A Penicillin Selective Enzyme Electrode G. J. Papariello, A. K. Mukherji, and C. M. Shearer Analytical and Physical Chemistry Section, Wyeth Laboratories, Inc., P.O. Box 8299, Philadelphia, Pa. 19101
In the field of pharmaceutical analysis it would, of course, be most useful and advantageous to have available ion selective electrodes for the analysis of certain drugs or classes of drugs. With this end in mind, a variety of approaches were explored toward the development of an electrode for the analysis of a most important drug class, penicillins. Ion selective membrane electrodes have been successfully developed for a variety of inorganic cations and anions (1, 2). There has also been some limited success in the fabrication and testing of selective electrodes for the analysis of organic substances (3-5). However, without a doubt, the greatest success to date in the determination of specific organic species by use of selective electrodes has been as a result of the development of the enzyme electrode. Guilbault and Montalvo (6, 7) first introduced the concept of the enzyme electrode by describing an electrode responsive to urea prepared by immobilizing the enzyme urease in a polymer film about an ammonium ion selective electrode. They went on to prepare enzyme electrodes for the analysis of l-amino acids and glucose (8, 9). Llenado and Rechnitz (201 developed an enzyme electrode for the determination of amygdalin. Following this lead, an enzyme electrode has been developed which utilizes penicillin p-lactamase (penicillinase) and is responsive to intact penicillin. This electrode is prepared by immobilizing the penicillinase in a thin membrane of polyacrylamide gel molded around and in intimate contact with a hydrogen ion glass electrode. When this electrode is exposed to an aqueous solution of penicillin adjusted to a p H of 6.4, the immobilized enzyme hydrolyzes the penicillin to produce the corresponding penicilloic acid, as shown below:
PENICILLOIC A C I D
The increase in hydrogen ion concentration from the penicilloic acid is sensed by the glass electrode and a potentiometric response is recorded. EXPERIMENTAL Apparatus. T h e electrode was prepared in a manner similar t o that described by Montalvo a n d Guilbault f l l ) , and if information beyond that supplied here is desired, one should refer to t h a t work. R. P. Buck, Ana/. Chem , 44, 270R (1972) R. A. Durst. E d . , "Ion Selective Electrodes,' N a t . Bur. S t a n d . i U S I Snec. Pub/. 314 (Nov. 1969). G . Baum, Anal. L e t t . 3, 105 (1970). M . Matsul and H Freiser, Anal. L e t t . 3, 161 (1970). T . Higtichi, C . K lllian, and J. L. Tossounian, Anal Chem.. 42, 1674 (1970) G . G . Guilbault and J. G . Montalvo, J Amer Chem Soc.. 91, 264
(1969) G G . Guilbault and J. G . Montalvo, Anal L e t t . 2, 283 (1969). G . G . Guilbault and E. Hrabankova, A n a / . Chem , 42, 1779 (1970) G . G . Guilbault and G . J . Lubrano, A n a / Chim. A c t a . 60, 254 (1972) R . A. Llenado and G . A. Rechnitz, Anal. Chem , 43, 1457 (1971). J. G . Montalvo and G . G . Guilbault, Anal C h e m . 41, 1897 (1969).
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ANALYTICAL CHEMISTRY, VOL. 45, N O . 4 , APRIL 1973
Three grams of acrylamide ( E a s t m a n ) and 0.58 gram of N,N'methylenebisacrylamide ( E a s t m a n ) were dissolved in 25 ml of 0.1M tris( hydroxymethy1)aminomethane buffer a t p H i. Three mg each of riboflavin a n d potassium persulfate were added t o catalyze photopolymerization. To one ml of the above solution, 125 mg of Penicillinase A ( B . cereus p-lactamase 6300 units/mg from Riker Laboratories) was added. A glass electrode (Beckman No. 39303 or 39301) was washed well with distilled water, wiped dry with tissue paper, and mounted upside down. A 1-in. x 1-in. piece of Nylon net (350 km) was placed over the glass bulb of the electrode and held in place with a thin wire wrapped just below the glass bulb. T h e electrode was mounted inside a glass tube ( 2 cm i.d.) which was continuously flushed with nitrogen. A 500-watt G E reflector lamp (PH500132Ri) was used for photopolymerization. T o prevent any heat transfer from the l a m p to the electrode, a glass tank 9 cm thick filled with water was placed between them. T h e enzyme-gel solution was added dropwise t o the electrode. Normally a total of only 8-10 drops was needed. During the addition of the enzyme-gel solution and for approximately 40 minutes thereafter, the electrode was exposed t o t h e light source. After polymerization was complete, a second piece of nylon netting was placed over the gel layer and held in place with an O-ring. T h e electrode was then equilibrated in p H 7 tris buffer for a period of not less than 24 hours prior t o use. T h e electrode was stored in a refrigerator to preserve enzyme activity. Reagents. Stock solutions (0.1M) of sodium ampicillin, sodium nafcillin monohydrate, potassium cyclacillin (WY-4508), potassium penicillin G (potassium benzylpenicillin), potassium penicillin V (potassium phenoxymethyl penicillin), sodium dicloxacillin monohydrate, and 1-aminocyclohexanepenicilloic acid were prepared. These stock solutions were prepared on the day of use. In order to obtain t h e solution with the penicillin concentration desired, these stock solutions were diluted with water in the appropriate manner. They were then adjusted t o a p H of 6.4 by use of dilute hydrochloric acid or dilute sodium hydroxide. Procedure. T h e enzyme coated electrode was connected to the indicating electrode terminal of a Corning Model 12 Expanded Scale p H meter. A Beckman Permaprobe Solid State electrode (39406) served as the reference electrode. All the measurements were made at ambient room temperature without stirring. except where otherwise indicated.
RESULTS AND DISCUSSION Effect of Substrate Concentration. The electrode system senses the hydrogen ions produced at the membrane via the penicillinase catalyzed hydrolysis of the penicillin. It is clear from the reaction shown in Equation 1 that the potential of the electrode can be related to the concentration of the penicillin in the following manner. E = E" + 2.3 R T / F log aHi
E
=
E"
t
2.3 R T i F log [penicillin]
(3)
Equation 3 predicts a slope of 59 mV per decade change in penicillin concentration when log [penicillin] is plotted cs. E However, it has been determined that the response of this electrode is not completely Nernstian. Table I lists the typical slopes obtained for a variety of penicillins on a given day at ambient room temperature. A t this time, no explanation can be offered for the observed differences in slopes for the various penicillins. However, a very recent publication by Blaedel et al. (12) has attempted to explain in a rigorous mathematical manner, the kinetic be(12) W J Blaedel. T R Kissel and B C Boguslaski, Ana/ Chem 2030 ( 1 9 7 2 )
44.
270 r
Table I. Slopes Obtained for Various Penicillins By Plotting Log (Penicillin) vs. Enzyme Electrode Potential Response Penicillin Sodium Ampicillin Sodium Nafcillin Monohydrate Potassium Penicillin G Potassium Penicillin V Potassium Cyclicillin Sodium Dicloxacillin Monohydrate
Slope, mV per decadechange in concentration
52 42 44 40 40 38
240 210
25 -
150
i
1M u'
w to 30 0 10'1
10-2 PENICILLIN CONCENTRATION, M
havior of easily definable fixed enzyme systems. An extension of their work and further experimentation in these laboratories may produce the answer. T h a t the slopes are less than that which theory predicts is not unexpected, since it is quite probable that not all the hydrogen ions reach the electrode surface. One must also recognize that it is quite improbable that one would obtain the exact slope for a given penicillin using two different enzyme electrodes. In fabricating the electrode, the thickness of the gel layer is not precisely controlled, and, therefore, variations in this gel layer lead to differences in slope. The slope of an electrode is also affected by the age and frequency of use of the electrode. Figure 1 illustrates the type of response obtained for different penicillins. It can be seen that the electrode is analytically useful in the penicillin concentration range of lo-' to 5 x 10-2M. Response Time. This enzyme electrode exhibits a relatively quick response to most penicillins. Typically, the response time for a new electrode is 15 to 30 seconds. Figure 2 illustrates the type of response obtained for different sodium ampicillin concentrations. The penicillin concentration of the solution being tested does have some effect on the response time. As might be expected, the particular penicillin species being measured will also have an effect on the response time. That is, penicillins which are less susceptible to penicillinase catalyzed hydrolysis, such as sodium nafcillin monohydrate and sodium dicloxacillin monohydrate (131, will come to an equilibrium value only after one to two minutes. On aging, the response of an electrode is slowed. An electrode which is two weeks old will normally take three to five times longer to respond than it did when it was new. Effect of pH. The pH a t which the sample solution is adjusted prior to introducing the enzyme electrode into the solution has been carefully chosen, for it has a marked influence on the total system. The pH affects the solubility of the penicillin, the stability of the penicillin, and the reactivity of the penicillinase. Thus, one could not operate this electrode a t a p H for example below 5 because of the low solubility of the penicillins in acid solution. Also, one could not operate a t a pH above 8 because of the poor stability of the penicillin in basic solution. Others have reported ( 1 3 ) that the optimum activity for penicillinase falls in the pH range of 5.8 to 6.8 for all the penicillins considered in this work. Our own experience has been that the electrode is most sensitive and responsive in the p H range of 6 to 7. An operating p H of 6.4 is recommended as a convenient compromise. Effect of Amount of Enzyme and Temperature. It can safely be assumed that there is a swamping amount of enzyme in the electrode membrane. If the electrode is prepared as recommended, there is enough penicillinase to react with more than 0.5 mole of a penicillin. T o prove 1 3 ) J . P H o u and J. W. Poole, J Pharm Sci
61. 1594 (1972).
Figure 1. Calibration curves for different penicillins
0-0 Sodium Ampicillin
0-0 Sodium Dicloxacillin Monohydrate X-X
Sodium Nafcillin Monohydrate
Figure 2. Electrode response times concentrations.
at
various sodium ampicillin
this point, an electrode was prepared using one-fifth the amount of penicillinase normally recommended. This electrode behaved in a perfectly typical fashion. It should be noted, however, that the amount of penicillinase present in the electrode membrane will have an effect on the useful life of the electrode. Electrodes prepared in the normal manner have functioned in an acceptable manner for a period of up to two weeks. In that period, a hundred or more measurements may have been made with the electrode. In order to determine if there would be some advantage a t operating this enzyme electrode at an elevated temperature, a study was made a t 37 "C. Although the response time was somewhat more rapid, there was no great improvement in general operation over the normal uncontrolled room temperature condition. Specificity in Analysis. In drug analysis, one is always confronted with the need for an analytical method which will clearly distinguish the intact drug from its degradation products. This electrode has such specificity built into it. One adjusts the p H of the sample solution using a standard glass-calomel electrode system to 6.4 prior to immersing the enzyme electrode. Consequently, the penicilloic acid which is present prior to the introduction of the enzyme electrode has no effect on the change in potential recorded after the electrode immersion. To prove this, a solution which was 0.01M with respect to I-aminocyclohexanepenicilloic acid and 0.01M with respect to sodium cyclacillin was measured in the normal manner. The potential obtained with such a solution did not differ from that obtained with a 0.01M sodium cyclacillin solution. Since a major route of penicillin degradation is through the penicilloic acid, one can conclude that this enzyme electrode would be useful in stability studies where partially degraded systems are measured. ANALYTICAL CHEMISTRY, VOL. 45, NO. 4 , APRIL 1973
0
791
Table II. Results of Analysis of 0.5 mg/ml Sodium Ampicillin Solution Using the Enzyme Electrode Found. rnolml Day
1
2
3
Average Rei std. dev
Electrode 1
0.48 0.46 0.46 0.66 0.40 0.55 0.48 0.50 16.7
Electrode 2
Electrode 3
0.52 0.46
0.63 0.55
0.44
0.48
0.47
0.55
8.9
13.6
Application to Penicillin Analysis. This enzyme electrode has a linear response down to a penicillin concentration of approximately lO-4M. Thus, it is competitive in terms of its sensitivity capabilities with the two most widely used chemical methods for penicillin analysis, namely the iodometric titration method and the hydroxamic acid colorimetric procedure. An attempt was made to evaluate the general utility of this electrode for penicillin analysis.
This work was done over a period of three days using three different enzyme electrodes and sodium ampicillin as the model compound. Calibration curves for all three electrodes were obtained on a daily basis. Analysis of a known, namely a 0.5 mg/ml sodium ampicillin solution, was attempted several times during the day using the three electrodes. The results of this work are summarized in Table 11. As can be seen, the reproducibility is poor. This is, of course, in part a reflection of the manner in which the data is plotted, that is, log [penicillin] us. observed potential. Thus, any small change in potential has a tremendous effect on the concentration value recorded. A possible source of' error is the contamination of the electrode by retention of part of the previous sample in the membrane. However, careful water washing and soaking in water for several minutes in between each measurement was performed, which should have been sufficient to overcome this problem. Further development is under way in these laboratories to improve the reproducibility of the penicillin electrode. In particular, a study of the geometry and configuration of the electrode is being pursued. Received for review November 9, 1972. Accepted December 21, 1972. This work was presented a t the Eastern Analytical Symposium, Atlantic City, N. J., November 2, 1972.
X-Ray Microdetermination of Chromium, Cobalt, Copper, Mercury, Nickel, and Zinc in Water Using Electrochemical Preconcentration B. H. Vassos,' R . F. Hirsch, and H. Letterman2 Department of Chemisfry. Seton Hall University, South Orange, N.J. 07079
X-Ray fluorescence exhibits good specificity and reasonable freedom from interferences, but its sensitivity in dealing with aqueous solutions is limited. A vast extension of the range for analysis a t trace levels can be obtained by preconcentration ( 1 -5). This paper describes a method in which the preconcentration step consists of electrodeposition of the metals to be determined onto a pyrolytic graphite electrode. In this way, small amounts of reducible metal ions can be separated from large volumes of dilute solutions. The desired metals are isolated in a form particularly suitable for analysis by X-ray fluorescence. After the deposition step, a thin disk is cleaved from the electrode surface (an operation possible only with pyrolytic graphite) and analyzed by X-ray spectrometry. The electrodeposited film behaves analytically as if it were infinitely thin. (The thickness of the deposit ranges 'Present address, Department of Chemistry, Colorado State University, Fort Collins. Colo. 80521. 'Present address, Bristol-Myers Products, Hillside. N.J. 07207. ( 1 ) W. T. Grubband P. D . Zemany, Nature. 76, 221 (1955). (2) C. L. Luke, Anal. Chim Acta. 41, 237 (1968). (3) K . Beyerman, H. J. Rose, J r . , and R . P. Christian, Anal. Chim. Acta. 45,51 (1969). (4) T. E. Green, S . L. Law, and W. J. Campbell, Ana/. Chem.. 42, 1749 (1970). (5) A . T. Kashuba'and C R . llines, Anal. Chern.. 43, 1758 (1971).
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
from 10 to 20 A per pg of metal.) The graphite disks are durable and easy to store. Because of their small thickness and great purity, they generate a minimum of background interference (ti, 7). Previous attempts to electrodeposit on metal electrodes (8,. 9) have been seriously handicapped in sensitivity by the background interference from the electrode material. By utilizing our approach, however, the method is in principle limited in its over-all sensitivity only by the electrolysis time, if large volumes of sample are available. (This trade-off between sensitivity and electrolysis duration was shown to be true for a t least one order of magnitude beyond the standard conditions described below.) We have evaluated the method for solutions of low electrolyte concentration (approximating fresh water) and for samples with conventional levels of added supporting electrolyte.
EXPERIMENTAL Apparatus. T h e electrolysis cell uses I-cm diameter graphite rods of l-cm length(union Carbide, carbon products~ i ~ i ~ i N e w York, N.Y.), c u t along t h e crystallographic C axis f r o m p l a t e ( 6 ) 6. H. Vassos. F. J. Berlandi, T. E. Neal. and H. B. Mark, Jr., Anai. Chem.. 37, 1653 (1965). (7) B. H. Vassos. R. F. Hirsch, and D. G. Pachuta, Ana/ Chern.. 43, 1503 (1971). (8) J. Natelson and P. K. De, Microchem. J , 7 , 448 (1963) (9) I . W. Mitchell. N. M . S a m and C. L. Hiltrop, Noreico R e p , 11, 39 (1964).
