INTRODUCTION:

Fluoroquinolones drugs, for example Ciprofloxacin CIP are synthetic antimicrobial drugs used to treat various infections. Ciprofloxacin is one of the fluoroquinolone second-generation drugs¹, whose chemical name is 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid².

Glucocorticoids are steroid drugs that have many uses such as anti-inflammatory effects, and Fluocinolone Acetonide FluA is one of them¹.

Fluocinolone Acetonide’s chemical name is (6α,11β,16α)-6,9-Difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]-pregna-1,4-diene-3,20-dione².

CIP and FluA is formulated as otic drops to treat acute otitis media caused by many bacteria, for example Staphylococus aureus, Streptococus pneumonia and Pseudomonas aeruginosa.

In the literature, many analytical methods were developed to determine CIP and FluA individually or with other drugs. For instance, determination of CIP in pharmaceutical preparation is performed using HPLC³, spectrophotometry^4-6, TLC^7,8, Electrochemistry^9-11. Determination of FluA in formulated products is carried out using HPLC¹², UV¹³, GC¹⁴, TLC¹⁵. There is only one work studied the simultaneously determination of CIP and FluA using a spectrophotometric method¹⁶. However, there is no Echo-Friendly chromatographic method developed to determine them simultaneously.

The aim of this study is to develop the first simple and rapid, Echo-Friendly isocratic HPLC method to determine both CIP and FluA simultaneously in bulk solutions and a new otic formulation.

EXPERIMENTAL:

MATERIALS:

Ciproﬂoxacin Hydrochloride (CIP) is obtained from AARTI drugs LTD, India. Fluocinolone Acetonide (FluA) is obtained from SWATI spentose Pvt. LTD, India. HPLC grade methanol was purchased from (Scharlau, Spain). Phosphoric acid was purchased from Surchem, UK. Heptanesulfonic acid was obtained from Tedia co., USA. Formulated Otovel® otic vials (batch number: 24338-080-14) is obtained from Arbor Pharmaceuticals, Barcelona (Spain).

Instrumentation:

A high performance liquid chromatography system (Shimadzu) equipped with a degasser (DGU 20 A3), a pump (LC-20AT), an autosampler (SIL-20A), an oven (CTO-20A), and Shimadzu photo diode array detector (SPD-M20A). Ultrasonic bath (Hwashin technology co., Korea). A Sartorius balance (BP221S d = 0.1 mg, Spain). pH meter (Crison, Spain).

Chromatographic Conditions:

For chromatographic analysis, a C18 column (EC 250/4.6 NUCLEODUR 100-5 C18 ec) was used. Separation was performed by isocratic elution. The mobile phase consists of 65:35 methanol: phosphate buffer (pH 3). The buffer contains 0.0125 M Heptanesulfonic acid as an ion- pair agent. Mobile phase was filtered under vacuum from 0.45 membrane filter and degassed in ultrasonic bath for 30 min before passing through the instrument. The injection volume was 10µl and the flow rate was 1ml/min. The analytes were measured with a UV-photo diode array detector at 239 nm. Chromatographic separations were carried out at 40˚C.

Preparation of Standard Solution:

Stock standard solutions of CIP and FluA were prepared in the mobile phase at a concentration of 1000µg/ml. Working standard solutions was prepared by dilution of stock solutions with the mobile phase.

Preparation of Sample Solution:

Sample solution of the otic drop were prepared in the mobile phase at a concentration of 84µg/mL and 7µg/mL for CIP and FluA, respectively by dilution of otic solution with the mobile phase.

Method Validation:

The developed analytical method was validated according to ICH guidelines¹⁴ for the following parameters: linearity, precision, specificity, accuracy, and system suitability.

Linearity:

Eight working standard solutions were prepared and injected three times on the same day and the concentrations were at range of 20 – 250µg/mL for CIP and of 2 – 600 µg/mL for FluA. Calibration curves where established by plotting peak area versus concentration.

Precision:

The precision was tested through intra and inter-day precision. For intra-day precision, three different concentrations of CIP and FluA within the linearity range were prepared and tested at the same day. For inter-day precision, three different concentrations of CIP and FluA within the linearity range were prepared and tested in two subsequent days.

Accuracy:

Accuracy of the method was established by performing recovery studies according to the ICH guidelines. Samples were performed by preparing solutions at three different concentration in triplicate. Mean percentage recovery values at three different concentrations of the two drugs were calculated.

Specificity:

Specificity of an analytical method may be defined as the ability of the method to measure accurately and specifically the analyte in presence of additional components such as matrix, impurities, degradation products and other related substances. Sample solution with known concentration containing all excipients present in the formulation were injected into the system and chromatogram was recorded.

System Suitability:

System suitability are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. Suitability parameters, such as retention time, resolution factor and theoretical plates number were investigated.

RESULTS AND DISCUSSION:

Scanning of ultraviolet absorption of the two drugs:

The UV-spectrum of CIP (Figure 1) and FluA (Figure 2) were obtained and the lambda max of CIP and FluA were found to be 278nm and 239nm, respectively. However, selected wavelength was 239nm for both CIP and FluA because it gave more for the minor component FluA. The chromatogram of mixed CIP and FluA solution were determined at 239nm (figure 3).

Mobile Phase Optimization:

Chromatographic conditions were optimized to obtain a short HPLC analysis time (t_R < 10 min) and resolution (R_s > 2) for simultaneous determination of CIP and FluA. Various ratios of methanol, acetonitrile and water were tried in mobile phase optimization. The optimum conditions were obtained when water/methanol ratio was 35/65, the water part contains phosphate buffer (pH 3) and Heptanesulfonic acid (0.0125M), oven temperature was 40˚ and flow rate was 1mL/min. The retention times were 3.1 and 6.8 for CIP and FluA, respectively. The detection wavelengths were.

Validation:

Calibration curves were constructed by plotting the peak area versus their corresponding concentrations. Linearity was in the range of 20 – 250 µg/mL (R² = 0.9996) and 2.5-600 µg/mL (R² = 0.9998) for CIP and FluA, respectively. The results are presented in table 1. The method’s precision was evaluated by calculation of relative standard deviation (RSD) values. The %RSD values have shown that the precision of the method was within the limits (%RSD < 1 for intra-day precision and %RSD < 2 for inter-day precision). The results are shown in table 2. The accuracy of the method was determined by recovery studies. The recovery values are closed to 100% within the range of 98-102% for CIP and FluA. The recovery values are shown in table 3. System suitability parameters are shown in table 4.

Figure 1: FluA UV-spectrum.

Table 1: Linearity by regression analysis (n=6).

Substance	R2	Slope	B	Conc. Range (µg/mL)
CIP	0.9996	13338	-33340	20 – 250
FluA	0.9998	23545	-9222	2.5 – 600

Figure 2: CIP UV-spectrum.

Figure 3: Peak 1 (left peak) for CIP at 3.1 min and peak 2 (right peak) for FluA at 6.8 min.

Table 2. Intra-day and inter-day precision.

Parameter	Conc. Range (µg/mL)	% RSD (Intra-day N=3)	% RSD (inter-day N=6)
CIP	50	0.74	0.83
	120	0.55	0.94
	250	0.27	0.43
FluA	5	0.31	0.48
	100	0.03	1.50
	400	0.04	0.06

Table 3. recovery studies.

Substance	Conc. Range (µg/mL)	Amount recovered. Range (µg/mL)	% recovery	% RSD
CIP	50	50.72	101.44	0.74
	120	118.98	99.15	0.75
	250	251.15	100.46	0.27
FluA	5	5.09	101.80	0.38
	100	99.50	99.50	0.70
	400	399.60	99.90	0.25

Table 4. System suitability parameters (n=5).

Parameter	CIP	FluA
Retention Time (min)	3.18	6.82
Resolution (RS)	-	6.77
Tailing Factor (T)	1.16	1.14
Capacity Factor (k’)	11.95	89.35
Number of Theoretical plates (N)	3407	6750
Precision (%RSD)	0.60	0.91

Assay of Otic Drop:

According to ICH in terms of of assay, demonstration of specificity requires that the procedure is unaffected by the presence of impurities or excipients. The assay value of the formulated product was found to be within the limits. Chromatogram of the sample shows that there was no overlap from the excipients present in the formulation (Fig. 2), and the peak purity profile shows that there was no impurity for each peak; this indicates the specificity of the method. The results are shown in table 5.

Figure 4: Recorded chromatogram for the sample solution of CIP and FluA at 239 nm.

Table 5. Assay of Otic Drop (n=5).

Drug	Label claim (µg/mL)	Amount found (µg/mL)	Mean %recovery	Relative standard deviation (%RSD)
CIP	84	85.59	101.89	0.73
FluA	7	7.12	101.71	0.49

CONCLUSION:

In the present work, the first and the only Echo-Friendly reversed phased HPLC method has been developed and validated for simultaneous estimation of CIP and FluA. It makes use of fewer amounts of solvents so it is echo friendly and change of set condition requires in a short time. This method is simple, rapid and sensitive, and can be suitable analyzed for the routine analysis of CIP and FluA in bulk and its otic dosage form.

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Received on 14.07.2019 Modified on 18.08.2019

Research J. Pharm. and Tech 2020; 13(2):715-718.

DOI: 10.5958/0974-360X.2020.00136.5