Measuring ofloxacin in pharmaceuticals using the differential pulse voltammetric method

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol. 58, No. 9, pp. 77-84 This paper is available online at MEASURING OFLOXACIN IN PHARMACEUTICALS USING THE DIFFERENTIAL PULSE VOLTAMMETRIC METHOD Tran Quang Hai1, Duong Quang Phung2, Vu Thi Huong2 and Tu Vong Nghi3 1Faculty of Chemical Technology, Hanoi University of Industry 2Faculty of Chemistry, Hanoi National University of Education 3Faculty of Chemistry, Hanoi University of Science, Vietnam National University, Hanoi Abstract. The differential pulse voltammetric method at a hanging mercury drop electrode was used to determine the presence of ofloxacin. In this study, the conditions under which differential pulse voltammetric measurement could be conducted were examined and established. The proposed method exhibited a sensitive response to ofloxacin in the range from 2.0 to 11.0 µg/mL with a detection limit of 0.19 µg/mL. The differential pulse voltammetric method was applied to measure ofloxacin in pharmaceuticals with high degree of precision and accuracy. Keywords: Ofloxacin, pulse voltammetric method, voltammetry. 1. Introduction Ofloxacin is a synthetic chemotherapeutic antibiotic of the fluoroquinolone drug class considered to be a second-generation fluoroquinolone [8] which has been widely used to inhibit the DNA gyrase of most Gram-negative bacteria, many Gram-positive bacteria and some anaerobes. It is popular because it is rapidly absorbed, it’s a potent inhibitor and it is inexpensive [7]. In Vietnam, ofloxacin is produced by a number of medical companies. The use of ofloxacin at a suitable dosage can prevent viruses from attaching, but an overdose is harmful. Therefore, determining of amount of ofloxacin that is present in a medicine is necessary to control the amount of ofloxacin that is taken into the body. Over the years, spectrophotometry [5], High Performance Liquid Chromatography (HPLC) [2], molecularly imprinted solid-phase extraction - Liquid Chromatography Received August 10, 2013. Accepted October 14, 2013. Contact Tran Quang Hai, e-mail address:: haitranquang07@gmail.com. 77 Tran Quang Hai, Duong Quang Phung, Vu Thi Huong and Tu Vong Nghi [9], capillary electrophoresis [6], chemiluminescence [4], spectro-fluorimetry [1] and microbiological assay [10] have been used to detect ofloxacin. The HPLC method is used by most medical companies around the world to detect ofloxacin because this method gives a precise and accurate result [3]. However, use of the HPLC method is limited due to the high cost of the necessary equipment, the complexity of the sample treatment method and the resulting solvent is hazardous of disposed of directly into the environment. Therefore, it is necessary to find a method that uses inexpensive equipment and has a simple sample treatment procedure that provides a measurement in short time. Recently, the electrochemical method has been used widely to measure both inorganic and organic compounds because of its accuracy and ease of sample treatment. In this study, the differential pulse voltammetry technique (DPV) using mercury a hanging drop electrode was used to measure the ofloxacin in medical products. 2. Content 2.1. Experiments * Chemicals and reagents A standard ofloxacin solution was supplied by the Testing Institute, Ministry of Health and used without further purification. Medicine containing ofloxacin were purchased from many medical companies. All other chemicals that were used in this study were purchased from Merck (Germany). All of the aqueous solutions were prepared with twice distilled water. * Apparatus All electrochemical measurements were carried out using a 757VA Computrace (Metrohm, Switzerland) in a three electrode cell consisting of a Ag/AgCl (3 M NaCl) reference electrode, a platinum rood auxiliary electrode and a mercury electrode used as the working electrode (Metrohm, Switzerland). All electrochemical measurements were taken at room temperature. * Optimum conditions for determination After the investigation, the optimal conditions for determining ofloxacin levels using 797VA Computrace equipment were found and listed in Table 1. All solutions were prepared in a Photphat buffer solution pH 6.50 and deoxygenated by purging it with very pure nitrogen. Table 1. Equipment parameters Working electrode HMDE Pulse amplitude (V) 0.05 Technique DP Pulse time (s) 0.04 s Drop size 4 Scan rate (mV/s) 12.5 Purging time (s) 120 Potential range (V) -1.1 ÷ -1.5 * Estimating the accuracy of the method 78 Measuring ofloxacin in pharmaceuticals using the differential pulse voltammetric method The accuracy of the method was estimated as follows: A typical procedure involved preparing several solutions which each contained the same amount of ofloxacin but a different amount of standard (at three addition concentrations). The accuracy of the method was estimated by the calculating the recovery percentage (Rev) using the proposed method. According to the Ministry of Health, recovery must be in the range 98 - 102%. * Investigating the repeatability of the analytical method In order to check the reproducibility of the method for measuring ofloxacin at the mercury electrode, the response of ofloxacin was measured at three concentrations making six measurements for each concentration. The repeatability of the method was estimated based on the repeatability of calculated concentration from the standard curve. Standard deviation (S) and relative standard deviation (RSD%) was used to estimate the repeatability of the analytical method. * Determining the amount of ofloxacin present in medicines sold in Hanoi The concentration of ofloxacin in two kinds of 0.3% (15 mg/5mL) eye drops manufactured by the Traphaco Pharmaceutical Company (Product number 290 811) and 200 mg ofloxacin tablets manufactured by the Imexpharm Pharmaceutical Company (Product number 020 512) were determined by using proposed method. 2.2. Results and discussion 2.2.1. Dynamic voltammetry response of ofloxacin at the mercury electrode After the optimal conditions were selected, a calibration curve was made by measuring the response of ofloxacin at the electrode using solutions prepared at different concentrations (from 2.0 µg/mL to 11.0 µg/mL) in a PBS buffer with a pH of 6.50. The results are shown in Figures 1 and 2. Figure 1. DPV voltammograms for various ofloxacin concentrations in a PBS buffer solution of pH 6.50 (A ofloxacin concentration 2.0 to 11.0 µg/mL at hanging mercury drop electrode) (Measurement temperature: 250C) 79 Tran Quang Hai, Duong Quang Phung, Vu Thi Huong and Tu Vong Nghi Figure 1 shows the dependency of the ofloxacin concentration on the peak current measured with the mercury hanging drop electrode. The linear regression equation I(nA) = (23.03 ± 0.18) × Cµg/mL) + (10.10 ± 1.17) showed linearity from 2.0 to 11.0 µg/mL (Figure 2) with a correlation coefficient of 0.9995. The detection limit of the method was found to be 0.19 µg/mL, equal 5.21× 10−7 M, and the quantity limit of the method was found to be 0.63 µ/mL, equal 1.74 ×10−6 M. The proposed method could be used to determine the amount of ofloxacin present in a medical product. Figure 2. The relationship between peak current height and ofloxacin concentration (The data were converted from the results shown in Figure 1) 2.2.2. Estimation of accuracy Table 2. Ofloxacin determination and estimation of accuracy Additional percentage (%) Additional conc. (µg/mL) Calculated conc. (µg/mL) Average conc. (µg/mL) RSD(%) Recovery Rev (%) 80% 3.20 3.20 3.18 3.13 3.09 3.15 1.58 98.48 100% 4.00 4.10 4.21 4.01 3.98 4.08 1.29 101.92 120% 4.80 4.72 4.79 4.70 4.81 4.76 1.13 99.06 80 Measuring ofloxacin in pharmaceuticals using the differential pulse voltammetric method The accuracy of the method was estimated using a standard addition method as follows: three levels of standard concentrations of ofloxacin (3.2 µg/mL; 4.0µg/mL; 4.8µg/mL) were placed in three solution containing the same electrolytics. The electrochemical response of ofloxacin was measured. The calculated data are shown in Table 2. The data show that the proposed method is highly accurate, from 98.48% to 101.92% (average 99.82%), within the 98 - 102% range limitation shown by the Ministry of Health. This method can be used to determine the amount of ofloxacin that is present in an antibiotic. 2.2.3. Estimation of repeatability The repeatability of the method was estimated as follows: the ofloxacin solutions were measured at three levels of standard concentrations (3.0 µg/mL, 5.0 µg/mL, 9.0 µg/mL). The measurement were performed six times independently using six independent samples for each concentration, all other conditions being the same. The measured and calculated results using the standard curve method are presented in Table 3. Table 3. The repeatability of method for measuring ofloxacin Sample Standard conc. (mug/mL) Measured conc. (µg/mL) Average conc. (µg/mL) RSD (%) 1 2 3 4 5 6 3.00 3.00 3.00 3.00 3.00 3.00 2.98 2.89 2.80 2.95 2.99 3.06 2.95 1.27 1 2 3 4 5 6 5.00 5.00 5.00 5.00 5.00 5.00 4.86 4.87 4.99 4.87 4.82 4.91 4.89 1.16 1 2 3 4 5 6 9.00 9.00 9.00 9.00 9.00 9.00 9.12 9.20 8.79 8.77 8.85 8.91 8.94 0.81 The experimental results show that the method is repeatable with a relative standard deviation (RSD%) of less than 2%. This indicates that this proposed method can be applied to quantitatively analyze the presence of ofloxacin in drug samples. 81 Tran Quang Hai, Duong Quang Phung, Vu Thi Huong and Tu Vong Nghi 2.2.4. Determining the amount of ofloxacin present in commercial medicines sold in Hanoi The process for measuring the amount of ofloxacin in an eye drop solution: Referring to a current method to determine the presence of ofloxacin in eye drop solutions [10, 11], 5 bottles of eye drops with 0.3% ofloxacin (15 mg/mL) were poured into a clean plastic bottle, the average volume of one bottle was calculated and it was shaken to mix thoroughly. Then, the entire solution was transferred into a 50 mL flask and twice distilled water was added to a mark to obtain solution 1. Exactly 5.00 mL were transferred to a 25 mL flask and distilled water was added to a mark to obtain solution 2A, a solution that has a concentration equivalent to 0.3 mg/mL). A micropipette was used to transfer 250.0 µl of solution 2A into a 25 mL flask, adding 5 mL of phosphate buffer with a pH of 6.5, and then distilled water was added to the mark. The obtained solution was shaken well„ transferred into an electrolytic cell, and finally measured under optimal conditions. The concentration of ofloxacin was calculated using the standard addition method. The process for measuring the amount of ofloxacin in a tablet: Based on a process for determining the amount of ofloxacin in tablets and capsules [10, 11], using 20 tablets, the average mass of one tablet was calculated, they were finely ground and then mixed well. An amount of powder that was to correspond to 50 mg of ofloxacin was put onto a 100 mL flask and distilled water was added to the mark. The solution (called solution 1) was shaken well and allowed to stand. Exactly 25.00 mL of solution 1 was placed into a 50 mL flask and it was filled to the mark with distilled water to create solution 2B, a solution that has a concentration equivalent to about 0.25 mg/mL. A micropipette was used to transfer 300 µl of 2B solution into a 25 mL flask to which 5 mL of phosphate buffer pH = 6.50 was added, then adding distilled water to the mark. This was shaken well, transferred into the electrochemical cell and measured under selected conditions. The concentration of ofloxacin was determined using standard addition methods. The amount of ofloxacin in one bottle can be estimated using the following equation: Amount of ofloxacin/bottle = 25× CX × 25 0.25 × 50 5 × 1 5 × 10−3 (mg). The amount of ofloxacin in one tablet can be estimated using following equation: Amount of ofloxacin/tablet = 25× CX × 25 0.3 × 100 50 × 200 50 × 10−3 (mg). The result of six measurements are displayed in Tables 4 and 5. 82 Measuring ofloxacin in pharmaceuticals using the differential pulse voltammetric method Table 4. The amount of ofloxacin in 0.3% (15 mg/5 mL) eye drops produced by the Traphaco Pharmaceutical Corporation Sample Conc. (µg/mL) Amount of ofloxacin/bottle Amount of ofloxacin in compared to manufacturer’s label (%) Calculated result 1 2.837 14.185 94.57 X = 14.067 (93.80%) SD = 0.20 RSD = 1.43 % m = 14.067 ± 0.210 (Confidence level 95%) 2 2.768 13.840 92.27 3 2.7618 13.809 92.06 4 2.8473 14.2365 94.91 5 2.8541 14.2705 95.14 6 2.8154 14.077 93.85 Table 5. Amount of ofloxacin in 200 mg ofloxacin tablets produced by the Imexpharm Pharmaceutical Corporation No. Conc (µg/mL) Amount of ofloxacin/tablet Percentage (%) Calculated data 1 2.9548 196.987 98.49 X = 196.463 (98.23%) SD = 3.33 RSD = 1.69 % m = 196.463± 3.495 (Confidence level 95%) 2 3.024 201.600 100.80 3 2.9816 198.773 99.39 4 2.9048 193.653 96.83 5 2.8924 192.827 96.41 6 2.9241 194.940 97.47 The estimate of the amount of ofloxacin in the two drugs measured showed a relative standard deviation of < 2%, and a 2% deviation is allowed by the Vietnam Ministry of Health. The percentage of ofloxacin calculated compared the level indicated on the label by the Traphaco Pharmaceutical Corporation is in the range of 92.06 - 95.14%; and its in the range 96.41 - 100.80% for the drugs produced by the Imexpharm Pharmaceutical Joint Stock Company. These amounts are all within the standard presented in the Vietnamese Pharmacopoeia 4. 3. Conclusion In this study, optimal conditions for measuring ofloxacin using differential pulse voltammetry (DPV) techniques with hanging mercury drop electrode were successfully investigated. The proposed method was also used to measure the amount of ofloxacin in samples of eye drop solution and antibiotics that are sold in Hanoi. The results obtained from this study indicate the suitability of applying electrochemical methods to control medicine quality in the laboratory and pharmaceutical testing centers. 83 Tran Quang Hai, Duong Quang Phung, Vu Thi Huong and Tu Vong Nghi REFERENCES [1] O. Ballesteros, J.L. Vílchez, A. Navalón, 2002. Determination of the antibacterial ofloxacin in human urine and serum samples by solid-phase spectrofluorimetry. Journal of Pharmaceutical and Biomedical Analysis 30, 1103. [2] N.E. Basci, S. Hanioglu-Kargi, H. Soysal, A. Bozkurt, S.O. Kayaalp, 1997. 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