration was reached in 3 h, as evidenced by the leveling off of i,. Equilibration for 10 and 0.5 pg/mL antibody coating concentrations was achieved in 3 and 5 h, respectively. Assays can be conducted a t times shorter than required for equilibration provided the incubation time for all standards and samples is the same. However, the use of shorter incubation intervals results in a decrease in the assay sensitivity. The difference in sensitivity for assays carried out under identical conditions except for variation in the digoxin/antibody incubation time can be seen in Table 1. The effect of shorter incubation times was most pronounced for the lower concentration ranges (0.5-0.0 ng/mL) where substantial increases in the differences in i, were obtained. One has the option of choosing increased assay sensitivity or decreased analysis times. Antibody coating concentrations of 0.5,5.0, and 10 pg/mL were used in conjunction with alkaline phosphatase conjugate dilutions of 1/125,1/250, and 1/500 to determine the optimal assay values. Highest assay sensitivity was achieved with an antibody coating concentration of 10 pg/mL and a digoxinalkaline phosphatase conjugate dilution of 1/125. The measurement of the antibody-bound alkaline phosphatase label should be made in substrate excess to give zero-order enzyme kinetics, which assures accurate quanti-
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tation. To determine the appropriate incubation interval for the alkaline phosphatase reaction, the various conjugate dilutions were incubated in antibody-coated cuvettes in the absence of digoxin. The conjugate solutions were then aspirated, the substrate solution was added to the cuvettes, and the enzyme reaction was allowed to proceed for varying lengths of time. The production of phenol was linear with time from 0 to 50 min for all conjugate dilutions. The length of the substrate reaction interval can also alter the assay sensitivity. In Figure 5 the results of conducting the heterogeneous assay for 2.0,0.5, and 0.0 ng/mL digoxin standards in human serum with varying substrate reaction intervals are shown. The slopes of the i, vs. time plots increased with decreasing concentrations of the digoxin standard. When the substrate reaction was allowed to proceed for longer times, larger differences could be achieved. One again has the option of increased sensitivity vs. shorter analysis time by adjustment of the substrate reaction time. LCEC Enzyme Immunoassay. Representative LCEC assay chromatograms for digoxin standards in plasma are shown in Figure 3. The peak current is proportional to the amount of phenol produced by the antibody-bound digoxinenzyme conjugate, which is inversely proportional to the amount of digoxin present in the standard. The LCEC method in conjunction with the optimal assay parameters resulted in a very sensitive immunoassay for digoxin in plasma throughout its therapeutic range, with a detection limit of 50 pg/mL. A standard curve is shown in Figure 6. The relative standard deviation for i, obtained for any given digoxin standard ranged from 4 to 12% on various days and is comparable to results ordinarily obtained by heterogeneous enzyme immunoassays. Approximately 20 samples can be injected per hour. As a result of the inverse binding relationship, sufficient enzyme-labeled material was bound at the lower digoxin concentrations (0.5-0.0 ng/mL) to generate phenol concentrations in the 10” M range. Phenol at this level is well above the demonstrated detection limit of the LCEC technique and therefore the measurement of the enzyme activity was not a limiting factor. For the higher digoxin concentrations (0.5-2.0 ng/mL) less of the enzyme label was bound and consequently a smaller concentration of phenol (low M) was generated, which required the low-level quantitative capabilities of the LCEC detection system. Assays can be conducted at times shorter than required for equilibration provided the incubation time for all standards and samples is kept the same. The use of a 15-min rather than
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Figure 7. Heterogeneousenzyme immunoassay with FIAEC detection for a series of digoxin standards in pooled human plasma. Concentrations of digoxin in plasma were (A) 5.0, (B) 2.0, (C) 1.0, (D) 0.5, and (E) 0.0 ng/mL. Conditions are the same as in Figure 3.
a 4-h digoxin/antibody incubation period yielded a usable but much less sensitive digoxin immunoassay throughout its therapeutic range. The concentration of phenol produced during the shorter incubation period was in the low M range and, therefore, also necessitated the use of LCEC. An analogous enzyme immunoassay with detection of phenol by LCEC has been developed for rabbit immunoglobulin G, IgG (14). The detection limit achieved for IgG was 5 ng/mL (32 pM), which is roughly comparable to the detection limit obtained for digoxin (50 pg/mL or 100 pM). These results suggest that competitive enzyme immunoassays with detection of phenol by LCEC are applicable to both large and small molecules. A sandwich immunoassay based on the determination of phenol by LCEC was also developed for rabbit IgG (14), for which a detection level of 10 pg/mL or 66 fM was achieved. The sandwich assay is capable of a 1000-fold improvement in detection limit compared to the competitive assay. However, the sandwich assay can only be used for the determination of molecules that are sufficiently large to bind to two or more antibody molecules. Digoxin is too small to fulfill this requirement. FIAEC Enzyme Immunoassay. The phenol concentrations produced under the optimal assay conditions are within the demonstrated detection range of FIAEC, and consequently the assay was also evaluated by using this method. Traces for the FIAEC analysis of digoxin standards in plasma using optimal assay conditions are shown in Figure 7. It can be seen that an assay for digoxin was obtained throughout its
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EIA-FIAEC, ng/mL Figure 8. Correlation between digoxin concentration in patients by R I A and competitive heterogeneous enzyme immunoassay liquid chromatography/electrochemistry(EIA-LCEC). A perfect correlation is represented by the solid line.
therapeutic range. The i,'s obtained for the 5.0 and 0.0 ng/mL digoxin standards corresponded to phenol concentrations in the M and lod M range, respectively. The i, of the blank signal for the substrate solution was 6% of the total peak signal obtained for the 5.0 ng/mL digoxin standard. The effect of the blank signal could be minimized by dilution of the standards. Approximately 2.5 samples/min could be analyzed by the FIAEC method. FIAEC could not be used in the shortened assay format since the amount of phenol produced for all digoxin standards under these conditions is too near the detection limit of the technique. Comparison of Electrochemical Enzyme Immunoassays with RIA. Samples from patients receiving digoxin therapy were analyzed by the heterogeneous immunoassay LCEC method using the optimized assay parameters. Samples were diluted 5/1 with pooled human plasma in order to eliminate an observed antibody matrix effect. Digoxin standards in pooled human plasma were treated similarly and the results used to construct a standard curve. Digoxin levels in patient samples were determined by reference to the standard curve. The values obtained for the samples by the electrochemical enzyme immunoassay method were compared to those obtained by RIA. The results for the 54 samples analyzed are presented in Figure 8. A good correlation was obtained between the two methods (r = 0.93). The optimized assay parameters were also employed for the analysis of patient samples by the heterogeneous enzyme FIAEC method. Although only a small number of samples was analyzed, a good correlation (r = 0.95) was found for the FIAEC immunoassay and RIA, as shown in Figure 9.
CONCLUSIONS The excellent detection limit of LCEC for phenol is the basis of a very sensitive enzyme immunoassay for digoxin. A detection limit of 50 pg/mL for digoxin was achieved in plasma samples, which is substantially lower than the therapeutic range for this drug. Thus, enzyme immunoassay with LCEC detection is potentially applicable to the low- and sub-picogram level of drugs. Since the detection of phenol was not a limiting factor in this work, even lower detection limits should be achievable by this methodology with antibody-hapten systems having a sufficiently large binding constant. The detection of phenol by FIAEC with electrochemical detection is limited to the low M range due to the presence
Figure 9, Correlation between digoxin concentration in patients by R I A and heterogeneous enzyme immunoassay flow injection analysis electrochemistry (EIA-FIAEC).
of a blank signal for the substrate solution. However, sufficient phenol was generated under optimal assay conditions to use FIAEC for the determination of digoxin throughout its therapeutic range. This method also possesses the advantage of rapid sample throughput and may therefore be preferable to the LCEC detection method when lower detection capabilities are not required and high sample throughput is important.
ACKNOWLEDGMENT The authors acknowledge Julius Zodda and Peter T. Kissinger for their useful comments and Douglas Fast and the Centers for Disease Control for generously providing the digoxin antisera. Registry No. Digoxin, 20830-75-5. LITERATURE CITED (1) Skelly, D. S.; Brown, L. P.; Besch, P. K. Clin. Chem. (Winston-Salem, N.C.)1973, 19, 146-148. (2) Charlton, J. C. Antlbiot. Chemother. (Basel) 1979, 2 6 , 27-37. (3) Jarvis, R. F. Antibiot. Chemother. (Basel) 1979, 2 6 , 105-117. (4) Leute, R. K.; Ullman, E. F.; Goldstein, A.; Herzenberg. L. A. Nature (London), New Biol. 1972 236, 93-94. (5) Montgomery, M. R.; Holtzman, J. L.; Leute, R. K.; Dewees, J. S.; Bok, G.; Clln. Chem. (Wlnston-Salem, N.C.)1975, 2 1 , 221-226. (6) O'Donnell, C. M.; Suffln, S. C. Anal. Chem. 1979, 57, 33A-40A. (7) Nakamura, R. M.; Dito, W. R. Lab. Med. 1980, I f , 807-811. (8) Doyle, M. J.; Halsall, H. B.; Helneman, W. R. Anal. Chem. 1984, 5 4 , 23 18-2322. (9) Wehmeyer, K. R.; Halsall, H. B.; Heineman, W. R. Clin. Chem. (Winston-Salem, N . C . ) 1982, 28, 1968-1972. (10) Weber, S. G.; Purdy, W. C. Anal. Left. 1979, f 2 , 1-9. (11) Wisdom, G. B. Clin. Chem. (Wlnston-Salem, N . C . ) 1978, 2 2 , 1243-1 255. (12) O'Beirne, A. J.; Copper, H. R. J . Histochem. Cytochem. 1979, 27, 1148-1182. (13) Doyle, M. J.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1984, 56, 2355-2360. (14) Wehmeyer, K. R.; Halsall, H. B.; Heineman, W. R. Clin. Chem. (Winston-Salem, N . C . ) , In press. (15) Smith, T. W.; Haber, E. J. Clin. Invest. 1970, 49, 2377-2382. (18) Wehmeyer, K. R.; Doyle, M. J.; Wrlght, D. S.; Eggers, H. M.; Halsall, H. B.; Heineman, W. R. J. L i 9 . Chromatogr. 1983, 6 , 2141-2156. (17) Herbert, G. A.; Pelham, P. L.; Pittman, B. Appl. Microblol. 1973, 2 5 , 26-36. (18) Levy, H. B.; Sober, H. A. Roc. SOC. Exp. Biol. Med. IQSO, 103, 250-252. (19) Chen, I . W.; Maxon, H. R.; Heminger, K. S.; Ellis, K. S.; Volle, C. P. J . NUCl. Med. 1980, 2 1 , 1162-1168. (20) Standefer, J. C.; Saunders, G. C. Clin. Chem. (Winston-Salem, N . C . ) 1978, 24, 1903-1907.
RECEIVED for review July 19,1984. Resubmitted June 7,1985. Accepted August 26, 1985. K.R.W. acknowledges support received as a Laws Fellow (Chemistry Department, University of Cincinnati) and a Summer Fellowship (University Research Council). This work was supported by NSF Grants CHE8217045 and CHE-79-11872.
