ANALYST, AUGUST 1991, VOL. 116 803 Voltammetric Behaviour of Salicylic Acid at a Glassy Carbon Electrode and Its Determination in Serum Using Liquid Chromatography With Amperometric Detection Denley Evans Department of Pathology, West Wales General Hospital, Carmarthen, Dyfed SA3I ZAF, UK John P. Hart* Department of Science, Bristol Polytechnic, Coldharbour Lane, Frenchay, Bristol, Avon BS16 IQY, UK Glan Rees Department of Haematology, West Wales General Hospital, Carmarthen, Dyfed SA31 2A F, UK Cyclic voltammetry was used t o investigate the oxidation of salicylic acid at a planar glassy carbon electrode. The electrode reaction was found t o be dependent on the pH and ionic strength of the acetate buffer, which contained 35% methanol. Under these conditions the maximum electrochemical signal was obtained with a supporting electrolyte of 0.06 mol dm-3 acetate buffer in 35% methanol (pH 5.0).The peak current value (i,) increased by approximately 10% when the methanol concentration was decreased to 8%. The substance was found t o undergo an irreversible reaction involving one electron and possibly two protons in the initial oxidation step with at least one possible quasi-reversible follow-up reaction. The optimum mobile phase for the liquid chromatography, with amperometric detection, of the serum extracts was found to be 0.06 mol dm-3 acetate buffer in 8% methanol (pH 5.0); the acidified serum was extracted with a mixture of chloroform and acetonitrile (60 + 40), prior t o injection onto a reversed-phase column. The peak current was measured at +1.35 V and the calibration graph was found t o be linear in the range 4-200 ng of sample injected.The average recovery from serum was found to be 60% with a relative standard deviation of 5.8%. A pharmacokinetic study was carried out and the results obtained were comparable t o those found in the literature. It was concluded that the method developed had possible application for the measurement of trace levels of salicylic acid in clinical studies. Keywords: Salicylic acid; amperometric detection; cyclic voltammetry; serum Aspirin, the prototype of the salicylates, is a non-steroidal anti-inflammatory agent and is known as acetylsalicylic acid.' Zrz vivo, the drug rapidly hydrolyses to salicylic acid and acetate. The continued interest in the methods of analysis of salicylic acid is undoubtedly due to its consumption as a therapeutic drug.Thus, its therapeutic action and its toxic effects make it a drug that is subjected to continuous research. The techniques described for the determination of salicylic acid are many and varied and include colorimetry, titrimetry, ultraviolet (UV) spectrophotometry, gas chromatography, direct potentiometry and high-performance liquid chromato- graphy (HPLC).2 Of these, the titrimetric methods are subject to interference from reducing agents and, hence, are non- specific. The colorimetric methods, which involve the forma- tion of complexes with iron(IrI), are insensitive for the determination of salicylic acid at sub-nanogram levels. Several chromatographic methods have been developed including liquid chromatography (LC) usually using either UV or fluorescent detectors. To date, there are only a few reports on the use of amperometric detection for the assay of salicylic acid,3 and no systematic studies on the optimization of the electrochemical conditions for liquid chromatography with electrochemical detection (LCEC), or the nature of the oxidation reaction at a glassy carbon electrode, have been reported.The purpose of thc first part of this work was to carry out detailed studies on the electrochemical behaviour of salicyclic acid at a glassy carbon electrode using cyclic voltammetry and a variety of solution conditions. This information was then used in the development of a selective and sensitive assay involving LCEC, which was required to be suitable for use in pharmacokinetic studies following a single 300 mg oral dose of aspirin and for the quantitative measurement of salicylic acid * To whom correspondence should be addressed.in human blood serum in the nanogram range after low doses of aspirin. Experimental Reagents All reagents were of analytical-reagent grade unless stated otherwise. Salicylic acid was purchased from Sigma and the solvents used for HPLC were of HPLC grade and purchased from Merck. The supporting electrolytes used for the cyclic voltammetry studies were prepared from stock solutions containing either 2 mol dm-3 sodium acetate or acetic acid. These were mixed to give solutions of the required pH (a pH meter was used) and the resultant acetate buffers were diluted with methanol and water to the desired concentrations.The methanolic acetate buffer was used as the mobile phase in the LCEC studies and was prepared by mixing 0.6 mol dm-3 sodium acetate-acetic acid buffer solution (pH 5.0) and methanol to give a final electrolyte concentration of 0.06 mol dm-3. Stock dilutions of salicylic acid were prepared in methanol and were protected from the light during all of the investigations. The mobile phase was filtered through glass micro-fibre membranes (Whatman GFE) and subsequently de-gassed with helium. Apparatus An Oxford electrode portable potentiostat, equipped with a Gould Series 60 OOO x-y plotter, was used for recording the cyclic voltammograms. A three-electrode cell was employed incorporating a glassy carbon working electrode, a saturated calomel reference electrode (SCE) and a platinum-wire counter electrode.The LCEC was performed using an LDC Constamatic Model 111 reciprocating pump plus pulse damperANALYST, AUGUST 1991, VOL. 116 and an EDT LCA15 amperometric electrochemical detector together with a Bioanalytical System TL-5 thin-layer cell containing a glassy carbon electrode. Voltammetric Procedure Cyclic voltammetry (CV) was performed on solutions containing 5 X 10-4 mol dm-3 salicylic acid, 35% methanol and 0.05 mol dm-3 (pH 3.0-7.0) acetate buffers, in order to investigate the effect of pH. The voltammetric conditions were as follows: initial potential, 0 V; scan rate 50 mV s-1; and final potential 1.50 V. The effect of the ionic strength of the acetate buffer was studied in the concentration range 0.01-1 .O mol dm-3 using a scan rate of 50 mV s-1.In between the successive runs the working electrode was cleaned by washing it with distilled water, polishing the surface with aluminium oxide, rinsing again with distilled water and finally drying with tissue paper. The effect of the concentration of methanol was investigated by dissolving 5 x 10-4 mol dm-3 salicylic acid in solutions of 8-35% methanol-0.06 mol dm-3 acetate buffer, pH 5.0, and performing CV using the conditions described above. The phenomenon of adsorption was investigated by varying the scan rate for a 5 x 10-4 mol dm-3 salicylic acid solution in 35% methanol-0.06 mol dm-3 acetate buffer (pH 5.0). In order to obtain the hydrodynamic voltammograms, 20 pl sample volumes, containing 5 x 10-4 mol dm-3 salicylic acid in 35% methanol-0.06 mol dm-3 (pH 5.0) acetate buffer were injected onto the column and the potential was varied between 0.50 and 1.60 V.A flow rate of 1 ml min-1 was used. Hydrodynamic voltammograms were constructed by plotting the recorded peak currents against the applied potential. The optimum potential for the determination of salicylic acid was found from the position of the plateau on the hydrodynamic wave. Determination of Salicylic Acid in Human Serum by LCEC Serum samples (0.5 ml) were extracted using the method described by Tebbett and Omile.4 Briefly, this involved acidification of the serum (0.5 ml) with 0.1 ml of 1 mol dm-3 HCl and extraction with 2 ml aliquots of a solvent mixture consisting of chloroform and acetonitrile (60 + 40).The serum sample was extracted three times and the extracts combined. The organic phase was evaporated to dryness under a stream of nitrogen and the residue redissolved in 100 p1 of the mobile phase. Samples of 20 p1 volume were injected onto the column. Calibration, Recovery and Precision of LCEC A calibration graph of peak current versus mass of salicylic acid injected was constructed over the range &lo00 ng per 0.5 ml of serum. The recovery of salicylic acid was determined by spiking the serum, which was known to be free from salicylic acid, with concentrations in the range 30-500 ng per 0.5 ml of serum. In order to keep the volumes constant throughout the experiment, a known concentration of salicylic acid was added to the 100 pl of 1 mol dm-3 HCl used for the acidification step.Further samples containing 50 ng per 0.5 ml of serum were analysed to investigate the precision of the assay. Pharmacokinetic Study Two healthy male volunteers participated in the study. The subjects had not taken aspirin or any other drug for at least 10 d prior to the study. Both subjects fasted overnight and in the morning 5 ml of venous blood was drawn through an in-dwelling polytetrafluoroethylene catheter inserted into the I 4 r I 1 0 0.5 1 .o 1.5 EN versus SCE Fig. 1 Cyclic voltammogram of 5 x rnol dm-3 salicylic acid in 35% methanol4.05 mol dm-3 acetate buffer solution (pH 5.0), using a glassy carbon electrode. Initial potential, 0 V; scan rate, 50 mV s-1. 1F and 2F, first and second forward scan; lR, first reverse scan 45 l5 i I I I 10 1 I 0.8 3 4 5 6 7 Fig.2 Effect of pH on peak current and peak tential for peak Ia (see Fig. 1) using 5 x 10-4 mol dm-3 sacylic acid in 35% methanol-0.05 mol dm-3 acetate buffer PH cephalic vein and collected into a plain serum container. Both subjects were then given 300 mg of aspirin orally, which was swallowed with water. Further blood samples were taken every hour throughout the day for 7 h. The samples were centrifuged within 10 min of collection and the serum separated, quickly frozen and stored at -20 "C. All of the samples were extracted and chromato- graphed the following day, using the same procedure as for the spiked serum. Results and Discussion Voltammetric Behaviour of Salicylic Acid at a Glassy Carbon Electrode and Optimization of Conditions Cyclic voltammograms were recorded for salicylic acid solutions in the pH range 3.0-7.0.In all instances, the first forward scan exhibited a single anodic peak (Fig. 1 Ia) owing to the oxidation of the salicylic acid, probably at the phenolic moiety. The oxidation process is irreversible; however, a reduction reaction does occur, as seen from peak IIc (Fig. 1). The reaction giving rise to this peak is quasi-reversible as indicated by the appearance of peak IIa on the second forward scan (Fig. 1). These results suggest that on the first anodic scan salicylic acid undergoes an electron transfer-chemical reaction-elec- tron transfer reaction pathway at the glassy carbon electrode. The final product(s) of this pathway might be of quinone-typeANALYST, AUGUST 1991, VOL.116 structure , which might be expected to undergo the quasi- reversible redox reactions producing peaks IIa and IIc (Fig. 1). Such reactions have been reported for a structurally- related compound containing a phenolic moiety.5 The variation of peak potential (E,) and peak current (i,) with pH for peak Ia is shown in Fig. 2. A break appears in the E, versus pH graph indicating a pk’ value of 5.0. Below this break the E, value obeys the relationship: E, = 1.50 - 0.11 pH. It should be noted that the pk, values of salicylic acid are 2.97 and 13.40, corresponding to dissociation of carboxylic acid and phenolic groups, respectively.6 Therefore, the pk’ is probably a manifestation of the dissociation of the carboxylic acid group. The resultant anion is stabilized by intramolecular hydrogen bonding with the adjacent phenolic group;7 such stabilization might be expected to alter the electrochemical behaviour of this group (see below).Values of an, (where a is the transfer coefficient and n, is the number of electrons involved in the rate determining step) were calculated at various pH values for peak Ia, as shown in Table 1, according to the following equation: 0.048 an, = E,-(E$) Where E,/2 is the potential at half peak current (442). The an, values calculated for the oxidation reaction producing peak Ia suggest that one electron is involved in the rate determining step. The slope of the Ep versus pH plot below the pk‘ value suggests that two protons might be involved in this step.If this is true, then protons from both the phenolic and carboxylic acid group might be controlling the rate of oxidation. In addition, the oxidation process became independent of pH above the pk’ value; these effects can be associated with intramolecular hydrogen bonding as discussed above. Peak Ia (Fig. 1) achieved maximum current with the methanolic acetate buffer at pH 5.0; therefore, this pH was used throughout the remainder of the study. The effect of the ionic strength of the acetate buffer (at pH 5) on the voltammetric peaks was investigated; the largest signal was obtained with a supporting electrolyte strength of 0.06 rnol dm-3, as shown in Fig. 3. In order to determine whether or not the initial oxidation process was accompanied by adsorption phenomena, plots of Table 1 Values of ena for salicylic acid (peak Ia) PH aria 3.0 0.32 4.0 0.48 5.0 0.80 6.0 0.69 7.0 0.69 t .s 0 0.02 0.04 0.06 0.08 1.0 Ionic strength of acetate bufferhol dm-3 Fig. 3 Effect of ionic strength on peak current for peak Ia (see Fig. 1) using acetate buffer as the supporting electrolyte, containing 35% methanol, and 5 X 10-4 mol dm-3 salicylic acid 805 idCV versus V: were constructed for peak Ia, where C = concentration and V = scan rate. That adsorption did not occur at the electrode surface as no increase of current function (idCV) with V was apparent, is indicated in Fig. 4. The effect of the concentration of methanol on the peak current was examined over the range 8-35% v/v; the acetate buffer composition was fixed at a pH of 5.0 and an ionic strength of 0.06 rnol dm-3.The magnitude of peak Ia was found to increase by about 10% when the methanol concentra- tion was decreased from 35 to 8%. Therefore, over the range studied, the methanol concentration had little effect on the oxidation process; the small increase observed might be because of an increase in the solubility of salicylic acid, or changes in the conductivity of the electrolyte. These observations suggest that the mobile phase compo- sition, for the LCEC assay of salicylic acid, should be 0.06 mol dm-3 acetate buffer (pH 5.0), and that the methanol concentration can be varied between 8 and 35% with only a small change in the magnitude of the signal. Optimization of LCEC Conditions In order to determine the optimum applied potential for electrochemical detection, following HPLC, a hydrodynamic voltammogram was constructed for salicylic acid. The maxi- mum current was obtained at a potential of +1.5 V (versus an Ag-AgC1 electrode) or greater as shown in Fig.5. However, it is apparent that the background current is rather high at 1.5 V, which results in significant noise levels when achieving high sensitivities. It was found that much lower noise levels could be achieved with an applied potential of +1.35 V and only a small decrease in signal was observed. This resulted in improved signal-to-noise (S/N) ratios and consequently lower limits of detection. !2 11 3 10 A s 0 9 3 4 5 6 7 8 9 10 v: Fig. 4 Current function versus Vh for 5 x 10-4 rnol dm-3 salicylic acid in 35% methanol-0.06 rnol dm-3 acetate buffer, pH 5 1000 800 600 2 .3 400 200 0 0.8 1.10 1.2 1.4 1.6 1.8 E a pp I i e d N Fig.5 Hydrodynamic voltammogram for salicylic acid in 35% methanol-0.06 rnol dm-3 acetate buffer, pH 5.0. Concentration of salicylic acid standard, 5 x mol dm-3. Solid line, hydrodynamic wave for salicylic acid; and broken line, background current806 ANALYST. AUGUST 1991, VOL. 116 I 18 10 5 Ti me/m i n 0 Fig. 6 Chromatogram obtained by LCEC for extract from serum spiked with 50 ng of salicylic acid. Serum extract was dissolved in 0.1 ml of the optimum mobile phase consisting of 8% methanol-0.06 mol dm-3 acetate buffer, pH 5.0. Volume injected, 20 pl. SA = salicylic acid 1400 E $1200 c - E q 1000 0 Q) Q L 800 \ 0 2 600 .- 4- 8 400 E m V .- 6 200 .- - m v 0 I I I I I I I 1 2 3 4 5 6.7 8 Time/h Fig. 8 Concentration-time relation after a 300 mg oral administra- tion of aspirin on two volunteers (both men): solid line, volunteer 1; and broken line, volunteer 2 j L ” J l , , , , 1 0 200 400 600 800 1000 Salicylic acid added/ng per 0.5 ml of serum Fig. 7 &loo0 ng per 0.5 ml of serum Recovery of salicylic acid added to serum over the range The variation of retention time of salicylic acid with different percentage methanol composition (8-35%) was examined. The shortest retention time (4 min) occurred with a mobile phase containing 35% methanol-0.06 mol dm-3 acetate buffer, pH 5.0. However, when serum extracts were analysed with this eluent the analyte peak was not resolved from the interference peaks. In order to resolve the salicylic acid peak from interfering peaks, the methanol concentration was reduced to 8% (Fig.6). These conditions were then considered suitable for the assay of salicylic acid following oral doses of aspirin. Calibration, Recovery and Precision of LCEC Assay The calibration graph of peak current versus the mass of salicylic acid injected was linear over the range 4-200 ng of mass injected. The recovery of salicylic acid added (30-500 ng per 0.5 ml of serum) to serum was linear, with a mean recovery of 60% (Fig. 7) and the relative standard deviation for 5 samples spiked with 100 ng per 0.5 ml of serum and for 5 samples spiked with 10 ng per 0.5 ml of serum was 2.2 and 5.9% , respectively. The limit of detection was found to be 4 ng of salicylic acid injected; this value was based on an S/N ratio of 3 : 1 and a full-scale deflection of 10 nA.Analytical Application It was of particular interest to ascertain whether the present method was suitable for studies on the absorption rate of salicylic acid following an oral dose of aspirin. The pharmaco- kinetic curves obtained for two healthy male subjects follow- ing a 300 mg oral dose of aspirin are shown in Fig. 8. These results are in good agreement with previously published reports.8 This indicates the reliability of the present method at the dosage level administered. It has recently become desirable to determine the minimum level of serum salicylic acid able to reduce platelet activity,gJo which becomes raised in coronary heart disease. Thus, a sensitive assay for low levels of this drug could be of major importance in such clinical studies. Bearing in mind that the present assay has a detection limit of 4 ng injected, we intend applying it to patients undergoing treatment with low level doses of aspirin. The authors thank M. Norman and R. Shadwick of Bristol Polytechnic for technical assistance. \ \\ 1 2 3 4 5 6 7 8 9 10 References Jackson, J. V., Moss, M. S., and Widdop, B., Clarke’s Isolation and Identification of Drugs, Pharmaceutical Press, London, 1984, p, 965. Stewart, M. J., and Watson, I. D., Ann. Clin. Biochem., 1987, 24,552. Selinger, K., and Purdy, W. C., Anal. Chim. Actu., 1983, 149, 343. Tebbett, I. R., andOmile,C. I.,J. Chromatogr., 1985,329,196. Hart, J. P., and Hayler, P. J., Anal. Proc., 1986, 23, 439. Handbook of Chemistry and Physics, ed. Weast, R. C., CRC Press, Boca Raton, FL, 1974, p. D-129. Sykes, P. , A Guidebook to Mechanisms in Organic Chemistry, Longman, London, 1970, p. 62. Bochner, F., Williams, D. B., Morris, P. M., Siebert, D. M., and Lloyd, J. V., Eur. J. Clin. Pharmacol., 1988, 35, 287. Hirsch, J., Stroke, 1985, 16, 1. Sinzinger, H., Kaln, J., Fischa, P., O’Grady, J., Prostaglandins Leukotrienes and Essential Fatty Acids, 1988, 34, 89. Paper I I009396 Received February 2nd, I991 Accepted April 2nd, 1991