Stability of Vancomycin 31 mg/ml in Extemporaneous Eye Drops Determined with Capillary Electrophoresis

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1 Chiang Mai J. Sci. 2011; 38(4) 533 Chiang Mai J. Sci. 2011; 38(4) : Contributed Paper Stability of Vancomycin 31 mg/ml in Extemporaneous Eye Drops Determined with Capillary Electrophoresis Anutra Khangtragool*[a], Sineenart Santidherakul [b] and Phuriwat Leesawat [c,d] [a] Division of Pharmacy, Faculty of Medicine, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai University, Chiang Mai 50200, Thailand. [b] Medical Science Research Equipment Center, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai University, Chiang Mai 50200, Thailand. [c] Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand. [d] Biomedical Engineering Center, Chiang Mai University, Chiang Mai 50200, Thailand. *Author for correspondence; akhangtr@med.cmu.ac.th Received: 9 March 2011 Accepted: 4 June 2011 ABSTRACT This study evaluates the stability of extemporaneous ophthalmic antibiotic preparations made from a commercially available injection of vancomycin of 31 mg/ml and stored at 2-8 o C and 28 o C for 3 months. After various storage times, the stability was determined by capillary electrophoresis. Solutions of vancomycin 31 mg/ml in Tear Naturale II TM were found to be stable after storage at 2-8 o C and 28 o C for 28 days and 10 days, respectively. Vancomycin 31 mg/ml in extemporaneous eye drops prepared and supplied by a pharmacy should be stored at 2-8 o C for less than 28 days. Keywords: extemporaneous eye drops, stability, vancomycin, capillary electrophoresis. 1. INTRODUCTION Vancomycin is a glycopeptide antibiotic with a primarily bactericidal action against a variety of gram-positive bacteria (including methicillin-resistant staphylococci) and no cross-resistance with other antibiotics [1, 2]. The seriousness of ophthalmic bacterial keratitis (ulceration of the cornea) requires a high concentration of antibiotic eye drops [3]. These highly concentrated preparations are not readily available. Ross et al.[4] reported that Staphylococcus aureus and Staphylococcus epidermidis are organisms that frequently cause conjunctivitis or blepharoconjunctivitis. They successfully treated a patient with methicillinresistant S. aureus and S. epidermidis conjunctivitis by using an extemporaneously prepared topical ophthalmic solution of vancomycin 31 mg/ml [4]. The division of Pharmacy at the Maharaj Nakorn Chiang Mai Hospital has often prepared extemporaneous ophthalmic solutions of vancomycin 31 mg/ml. However, this preparation lacks storage stability. In the literature, spectrophotometry [5, 6], highperformance liquid chromatography (HPLC) [7, 8] and capillary electrophoresis (CE) [9, 10] methods have been reported as

2 534 Chiang Mai J. Sci. 2011; 38(4) methods suitable for the determination of vancomycin. CE is a relatively new analytical technique which has been increasingly used within the pharmaceutical industry due to its simplicity, ease of method transfer, reduction in analysis time and reduced costs of solvent and columns when compared with HPLC [11]. In recent years, CE has attracted great interest for the analysis of pharmaceutical compounds [12, 13]. Its popularity is mainly due to its low cost, high efficiency, reduced solvent consumption and high versatility. Although CE is increasing in population in drug analysis, it has a weak point in the adsorption of the analyte on the inner surface of the capillary [14]. However, dynamicallycoated capillaries can help to prevent adsorption of the analyte on the capillary wall and provide significant improvements in separation times, precision and selectivity [15]. Dynamicallycoated capillaries, using an initial coating with a polymeric cation and subsequent coating with a polymeric anion or micelle, offer faster separation times, improved precision, and increased selectivity for the analysis of basic, acidic, and neutral drugs [15]. This study has aimed to develop a method to determine the stability of vancomycin in an extemporaneous solution by capillary electrophoresis using dynamicallycoated capillaries. 2. MATERIALS AND METHODS Vancomycin (hydrochloride salt) 500 mg was purchased from Lek (Zuellig). Tears Naturale II TM was purchased from Alcon/ U.S. Summit. Standard vancomycin was purchased from Sigma Chemicals. CElixir TM Initiator A and Accelerator B (ph 2.5) were purchased from MicroSolv Technology (Long Branch, NJ, USA). 2.1 CE Instrumentation and Conditions The CE instrument used was a Beckman Coulter Capillary Electrophoresis, using 32 Karat software and coupled with a photodiode array (PDA) detector. Separations were performed using a pre-made fused silica untreated capillary of 50.2 cm, ID 75 μm (effective length: 40 cm). The detection wavelength was 210 nm. A constant voltage of 25.0 kv was applied with the capillary maintained at 25.0 o C. Before use, new capillaries were rinsed successively with 1 M NaOH for 1 minute, 0.1 M NaOH for 4 minutes, 1 M HCl for 1 minute, 0.1 M NaOH for 4 minutes, water (20 psi) for 1 minute and water (20 psi) for 4 minutes. For the separation of vancomycin, the various steps were: rinse with 0.1M NaOH (20 psi) for 1 minute, water (20 psi) for 1 minute, apply the initial coating of the capillary with initiator solution (A) (20 psi) for 30 seconds, apply the final coating of the capillary with accelerator solution (B) ph 2.5 (20 psi) for 1 minute, inject the sample (0.5 psi) for 5 seconds, inject a water plug (0.10 psi) for 10 seconds and then separate the sample for 10 minutes. At the end of each working day, the capillary was rinsed with water for 5 minutes, 0.1M NaOH for 5 minutes and finally water again for 10 minutes. 2.2 Method Validation Preparation of stock solution of standard vancomycin 31 mg/ml in Tears Naturale II TM Standard vancomycin 0.01 gm was weighed and 100 μl water was added. This standard vancomycin solution was added to Tears Naturale II TM 223 μl and mixed.

3 Chiang Mai J. Sci. 2011; 38(4) Preparation and linearity of calibration curve Quantification of the method was performed using the peak areas of vancomycin standard solutions. Six standard solutions were prepared and subjected five times to analysis by CE. A linear calibration curve was prepared from working standards over a concentration range of μg/ml by pipetting a known amount of a stock solution of 31 mg/ml standard vancomycin and then adding water to give a final volume of 1,500 μl Precision An assay of method precision (intraday precision) was evaluated by carrying out six independent assays of standard vancomycin. The interday precision of the method was also evaluated on five different days in the same laboratory. Precision was determined from standard vancomycin (intraday and interday assays) prepared from working standards in concentrations of 60, 240 and 360 μg/ml by pipetting a measured of a stock solution of 31 mg/ml standard vancomycin and then adding water to give a final volume of 1,500 μl Accuracy (Recovery Test) The accuracy of a method is the closeness of agreement between the found value and a known reference value. The accuracy of this CE method was studied by recovery experiments and performed by adding known standard vancomycin. The recovery was performed at three concentration levels: 60, 240 and 360 μg/ml obtained from intraday and interday assays. The percentage recoveries were calculated from the calibration curve. 2.3 Preparation of Vancomycin Eye Drops for Stability Studies Six solutions of vancomycin for injection were each reconstituted with water to a concentration of 50 mg/ml and diluted with Tears Naturale II TM to 31 mg/ml in Tears Naturale II TM containers. The vancomycin 31 mg/ml in Tears Naturale II TM eye drops were divided into two groups. Three bottles were stored at room temperature (28 o C) while the other three bottles were kept at 2-8 o C in a refrigerator. On days 0, 3, 7, 10, 14, 21, 28, 60 and 90, the stability of each solution was determined by dilution with water to 300 μg/ ml prior to CE analysis. 3. RESULTS AND DISCUSSION The aim of this study that was to use CE with dynamically-coated capillaries to determine the stability of vancomycin in extemporaneous eye drops. Dynamicallycoated capillaries provide highly reproducible and reliable electro-osmotic flow (EOF) in CE [15]. Compared to classical CE, the double-layer coating technology produces an increased EOF while preventing adsorption of the analyte to the capillary wall. Musenga et al. [9] used ph 2.5 phosphate buffer to determine vancomycin with CE giving good results in terms of peak effiency and migration times. In this present study, a method has been developed using capillaries dynamically coated with CElixir TM initiator A and accelerator B (ph 2.5). CElixir TM should be attributed to the adsorption of the vancomycin onto the capillary wall because of the electrostatic interaction between the positive charged vancomycin and the negative charged capillary wall. These conditions gave a peak for standard vancomycin with a retention time of approximately 6 minutes. With this retention time, sequential analyses could be completed in less than 10 minutes. Considering these results to be satisfactory, validation experiments were completed to ascertain if the method was suitable for the analysis of vancomycin in extemporaneous eye drops.

4 536 Chiang Mai J. Sci. 2011; 38(4) 3.1 Method Validation Specificity Specificity is the validation procedure which confirms that the method can clearly assess the vancomycin in the presence of other components in the extemporaneous eye drops. The electropherograms of standard vancomycin and blank, as shown in Figures 1 and 2, demonstrate the ability of the CE method to detect standard vancomycin with no interference from other constituents in the solution. The determination of peak homogeneity from the diode array UV spectra (190 to 300 nm) of standard vancomycin in Figure 3 show that the absorption spectra, each with maxima around 210 nm are very similar in appearance, both in terms of the size and shape of the curves. This data confirms that the peak from Figure 1 is specific for standard vancomycin Calibration and linearity The calibration curve showed good linearity. The response of the drug was verified at 50 to 500 μg/ml and was found to be linear. Peak area was plotted against concentration over the concentration range from 50 to 500 μg/ml in order to construct the calibration curve which was calculated by linear regression analysis. The correlation coefficient (r 2 ) of determination was The calibration and linearity data for standard vancomycin (50 to 500 μg/ml) are given in Table Precision and accuracy The results of the intraday and interday assays were found to have good accuracy, indicating an agreement between the true Figure 1. Electropherogram of standard vancomycin. Figure 2. Electropherogram of Tears Naturale II TM.

5 Chiang Mai J. Sci. 2011; 38(4) 537 Figure 3. Diode array UV spectra of standard vancomycin from Figure 1 scanned over the wavelength range from 190 to 300 nm. Table 1. Calibration and linearity data for standard vancomycin (range 50 to 500 μg/ml). No Regression equation Y = ax + b Regression coefficient (r 2 ) 1 y = x y = x y = x y = x y = x Table 2. Intraday and interday precision and recovery of standard vancomycin. Standard vancomycin (μg/ml) 60 μg/ml 240 μg/ml 360 μg/ml Intraday (n=6) % Recovery Interday (five days, each day n=2) % CV % Recovery % CV value and the value found. From Tables 2, all of the intraday and interday assays were determined as coefficients of variation and were less than 5%. The overall mean recoveries of the intraday and interday assays were found to be 97.4% and 96.4%, respectively. Electropherograms of vancomycin in Tears Naturale II TM in the day of preparation (day 0) (Figure 4a) showed a single peak but in day 7 (Figure 4b) and day 10 (Figure 5a) after storage at 28 o C showed an additional small peak after the vancomycin peak. Thus,

6 538 Chiang Mai J. Sci. 2011; 38(4) Figure 4. Electropherograms of vancomycin in Tears Naturale II TM taken on (a) day of preparation and (b) day 7 after storage at 28 o C. Figure 5. Electropherograms of vancomycin in Tears Naturale II TM on (a) day 10 after storage at 28 o C, after standard vancomycin was heated in an incubator at 80 o C for 16 hours (b) vancomycin (c) degradation of vancomycin, after standard vancomycin was heated in an incubator at 80 o C for 16 hours and standard vancomycin added (d) vancomycin (e) degradation of vancomycin.

7 Chiang Mai J. Sci. 2011; 38(4) 539 degradation of the vancomycin was shown by this small peak in the electropherogram. This demonstrated that this CE method for determining vancomycin in an extemporaneous solution can differentiate between undegraded and degraded vancomycin. The effects of temperature are shown in Figure 5c and reveal that, when the standard vancomycin was heated in an incubator 80 o C 16 hours, a degradation product was formed. After standard vancomycin was added to the heated standard vancomycin, the peak area of the heated standard vancomycin plus the standard vancomycin (Figure 5d) was found to be more than that of the heated standard vancomycin alone. Thus, from such accelerated conditions, this CE method was able to detect and determine vancomycin specifically and independently of any degradation product. 3.2 Stability of Vancomycin The percent labeled amount of vancomycin is calculated as the ratio of the amount of vancomycin found during analysis to the amount of standard present in the test solution, expressed as a percentage. Stability was defined as the retention of a mean of 90% of the initial vancomycin concentration [16]. Figure 6 (percentage graph of the labeled amounts) shows extemporaneous vancomycin 31 mg/ml in Tears Naturale II TM from which it could be calculated that the percentage of labeled amounts of vancomycin 31 mg/ml retained was at least 90% of the initial drug concentration after storage at 2-8 o C and 28 o C for 28 days and 10 days, respectively. Figure 6. Graphs of the percentage of the labeled amounts of vancomycin eye drops at 31 mg/ml stored at 2-8 o C and 28 o C. 4. CONCLUSION This CE approach is both time-saving and easy to perform for determining the stability of vancomycin eye drops. Vancomycin 31 mg/ml in Tears Naturale II TM was stable for 28 days at 2-8 o C and 10 days at 28 o C.

8 540 Chiang Mai J. Sci. 2011; 38(4) ACKNOWLEDGEMENT This research was supported by a Young Researchers Grant from Chiang Mai University, Chiang Mai, Thailand. REFERENCES [1] Martindale W. and Reynolds J.E.F., Martindale : The Extra Pharmacopoeia, 30th Edn., Singapore: Info Access & Distribution, [2] Fiscella R.G., Vancomycin use in ophthalmology, Arch. Ophthalmol., 1995; 113: [3] Steinert R.F., Current therapy for bacterial keratitis and bacterial conjunctivitis, Am. J. Ophthalmol., 1991; 112: 10S-14S. [4] Ross J. and Abate M.A., Topical vancomycin for the treatment of Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus conjunctivitis, DICP Ann. Pharmacother., 1990; 24: 1050, [5] Arici M.K., Sumer Z., Guler C., Elibol O., Saygi G. and Cetinkaya S., In vitro potency and stability of fortified ophthalmic antibiotics, Aust. N.Z.J. Ophthalmol., 1999; 27: [6] Vercaigne L.M., Sitar D.S., Penner S.B., Bernstein K., Wang G.Q. and Burczynski F.J., Antibiotic-heparin lock: in vitro antibiotic stability combined with heparin in a central venous catheter, Pharmacotherapy, 2000; 20: [7] Abu-Shandi K.H., Determination of vancomycin in human plasma using highperformance liquid chromatography with fluorescence detection, Anal. Bioanal. Chem., 2009; 395: [8] Jesus Valle M.J., Lopez F.G. and Navarro A.S., Development and validation of an HPLC method for vancomycin and its application to a pharmacokinetic study, J. Pharm. Biomed. Anal., 2008; 48: [9] Musenga A., Mandrioli R., Zecchi V., Luppi B., Fanali S. and Raggi M.A., Capillary electrophoretic analysis of the antibiotic vancomycin in innovative microparticles and in commercial formulations, J. Pharm. Biomed. Anal., 2006; 42: [10] Bonnici P.J., Damen M., Waterval J.C. and Heck A.J., Formation and efficacy of vancomycin group glycopeptide antibiotic stereoisomers studied by capillary electrophoresis and bioaffinity mass spectrometry, Anal. Biochem., 2001; 290: [11] Altria K.D. and Hadgett T.A., An Evaluation of the Use of Capillary Electrophoresis to Monitor Trace Drug Residues Following the Manufacture of Pharmaceuticals, Chromatographia., 1995; 40: [12] Suntornsuk L., Recent advances of capillary electrophoresis in pharmaceutical analysis, Anal. Bioanal. Chem., 2010; 398: [13] Holland L.A., Chetwyn N.P., Perkins M.D. and Lunte S.M., Capillary electrophoresis in pharmaceutical analysis, Pharm. Res., 1997; 14: [14] Znaleziona J., Petr J., Knob R., Maier V. and ev k J., Dynamic Coating Agents in CE, Chromatographia. Supplement., 2008; 67: S5-12. [15] Lurie I.S., Hays P.A. and Parker K., Capillary electrophoresis analysis of a wide variety of seized drugs using the same capillary with dynamic coatings, Electrophoresis, 2004; 25: [16] Barnes A.R., Near-patient stability studies, J. Clin. Pharm. Ther., 1996; 21: