Ion-pair complexes formation between fluoroquinolone antibiotics and methyl red and their use for the extraction-spectrophotometric analysis

Abstract

The new spectrophotometric methods of fluoroquinolones: enrofloxacin, norfloxacin, and ofloxacin determination with the methyl red are based on ion-pair complex formation between fluoroquinolones and methyl red in acidic medium at pH 3–4 and subsequent three times extraction of the reaction products by chloroform. The obtained orange extract has an absorbance maximum at λ_max = 492 nm. Optimum conditions for the formation as well as for the extraction of ion-pair complexes between fluoroquinolones and methyl red at the presence of acetate buffer and potassium chloride solutions have been established: C (CH₃COONa) = 0.85 M, C (KCl) = 2 M, pH = 3.8. Effective molar absorbtivity of ion-pair complexes chloroform extracts is ɛ₄₉₂∼3·10³l mol⁻¹cm⁻¹. New extraction-spectrophotometric methods for fluoroquinolone determination with methyl red were developed on the basis of the optimum reaction conditions. Concentration range for the system fluoroquinolone – methyl red is (2.5–25)·10⁻⁶ M; limit of detection for enrofloxacin is C_min = 2.48·10⁻⁶ M, for norfloxacin – C_min = 3.07·10⁻⁶ M, for ofloxacin – C_min = 3.17·10⁻⁶ M.

Keywords

Fluoroquinolones methyl red ion-pair complex extraction-spectrophotometric analysis drugs

Introduction

Fluoroquinolones (FQs) are a group of synthetic chemotherapeutic agents that are widely used in medicine and veterinary practice. They 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. Due to the number of fluorine atoms in the molecule FQs are divided into monofluoroquinolones, difluoroquinolones, and trifluoroquinolones. Today more than 30 drugs of FQ antibiotics have been developed. They are divided into four generations based on time when they were created (Treskach and Kucherenko, 2004). Pharmacodynamics of FQs has broad-spectrum antibacterial (bactericidal) postantibiotic and immunomodulative effects (Melenteva and Antonova, 1985; Shchekina, 2007).

The safe and effective use of medications requires multilevel quality control at all stages of drug manufacturing from the synthesis of the raw materials and ending with the obtaining of the final products. FQ antibiotics are quantified using analytical methods of the titrimetry, spectrophotometry, fluorescence, potentiometry, capillary electrophoresis, chromatography, and immunoenzymatic analyses.

According to regulatory documents quantification of FQs is carried out by nonaqueous titration. The disadvantages of this technique are the needs to prestandardize perchloric acid solution and the duration and complexity of the titration procedure. The analysis of the numerous publications on the FQ antibiotics determination revealed that the priority in the choice of antimicrobials analysis method is determined by the sample’s nature. For FQs determination in pure substances and simple drugs, the own absorbance in UV spectrum range (Nebsen et al., 2013; Ötker and Akmehmet-Balcıoğlu, 2005) is used. For their analysis in multicomponent drugs, the high performance liquid chromatography (HPLC) is applied. HPLC can get reliable results, but the high cost of equipment and reference materials, use of the toxic solvents, as well as the need in highly qualified specialists restrict its use for mass analysis.

For quantification of drugs component, use of the method of spectrophotometry is very promising. It does not require titrant standardization, reduces the time of sample preparation; thus rapidity and good validation characteristics constitute obvious advantages of the method.

It is known that alkaline earth and transition metals (Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Fe²⁺, Co²⁺, A1³⁺, Fe³⁺ etc.) form colored complexes with FQs (El Khateeb et al., 1998; García et al., 2005; Pan et al., 2012; Uivarosi, 2013). Spectrophotometric methods of enrofloxacin (EF) determination with eosin, 2,3,4,5-tetrachloronitrobenzene, and 2,3-dichloro-5,6-dicyano-1,4-benzoquinonе with formation of complexes with charge transfer as well as with bromocresol purple, bromophenol blue, and methyl orange with formation of ion-pair complexes, which are extracted by chloroform have been developed (Mostafa et al., 2002a). For the spectrophotometric determination of EF, perchloric acid and tetracyanoethylene, which form transient complexes with EF, are used (Mostafa et al., 2002b). Belal et al. (1999) used the method of norfloxacin (NF) determination based on spectrophotometric measurement of chromogenic reagent green solution absorbance formed by the interaction of NF with CeNH₄(SO₄)₂ and 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate. For NF determination, the absorbance of yellow colored complexes with bromocresol green in dichloromethane and with tetracyanoethylene in acetonitrile is used (El-Brashy et al., 2004). Ni et al. (2008) described a spectrophotometric method for NF determination, which is based on monitoring of the kinetic spectrophotometric reaction of two analytes with potassium permanganate as oxidizer. Study of the existent literature on the subject revealed that spectrophotometric method of FQs determination has not been given appropriate attention. Thus, it is important to look for promising new available reagents for FQ determination.

We investigated the interaction of three representatives of second-generation FQs: EF, ofloxacin (OF), and NF (Table 1) with the acid–base indicator monoazo dye methyl red (MR), which forms ion-pair complexes with FQs. We used this type of analytes to develop the extraction-spectrophotometric methods for FQ determination in drugs.

Table 1.

Molecular structure of studied fluoroquinolones.

Name	Structural formula
Norfloxacin 1-ethyl-6-fluoro-4-oxo-7-piperazin-1-yl-1H -quinoline-3-carboxylic acid Formula: C₁₈H₂₀FN₃O₄ CAS number: 70458-96-7. M.W. = 319.33 g mol⁻¹.
Ofloxacin (RS)-7-fluoro-2-methyl-6-(4-methylpiperazin-1-yl)- 10-oxo-4-oxa-1-azatricyclo[7.3.1.05,13] trideca- 5(13),6,8,11-tetraene-11-carboxylic acid; (±)-9-fluoro- 2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)- 7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine- 6-carboxylic acid Formula: C₁₈H₂₀FN₃O₄ CAS number: 82419-36-1. M.W.=361.4 g/mol⁻¹.
Enrofloxacin 1,4-dihydro-1-cyclopropyl-7-(4-ethyl-1-piperazinyl)- 6-fluoro-4-oxo-3-quinolinecarboxylic acid; 1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo- 1,4-dihydroquinoline-3-carboxylic acid Formula: C₁₉H₂₂FN₃O₃ CAS number: 93106-60-6. M.W. = 359.4 g mol⁻¹.

Azo dye MR is a pH indicator; at pH ≤4.4 it is red, at pH > 6.2 it is yellow, and at 4.4 <pH 6.2 it is orange, pKa is 5.1 (Zhang et al., 2012). MR is used for titration of aliphatic amines in 1,4-dioxane and chloroform as well as it is used in argentometry as an adsorption indicator (color transition from yellow to orange-red). Sodium salt of MR is water soluble.

Experimental

Reagents and equipment

All aqueous solutions were prepared using distilled water.

FQs were purchased from Sigma (USA). Solutions of EF was prepared by dissolving appropriate amounts of the reagents of pharmacopoeia grade in 0.1 M sodium hydroxide solution; solutions of OF – in 0.01 M sodium hydroxide solution; solutions of NF – in 0.1 M hydrochloric acid. All solutions were stored at a room temperature in a dark place.

Solutions of MR (Shostkinsky Chemical Plant, Ukraine) were prepared by dissolving appropriate amounts of the reagent of analytical grade ≥98% purity in distilled water.

The solutions of hydrochloric acid, sodium hydroxide, potassium chloride, sodium acetate, and sodium sulfate were prepared from the chemicals of the analytical grade.

Organic solvents of the analytical grade: toluene, benzene, hexane, chloroform, and amyl alcohol were used in the experiment.

UV–Vis measurements were performed with UV–Vis scanning spectrophotometer SPECORD M-40, Carl Zeiss Jena, Germany (spectral range 190 – 1100 nm with resolution of 1 nm; absorbance range −0.3 to 2; standard deviation of absorbance measurement 0.5% at A = 1) and photometer KFK-2 MP, Zagorsky mechano-optical plant, Russia (spectral range 315–980 nm; width of absorption filters transmission band from 25 ± 5 to 45 ± 5 nm depending on a filter; absorbance range 0–2; standard deviation of separate absorbance measurement up to 0.3%).The path length of cuvettes was 1 cm for spectrophotometers and in the range of 1–5 cm for optimal measurements with the photometer. All absorbance measurements were performed at 20–25℃.

The pH value was measured by pH-meter model pH 150M (Gomelsky Plant of Measuring Devices, Belarus), equipped with a combination electrode, which incorporates both glass and reference silver chloride electrodes into one body. The required pH of each solution was adjusted using diluted HCl and NaOH solutions.

Procedure of FQs determination

Procedure of acetate buffer solution preparation

Sample weights of sodium acetate (7 g) and potassium chloride (12.5 g) were dissolved in 70 ml of distilled water and glacial acetic acid was poured up to pH of 3.8 using a pH meter, and then distilled water was added to the full volume of 100 ml.

Procedure of oral solution “Enroflox 10%” preparation for the EF determination

Aliquot of solution containing 100 mg of EF was placed into a 100 ml volumetric flask and 0.1 M NaOH was added to complete the volume. Then the solution was mixed thoroughly and was 10-fold diluted by the same solvent (working solution).

Procedure of EF determination in oral solution “Enroflox 10%” by the own absorbance in UV spectrum range at λ = 271 nm at pH = 10

Aliquot of 1 ml of working solution of “Enroflox 10%” was placed into a 25 ml volumetric flask. Solution was diluted by distilled water and the pH of solution was adjusted to pH = 10 and next distilled water was added to the full volume of 25 ml. Then the solution was mixed thoroughly and the absorbance measurements (at the room temperature ∼20℃) were carried at 271 nm in 1.0 cm cuvette. EF concentration was calculated using the methods of single-point standardization.

General procedure of EF extraction-spectrophotometric determination with MR

Four milliliters of one acetate buffer mixture with pH 3.8 ((C(CH₃COONa) = 0.85 M, C(KCl)=2 M) was placed into a 25 ml volumetric flask. Then a sample of solution containing (2.5–25)·10⁻⁶ M of EF (in final volume) was added. Next 1 ml of 1.25·10⁻³ M MR solution was added into the flask. Next distilled water was added to the full volume of 25 ml. Then the solution was mixed thoroughly and three times extraction by 7 ml of chloroform was performed using separating funnel. The extract was collected into a 25 ml dry volumetric flask and subsequently the same solvent was added to the full volume of 25 ml. The extract was dried with anhydrous sodium sulfate during 5 min. Then the solution was mixed thoroughly and the absorbance measurements (at the room temperature ∼20℃) were carried out against chloroform extract of all corresponding reagents blank solution at 492 nm in 1.0 cm cuvettes. EF concentration was calculated using the methods of calibration curve and single-point standardization.

Results and discussion

To find effective analytic forms for the FQs determination, we studied their interaction with monoazo dye MR in aqueous solution depending on the pH. As follows from the experimental data, the orange precipitates for products of the MR interaction with all FQs at pH 3–4 have been formed, which most probably indicates the formation of ion-pair complex. This effect is promising for further research to develop the methods for FQs determination.

The choice of organic solvent for extraction of ion-pair complexes of FQs with MR

FQs have two proton bonded position (piperazine and carboxyl groups). In aqueous solutions they have four protonated forms (Guyot et al., 1995). Cationic form dominates at pH 4–5, zwitterion (neutral form) at pH 7–8, anionic form at a pH > 9–10 (Yang et al., 2012). Therefore, EF in acidic medium is a positively charged amino compound (pK_a2 = 8.51) (Yang et al., 2012). The intense absorbance maximum at pH = 3–4 for EF at λ = 277 nm as well as split maximum at λ = 317 and 329 nm are observed.

In aqueous solution MR is zwitterion and has two resonance structures (Tobey, 1958; Zhang et al., 2012):

Absorbance maximum for HMR (pK_a = 4.76) is at 520 nm, and for MR at 425 nm. The pH value range of color change of MR in aqueous solution is well known as 4.4–6.2. Therefore, at pH <4 MR has a negative charge due to the dissociation of the carboxyl group. The negatively charged indicator and positively charged FQ form ion-pair complex in acidic medium. BA-type electrolyte dissociates in an aqueous medium according to the equation BA ↔ B⁺+A⁻. The equilibrium of BA-type electrolyte dissociation may shift to the left (association) if ion-pair complex is removed by extraction using a solvent that does not mix with water (Mostafa et al., 2002a).

The next stage was to study the extraction of slightly soluble products of MR interaction with EF by the various organic solvents: chloroform, toluene, benzene, amyl alcohol, and hexane. In almost all cases, there was extraction of both the dye and the product. Optical characteristics of obtained extracts of ion-pair complex of MR with EF are listed in Table 2.

Table 2.

Optical characteristics of various extracts of enrofloxacin ion-pair complex with methyl red. C_MR = 5·10⁻⁵ M, C_FQ = 5·10⁻⁶ M, pH = 3.8.

Solvent	Toluene	Benzene	Hexane	Chloroform	Amyl alcohol
λ = 540 nm	0.236	0.205	0.013	0.322	0.629
λ = 490 nm	1.384	1.341	0.416	1.429	1.543

As shown by previous studies all these extracts absorb in the range 435–520 nm. The data, which are presented in Table 2, indicate that the optical characteristics of benzene, toluene, and chloroform extracts of FE ion-pair complex with MR are similar. All of these reagents are used for extraction, but absorbance of chloroform extract is somewhat greater, so we chose it for further research.

Figure 1 shows the spectra of chloroform extracts of the methyl red as well as product of its interaction with enrofloxacin at pH = 3.8.

Figure 1.

Absorption spectra of chloroform extracts of methyl red and product of its interaction with enrofloxacin at pH = 3.8. C_MR = 5·10⁻⁵ M, C_FQ = 6·10⁻⁶ M.

As follows from Figure 1 absorbance maxima at 285 and 490 nm are present on spectrum of chloroform extracts of MR as well as on spectrum of chloroform extracts of product of MR interaction with EF, but for the product they are much higher. Therefore, a further step of our research was to determine the linearity range for EF determination at the 492 nm and to select the buffer mixture for improvement of the analysis rapidity.

Selectivity of interaction in the system FQs – MR in the presence of sodium acetate and potassium chloride

In order to create stable conditions for the formation of ion-pair complexes, to maintain the optimum pH medium, and to improve the rapidity of the methods for FQs determination by MR it is necessary to pick up a buffer mixture. As the ion-pair complex is formed at pH 3–4, the acetate buffer can be used to achieve the optimal reaction conditions. Therefore, we examined the influence of the sodium acetate concentration on maximum yield of ion associate and its further extraction (Figure 2(a)). Better separation of liquid phases that do not mix is achieved in the presence of strong electrolytes, so we investigated the effect of potassium chloride concentration on the extraction efficiency and absorbance value of the obtained extracts (Figure 2(b)).

Figure 2.

Effect of potassium chloride (a) and sodium acetate (b) concentration in aqueous solution on the absorbance of chloroform extracts of the FQs ion associates with MR pH = 3.8, C_MR = 5·10⁻⁵ M, C_FQ = 5·10⁻⁶ M, l = 1 cm, λ_max = 540 nm.

As follows from Figure 2, the presence in solution of both sodium acetate and potassium chloride stabilizes the extractive system and increases the absorbance of ion associates extracts of all three FQs. In further studies we used acetate buffer solution.

In order to achieve the maximum value of the analytical signal, we determined the optimal amount of acetate buffer required for obtaining of the pH 3.8 (Figure 3).

Figure 3.

Effect of the volume of acetate buffer (C(CH₃COONa) = 0.85 M, C(KCl) = 2 M, pH = 3.8), on the absorbance of chloroform extracts of the FQs ion associates with MR, C_MR = 5·10⁻⁵ M, C_FQ = 5·10⁻⁶ M, l = 1 cm, λ_max = 540 nm.

As one can see from Figure 3, using 4–6 ml of acetate buffer with pH 3.8 the maximum absorbance of ion associates extracts of all FQs has been attained.

Validation and spectroscopic characteristics of the developed method of the FQs determination using MR

To develop the spectrophotometric method the absorbance time stability of analytic form is important. So we found that the absorbance of the chloroform extract of EF interaction product with MR is stable for 1 h (Table 3). This is long enough for the necessary measurements of absorbance and determination of analyte content.

Table 3.

Spectroscopic characteristics of the chloroform extract of ion pair complexes of the fluoroquinolones with methyl red and validation results of extraction-spectrophotometric determination of fluoroquinolones with MR; C_MR = 5·10⁻⁵ M, C(CH₃COONa) = 0.85 M, C(KCl) = 2 M, pH = 3.8, λ_max = 492 nm, l = 1 cm, n = 5, P = 0.95.

Parameters/characteristics	EF	NF	OF
λ_max (nm)	492
Stability (h)	1
Optimum photometric linear range, C_FQ·10⁶ (M)	2.5–25.0
Limit of detection·10⁶ (M)	2.48	3.07	3.17
Limit of quantification·10⁶ (M)	7.44	9.21	9.51
Molar absorptivity, ɛ₄₉₂·10⁻³ (M⁻¹cm⁻¹)	3.56	3.42	3.94
Regression equation (A)^a slope (b)	1851.84	2539.69	3401.19
Intercept (a)	0.0212	0.0113	−0.0011
Correlation coefficient (R)	0.9989	0.9979	0.9904
Standard deviation (S₀)	0.0009	0.0017	0.0049
S_a	0.0007	0.0013	0.0038
S_b	49.170	95.677	274.429

EF: enrofloxacin; NF: norfloxacin; OF: ofloxacin.

A = bc+a, where c is the concentration of fluoroquinolones in M.

It was established that the absorbance of the chloroform extract of FQs interaction product with MR linearly depends on FQ concentration in the solution. On the basis of the optimal conditions of FQ interaction with MR the new extraction-spectrophotometric technique of FQ determination has been developed. Validation and spectroscopic characteristics of determination of three FQs by means of MR are presented in Table 3.

As follows from Table 3 the developed methods for the extraction-spectrophotometric determination of all tested FQs with MR possess wide linear ranges; they are simple, rapid, and sensitive. Sensitiveness of EF determination was higher, for remainder FQ it was practically the same.

Approbation of the methods of extraction-spectrophotometric determination of EF with MR in drugs analysis

Proposed method of FQs determination by mean of MR was tested for quantification of EF in one-component drug, oral solution “Enroflox 10%” (INVESA, Spain) produced in 1 ml ampoules. Each ampoule contains 100 mg of active substance EF and excipients: benzyl alcohol (14 mg), potassium hydroxide, purified water. It is antimicrobial veterinary medicine for system use, predominantly for treatment and prevention of poultry diseases caused by bacterial flora, sensitive to EF: colibacillosis, salmonellosis, necrotic enteritis, streptococcosis, micoplasmosis, other diseases of inflectional etiology.

The assay results are presented in Table 4.

Table 4.

Determination of enrofloxacin in oral solution “Enroflox 10%” (INVESA, Spain). C_MR = 5·10⁻⁵ M, C(CH₃COONa) = 0.85 M, C(KCl) = 2 M, pH = 3.8, λ_max = 492 nm l = 1 cm, n = 5, P = 0.95).

Enrofloxacin (regulated content in preparation)	Amount of found drug $\bar{x} \pm Δ x$ and relative standard deviation (S_r)
	Spectrophotometric method by own absorbance at λ = 271 nm (pharmaceutical method)		Extraction-spectrophotometric with MR (proposed method)
	$\bar{x} \pm \frac{S \cdot t_{α}}{\sqrt{n}}$ ^a	S_r	$\bar{x} \pm \frac{S \cdot t_{α}}{\sqrt{n}}$	S_r
(100 ± 10 mg/ml⁻¹)	101.2 ± 1.15	0.009	99.8 ± 1.98	0.012

S is the standard deviation of five determinations (n). The tabulated value of t_α at α = 95% confidence limit is 2.78.

According to Table 4, the EF content in medicine obtained due to the developed technique using MR correlates rather well with the content of the EF specified by the manufacturer as well as with the results obtained according to the official methods of own absorbance in UV spectrum range at 271 nm.

For determination of the FQs in multicomponent drugs, use of the HPLC is recommended. However, the developed method is much simpler and it is characterized by high reproducibility and rapidity. Sensitivity of this technique is higher than in some other extraction-spectrophotometric methods of FQ determination. The value of S_r does not exceed common values of spectrophotometry errors.

Conclusion

It was established that orange color precipitates are formed during FQs interaction with MR at pH 3–4, which indicates the formation of ion pair complexes. The precipitates are extracted by some organic solvents, including chloroform. We have investigated optimum conditions to obtain proper pH value and constant ionic strength for the formation and the extraction of ion-pair complexes between FQs and MR depending on the concentration of acetate buffer and potassium chloride solutions.

It was found that the absorbance of ion-pair complex chloroform extracts is constant for more than 1 h. Effective molar absorbtivities of ion-pair complex chloroform extracts are ɛ₄₉₂∼3·10³ M⁻¹ cm⁻¹. New extraction-spectrophotometric methods for FQ determination with MR were developed on the basis of the optimum reaction conditions. Linearity range for the system FQ – MR is (2.5–25)·10⁻⁶ M (C(CH₃COONa) = 0.85 M, C(KCl) = 2 M, pH = 3.8, λ_max = 492 nm); limit of detection for EF is C_min = 2.48·10⁻⁶ M, for NF – C_min=3,07·10⁻⁶ M, for OF – C_min = 3,17·10⁻⁶ M.

The proposed methods of EF spectrophotometric determination with MR have been tested during the analysis of one-component veterinary drug. The obtained result correlates well with the official pharmaceutical methods, and the value of S_r does not exceed common values of spectrophotometry errors. Consequently, the elaboration method of extraction-photometric determination of FQs in the form of ion-pair complex with MR can be recommended for the determination of FQ in veterinary drugs.

Footnotes

Acknowledgement

The paper “Ion-pair complexes formation between fluoroquinolone antibiotics and methyl red and their use for the extraction-spectrophotometric analysis” was first presented at the 15th Ukrainian–Polish Symposium on Theoretical and Experimental Studies of Interfacial Phenomena and their Technological Applications, Lviv, Ukraine, 12–15 September 2016.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Belal

Al-Majid

Al-Obaid

(1999) Methods of analysis of 4-quinolone antibacterials. Talanta 50(4): 765–786.

El-Brashy

Metwally

ME-S

El-Sepai

(2004) Spectrophotometric determination of some fluoroquinolone antibacterials through charge-transfer and ion-pair complexation reactions. Bulletin of the Korean Chemical Society 25(3): 365–372.

El Khateeb

Razek

SAA

Amer

(1998) Stability-indicating methods for the spectrophotometric determination of norﬂoxacin. Journal of Pharmaceutical and Biomedical Analysis 17(4): 829–840.

García

Albero

Sánchez-Pedreño

et al. (2005) Flow injection spectrophotometric determination of oﬂoxacin in pharmaceuticals and urine. European Journal of Pharmaceutics and Biopharmaceutics 61(1): 87–93.

Guyot

Fawaz

Bildet

et al. (1995) Physicochemical characterization and dissolution of norfloxacin/cyclodextrin inclusion compounds and PEG solid dispersions. International Journal of Pharmaceutics 123(1): 53–63.

Melenteva GA and Antonova LA (1985) Pharmaceutical Chemistry. Moscow: Medicine. Available at: http://med-books.by/chimiya/3638-farmacevticheskaya-himiya-melenteva-ga-antonova-la-1985-god-480-s.html (accessed 20 March 2015).

Mostafa

El-Sadek

Alla

(2002a) Spectrophotometric determination of enroﬂoxacin and peﬂoxacin through ion-pair complex formation. Journal of Pharmaceutical and Biomedical Analysis 28(1): 173–180.

Mostafa

El-Sadek

Alla

(2002b) Spectrophotometric determination of ciproﬂoxacin, enroﬂoxacin and peﬂoxacin through charge transfer complex formation. Journal of Pharmaceutical and Biomedical Analysis 27(1–2): 133–142.

Nebsen

Elsayed Gh

Kawy

et al. (2013) Determination of oﬂoxacin and dexamethasone in dexaﬂox eye drops through different ratio spectra manipulating methods. Bulletin of Faculty of Pharmacy, Cairo University 51(2): 175–184.

10.

Wang

Kokot

(2008) Multicomponent kinetic spectrophotometric determination of peﬂoxacin and norﬂoxacin in pharmaceutical preparations and human plasma samples with the aid of chemometrics. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 70(5): 1049–1059.

11.

Ötker

Akmehmet-Balcıoğlu

(2005) Adsorption and degradation of enroﬂoxacin, a veterinary antibiotic on natural zeolite. Journal of Hazardous Materials 122(3): 251–258.

12.

Pan

Han

et al. (2012) Temperature dependence of oﬂoxacin ﬂuorescence quenching and complexation by Cu(II). Environmental Pollution 171(12): 168–173.

13.

Shchekina

(2007) Fluoroquinolones: Modern concept of application. Provisor 21: 19–22.

14.

Tobey

(1958) The acid dissociation constant of methyl red. A spectrophotometric measurement. Journal of Chemical Education 35(10): 514–520.

15.

Treskach

Kucherenko

(2004) General characteristic of derivatives of fluoroquinolones. Ukrainian Medical Almanac 7(5): 157–162.

16.

Uivarosi

(2013) Metal complexes of quinolone antibiotics and their applications: an update. Molecules 18(9): 11153–11197.

17.

Yang

Zheng

et al. (2012) Adsorption behaviour and mechanisms of norﬂoxacin onto porous and carbon nanotube resins. Chemical Engineering Journal 179: 112–118.

18.

Zhang

J-H

Liu

Chen

Y-M

et al. (2012) Determination of acid dissociation constant of Methyl Red by multi-peaks Gaussian fitting method based on UV-Visible absorption spectrum. Acta Physico-Chimica Sinica 28(5): 1030–1036.