Enantioselective Determination of Ofloxacin in Pharmaceutical formulation by Capillary Electrophoresis using CM--CD as a chiral selector. Abdalla A.

Elbashir, Bahruddin Saad, Abdussalam Salhin Mohamed Ali and Muhammad Idiris Saleh

School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia Abstract Ofloxacin, ()-9-fluro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-

oxo7H-pyrido[1,2,3-de]-1,4-benzooxazine-6-carboxylic acid is a member of the fluoroquinolones, a class of synthetic antimicrobial agents. Pharmaceutical research has shown that the antibacterial activity of the levo-enantiomer of ofloxacin (S-(-)-ofloxacin) or levofloxacin) is 8-128 times higher than that of the racemate. Currently, regulatory authorites in the USA, Europe, China and Japan provide clear guidelines which stipulate that preferably only active enantiomer (eutomer) of a chiral drug should be brought to the market. A capillary electrophoresis (CE) method has been developed allowing the separation and quantition of ofloxacin enantiomers using carboxymethyl--cyclodextrin (CMCD) as chiral selectors. The influence of chemical and instrumental parameters on the separation such as type and concentration of cyclodextrin, buffer concentration, and pH, applied voltage, capillary temperature, and injection time were investigated. Good chiral separation of the racemic mixture of ofloxacin was achieved in 10 minutes. Validation criteria for sensitivity, linearity, precision, and accuracy/recovery were also studied. Possible mechanism that leads to the successful separation of the enantiomers will also be discussed. The proposed CE method show considerable promise to be applied to the assay of ofloxacin in pharmaceutical formulation.

Keywords: ofloxacin, enantiomers, capillary electrophoresis, cyclodextrin derivatives

1 Introduction Ofloxacin (Figure 1), having the chemical name ()-9-fluro-2,3-dihydro-3methyl-10-(4-methyl-1-piperazinyl)-7-oxo7H-pyrido[1,2,3-de]-1,4-benzooxazine-6carboxylic acid, belongs to the quinoline class of antibiotics. Pharmaceutical research has shown that the antibacterial activity of the levo-enantiomer of ofloxacin (S-(-)-ofloxacin) or levofloxacin) is 8-128 times higher than that of R-(+)-enantiomer and twice as potent as that of the racemate [1,2]. Currently, the drug is used therapeutically as a racemic mixture composed of equal amounts of R-(+) and S-(-) enantiomers and S-(-)-isomer. Therefore works on the separation of racemic ofloxacin and its application in pharmaceutical formulation are important. Based on this, our work allows an enantioseparation using CM--CD as chiral selector. Chiral separation of ofloxacin in biological fluid has been achieved by HPLC after addition of chiral selector to the mobile phase [3, 4]. However disadvantages of these methods include tedious sample preparation procedure extraction and/or derivatization steps, and relatively expensive chiral columns. A number of CE methods have been proposed for the qualitative chiral separation of ofloxacin enantiomers using different chiral selectors, cyclodextrins (CDs) [5-7], vancomycin [8], bovine serum albumin [9, 10] or combination of -CD and Dphenylalanine and zinc sulphate as chiral selectors [11]. In some experiment the resolution factor were poor, and lack of reproducibility and impracticability for migration time of analytes. Quantitative assay of ofloxacin enantiomers and it metabolites in human urine using laser induced fluorescence detection has been reported [12]. However laser-induced fluorescence detectors are very expensive and not widely use. Quantification method of ofloxacin enantiomers in Hank,s balanced salt solution (HBSS) as physiological solution for pharmacokinetic studies has been optimized [13]. Up to know, no CE method for chiral separation of R-(+) and S-(-) enantiomers in pharmaceutical formulation has been published. Therefore a CE method for analysis R-(+) and S-(-) enantiomers in pharmaceuticals formulation was developed. In this study easy alternative method for enantiomeric separation of ofloxacin, using low concentration of carboxymethyl--cyclodextrin in normal polarity mode was

described. Conditions such as pH, buffer concentration, cyclodextrin concentration, applied voltage were optimized in order to obtain high enantiomeric resolution with the short analysis time. Further, under optimum condition the developed method has been

applied to chiral separation in the pharmaceutical formulation of the drug.

2. Materials and Methods

2.1 chemicals and reagents Ofloxacin, ()-9-fluro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo7Hacid, (s)-ofloxacin and

pyrido[1,2,3-de]-1,4-benzooxazine-6-carboxylic

tris(hydroxymethyl)aminomethane were purchased from Sigma-Aldrich. -cyclodextrin (-CD), -cyclodextrin (-CD), 2-hydroxypropyl--cyclodextrin (HP--

CD),hydroxypropyl--cyclodextrin (HP--CD) hydroxypropyl- -cyclodextrin (HP-CD) and caroboxymethyl--cyclodextrin (CM--CD), were obtained from Fluka. Citric acid anhydrous was purchased from Sigma. Commercial pharmaceutical preparations in the form of tablets, (claimed to contain 200 mg and 100mg active ingredient) were purchased from the campus drug-store and was manufactured by different pharmaceutical companies in and outside Malaysia.

2.2 Instrumentation and Electrophoretic conditions The analysis was carried out on a Waters Capillary Ion Analyzer (Milford, MA, USA) which was interfaced to a Waters PC 800 Workstation. Uncoated fused-silica capillary (total length, 35 cm and internal diameter, 50 m) were used for the enantiomeric separation. The separation were carried out at 25 C and applying voltage of 10 kV. Samples were injected hydrostatically for 25 seconds. Detection was achieved at 254 nm . New uncoated fused-silica capillary was conditioned by flushing with 1 M NaOH for 30 minutes, then 0.1 M NaOH for 10 minutes and Milli-Q water and buffer each for 15 minutes. The running buffer consisted of 50 mM citric acid that had been adjusted to the desired pH with 1 M Tris solution. The running buffer solution was passed through

0.2 m cellulose nitrate membrane filters (Whatman International, Maidstone, England) and degassed by sonication prior to use. Prior to each analysis, capillary column was rinsed with 0.1 M NaOH for one minute, and then purified water, followed by the carrier electrolyte, each for 2 minutes between the runs.

2.3 Stock and standard solutions

Standard stock solution (1000 g mL) of racemic ofloxacin and S-(-)-ofloxacin were prepared in 0.1 M NaOH and was kept refrigerated. Working standard solutions were prepared daily by diluting suitable aliquots of the stock solution with Milli-Q water. The standard solutions were stored in brown glass vial to protect the analytes from light.

2.4 Pharmaceutical sample preparation Five tablets were weighed, ground and mixed in a mortar, and an appropriate amount of the powder was taken and dissolved in 25 mL 0f 0.1 M sodium hydroxide by ultrasonic for three minutes and diluted to 100 mL with Milli-Q water. The sample was filtered through a membrane (0.45 m) an amount of 1 ml of the filtrate was diluted with Milli-Q water to 10 ml. This solution was introduced to the capillary electrophoresis system for the separation.

3 Result and discussion 3.1 Optimization of separation conditions 3.1.1 Choice of Chiral selector In a previous study [12] on ofloxacin chiral separation indicated that electrophoretic separation of ofloxacin was possible at nearly all pH values except at their isoelectric points which is 7.4 for ofloxacin [14]. So pH 4.0 was chosen for preliminary studies. The chiral separation of the enantiomers was attempted using different cyclodextrins, cyclodextrin, namely native -cyclodextrin, -cyclodextrin, hydroxypropyl-and

hydroxypropyl--cyclodxtrin,

hydroxypropyl--cyclodxtrin,

carboxymethyl--cyclodextrin. No chiral recognition were observed with -CD, -CD and HP--CD , where as HP--CD, HP--CD, and CM--CD where able to separate the enantiomers with a resolution of 1.13, 1.14 and 2.4 respectively.

3.1.2 Choice of pH The effect of pH on the resolution and migration time was investigated over the pH range 3.5-7, using 50 mM buffer solutions prepared at different pH containing 3.0 mg/mL CM--CD. The results are shown in Figure 2. A increase in resolution was observed when the pH values were increased from 3.5 to 4.5 and as a consequence, pH 4.5 was chosen as the optimum, since at this value a high resolution with reasonable migration time was obtained. 3.1.3 Choice of buffer concentration The effects of buffer concentration (25-100 mM) were investigated, by maintaining 3 mg/mL CM--CD in the background electrolyte (BGE) at pH 4.5. As the buffer concentration increase the resolution slightly decrease, (data not shown). A 50 mM buffer was selected to achieve good resolution without sacrificing the migration time. 3.1.4 Choice of cyclodextrin concentration The effect of CM--CD of different concentration (1-4 mg/mL) on the resolution (Rs) and migration times were investigated. Increasing the concentration of the CM--CD causes the resolution and the migration time (but to a lesser extent) to increase (Figure 3). This is probably due to favourable complexation with the chiral selector. 3mg/mL CM-CD was used as a compromise between resolution and speed of analysis. 3.1.5 Choice of Applied voltage The effect of applied voltage (8-20 kV) on peak resolution was investigated. Resolution with good peak shape and reasonable migration time was found when operated at 15 kV 3.1.6 Choice of injection time In order to further reduce the detection limits, the injection time was varied from 15-35 second. Using hydrostatic injection, 25 seconds injection time was selected as the optimal value.

Details of the optimized electrophoretic separation conditions are summarized in Table 1, while Figure 4 shows typical electropherogram obtained when a standard solution of 100 g /mL of racemic ofloxacin were injected under the optimized conditions.

3.2 Method validation 3.2.1. Precision Intraday precision was assessed by injecting racemic ofloxacin standard at three concentrations (50,150 and 250 g/mL) six times. In all cases, RSD for migration time and corrected peak areas were less than 1.5 and 4.0, respectively. (Table 2) The interday precision was assessed with three concentrations of the standard (50,150 and 250 g/mL) injected six times for three consecutive days. In all cases, RSD for migration time and corrected peak areas were less than 5.0 and 4.0, (Table 2) 3.2.2. Accuracy The accuracy of the method was evaluated for placebo by recovery studies. Several a liquots of the racemic ofloxacin at three different concentrations (50,150 and 250 g/mL) were added to analytical placebo prepared from the excipients. The obtained results are summarized in table 3. As it can be observed, recoveries between 98.36 and 103.38 were reached in all cases. 3.2.3 Linearity The calibration graph was constructed by plotting corrected peak areas as a function of analytes concentration in g/mL. Six standard solutions containing racemic ofloxacin in range of 25 to 250 g/mL. The linear regression equations obtained are summarized below S-ofloxacin: y=37.59x-7.5319, R2 = 0.9983 R-ofloxacin: y= 34.93x-5.6493, R2 = 0.9981 respectively.

3.3. Analytical Application Our method was applied to the chiral analysis of ofloxacin in different pharmaceutical formulations commercially available in Malaysia (ofl-200, prifloxacin

and tarivid. The preparation of the sample was described in the section 2. Triplicate determinations of the content of ofloxacin enantiomers in the pharmaceutical formulation were carried out. The results obtained are summarized in table 4. 4 concluding remarks In this paper a capillary electrophoresis method was developed for the chiral separation of ofloxacin using low concentration of CM--CD as a chiral selector. Several experimental parameters that affect chiral separation were investigated. The optimum condition were found to be 50 mM Tris-phosphate, pH 4.5, containing 3mg/mL CM-CD, applied voltage 12 KV, and capillary temperature 25 C and injection time 25 seconds . The chiral CE method using the selected optimum condition was validated to show the quality, reliability, and consistency of its results. Good performance with regards to linearity, repeatability, reproducibility, and accuracy was achieved. The validated method was applied to determine the amount of ofloxacin enantiomers in three different commercial tablets, being suitable for such purpose.

5 References [1] Fujimoto, T. Misuhashi, Chemotherapy 1990, 36, 268 [2] Hayakawa, I., Atarashi, S., Yokohama, S., Imamura, M., Sakano, K., Furukawa, M., Antimicrob. Agents Chemother. 1986, 29, 163-164. [3] Karl-Heinz, L., Damm,P., J. Chromatogr. 1988, 425, 153-161. [4] Arai, T.,Koike, H., Hirata,K., Oizumi, H., J. Chromatogr. 1988,448, 439-444. [5] Koppenhoefer, B., epperlein, u., Schlunk, R., Zhu, X., Lin, B.,J. Chromatogr. A,1998,793,153-164. [6] Zhou, S., Ouyang, J.,Baeyyens, W.R.G., Zhao, H., Yang,Y., J. Chromatogr. A [7] Boer, T.D.,Mol,R.,Zeeuw,R.A.D.,Jong,G.J.D.,Ensing, K., Electrophoresis

2001,22,1413-1418. [8] Arai, T., Nimura, N., Kinoshita, T., J. Chromatogr.A 1996,736,303-311. [9] Zhu, X.F., Ding,Y.S.,Lin,B.C., Jakob.,A., Koppenhoefer,B., Electrophoresis 199,20,1869-1877. [10] Arai, T., Ichinose, M., kuroda, H.,Nimura, N.,Kinoshita, T.,anal. Biochem. 1994,217,7. [11] Horimai, T., Ohara, M., Ichinose, M., J. Chromatogr. A 1997, 760,235-244. [12] Horstkotter, C., Blaschke,G., J. Chromatogr. B 2001,754,169-178. [13] Awadallah, B., Schmidt, P. C.,Wahl, M. A., J. Chromatogr. A 2003, 988, 135-143. [14] Sun,S.,W.,Chen,L.Y., J. Chromatogr. A 1997, 766, 215-224.

O F

O OH

N N H3 C O

N
*

CH3

Figure 1. The chemical structure of ofloxacin; asterisk indicate chiral centre

3 R s lu n(R ) e o tio s 2.5 2 1.5 1 0.5 0 3.5 4.5 5.5 pH 6.5 7.5

Figure2. Effect of pH on the resolution of ofloxacin enantiomer (buffer concentration 50 mM, CM--CD concentration: 3 mg/mL, temperature 25 C and applied voltage 12 kV).

3.5 Resolution (Rs) 3 2.5 2 1.5 1 1 2 3 4 5

CM--CD conc. [mg/mL]

Figure 3. Effect of CM--CD concentration on the resolution of ofloxacin enantiomers. Please refer to Table 1 for CE conditions

A
-3.00 mV -4.00

-5.00

1.00
-2.50

2.00

3.00

4.00

5.00 Minutes

6.00

7.00

8.00

9.00

10.00

B
-3.00 -3.50 mV -4.00 -4.50 -5.00 2.00 4.00 6.00 Minutes 8.00 10.00 12.00 14.00

Figure 4. Electropherogram obtained from the injection of (A) 100 g/ml STD racemic ofloxacin (B) 100 g/ml racemic ofloxacin formulation . Please refer to Table 1 for CE conditions.

Table 1 Optimum CE operating conditions

Electrolyte Applied voltage Sample injection Capillary Temperature Fused silica capillary

50 mM Tris-citrate buffer, pH 4.5 3mg/mL CM--cyclodextrin 12 KV Hydrostatic, 25 s 25 C 35 cm total length (27.5 cm effective length) x 50 m 254 nm

i.d
Detection wavelength

Table 2 Within-day and inter-day reproducibility for the repeated injection of different concentration of racemic ofloxacin standard. RSD (%) Migration time S-ofloxacin Intraday precision (n=6) 50 1.29 150 0.312 250 1.49 Interday precision (n=18) 50 4.17 150 3.146 250 3.67 Factor Conc. (g/mL) RSD (%) Corrected peak areas S-ofloxacin R-ofloxacin 3.67 1.978 3.018 3.44 0.763 2.41

R-ofloxacin 1.41 0.332 1.36

4.918 3.469 4.185

3.784 2.254 3.7477

3.266 1.8626 3.64

Table 3 Recoveries obtained from the determination of ofloxacin in placebos that contained different levels of spiked standards

Sample

racemic ofloxacin (g/mL)

Recovery (%) (meanS.D) S-ofloxacin 98.361.72 103.383.52 102.951.077 R-ofloxacin 99.281.45 101.240.214 99.450.552

1 2 3

50 150 250

Table 4 Analysis results for the pharmaceutical formulations containing racemic ofloxacin Commercial formulation Prifloxcin 200mg Oflo 200mg Tarivid 100mg S-ofloxacin (mg) 100.763.29 102.272.39 53.110.964 R-ofloxacin (mg) 99.220.411 104.561.51 51.321.12 total weight (mg) 199.963.7 206.833.9 104.432.08