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Anal. Chem. 1984, 56,849-852
terminations for each value given. It can be seen that each of the eight NAs adsorb at one or more concentrations of perchloric acid-Celite, but with the hydrochloric acid-Celite only DMN and NPYR adsorb at the 6 N strength. The adsorption of the NAs on acid-Celite appears to be a function of the base strength of each and the particular acid concentration needed to protonate the NA. It has been reported that strong acid and/or chloride ion will destroy NAs (10). To confirm this and explain the performance of the perchloric acid-Celite chromatographic material, an acidCelite mixture was made with 6 N HCIOl and 3 N NaC1. All eight NAs subjected to chromatography on this material were subsequently destroyed. Therefore, it is apparent that increasing the acid strength of hydrochloric acid-Celite will not extend the capabilities of that method. The information in Table I can be used by the analyst as a guide in preparing acid-Celite chromatographic material. If a single NA is to be determined, chromatographic conditions could be optimized for the analysis. For DMN only the 3 N material would be used, whereas for DPN only the 9 N material would be used, each giving 95% recovery. For NAs that chromatograph poorly on a single acid-Celite, stacking would be the method of choice. DEN would be analyzed best with a stacked set of 3 N-4 N-6 N acid-celite. “PIP would require only a stacked set of 4 N-6 N acid-Celite to achieve a recovery of 100%. This could “tune” the technique for a specific NA. For a mixture of NAs, the stacking method of preparation must be selected. Figure 1illustrates four GC-TEA runs on a single sample containing 0.2 ng each of eight NAs. The top chromatogram is the combined extract from a stack set of 3 N-6 N-9 N acid-Celite. The next three chromatograms are the individual fractions. Analysis of a mixture can not guarantee 95-100% recovery of every NA present, but as is seen in Figure 1, splitting fractions can be advantageous, especially for GC/MS (3, 9). A single analysis with this stacking method could also be achieved by reverse elution of the stack, saving time and solvent quanity. Upon further examination of Table I it is seen that comparing the percent recoveries of the NAs in the 3 N HC104
column to the 6 N HC1 column, the recoveries for DMN and NPYR are better by 10%. This would indicate that a change in acid from HC1 to HCIOl would improve results for any analyst currently using hydrochloric acid-Celite. It is well documented that hydrochloric acid-Celite produces “clean” sample extracts (2, 7-9) suitable for analytical techniques requiring highly concentrated final extracts, such as GC/MS (3) or capillary gas chromatography ( 4 ) . In this regard perchloric acid-Celite would be wholly comparable but have a higher efficiency and broader analytical range for NAs, making it a better choice for a chromatographic cleanup method.
ACKNOWLEDGMENT The authors thank Walter I. Kimoto of the U.S. Department of Agriculture, Agricultural Research Service, Philadelphia, PA, for many helpful discussions. Registry No. 1,62-75-9;2,10595-95-6;3,55-18-5;4,621-64-7; 5 , 100-75-4; 6, 930-55-2; 7, 59-89-2; 8, 932-83-2; perchloric acid, 7601-90-3. LITERATURE CITED (1) Magee, P. N.; Barnes, J. M. Br. J . Cancer 1956, 10, 114-122. (2) Hotchkiss, J. H. J. Assoc. Off. Anal. Chem. 1981, 6 4 , 1037-1054. (3) Hotchkiss, J. H.; Libbey, L. M.; Scanlan, R. A. J. Assoc. Off. Anal. Chem. 1980, 6 3 , 74-79. (4) Chamberlain, W. J.; Arrendale, R. F. J. Chromatogr. 1982, 234, 478-48 1. (5) Havery, D. C.; Fazio, T.; Howard, J. W. J. Assoc. Off. Anal. Chem. 1978, 61, 1374-1378. (6) Fine, D. H.; Roundbehler, D. P. J. Chromatogr. 1975, 109, 271-279. (7) Howard, J. W.; Fazio, T.; Watts, J. 0. J. Assoc. Off. Anal. Chem. 1970, 53, 269-274. ( 8 ) Pensabene, J. W.; Miller, A. J.; Greenfield, E. L.; Fiddler, W. J. Assoc. Off. Anal. Chem. 1982, 6 5 , 151-1513, (9) Fazlo, T.; Havery, D. C.; Howard, J. W. IARC Sei. Publ. 1980, No. 3 1 , 419-433. (lo) Buglass, A. J.; Challis, B. C.; Osborne, M. R. IARC Sci. Pub/. 1975, NO. 9 , 94-100.
RECEIVED for review September 28,1983. Accepted December 28,1983. This investigation was supported, in part, by Grant No. CA18618, awarded by the National Cancer Institute, DHEW.
Extractive Preconcentration of Organic Compounds at Carbon Paste Electrodes Joseph Wang* and Bassam A. Freiha Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003 In recent years there has been an increasing interest in the development of new approaches for accumulating electroactive analytes on electrode surfaces prior to their voltammetric quantitation. For analytes that cannot be preconcentrated by electrolysis (as in conventional stripping analysis), alternative schemes based on preferential accumulation at ordinary and modified electrodes have been proposed (1-14). These new preconcentration/voltammetric methods offer both signal enhancement and a high degree of selectivity, compared to conventional (solution-phase) voltammetry. One of the most useful ordinary electrodes for spontaneous accumulation of organic compounds has been the carbon paste electrode. The preconcentration of important compounds such as chlorpromazine (5, IO),adriamycin (11),9,10-phenanthrenequinone (61, butylated hydroxyanisole (131,or uric acid (14)at carbon paste electrodes has been utilized for direct measurements in complex samples such as body fluids. In most cases, de-
tection limits at the nanomolar concentration level have been reported. An interesting and important fundamental question to be posed is whether the accumulation process at carbon paste electrodes involves adsorption at the electrode interface, extraction into the electrode, or combination of both. Since the carbon paste electrode is composed of a matrix of graphite particles with water-immiscible organic pasting liquid, there is always a possibility that some organic compounds will be extracted into the organic phase of the electrode. Such extractive preconcentration schemes may extend the scope of voltammetric measurements at solid electrodes, as the pasting liquid and the experimental conditions can be adjusted (based on principles of solvent extraction) to obtain the preferential accumulation of a certain compound, or group of compounds. Work in the mid 1960s indicated possible extraction of electroactive species into carbon paste electrodes. Chronopotentiometric study of the bromide-bromine couple at the
0003-2700/84/0356-0849$01.50/00 1984 American Chemical Society
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ITD C
/
/
B
A Flgure 1. The carbon paste electrode: (A) carbon paste; (B) Plexiglas sleeve; (C) brass rod; (D) electrical contact.
Flgure 2. Differential pulse voltammograms for 5 X lo-' M BHA (A) and 1 X lo-' M chlorpromazine (B) obtained after 15 min of preconcentration (stirring, 500 rpm), removal of 0.2 (a)and 0.4 (b) mm thick outer layers of the carbon paste, and placing the electrode in an electrolytic blank solution. Differential pulse conditions were as follows: scan rate, 10 mVls; amplitude, 50 mV. Supporting electrolyte was 0.05 M phosphate buffer.
carbon paste electrode indicated that bromine could penetrate to the interior (15). Kuwana and French (16) dissolved organic compounds in the pasting liquid and added graphite to make electrodes containing the depolarizers. They suggested that the carbon paste electrode functions for organic compounds in a manner analogous to a mercury electrode for metals. Chambers and Lee (17 ) used a radioactive tracer to study the accumulation of N,N-dimethylaniline into carbon paste electrodes. In addition, the use of carbon paste electrodes of different compositions (13) and chronocoulometry ( 7 ) has provided recently indicat,ions of extractive accumulation. In this paper, a new and simple approach for obtaining evidence of extractive accumulation into carbon paste electrodes is presented. For this purpose a pistonlike configuration of the electrode is employed, allowing the removal of the desired amount of the paste. Thus, if after the preconcentration period an outer layer of the paste is extruded, the subsequent voltammetric measurement-recorded in an electrolytic blank solution-is performed on the previously inner part of the electrode (not exposed to the solution during the accumulation step). As a result, new insights into the interior of the electrode can be achieved in a rapid and convenient way during the voltammetric measurement. As will be discussed in the following sections, this provides direct evidence of extractive accumulation and additional information on the nature of the preconcentration step.
(Acheson 38, Fisher) and Dow Corning silicone grease (37% grease by weight). Other compositions of the paste were also examined, as described below. The geometrical area of the electrode was approximately 0.13 cm2. As the brass rod is rotated its tip forces the removal of the paste. Two lines (one on the brass rod head, the second on the Plexiglas tube) serve as guides, controlling the amount of paste extruded. Reagents. Deionized water was used to prepare all solutions. Millimolar stock solutions of chlorpromazine, butylated hydroxyanisole, and uric acid (Sigma Chemical Co.) were prepared each day. The supporting electrolyte (of the sample and exchange solutions) was 0.05 M phosphate buffer (pH 7.4) prepared from a 1:4 mixture of KH2P04and K2HP04.A 0.2 M KHzPOl solution, acidified to pH 3 with phosphoric acid, served as the supporting electrolyte for the preconcentration of uric acid. Procedure. For the preconcentration step, the carbon paste electrode (with a fresh and smoothed surface) was immersed in a stirred 10-mL sample solution for a given period of time; the preconcentration proceeded at an open circuit. The electrode was then removed from the sample solution and rinsed with water for 5 s. The required amount of the carbon paste was extruded by rotating the brass rod and cutting the paste with a razor blade. The new surface was smoothed on a deck of computer cards with a circular motion, rinsed with water for 5 s, and reimmersed in the electrolytic blank solution. The initial potential was then applied, and after 15 s for the current decay, the voltammogram was recorded by scanning the potential in the anodic direction.
EXPERIMENTAL SECTION Apparatus. Two 10-mL cells (Model VC-2, Bioanalytical Systems), one containing the sample solution and the other containing the electrolytic blank solution, were used. The carbon paste working electrode, the Ag/AgCl (3 M NaC1) reference electrode, and the platinum wire auxiliary electrode were placed in the cells through holes in the covers. The preconcentration cell was placed on a magnetic stirrer (Sargent-WelchModel 76490) and a 1.6 cm long stirring bar was placed in the cell bottom. The three electrodes were connected to a Princeton Applied Research Model 174 polarographic analyzer, the output of which was displayed on a Houston-Omniscribe strip-chart recorder. The carbon paste electrode is shown in Figure 1. It consists of a Plexiglas tube (5.9 cm long, 2 mm i.d., 5 mm 0.d.) and a threaded brass rod (7.5 cm long, 2 mm diameter) arranged in a pistonlike configuration so that the paste could easily be extruded. The brass rod accurately matches the Plexiglas tube. The electrical contact is made through the head of the brass rod. The carbon paste is placed at the end of the Plexiglas tube, filling the tip to a height of about 13 mm. The paste used for the majority of the work was made by thoroughly mixing graphite powder
A transfer of an electrode (between the preconcentration and measurement steps) is commonly used for examining adsorptive accumulation processes and improving the selectivity of the measurement (5,6,11). By extruding the outer layers of the surface, during the electrode transfer, we perform the voltammetric measurement on an inner part of the paste. Thus, contributions from adsorbable species, present a t the electrode-solution interface are eliminated, and the response will be solely due to extractable species present in the interior of the electrode. Three compounds, known to accumulate a t carbon paste electrodes, chlorpromazine (5), butylated hydroxyanisole (13),and uric acid (11, 14),were employed for evaluating the proposed method. The electrode used for the majority of the work composed of silicone grease as the pasting liquid (37% by weight). Figure 2 illustrates differential pulse voltammograms at a carbon paste electrode which has been immersed in stirred BHA (A) and chlorpromazine (B) solutions and transferred to an electrolytic blank solution after extruding 0.2 (a) and 0.4 (b) mm thick outer layers of the
RESULTS AND DISCUSSION
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
851
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0.4
0.8
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Figure 3. Differential pulse voltammograms for 5 X M BHA obtained after 3 (a), 5 (b), 7 (c), and 10 (d) min preconcentration periods, removal of 0.2 mm thick outer layer of the carbon paste, and placing the electrode in an electrolytic blank solution. Supporting electrolyte, stirring, and differenthl pulse conditions are given In Figure
2.
electrode. As the measurement is performed on the inner part of the electrode, the differential pulse response provides evidence of extractive accumulation. No response was observed when the same procedure was applied to the blank solution. AE the thickness of the electrode layer extruded increases from 0.2 to 0.4 mm, the peak current decreases as expected from the smaller amount of the extractable species present at deeper parts below the surface. Apparently, a 15 min preconcentration period is sufficient for the extractable species to penetrate to an appreciable depth below the surface. Longer preconcentration periods result with deeper penetrations, as will be discussed below. The response at the original electrode surface-following transfer to a blank solution-is about 10fold larger than that obtained after removing a 0.2 mm layer (not shown). No response from the interior of the electrode was obtained with uric acid as the accumulated analyte (not shown). This indicates that the accumulation of uric acid involves adsorption at the surface rather than extraction into the bulk of the electrode. This observation and the fact that solution-phase species (e.g., ascorbic acid) do not contribute to the response following medium exchange (5) indicate a close contact between the carbon paste and the Plexiglas tube; thus, no solution creeping between the paste and the walls occurs and contributes to our response. Data similar to those of Figure 2A were obtained for the accumulation of BHA at the Nujol oil based carbon paste electrode, indicating that both silicone grease and Nujol oil serve as extractive phase in the accumulation of organic compounds from aqueous solutions. Figure 3 shows differential pulse voltammograms at a carbon paste electrode which has been immersed in a stirred 5X M BHA solution for increasing periods of time and transferred to an electrolytic blank solution after extruding a 0.2 mm thick outer layer of the electrode. As expected, the amount of extractable species, a t a given depth of the electrode, increases by increasing the preconcentration period. A plot of the peak current vs. the square root of the preconcentration time was linear, as expected for a diffusion transport. A similar trend was obtained for the preconcentration of chlorpromazine (not shown). To estimate the depth of the extractive penetration, longer preconcentration periods were employed. Figure 4 illustrates voltammograms for BHA
Figure 4. Differential pulse voitammograms for 8 X M BHA (a) and 1 X lo-' M chlorpromazine (b) obtained after 120 (a) and 30 (b) min preconcentration,removal of 0.9 (a) and 1.2(b) mm thick outer layers of the carbon paste, and placing the electrode in an electrolytic blank solution. Supporting electrolyte, stirring, and differential pulse conditions are given in Figure 2.
(a) and chlorpromazine (b) obtained after the removal of 0.9 and 1.2 mm thick layers, respectively, between the preconcentration and voltammetric measurement. Well-defined peaks are observed. As indicated from voltammogram b, 30 min preconcentration is sufficient for penetration to an appreciate depth (1.2 mm) below the carbon paste surface. These distances indicate that the diffusion coefficients of the electroactive species in the pasting liquid are of the same order found in aqueous solutions. Another insight into the nature of the extraction process was achieved by utilizing different compositions of the paste. For the 28 and 44% silicone grease electrodes, the average peak currents, obtained after extruding a 0.2 mm thick layer between the preconcentration and measurement steps, were 36 and 87 nA, respectively (conditions: five multiple measurements using a l X lo-' M BHA solution; other conditions are given in Figure 3b). As expected, the extraction of organic compounds is enhanced by increasing the amount of organic phase in the carbon paste matrix. The reproducibility of the above data (RSDs of 26 and 14% respectively) indicates some variations in the response, as observed throughout this study. Variations in the response are expected for different surfaces of the same batch of paste (6); additional contributions to variations in the response may result from the stability of the stirring transport, temperature fluctuations associated with the stirrer operation, and the reproducibility of the paste removal and electrode transfer step. RSDs of 10-30% have been reported for adsorptive accumulation studies at carbon paste electrodes (without extruding the paste) (6, 2 1 ) . In conclusion, the procedure described in this study provides direct evidence of extractive accumulation into carbon paste electrodes, using a rapid and simple way. We believe that the scope and application of the extractive preconcentration/ voltammetric approach may be very wide, providing the establishment of appropriate experimental conditions (e.g., the use of different organic pasting liquids, as in solvent-solvent extraction). Studies in this laboratory are continuing to this direction. LITERATURE CITED (1) (2) (3) (4)
Cheek, G. T.; Nelson, R. F. Anal. Lett. 1978, 7 1 , 393-402. Kolpin, C. F.; Swofford, H. S. Anal. Cbem. 1978, 50, 916-920. Sirla, J. W.; Baldwin, R. P. Anal. Lett. 1980, 73,577-588. Prlce, J. F.; Baldwin, R. P. Anal. Chern. 1980, 52, 1940-1944.
852
(9) (10) (11) (12)
Anal. Chem. 1984, 56,852-854 Wang, J.; Freiha, B. A. Anal. Chim. Acta 1983, 148, 79-85. Cheng, H. Y.; Faiat, L.; Li, R. L. Anal. Chem. 1982, 5 4 , 1384-1388. Jarbawi, T. B.; Heineman, W. R . Anal. Chim. Acta 1982, f35, 359-362. Lubert, K. H.: Schnurrbusch, M.; Thomas A. Anal. Chim. Acta 1982, 144. 123-136. Kalvoda. R . Anal. Chim. Acta 1982. 738. 11-18. Wang, J.; Freiha, B. A. Anal. Chem. 1983, 55, 1285-1288. Chaney, E. N.; Baidwin, R. P. Anal. chem. 1983, 54, 2556-2560. Wang, J.; Freiha, 8. A. J . Nectroanal. Chem. 1983, 757, 273-275.
(13) (14) (15) (16) (17)
Wang, J.; Freiha, 6 . A. Anal. Chim. Acta 1983, 754, 87-94. Wang, J.; Freiha, B. A. J . Bioelectrochem. Bioenerg., in press. Davis, D. G.; Everhart, M. E. Anal. Chem. 1984, 36, 38-40. Kuwana, T.; French, W. G. Anal. Chem. 1964, 3 8 , 241-242. Chambers. C. A. H.; Lee, J. K. J . Nectroanal. Chem. 1987, 1 4 ,
--- - . ..
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RECEIVED
fori%view October 25, 1983. Accepted November
28, 1983.
Enzyme-Amplified Determination of Methotrexate with a pC0, Membrane Electrode Purneshwar Seegopaul and G. A. Rechnitz* Department of Chemistry, University of Delaware, Newark, Delaware 19711 Methotrexate, or N - [ 4 - [[(2,4-diamino-6-pteridinyl)methyllmethylaminol benzoyl]glutamic acid, is an antimetabolite used in the treatment of certain neoplastic diseases. Its principal mechanism of action is the competitive inhibition of the enzyme folic acid reductase, important in the process of DNA synthesis, and thus interferes with tissue cell reproduction. With careful dosage control, methotrexate may impair malignant growth without irreversible damage to normal tissues ( I ) . Methotrexate (MTX) has been measured by enzyme inhibition (2, 31, HPLC (4), ion-exchange chromatography (5), homogeneous and heterogeneous enzyme immunoassay (4,6-81, radioimmunoassay (4), fluorometry (9), and mass spectrometry (10). We now report the use of an enzyme cycling amplification procedure in conjunction with a pCOz membrane electrode for the determination of methotrexate. The method is based on the inhibition of dihydrofolate reductase enzyme which couples with 6-phosphogluconic dehydrogenase to recycle the NADP+/NADPH redox system (Figure 1). Inhibition of the reductase by methotrexate reduces the extent of cycling which is then directly related to the drug concentration.
EXPERIMENTAL SECTION Apparatus. Enzymatic reactions were carried out in a 10-mL jacketed glass cell thermcatated at 37 f 0.1 "C with a Haake Model FM constant temperature circulator. Potentiometric rate measurements were made with a Corning Model 12 Research pH/mV meter and recorded on a Heath Schlumberger Model SR-255B strip chart recorder operated at 5 mm min-' and a range of 100 mV. Carbon dioxide liberation from the enzymatic reaction was monitored by an Orion Model 95-02 carbon dioxide membrane electrode. Reagents. Reagent grade chemicals were used without further purification, unless otherwise noted, and deionized water was used to prepare all solutions. Enzymatic reactions were carried out in 0.2 M citrate buffer of pH 6.2 containing 0.3 M potassium chloride. 6-Phosphogluconic acid (catalog no. P-7877), @-nicotinamide adenine dinucleotide phosphate, reduced form, @-NADPH(catalog no. N-7505),and dithioerythritol, DTE (catalog no. D-8255), were all obtained from Sigma Chemical Co. The 0.01 M @-NADPH solutions were made up fresh each day in 0.1 M citrate buffer of pH 6.2 and stored in the dark in crushed ice. The 0.25 M 6phosphogluconic acid solution was stored in the dark at 4 "C following adjustment to pH 6.2. Dihydrofolic acid (Sigma, catalog no. D-7006) was bought in 25-mg ampules and kept at -20 "C. A stock solution was prepared by dissolving 25 mg of the substrate in 5 mL of water containing 5 mM HCl and 30 mM DTE. The resultant suspension was thoroughly stirred in the dark at 4 OC and then stored frozen in 0.5-mL aliquots. Working solutions were prepared each day by thawing the 0.5-mL aliquot in the dark followed by the addition of 0.5 mL of 0.3 M citrate buffer, pH 6.2, containing 0.2 M DTE
and stored in crushed ice. Excess working solutions were discarded after 8-10 h. Dihydrofolate reductase (E.C. 1.5.1.3) obtained in 5 units vials of 10 units mL-' (Sigma, catalog no. D-6385, bovine liver source) was stored at 4 "C in the dark and used undiluted. 6-Phosphogluconic dehydrogenase (E.C. 1.1.1.44) obtained in 25 unit vials of 47.2 units mL-' (Sigma catalog no. P-0632 yeast source) was stored at 4 OC and used undiluted. Methotrexate ((+)-amethopterin,Sigma catalog no. A-6770) was stored at -20 "C. A stock solution was made by dissolving 3 mg of methotrexate in 100 mL of water in the dark at 4 "C. Following dissolution, 5-mL aliquots in tightly sealed vials were stored frozen in the dark. Working solutions of 3000 pg L-' were prepared fresh each day by 1 in 10 dilution of the thawed stock solution and stored in the dark in crushed ice. Procedures. Determination of Optimum Dihydrofolate Reductase Activity Necessary for Inhibition Reaction with Methotrexate. A 1.5-mL portion of 0.2 M citrate buffer, pH 6.2, containing 0.3 M KC1 was added t o the thermostated glass cell containing a small Teflon-coated spinbar for solution mixing at 37 A 0.1 "C. The 100 pL of 0.25 M 6-phosphogluconic acid, 50 pL of 0.01 M fi-NADPH, and 35 pL of 6-phosphogluconic dehydrogenase (1.65 units) were added to the buffer solution. Various aliquots of dihydrofolate reductase (0.0054.08 units) were then added and the total solution volume was adjusted to 1.9 mL with deionized water. After a stable base line potential was M dihydrofolic acid was added obtained, 100 pL of 5.64 X to initiate the enzymatic reaction. The carbon dioxide produced was detected by the pCOz electrode and the initial rate in millivolts per minute was recorded. Blank determinations in the absence of the reductase enzyme were carried out for correction of the rate measurements. The initial rates were plotted against corresponding enzyme activities to provide standard curves. Similarly, for optimization procedures, each reagent concentration or kinetic parameter was varied in turn while maintaining all other conditions constant. Determination of Methotrexate. One hundred microliters of 0.25 M 6-phosphogluconic acid, 50 pL of 0.01 M @-NADPH,35 pL of 6-phosphogluconic dehydrogenase (1.65 units), and 2 pL of dihydrofolate reductase (0.02 units) were added to 1.5 mL of 0.2 M citrate buffer, pH 6.2, containing 0.3 M KCl. Following solution mixing at 37 f 0.1 "C, various aliquots of 3000 wg L-' working methotrexate standard (1-30 Fg L-') were added and the total solution adjusted to 1.9 mL with deionized water. After 5 min of preincubation, 100 pL of 5.64 X lo-* dihydrofolic acid was added to initiate the enzymatic reaction. Initial rates, in millivolts per minute, were then recorded. Blank determinations in the absence of methotrexate were carried out for correction of rate measurements. The initial rates were plotted against the corresponding methotrexate concentrations t o provide a standard calibration curve. Unknown methotrexate samples were similarly processed and their levels determined from the standard curve.
RESULTS AND DISCUSSION The kinetic parameters influencing the coupled enzyme reaction in the absence of methotrexate were first optimized
0003-2700/84/0356-0852$01.50/00 1984 American Chemical Society
