Analytica Chimica Acta, 158 (1984) 357-362 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

ENZYME ELECTRODE FOR THE DETERMINATION OF SALICYLATE

TEKUM FONONG and GARRY A. RECHNITZ*

Department of Chemistry, University of Delaware, Newark, DE 19711 (U.S.A.)

(Received 28th October 1983)

SUMMARY

Salicylate hydroxylase is used with a carbon dioxide sensor for the determination of salicylate in aqueous solution and pooled serum. The enzyme is physically entrapped with a dialysis membrane at the sensing tip of the carbon dioxide electrode. The enzyme catalyzes the stoichiometric formation of catechol and carbon dioxide from salicylate and reduced pyridine nucleotide in the presence of flavin adenine dinucleotide as a specific cofactor. The carbon dioxide is detected by the sensor and related to the concentration of salicylate via a calibration curve. The method compares favorably with the spec- trophotometric method for assay of salicylate. Although suitable for salicylate concen- trations in the range of 5-300 rg ml-‘, its response below 5 rg ml-l is limited by the detection limit of the carbon dioxide sensor.

Acetylsalicylic acid (aspirin) is widely used as an analgesic and anti- inflammatory agent. Its common metabolites are salicylic acid, salicyluric acid, and 2,5-dihydroxybenzoic (gentisic) acid. Because formation of sali- cyluric acid and salicylphenolic glucuronide is capacity-limited in the thera- peutic dose range [ 141, there is a disproportionate rise in plasma salicylate levels as the dose of aspirin increases. For design of safe and effective dosages for long-term therapy, it is important to be able to monitor the time course of plasma salicylate concentrations as a function of dose and frequency of aspirin administration.

Methods for the determination of salicylate include gas-liquid chromato- graphy [ 5-111, thin-layer chromatography [ 12, 131, spectrofluorimetry [ 14, 151, high-performance liquid-chromatography [ 16-191, calorimetry [20-221, and potentiometry [23]. An enzymatic or enzyme-based method has now become attractive owing to the recent commercial introduction of the salicylate hydroxylase enzyme.

Principle of the method The method utilizes the enzyme-catalyzed reaction in which salicylate is

stoichiometrically converted to catechol and carbon dioxide

a COOH OH

0 Salicylate

+NADH+H+ +02 - hydroxylase a 0 + NAD+ + Hz0 + Cop

OH OH

(1)

0003-2670/84/$03.00 0 1984 Elsevier Science Publishers B.V.

353

The carbon dioxide produced is sensed with a potentiometric pC02 mem- brane sensor.

EXPERIMENTAL

Reagents, solutions and instrumentation Sodium salicylate, acetylsalicylic acid, salicyluric acid (o-hydroxyhippuric

acid), sodium gentisate (sodium 2,5dihydroxybenzoate), p-nicotinamide adenine dinucleotide, and Type S 2385 salicylate hydroxylase (E.C. 1.14.13.1) were used (Sigma Chemical Company, St. Louis, MO).

Only reagent-grade chemicals were used. Distilled-deionized water was used throughout in making solutions. Stock solutions of salicylate (6.50 X lo-’ M), gentisate (6.84 X 16’ M), and salicyluric acid (6.84 X lob3 M) were prepared by dissolving the appropriate amount of each compound in water. Solutions of p-NADH were made in pH 6.00 (0.10 M) phosphate/EDTA buffer. The buffer (30 mM K2HP04/1 mM EDTA) was prepared by dis- solving appropriate amounts of dipotassium hydrogenphosphate and di- sodium-EDTA in 80 ml of water, adjusting the pH to 6.00 with hydrochloric acid, and diluting to a final volume of 100 ml with water [24].

An Orion Model 95-02 carbon dioxide sensor connected to a Corning Model 12 Research pH/mV meter was used with a Heath-Schlumberger Model SR 255 B strip-chart recorder to record the potentiometric data. The chart speed was set at 0.05 cm min -’ at a range of lOO-mV full scale. A lo-ml double-jacketed glass cell thermostated at 30.0 f O.l”C with a Haake Model FM constant-temperature bath was used in the measurements. Spectrophoto- metric data were obtained with a Hitachi Model 100-60 spectrophotometer.

Procedure In all measurements, the initial volume of solution was 3.00 ml (0.5 ml

of 0.062 M p-NADH and 2.50 ml of 0.10 M phosphate/EDTA buffer, pH 6.00). All solutions were allowed to reach thermal equilibrium (30°C) with constant stirring in the thermostated cell.

The enzyme was obtained as a lyophilized powder and was immobilized by physically entrapping 1.0 mg (4 units) at the tip of the carbon dioxide sensor with a Technicon Type C dialysis membrane. The electrode was inserted into the cell and after a steady baseline potential had been obtained (after about 20 min), the enzyme-catalyzedreaction was initiated by addition of salicylate.

RESULTS AND DISCUSSION

Effect of pH In acidic solutions (<pH 4.00), the activity of the enzyme decreases

rapidly with time; and at 9pH 5.50, p-NADH is almost instantly destroyed. Therefore, the choice of operating pH for the immobilized enzyme will

359

require a compromise between these limits and the inherent properties of the pCOl electrode itself. The overall pH profile obtained experimentally is shown in Fig. 1. Although the apparent pH for maximum activity is 5.50, all experiments were done at pH 6.00 to minimize the loss of PNADH. In confirmation, the absorbance of a solution of /3-NADH (1 X 10” M) at 340 nm was monitored at pH 6.00 for 1 h; no significant decrease was observed. Because of evidence that inhibitor can form in solutions of p- NADH without a decrease in absorbance at 340 nm [ 251, all solutions of fl-NADH were prepared and used within 1 h.

Calibration curves and lifetime of the electrode Calibration curves (Fig. 2) were constructed in solutions with initial

volumes of 3.00 ml thermostated at 3O”C, as described in the Procedure. Aliquots of stock salicylate solution (6.50 X low3 M) were added to initiate the enzyme-catalyzed reaction. Steady-state potentials were recorded after each addition. The response time was 8 min for the lowest salicylate con- centration (1.08 X 10e5 M) and 2 min for the highest salicylate concentration (8.23 X lo4 M).

The slope of the semilogarithmic plot was found to be 38 mV/decade for data between 7.50 X 10e5 and 7.30 X lo4 M. Lifetime studies of the elec- trode were taken by constructing calibration curves daily for 15 days. After 12 days, the slope decreased.

-100

-1 ia

O- -120

/

00 -13oL 4 00 6 00 6 00 I 00 3 00 5 4 T--- PH -Log [Compound] (M)

Fig. 1. pH profile of the immobilized salicylate hydroxylase electrode at 30°C.

Fig. 2. (A) npical calibration curve for salicylate with the immobilized salicylate hydroxy- lase electrode at 30°C and pH 6.00. (B) Response to gentisate under the same conditions.

360

Selectivity of the electrode Although salicylate is the major metabolite of aspirin, minor metabolites

such as gentisate and salicyluric acid are known to accumulate in the body after ingestion of large doses of aspirin. Therefore, gentisate and salicyluric acid were examined as substrates for the enzyme. It was found that only gentisate gave potential changes. The extent of interference is shown in Fig. 2. While gentisate is clearly a significant interference, the relative con- centrations of salicylate and gentisate in physiological situations are such that the gentisate interference is of no practical importance in clinical samples. There was no response of the electrode to aspirin itself.

Precision studies The precision of this determination of salicylate was tested in both

aqueous solution and pooled serum. Salicylate concentrations were randomly chosen in the range of 36.0-292.5 pg ml-i and quantified. Aliquots of a standard salicylate solution (6.50 X lo-’ M) were added to the aqueous or serum solution and the steady-state potential was measured for each added aliquot. The corresponding concentration obtained from the standard curve was compared to that of the sample added to the solution. Table 1 shows that the relative errors in these studies ranged from 1.2-2.4s. Within-run precision results are also given in Table 1. These values range in standard deviation from ?1.2% to ?4.1% with an average of *2.1%.

The results of recovery studies in aqueous and pooled-serum solutions are given in Table 2. These results show an average recovery of 97% for aqueous solutions and 98% for serum solutions.

Comparison of potentiometric and spectrophotometric methods Aqueous samples of salicylate were testedusing both the electrode method

and the spectrophotometric method outlined by Sigma Chemical Company. In the spectrophotometric method, the decrease in absorbance of /3-NADH was monitored for each aliquot of salicylate solution added. Linear regression analysis of the two sets of results showed that the potentiometric method

TABLE 1

Precision studies and relative errors in random salicylate assays at pH 6 .OO and 30°C

Salicylate concentration (rg mid)

Taken Found

36.0 36.9 61.0 62.4

204.1 200.8 248.8 245.9 292.5 287.2

*RSD calculated from ten results.

Relative Within-run error precision’ (%) (%)

+2.5 i4.1 +2.3 *3.0 -1.6 *l.O -1.2 *l.O -1.8 il.2

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TABLE 2

Recovery studies of salicylate in aqueous solutions and pooled serum

Average salicylate added (rg ml-i)

Recovery (%) Average salicylate Recovery (%)

Aqueous Pooled added (pg ml-‘)

Aqueous Pooled solution serum solution serum

5.2 98 109 61.0 98 97 10.4 97 102 110.5 98 96 15.5 96 98 204.1 97 96 25.8 98 96 270.9 97 98 36.0 96 95 313.6 97 96

agreed well with the spectrophotometric method. For ten samples ranging from 5.2 to 200 pg ml-‘, a plot of the potentiometric data (y) vs. the spec- trophotometric data (LX), gave a straight line defined by the equation y = (1.01 + 0.01)x + 2.24 + 0.02 with S,, = 1.5 and r = 0.98.

The results of this study show that the potentiometric method may pro- vide an attractive alternative for the determination of salicylate. The elec- trode shows useful sensitivity and selectivity while requiring minimal sample pretreatment compared to other methods [ 261.

We are grateful to NIH (Grant GM 25308) for support of this work.

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