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 ap- proaches were explored toward the development of an electrode for the analysis of a most important drug class, penicillins.

Ion selective membrane electrodes have been success- fully developed for a variety of inorganic cations and an- ions (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 elec- trode. Guilbault and Montalvo (6, 7) first introduced the concept of the enzyme electrode by describing an elec- trode responsive to urea prepared by immobilizing the en- zyme urease in a polymer film about an ammonium ion selective electrode. They went on to prepare enzyme elec- trodes 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 de- veloped which utilizes penicillin p-lactamase (penicillin- ase) 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 pH of 6.4, the immobilized en- zyme hydrolyzes the penicillin to produce the correspond- ing 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 po- tentiometric response is recorded.

EXPERIMENTAL Apparatus . The electrode was prepared in a manner similar to

that described by Montalvo and Guilbault f l l ) , and if informa- tion beyond that supplied here is desired, one should refer to tha t work.

R. P. Buck, Ana/. Chem , 44, 270R (1972) R. A. Durst. E d . , "Ion Selective Electrodes,' Nat . Bur. S tand . i U S I Snec. Pub/. 314 (Nov. 1969). G . Baum, Anal. Le t t . 3, 105 (1970). M . Matsul and H Freiser, Anal. Le 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, Ana/ . Chem , 42, 1779 (1970) G . G . Guilbault and G . J . Lubrano, Ana/ Chim. Ac ta . 60, 254 (1972) R . A. Llenado and G . A. Rechnitz, Anal. Chem , 43, 1457 (1971). J. G . Montalvo and G . G . Guilbault, Anal Chem. 41, 1897 (1969).

Three grams of acrylamide (Eas tman) and 0.58 gram of N,N'- methylenebisacrylamide (Eas tman) were dissolved in 25 ml of 0.1M tris( hydroxymethy1)aminomethane buffer a t p H i. Three mg each of riboflavin and potassium persulfate were added to 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. The 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 lamp to the electrode, a glass tank 9 cm thick filled with water was placed between them.

The enzyme-gel solution was added dropwise to the electrode. Normally a total of only 8-10 drops was needed. During the addi- tion of the enzyme-gel solution and for approximately 40 minutes thereafter, the electrode was exposed to the 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. The electrode was then equilibrated in p H 7 tris buffer for a period of not less than 24 hours prior to use. The 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), potassi- um penicillin G (potassium benzylpenicillin), potassium penicil- lin V (potassium phenoxymethyl penicillin), sodium dicloxacillin monohydrate, and 1-aminocyclohexanepenicilloic acid were pre- pared. These stock solutions were prepared on the day of use. In order to obtain the solution with the penicillin concentration de- sired, these stock solutions were diluted with water in the appro- priate manner. They were then adjusted to a p H of 6.4 by use of dilute hydrochloric acid or dilute sodium hydroxide.

Procedure. The 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 sys-

tem 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 concentra- tion 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. At this time, no explanation can be offered for the observed differences in slopes for the various penicillins. However, a very recent publication by Blaedel e t al. (12) has attempted to ex- plain in a rigorous mathematical manner, the kinetic be-

(12) W J Blaedel. T R Kissel and B C Boguslaski, Ana/ Chem 44. 2030 (1972)

790 ANALYTICAL CHEMISTRY, VOL. 45, NO. 4 , APRIL 1973

Table I. Slopes Obtained for Various Penicillins By Plotting Log (Penicillin) vs. Enzyme Electrode Potential Response

Slope, mV per decadechange

Penicillin in concentration

Sodium Ampici l l in 52 Sodium Nafcil l in Monohydrate 42 Potassium Penicil l in G 44 Potassium Penicil l in V 40 Potassium Cyclici l l in 40 Sodium Dicloxaci l l in Monohydrate 38

havior of easily definable fixed enzyme systems. An exten- sion of their work and further experimentation in these laboratories may produce the answer. That 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 rela- tively quick response to most penicillins. Typically, the response time for a new electrode is 15 to 30 seconds. Fig- ure 2 illustrates the type of response obtained for different sodium ampicillin concentrations. The penicillin concen- tration of the solution being tested does have some effect on the response time. As might be expected, the particu- lar penicillin species being measured will also have an ef- fect 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 solubili- ty of the penicillin, the stability of the penicillin, and the reactivity of the penicillinase. Thus, one could not operate this electrode a t a pH 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 sta- bility of the penicillin in basic solution. Others have re- ported ( 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 pH range of 6 to 7. An operating pH 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 en- zyme in the electrode membrane. If the electrode is pre- pared as recommended, there is enough penicillinase to react with more than 0.5 mole of a penicillin. To prove

1 3 ) J . P H o u and J. W. Poole, J Pharm Sci 61. 1594 (1972).

270 r

240

210

2 5 150

1M

w to

30

0

i - u'

10'1 10-2 PENICILLIN CONCENTRATION, M

Figure 1. Calibration curves for different penicil l ins

0-0 Sodium Ampicillin 0-0 Sodium Dicloxacillin Monohydrate X -X Sodium Nafcillin Monohydrate

Figure 2. Electrode response times at various sodium ampici l l in concentrat ions.

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 pres- ent 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 temper- ature, a study was made a t 37 "C. Although the response time was somewhat more rapid, there was no great im- provement in general operation over the normal uncon- trolled 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 degrada- tion products. This electrode has such specificity built into it. One adjusts the pH of the sample solution using a standard glass-calomel electrode system to 6.4 prior to im- mersing the enzyme electrode. Consequently, the penicil- loic 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-aminocyclo- hexanepenicilloic acid and 0.01M with respect to sodium cyclacillin was measured in the normal manner. The po- tential 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 par- tially 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 Electrode 1 Electrode 2 Electrode 3

1 0.48 0.52 0.63 2 0.46 0.46 0.55

0.46 0.66 0.40

3 0.55 0.44 0.48 0.48

Average 0.50 0.47 0.55 Rei std.

dev 16.7 8.9 13.6

Application to Penicillin Analysis. This enzyme elec- trode has a linear response down to a penicillin concentra- tion 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 hydrox- amic acid colorimetric procedure. An attempt was made to evaluate the general utility of this electrode for penicil- lin 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. ob- served 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 elec- trode 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 measure- ment 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 Decem- ber 21, 1972. This work was presented a t the Eastern Ana- lytical Symposium, Atlantic City, N. J., November 2, 1972.

X-Ray Microdetermination of Chromium, Cobalt, Copper, Mercury, Nickel, and Zinc in Water Using Electrochemical Preconcent rat ion

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 reason- able 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 preconcen- tration 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 sepa- rated 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 py- rolytic graphite) and analyzed by X-ray spectrometry. The electrodeposited film behaves analytically as if it were infinitely thin. (The thickness of the deposit ranges

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 prin- ciple limited in its over-all sensitivity only by the electrol- ysis 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 be- yond the standard conditions described below.) We have evaluated the method for solutions of low electrolyte con- centration (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 the crystallographic C axis f r o m p late

(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,

(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).

'Present address, Department of Chemistry, Colorado State Universi-

'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, Jr . , and R . P. Christian, Anal. Chim.

Acta. 45,51 (1969). 1503 (1971). (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).

ty, Fort Collins. Colo. 80521.

792 0 ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973