Determination of Fluoroquinolones in Pharmaceutical Formulations by Extractive Spectrophotometric Methods Using Ion-Pair Complex Formation with Bromothymol Blue

In this paper, we reported a new, simple, accurate, and precise extractive spectrophotometric method for the determination of fluoroquinolones (FQs) including ciprofloxacin (CFX), levofloxacin (LFX), and ofloxacin (OFX) in pharmaceutical formulations. The proposed method is based on the ion-pair formation complexes between FQs and an anionic dye, bromothymol blue (BTB), in acidic medium. The yellow-colored complexes which were extracted into chloroform were measured at the wavelengths of 420, 415, and 418 nm for CFX, LFX, and OFX, respectively. Some effective conditions such as pH, dye concentration, shaking time, and organic solvents were also systematically studied. Very good limit of detection (LOD) of 0.084 µg/mL, 0.101 µg/mL, and 0.105 µg/mL were found for CFX, LFX, and OFX, respectively. The stoichiometry of the complexes formed between FQs and BTB determined by Job's method of continuous variation was 1 : 1. No interference was observed from common excipients occurred in pharmaceutical formulations. The proposed method has been successfully applied to determine the FQs in some pharmaceutical products. A good agreement between extractive spectrophotometric method with high-performance liquid chromatography mass spectrometry (HPLC-MS) for the determination of FQs in some real samples demonstrates that the proposed method is suitable to quantify FQs in pharmaceutical formulations.


Introduction
Fluoroquinolones (FQs) are the important antibiotics used for the treatment of Gram-negative bacterial infections in both human and veterinary medicine. ey are derivatives of 4-quinolone, which have unsubstituted or substituted piperazine ring attached at the 7-position to the central ring system of quinoline as well as fluorine atom at the 6-position.
Ciprofloxacin (CFX), which is one of the secondgenerated groups of synthetic FQs, can exhibit greater intrinsic antibacterial activity and make a broader antibacterial spectrum. Ofloxacin (OFX) is a chiral compound that is widely used to treat above infections. Levofloxacin (LFX) is the pure (-)-(S)-enantiomer of the racemic drug substance ofloxacin. Figures 1(a)-1(c) show the chemical structures of CFX, LFX, and OFX, respectively.
Bromothymol blue (BTB) (Figure 1(d)) is an anionic dye and that can be protonated or deprotonated to form yellow or blue, respectively. e BTB was used to make ion-pair complex, which was applied to determine many pharmaceutical compounds by extractive spectrophotometric methods [21][22][23][24][25][26][27][28][29][30]. However, the ion-pair complex between BTB and FQs has not been studied. e method based on ion-pair complexes between analytes and BTB into a suitable organic solvent is also simple, fast, and cheap.
In the previous study, we used sulphonphthalein acid including bromophenol blue, bromocresol green, and bromothymol blue to determine ciprofloxacin pharmaceutical formulations and achieved good results [31].
In this paper, for the first time, we investigated extractive spectrophotometric method based on the formation of ionpair complexes between ciprofloxacin, levofloxacin, and ofloxacin with BTB subsequent extraction into chloroform. Some effective conditions on the formation of complexes such as pH, shaking time, organic solvent, and the concentration of dye were systematically studied. e present method was also applied to determine FQs in some pharmaceutical formulations including tablets and infusions.

Apparatus.
A double beam UV-visible spectrophotometer (SP-60, Biochrom Ltd., UK) with 1.0 cm of path length quartz cells was used to measure all sample absorbances. Inolab pH-meter instrument (Germany) was used to monitor the pH of solutions. ree standard buffers were used to calibrate the electrode before measuring pH of solutions. All measurements were conducted at 25 ± 2°C controlled by air conditional laboratory.

Materials and Reagents.
All chemicals used were of analytical grade and double-distilled water was used to prepare all solutions in the present study.
FQs were purchased from Sigma (Germany, with purity >99.0%), whereas bromothymol blue (BTB) was supplied by Maya-R, China, with purity >99%. e organic solvents including chloroform, dichloromethane, carbon tetrachloride, dichloroethane, benzene, toluene, and other chemicals are analytical reagents (Merck, Germany). e following dosage forms containing FQs were purchased from local pharmacy market and employed in the study: Hasancip and Kacipro tablets equivalent to 500 mg ciprofloxacin (Hasan-Dermapharm and Dong Nam manufacturing-Trading pharmaceutical Co., Ltd, Vietnam). Ciprofloxacin infusion equivalent to 200 mg ciprofloxacin/100 ml solution for infusion (Hebei Tiancheng Pharmaceutical Co., Ltd and Shandong Hualu Pharmaceutical Co., Ltd, China). Stada and DHG tablets equivalent  en, 4.0 mL of 0.025% BTB solution was added before thoroughly mixing. After that, a 10 mL of chloroform was added to each of the separating funnel. e contents were shaken for 2 min and allowed to separate the two layers. e yellow-colored chloroform layer containing the ion-pair complexes was measured at 420 nm for CFX, 415 nm for LFX, and 418 nm for OFX against the reagent blanks. At each concentration, the experiment was repeated 6 times. e colored chromogen complexes are stable for 24 h.

Sample Preparation.
Weigh and mix the contents of twenty tablets of each drug (CFX, LFX, and OFX), an accurately weighed amount of powder equivalent to 0.1 g of drugs transferred into a 100-mL beaker. A magnetic stirrer was used to completely disintegrate the powder in doubly distilled water. en, filter through a Whatman paper (No 40) and fill up to 100 mL with doubly distilled water in a volumetric flask. e working solution of the drugs containing 100 µg/mL was prepared by dilution and determined under optimum conditions.

Effect of Extracting Solvent.
Six organic solvents including chloroform, carbon tetrachloride, dichloromethane, dichloroethane, benzene, and toluene were used to study the effect of solvent to ion-pair formation between FQs and BTB. Figure 2 shows that chloroform is the most suitable solvent for the extraction of three FQs with low blank absorbance, highest absorbances, and lowest standard deviations. It implies that chloroform is the best extracting solvent to achieve a good recovery of the complexes with the shortest time to reach the equilibrium processes.

Effect of pH.
e pH of solution plays an important role in the complex formations. e effect of pH on the formation of ion pairs was examined by varying the pH from 2.0 to 6.0 by adjusting 1 M HCl and 1 M NaOH. e maximum absorbances were observed at pH 3.3, 3.4, and 3.5 for the complexes of BTB and OFX, CFX, and LFX, respectively ( Figure 3).
ese pH values correspond to the initial pH of the examined drug and the dye. erefore, it is not necessary to adjust the pH before extraction.

Effect of Dye Concentration.
e effect of dye concentrations was studied by adding different volumes of 0.025% BTB from 1.0 to 6.0 mL with a fixed concentration of FQs (10 μg/mL) ( Figure 4). Figure 4 shows that the maximum absorbance of the complex was achieved with 4.0 mL of 0.025% of BTB in each case and excess dye did not affect the absorbance of the complex. erefore, 4.0 mL of 0.025% of BTB is optimum dye volume and it is kept as constant for further studies.

Effect of Shaking Time.
e effect of shaking time on the formation and stability of the ion-pair complex was investigated by measuring the absorbance of the extracted ion associates with increasing time from 0 to 4.0 min. Figure 5 shows that the ion-pair complexes were formed 10 mL, whereas the absorbances were measured at 420, 415, and 418 nm, for CFX, LFX, and OFX, respectively. e absorbances were plotted against the mole fraction of the drugs. e stoichiometry for each drug-dye ion-pair complex was found to be 1 : 1 ( Figure 6).

Mechanism of Reaction and Absorption Spectra.
Fluoroquinolones can contain a secondary amino group (CFX) and a tertiary amino group (LFX and OFX) that can be easily protonated under acidic conditions. On the one hand, the sulphonic acid group in BTB, that is, the only group undergoing dissociation in the pH range 1-5. e colour of BTB is on the basis of lactoid ring and subsequent formation of quinoid group. It is suggested that the two tautomers are plausible in equilibrium due to strong acidic nature of the sulphonic acid group. us, the quinoid body must predominate. Finally, the protonated fluoroquinolones form ion pairs with BTB dye that could be quantitatively extracted into chloroform. e possible reaction mechanisms are proposed and given in a scheme in Figure 7.
e absorption spectra of the ion-pair complexes, which were formed between FQs and BTB, were measured in the wavelength range 350-500 nm against the blank solution and shown in Figure 8. Figure 8 shows that absorption maxima for CFX-BTB, LFX-BTB, and OFX-BTB in chloroform were observed at 420, 415, and 418 nm, respectively. e reagent blanks under similar conditions have insignificant absorbances. At wavelengths 420, 415, and 418 nm, absorption spectrum of BTB does not affect the absorption spectrum of ion-associate complexes of FQs. erefore, the selectivity of the proposed method for the determination of FQs is guaranteed.

e equation of association constant of ion-pair complex is
where A and A m are the observed absorbance and the maximum absorbance value when all the drug present is associated, respectively. C M is the molar concentration of the drugs at the maximum absorbance and n is the stoichiometry in which BTB ion associates with drugs. e conditional stability constants (K f ) of the ion-pair complexes according to Britton [32] for the cases of FQs were calculated from the continuous variation data using the following equation: e conditional stability constants (K f ) of the ion-pair complexes for FQs are indicated in Table 1. Table 1 shows that the log K f values of ion-pair associates for OFX-BTB, LFX-BTB, and CFX-BTB were 6.08 ± 0.46, 6.04 ± 0.58, and 5.91 ± 0.32, respectively (numbers of replicated experiments, n � 6). e obtained results confirmed that the ion-pair formation complexes are of high stability.

Validation of the Present Method.
e proposed methods are validated according to ICH recommendations Q2(R1) [33]. e parameters that have been investigated are indicated below.

Linearity, Sensitivity, and Limits of Detection and Quantification.
A linear relationship between the measured absorbance and the concentration range studied for each drug as shown in Figure 9 and the correlation coefficient (R) of at least 0.997 were achieved. e limit of detection (LOD) and quantification (LOQ) of the method are determined by 3.3(SD/b) and 10(SD/b), respectively, where SD is the standard deviation of blank absorbance values and b is the slope of the calibration curve equation.
e LOD and LOQ values, slope, and intercept of linear graphs for all the drugs and analytical parameters are indicated in Table 2. e molar absorptivities and Sandell's sensitivity of each methods were calculated and these values showed that the molar absorptivity of ion-pair complexes was in the order CFX-BTB > LFX-BTB > OFX-BTB.

Accuracy and Precision.
e accuracy and precision of the methods were determined by preparing solutions of three different concentrations of drug and analyzing them in six replicates. e precision of the proposed methods was evaluated as percentage relative standard deviation (RSD%) and accuracy as percentage relative error (RE%). e percentage relative error was calculated using the following equation: e accuracy and precision were summarized in Table 3. e low values of the RSD and RE confirm the high precision and the good accuracy of the present method.

Robustness and Ruggedness.
For the evaluation of the method robustness, some parameters were interchanged: pH, dye concentration, wavelength range, and shaking time. e capacity remains unaffected by small deliberate variations. Method ruggedness was expressed as RSD% of the same procedure applied by two analysts and using different instruments on different days. e results showed no statistical differences between different analysts and instruments, suggesting that the developed methods were robust and rugged (Table 4).

Selectivity and Effect of Interferences.
e effect of commonly utilized excipients in drug formulation was studied.
e investigated FQs were studied with various excipients such as magnesium stearate, glucose, lactose, starch, and sodium chloride which were prepared in the proportion corresponding to their amounts in the real drugs with a final dosage of 10 µg/mL FQ. e effect of excipients on the determination of FQs was evaluated by recovery when determining FQs analyzed with the proposed method in the presence of excipient (Table 5). e results in Table 5 show that the recoveries are in the range of 98.53-102.04, demonstrating that there is no interference of excipients when FQs in drugs are quantified by extractive spectrophotometric using ion-pair formation with BTB. In other words, the present method has a high selectivity for determining FQs in its dosage forms.

Comparison with Other Spectrophotometric Methods.
e proposed method compares with other reported methods. It has been observed that the extractive spectrophotometric method with BTB in the present study is of high sensitivity than other ones (Table 6). It also does not need heating, the product is stable for a longer time, and the interferences are minimum.

Analysis of Pharmaceutical Formulations.
e proposed method was applied successfully for the determination of studied drugs in the pharmaceutical formulations (tablets   Journal of Analytical Methods in Chemistry 7 and infusion) and the results are presented in Table 7. Six replicated determinations were measured. Table 7 shows that satisfactory recovery data were obtained and the recovery efficiency varies from 97.41% to 101.20%, indicating high accuracy of the present method in determining real pharmaceutical samples.

Comparison with HPLC-MS Method.
In order to validate the experimental data in determining some real drug samples, HPLC-MS was used with the conditions described on Section 2.6 according to the previously published paper [13]. e comparison between the results determined by the present method with HPLC-MS method was indicated in Table 8. Table 8 shows a good agreement between the proposed method and HPLC-MS where the relative differences of two methods were less than 11%. Furthermore, the standard deviation of the proposed method is almost lower than that of HPLC-MS. Our results indicate that the extractive spectrophotometric determination of FQs using BTB dye in chloroform is a very good method to quantify the FQ in pharmaceutical formulations.

Conclusions
We have reported a new method when using BTB as an anionic dyes for the extractive spectrophotometric determination of ciprofloxacin (CFX), levofloxacin (LFX), and ofloxacin (OFX) in different pharmaceutical drugs (tablets and infusions). e methods have the advantages of simplicity without heating, pH-adjustment, and high sensitivity. e limit of detection (LOD) values are 0.084 µg/mL for CFX, 0.101 µg/mL for LFX, and 0.105 µg/mL for OFX. No interference from common excipients was confirmed. e stoichiometry complexes of FQs and BTB determined by Job's method of continuous variation were found to be 1 : 1.
e developed and validated methods are indicated as the acceptable precision and accuracy, and recovery of the drugs and suitable for routine analysis of drugs in pharmaceutical formulations. e results of some real samples by the present method that were compared with HPLC-MS method with    Highly sensitive with wide linear dynamic ranges, no heating, and no pH-adjustment is study

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.