Abstract A new method was described for simultaneous determination of ciprofloxacin (CIP), ofloxacin (OFL) and norfloxacin (NOR) by capillary electrophoresis with a diode array detector at 280 nm. The separation conditions were investigated and optimized. A new medium, 30 mM sodium tetraborate buffer solution and 22 mM sodium dodecyl sulphate containing 5% (v/v) methanol (pH 8.7), was developed for the complete separation of the three quinolones. The excipients do not affect to separation and analysis. Calibration curves between peak area and concentration in the range 10-1000 mg/L for each compound have good linearity with a correlation coefficient (r) of greater than 0.999, and the detection limit£¨s/n£© was 7.90 mg/L for CIP£¬7.92 mg/L for OFL and 7.56 mg/L for NOR. This method has been successfully applied for simultaneous determination of CIP, OFL and NOR in the pharmaceutical preparations with rapid, accurate, and simple characteristics.
Keywords Capillary electrophoresis; Ciprofloxacin; Ofloxacin; Norfloxacin£»Pharmaceutical analysis

1 INTRODUCTION
Fluoroquinolones are chemotherapeutic agents that are widely used in human and veterinary medicine because of their safety with good tolerance and broad antibacterial spectrum, which exhibit bactericidal activity primarily by inhibiting bacterial DNA gyrase. Ciprofloxacin (CIP), ofloxacin (OFL) and norfloxacin (NOR) are three most important and widely used fluoroquinolones, and have a great potency against many common bacterial pathogens and high activity against Gram-negative and Gram-positive bacteria in vivo and in vitro.
As far as we know, many analytical methods concerning fluoroquinolones have been published. Most of them are based on high-performance liquid chromatography (HPLC) [1-5]. However, capillary electrophoresis (CE) has become a useful technique for pharmaceutical analysis because of its high separation efficiency, high resolution, high speed and small sample volume requirements. Some methods have been developed for the determinations of CIP, OFL and NOR by CE, respectively [6-8]. Simultaneous determination is rapidly growing in fluoroquinolone pharmaceutical analysis [9-15]. Josephine et al. described a capillary zone electrophoresis¨Celectrospray ionization tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics. Nine fluoroquinolones have been separated and detected in a single run by this technique [9]. However, the detector cannot be found in common lab. Simultaneous determination of 14 quinolone antibacterials was achieved by CE using overlapping resolution mapping scheme, but the separation system is complicated [10]. Fierens et al. used a phosphate buffer (pH 7.0, 125 mM) to study the analysis of 10 quinolones, but norfloxacin and ciprofloxacin cannot be separated in this way [11]. Obviously£¬ a more rapid, less cost and easier operation method is also required.
The aim of this study was to elaborate and validate a simple and efficient DAD-CE methods for the separation and simultaneous determination of CIP, OFL and NOR in pharmaceutical preparations.

2 EXPEREMENTAL
2.1 Apparatus and conditions
All the experiments were performed by an Agilent 3D CE system with air-cooling and a diode array detector (Agilent Technologies, Waldbronn, Germany). A 48.5 cm (40.0 cm to the detector) ¡Á 50 mm i.d. uncoated fused-silica capillary (Agilent) was utilized. Other conditions are: capillary temperature at 20 ¡æ£¬applied voltage at 15 kV and UV detection at 280 nm. The hydrodynamic injection was at a pressure of 50 mbar for 4 s. The capillary was conditioned at the beginning of each day with 0.1 M NaOH for 5 min, followed by water for 5 min and running buffer for 10 min. Between consecutive analyses, the capillary was flushed with water for 1 min and the running buffer for 3 min to guarantee good reproducibility.
2.2 Chemicals and reagents
All the chemicals used were of analytical reagents: Methanol (MeOH), boric acid, sodium tetraborate decahydrate, sodium dodecyl sulphate (SDS), sodium hydroxide and hydrochloric acid were obtained from Beijing Chemical Factory (Beijing, China); Ciprofloxacin, ofloxacin and norfloxacin were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The structures of the studied compounds are shown in Fig. 1. Doubly deionized water (DDW) was used throughout.

Fig. 1 The chemical structures of ciprofloxacin, ofloxacin and norfloxacin

2.3. Preparation of buffer and standard solutions
Three stock solutions (2000 mg/L) of CIP, OFL and NOR were prepared by dissolving 20 mg standards in 0.1 M HCl (10 ml) and stored under refrigeration. Reference solutions for quantitative analysis of CIP, OFL and NOR were prepared from the commercially available drugs by mixing the powder with the running buffer.
The electrophoretic solution was 30 mM sodium tetraborate and 22 mM SDS containing 5% (v/v) methanol at pH 8.7. The buffers with different concentrations were adjusted to the desired pH-values with 1 M NaOH and 1 M HCl. The suspensions were filtered through a membrane (0.22mm).

3. RESULTS AND DISCUSSION
   3.1. Optimization of capillary electrophoresis separation
   The aim of our experiments was to develop a simple and rapid method for the separation of CIP, OFL and NOR by CE. The pKa values of the three compounds are shown in Table 1[16].

Table 1 The pKa values of CIP, OFL and NOR

The given pKa corresponds to acidity of the carboxyl group and the nitrogen located at the distant end of the piperazine. The best separation based on this parameter is expected to be found at a pH (around 8.87), where all the compounds would be analyzed as anions. If analysis was carried out in 30 mM sodium tetraborate at different pH values, OFL, CIP and NOR could not be separated completely, as shown in Fig.2.

Fig. 2 Electropherogram of a mixture of CIP, OFL and NOR when using 30 mM sodium tetraborate at different pH values (a) pH 8.4£»(b) pH 8.7£»(c) pH 9.0; injection, 50 mbar for 4 s; separation voltage, 15 kV; capillary temperature, 20oC ; DAD detection at 280 nm.

The separation of CIP and NOR would be improved using the micellar electrokinetic chromatography (MEKC), which is based on the relative distribution of the analytes between the aqueous phase and the micellar phase added to the separation buffer (hydrophobicities of the analytes). SDS is the most routinely used anionic surfactant for the analysis of fluoroquinolones with MEKC [15-16]. At a concentration over the critical micellar concentration (CMC), spontaneous aggregation of the surfactant molecules is caused by the increase of hydrophobic interactions. The working range was set at 18¨C28 mM of SDS (Fig. 3), and the critical concentration of 22 mM SDS was chosen, which gave the better results, as shown in Fig. 3(b). The migration order of these compounds is thus governed by a combined effect of their hydrophobicities (incorporation into the hydrophobic sites of the micelles) and their positive charge (ionic interactions between the negatively charged micelle surface and the cationic part of the analytes).

Fig.3 Electropherogram of a mixture of CIP, OFL and NOR with (a) 18 mM£»(b) 22 mM£»(c) 28 mM SDS in 30 mM sodium tetraborate (pH 8.7); injection, 50 mbar for 4 s; separation voltage, 15 kV; capillary temperature, 20oC ; DAD detection at 280 nm.

From Fig. 3, it can be seen that CIP, OFL and NOR were completely separated. But the shape of the third peak was still bad. Organic solvents added to the running buffer can improve the shape of the third peak, which is due to the addition of solvents with a high dielectric constant and lipophilic character (lipophilic character increasing the solubility of the monomeric surfactant and the CMC value). Methanol is a typical protic solvent with little lipophilic character, does not appear to disturb the micellar system much, since the increase of the CMC is small even for volume percentages as high as 35% [17]. When methanol was added to the running buffer, the migration time of three peaks was increased (Fig. 4).
The best results were obtained when 5% methanol was added. Three peaks were separated completely and the peaks were very sharp (Fig. 4.b).

Fig.4 Electropherogram of a mixture of CIP, OFL and NOR with (a) 0£»(b) 5%£»(c) 10% MeOH in 30 mM sodium tetraborate and 22 mM SDS (pH 8.7); injection, 50 mbar for 4 s; separation voltage, 15 kV; capillary temperature, 20oC; DAD detection at 280 nm..

3.2. Quantitative determination in pharmaceutical preparations
The method was applied for the quantitative determination of CIP, OFL and NOR in the mixtures of their tablets and capsules. The electropherogram of mixture of CIP, OFL and NOR in tablets and capsules were shown in Fig. 5.b.

Fig.5 Electropherogram of mixture of CIP, OFL and NOR (a) in standard solution; (b) in tablets and capsules. A buffer consisting of 30 mM sodium tetraborate and 22 mM sodium dodecyl sulphate containing 5% (v/v) methanol (pH 8.7); injection, 50 mbar for 4 s; separation voltage, 15 kV; capillary temperature, 20oC ; DAD detection at 280 nm. 1. OFL£»2. .NOR£»3.CIP

It can be seen that the excipients (maize starch, lactose, magnesium stearate, etc.) in the pharmaceutical preparations do not have adverse effect on the determination of CIP, OFL and NOR.

3.3 Validation of the method
In most HPLC methods, large amount of organic solvent have to be used as mobile phase and long analytical time is usually a boring problem to be resolved. Comparing with HPLC, the proposed CE method is simple, fast, and much practical for the determination of CIP, OFL and NOR with 30 mM sodium tetraborate buffer containing 22 mM SDS solution 5% (v/v) methanol (pH 8.7) at 20 ¡æ and 15 kV. The whole analysis was achieved within 5 minutes.
The linearity for CIP, OFL and NOR analysis was evaluated with concentrations of calibration standards against measured peak areas under the optimal conditions. The equations of calibration curve and detection limits (S / N = 3) were listed in Table 2.

Table 2 Linearity and detection limits of CIP, OFL and NOR

It can be seen that the linearity was satisfactory with a correlation coefficient (r) greater than 0.999, and the detection limits for three compounds were 7.56-7.92 mg/L.
The mean value of the contents and relative standard deviation (RSD) were determined under the same operating conditions by determining 10 replicate samples on the same day. The result is shown in Table 3, The RSD is less than 2 %. The recovery for CIP, OFL and NOR are shown in Table 4. The recoveries are in the range of 95.3 -104.1%.

Table 3 Precision for determining 10 replicate samples

Table 4 The recoveries of CIP, OFL and NOR

4 CONCLUSION
The results suggest that 30 mM of borate buffer and 22 mM SDS containing 5% (v/v) methanol at pH 8.7 is a very efficient running buffer for separating CIP, OFL and NOR. The study demonstrates that capillary electrophoresis can be successfully applied for the qualitative and quantitative analysis of CIP, OFL and NOR in pharmaceutical formulations.