Talanta 50 (1999) 635 – 640

Determination of theophylline in tea and drug formulation using a Nafion®/lead–ruthenium oxide pyrochlore chemically modified electrode Jyh-Myng Zen a,*, Tung-yue Yu a, Ying Shih b b

a Department of Chemistry, National Chung-Hsing Uni6ersity, Taichung 402, Taiwan, ROC Department of Applied Cosmetology, Hung-Kuang Institute of Technology, Taichung 433, Taiwan, ROC

Received 17 December 1998; received in revised form 7 May 1999; accepted 7 May 1999

Abstract Square-wave voltammetry was used for the determination of trace amounts of theophylline in tea and drug formulation at a Nafion®/lead–ruthenium oxide pyrochlore chemically modified electrode. This chemically modified electrode exhibits a marked enhancement of the current response compared to a bare glassy carbon electrode. The calibration graph for the determination of theophylline was linear up to 100 mM in 0.1 M, pH 3 phosphate solution with a detection limit (S/N=3) of 0.1 mM. The results of 15 successive repetitive measurement-regeneration cycles showed a relative standard deviation of 1.3% for 10 mM theophylline indicating that the electrode renewal gives a good reproducible surface. Quantitative analysis was performed by the standard addition method for the theophylline content in commercially available tea and drug. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Theophylline; Tea; Drug formulation; Chemically-modified electrode

1. Introduction Theophylline (1,3-dimethyl-1H-purine-2,6dione) is a xanthine derivative with diuretic, cardiac stimulant, and smooth muscle relaxant activities. Accurate and rapid method is required for the determination of soluble theophylline in tea and tea products because the stimulating effect of tea beverage is due to the presence of purine bases, such as, caffeine, theobromine, and * Corresponding author. Fax: +886-4-2862547. E-mail address: [email protected] (J.-M. Zen)

theophylline. Meanwhile, theophylline has been widely used for the treatment of asthma and bronchospasm in adult [1]. The efficiency and toxicity of this drug can be modified by many factors [2]. Thus, in order to adapt dosing and to verify compliance, therapeutic drug monitoring is necessary. Previous approaches used for the determination of theophylline include high-performance liquid chromatography (HPLC) and gradient capillary HPLC [3–7], spectrophotometry [8], enzyme immunoassay [9], capillary electrophoresis [10], frit-fast atom bombardment mass spectrometry (LC-frit-FAB-MS) [11], and electro-

0039-9140/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 9 9 ) 0 0 1 6 0 - 5

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Fig. 3. The effects of preconcentration potential (a) and the preconcentration time (b) on the SW response for 20 mM theophylline at the CME. Other conditions are as in Fig. 1.

Fig. 1. SW voltammograms for 20 mM theophylline in 0.1 M phosphate solution (pH 3) at a bare GCE (a), the Nafion®coated GCE (b), and the CME (c). SW amplitude, 25 mV; SW frequency, 15 Hz; step height, 4 mV.

Fig. 2. Dependence of the anodic peak current on pH in SW voltammetry for 20 mM theophylline at the CME. SW parameters are as in Fig. 1.

chemical method [12]. Compare to the electrochemical method, the HPLC method is time consuming and the LC-frit-FAB-MS is not suitable for routine analysis. Enzyme immunoassay procedures are often used in therapeutic controls in hospital. The main prospect of this study is therefore to develop a good alternative method for this purpose. The previous electrochemical method used adsorptive cathodic stripping voltammetry for the determination of trace amount of theophylline at a hanging mercury drop electrode [12]. There is an interference problem from some purine compounds and metal ions. We report here a relatively rapid and selective electrochemical method for the determination of theophylline using a Nafion®/lead–ruthenium oxide pyrochlore chemically modified electrode (CME). This work describes a combined catalytic–adsorbing interface layer to modify an electrochemical electrode surface to measure theophylline in tea and tablet formulations. The optimal experimental conditions were thoroughly investigated. Practical analytical utility was illustrated by selective measurements of theophylline in commercially available drug (ampoule of aminophylline for i.v. injection) and tea.

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Fig. 4. Typical SW voltammetry responses for the determination of theophylline in (A) ampoule, (B) tablet, (C) black tea, and (D) green tea with spiking theophylline concentrations of (a) 0, (b) 20, (c) 40, (d) 60, (e) 80, (f) 100 mM. Other conditions are as in Fig. 1.

2. Experimental Nafion® perfluorinated ion-exchange powder, 5 wt% solution in a mixture of lower aliphatic alcohols and 10% water, was obtained from the Aldrich (Milwaukee, WI, USA). Theophylline (Aldrich) and all the other com-

pounds (ACS-certified reagent grade) were used without further purification. Aqueous solutions were prepared with doubly distilled deionized water. Electrochemistry was performed on a Bioanalytical Systems (West Lafayette, IN, USA) BAS50W electrochemical analyzer. A BAS VC-2

Table 1 Determination of theophylline in tea beverages* and pharmaceutical formulation* with the CME Original value (mM)

Spike (mM)

Detected value after spike (mM)

Recovery (%)

Ampoule a

40.4890.23

20 40 80

60.93 9 0.43 78.33 9 0.41 115.60 9 0.67

102.25 92.44 94.60 91.18 93.90 90.89

Tablet a

38.7390.39

40 60 80

79.23 90.54 98.66 9 0.78 118.03 90.91

101.25 91.68 99.90 91.45 99.10 91.24

Black tea b

9.209 0.15

40 60 80

48.67 90.68 67.42 90.89 89.65 91.15

98.68 91.75 97.00 91.50 100.60 91.45

Green tea b

7.329 0.15

40 60 80

45.45 9 0.79 66.17 90.95 86.16 9 1.21

95.32 92.00 98.10 91.60 98.60 9 1.50

a

Dilution factor: 6/1000. Dilution factor: 1/20. * Number of samples assayed, 3.

b

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electrochemical cell was employed in these experiments. The three-electrode system consisted of either a glassy carbon electrode (GCE) or a Nafion®/lead – ruthenium oxide pyrochlore CME working electrode, an Ag – AgCl reference electrode (Model RE-5, BAS), and a platinum wire auxiliary electrode. Since dissolved oxygen did not interfere with the anodic voltammetry, no deaeration was performed. The Nafion®/lead – ruthenium oxide pyrochlore CME was prepared as follows. In brief, the GCE was first polished with the BAS polishing kit and rinsed with deionized water, then further cleaned ultrasonically in 1:1 nitric acid and deionized water successively. Nafion®, polymer was spin-coated onto a cleanly polished GCE. Lead-ruthenium oxide pyrochlore particles were then synthesized in the Nafion® matrix by treatment of Ru3 + , Pb2 + -exchanged polymer in alkaline aqueous solution with purging of O2. Electrode were prepared with the optimum coating solution of 1.25 wt% Nafion® at a 3000 rpm spin-coating rate. The CME was equilibrated in the test solution containing theophylline before measurement. Square-wave (SW) voltammograms were obtained by scanning the potential from + 0.6 to +1.4 V (vs. Ag – AgCl) at a SW frequency of 45 Hz and SW amplitude of 30 mV. At a step height of 4 mV, the effective scan rate is 180 mV s − 1. The theophylline quantitation was achieved by measuring the oxidation peak current after background subtraction. A stock solution was prepared by dissolving 180.17 mg of theophylline in 100 ml of water. An aliquot was diluted to the appropriate concentrations with 0.1 M, pH 3.0 phosphate solution before actual analysis. Pharmaceutical samples were prepared by dissolving 2.0 ml ampoule or 1.0 g tablet in 100 ml water. They were then diluted by a factor of 6/1000 (v/v) for detection. Tea was prepared by infusion, using a tea-bag (2.0 g) immersed in 100 ml of boiling water for 3 min. It was then diluted by a factor of 1/20 (v/v). The standard addition method was used to evaluate the content of theophylline in real samples.

3. Results and discussion

3.1. Voltammetric beha6ior Fig. 1 demonstrates the catalytic function of the CME in the determination of theophylline by SW voltammetry. On scanning from + 0.4 V toward a positive potential at a bare GCE, only a much smaller anodic peak at + 1.26 V was observed for 20 mM theophylline (curve a). A slightly increase in anodic peak at + 1.24 V was observed when a Nafion®-coated GCE was used (curve b). Whereas, a large increase in the peak current at + 1.17 V was observed when the CME was used (curve c). The enhancement in current response and the shift in oxidation potential are clear evidences of the catalytic effect of the CME toward theophylline oxidation. Further investigation was made to the transport characteristics of theophylline in the CME. The linear scan voltammetry current response obtained at the CME was found linearly proportional to the scan rate, which illustrated that the process was adsorptivecontrolled. More evidences for the adsorption behavior of theophylline was demonstrated by the following experiment. When the CME was switched to a medium containing only supporting electrolyte after being used in measuring a theophylline solution, the same voltammetric signal was observed.

3.2. Analytical characterization Both the electrode and the detection aspects should be considered to arrive at the optimum conditions for theophylline determination. As to the electrode aspect, the optimum conditions generally follow those used in previous studies [13– 18]. The effect of pH on the voltammetric response of the CME was studied first and the results for 20 mM theophylline is shown in Fig. 2. As can be seen, the CME shows an optimum performance around pH 3. A 0.1 M, pH 3 phosphate solution is therefore used in subsequent studies. The effects of pre-concentration potential and the pre-concentration time on the SW response for theophylline were studied next. The results obtained are shown in Fig. 3(A) and Fig.

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3(B), respectively. As can be seen in Fig. 3(A), the peak current increases as the potential of the electrode becomes more negative in the experimental range. This behavior is explained by the fact that theophylline bears a positive charge in pH 3 environment; as a result, the accumulation of theophylline is favoured at more negative potentials. However, the peak current drops rapidly when the potential is less than − 0.2 V. The decrease in the current at potentials below − 0.2 V is evidently caused by competition from adsorption of hydrogen. This is another evidence that the analyte is pre-concentrated by adsorption. A pre-concentration potential of 0 V was therefore chosen in all the subsequent work. As to the effect of the pre-concentration time, for 20 mM of theophylline, the peak current increases as the pre-concentration time increases and starts to level off at around 15 s as shown in Fig. 3(B). It takes longer time for the peak current to level off for a lower concentration of theophylline. This phenomenon is as expected and further confirms the adsorption-controlled behavior of the CME. Therefore, in order to increase the sensitivity of detection, a longer time is needed for the lower concentration of theophylline. A pre-concentration time of 15 s was used in most of the subsequent work. The peak current obtained in SW voltammetry depends on various instrumental parameters such as SW amplitude, SW frequency, and step height. These parameters are interrelated and effect the response, but here only the general trends will be examined. It was found that these parameters had little effect on the peak potential. When the SW amplitude was varied in the range of 10 –50 mV, the peak currents were increased with increasing amplitude until 30 mV. However, when the amplitude was greater than 30 mV the peak width increase at the same time. Consequently, 30 mV was chosen as the SW amplitude. The step height together with the frequency defines the effective scan rate. Hence, an increase with either the frequency or the step height results in an increase in the effective scan rate. The response for theophylline increases with SW frequency; however, above 45 Hz the peak current was unstable and obscured by a large residual current. By

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maintaining the frequency as 45 Hz, the effect of step height was studied. At a step height of 4 mV, the response is more accurately recorded. Overall, the optimized parameters can be summarized as follows: SW frequency, 45 Hz; SW amplitude, 30 mV; step height 4 mV. The effective scan rate is 180 mV s − 1. Under optimal conditions, the SW voltammetric current response is linearly dependent on the concentration of theophylline between 0 and 100 mM in pH 3 phosphate solution with slope (mA/ mM), and correlation coefficient of 0.110 and 0.999, respectively. The detection limit (S/N=3) is 0.1 mM. To characterize the reproducibility of the CME, repetitive measurement-regeneration cycles were carried out in 10 mM theophylline. The electrode was removed from the test solution after measurement, washed thoroughly and introduced into buffer solution and potential sweep were carried out in the same potential window several times until the original background current was regained. The results of 15 successive measurements show a coefficient of variation of 1.34%. Thus, the electrode renewal gives a good reproducibility surface.

3.3. Sample analysis The CME was applied to the measurement of theophylline in commercially available drug and tea and typical results are shown in Fig. 4. The accuracy of the method was determined by its recovery during spiked experiments. A commercial drug of theophylline was spiked with theophylline standard solution at a concentration of 20 mM/spike or 40 mM/spike. The recoveries of theophylline from the drug matrices and tea matrices were satisfactory with values ranging from 94 to 102% and 95 to 101%, respectively. Confirming those quantitative and reproducible results can be obtained with this method (Table 1). This study has demonstrated that the CME can be applied to the detection of theophylline in pharmaceutical formulation and tea with excellent sensitivity and selectivity by SWV. The CME can be easily regenerated and the detection can be achieved without deoxygenating. Significant advantages have been achieved by combining the

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electrocatalytic function of the catalyst with charge exclusion and preconcentration features of Nafion®. The reliability and stability of the CME offers a good possibility for extending the technique in routine analysis of theophylline.

Acknowledgements The authors gratefully acknowledge financial support from the National Science Council of the Republic of China under Grants NSC 88-2113-M005-019 and NSC 88-2113-M-005-020.

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