INTRODUCTION:

Ciprofloxacin (CFN) chemically^1-3 is 1-Cyclopropyl-6-fluoro-1, 4 dihydro-4-oxo-7-(piperazin-1-yl) quinoline-3-carboxylic acid. Ciprofloxacin is second generation fluoroquinolone that has spawned many derivative antibiotics; it inhibits enzymes topoisomerase II and IV which are required for bacterial DNA replication, transcription, repair and recombination. It is formulated in oral dosage form and injectable form. Various analytical methods have been reported for estimation of CFN such as UV spectrophotometric method^4-9, potentiometric method^10,11, RP-HPLC¹², Bio analytical LC¹³ and LC-MS/MS¹⁴ alone or in combination with other drugs.

Metronidazole (MND) chemically 2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethanol^1-3is frequently used to treat gastrointestinal infections as well as trichomoniasis and giardiasis and amoebiasis, which are parasitic infections. It is formulated as tablets, injectable and topical preparations. It acts by inhibiting nucleic acid synthesis by disrupting the DNA of microbial cells. For estimation of MND UV spectrophotometric method^15-22, Novel spectrophotometric methods²³ alone or in combination with other drugs have been reported.

Both drugs are official in British Pharmacopoeia²⁴and Indian Pharmacopoeia²⁵. Chemical structures of both drugs are shown in (Fig. No. 1).

Fig No 1: Chemical structure of Drug molecule

Quality by design concept is applied for the development of pharmaceutical processes to assure a predefined product quality. QBD concepts are mentioned in ICH guidelines Q8 (R1) (Pharmaceutical development), Q9 (Quality risk management), and Q10 (Pharmaceutical quality system)^26-28 shown in Fig No 2. ICH guidelines Q8 (R2) defines QBD as a “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”²⁹. QBD approach in analytical method summarizes a complete understanding of how the analytical technique attributes and operating conditions affect the analytical performance. Factors to study in analytical quality by design (AQbD) approach may include the type of analytical technique chosen, reagents used and instrument parameters.

Fig. No. 2: Analytical QbD approach

There are similar advantages of applying QbD principles to analytical methods as to manufacturing processes and product³⁰. A QbD approach can be beneficial in the development of suitable, robust, low cost and eco-friendly (eco-friendly solvent, chemicals) method which is applicable at any stage of the lifecycle of the product. Also some regulatory guidelines have mentioned flexibility of changing analytical method without revalidation if the AQbD approach has been implemented during analytical method development. The first stage of AQbD approach is to fix an analytical target profile (ATP) for the method. ATP defines the goal of the analytical method development process and it is the sign of method performance^31,32. For analytical method validation ICH Q2 (R1) has given various method performance characteristics for an analytical method. Thus a QbD based UV spectrophotometric was developed, QbD approach was implemented with the study of the effect of method input variables on spectral shape, intensity of absorbance, and absorbance maxima λ_max and critical parameters were selected for the proposed method and method was validated as per ICH guidelines Q2 (R1).

MATERIALS AND METHODS:

Instrumentation:

Analysis was performed with a Shimadzu Double beam UV-Visible spectrophotometer (Shimadzu, Kyoto, Japan) with spectral bandwidth of 2nm and wavelength accuracy of ± 1nm with 10mm matched Quartz cells was used. Electronic balance Afcoset balance (The Bombay Burmah Trading corpo Ltd) with accuracy ±0.1mg Model No. ER 200A was utilised for weighing; and for degassing the solution, Digital Ultrasonic cleaner 1.8Ltr (Labman scientific Instruments Chennai) was used.

Reagents and Chemicals:

Pharmaceutically pure samples of CFN and MND from Cure Medicines, Pune Maharashtra were procured as a gift samples and the commercial formulation containing Ciprofloxacin 500mg and Metronidazole 300mg was procured from the local market.

AQbD approach application in method development:

AQbD approach was applied to study the influence of input variable parameters on spectrophotometric analytical method performance shown in (Fig No 3).

Fig No 3: Diagram showing the relationship between input variable parameters and the spectrophotometric method performance characteristics

Solvent selection:

CFN is freely soluble in acetic acid, soluble in water, methanol and ethanol; whereas MND is soluble in water, methanol, ethanol, DMSO and in dilute acids. Although the solubility of the procured drugs were studied in methanol, 0.1 N HCl and 0.1 N NaOH separately; and found that in 0.1 N NaOH both drugs were insoluble. Each solution with known conc of analyte was scanned in UV range of 200nm to 400nm. The recorded spectra in solvent are shown in Fig No 4, 5 and 6. It was found that suitable solvent is D/W with respect to low cost, robust and precise in producing result.

Fig. No. 4: UV-VIS spectrum of CFN in 0.1 N HCl

Fig. No. 5: UV-VIS spectrum of CFN in D/W

Fig. No. 6: UV-VIS spectrum of CFN and MND in 0.1 N HCl

Preparation of stock solutions and standard solutions:

10mg each of drug CFN and MND were separately and accurately weighed; and transferred into separate 50ml volumetric flask. Dissolved into distilled water and volume was made to 50ml with solvent. Subsequent standard solution of each drug with conc 10μg/ml was prepared by diluting aliquot 0.5ml of stock solution to 10 ml into 10ml capacity volumetric flask.

Selection of wavelength and conc range:

From UV spectra it was found that CFN has measurable absorbance at 271.5nm and less interference was observed by MND; similarly, MND has maximum absorbance at 319nm and negligible interference by CFN was accounted. Chemometric method using simultaneous equation method was applied and which was reasonable remedy to overcome interference at each other’s absorbance. From the nature of spectra to study linearity, working conc range 2 to 14μg/ml for CFN and 2 to 14μg/ml for MND was selected. Also combined drug solution was prepared simulated to marketed formulation. Selected critical parameters based upon above discussion, observations were listed in Table No. 1 and by using these; method was validated as per ICH guidelines and by analysing marketed preparations.

Table No. 1: Selected critical parameter for UV-VIS analytical method of CFN and MND

Parameter	Selected variables for simultaneous equation method CFN MND
Wavelength	271.5	319
Solvent	Distilled Water	Distilled Water
Scan speed	Fast	Fast
Sampling interval	0.2 nm	0.2 nm

Experimental Method for estimation:

From the overlain spectra simultaneous equation method was applicable for estimation of both the analytes from the combined dosage form.

Method for estimation of both the drugs by Simultaneous equation method:

CFN was shown maximum absorbance i.e. λ_maxat 271.5 nm where less interference by MND found and MND has maximum absorbance i.e. λ_maxat 319 nm where measurable interference by CFN observed. Hence these two wavelengths 271.5 and 319 nm were considered as 1 and 2 respectively for the said method. The equation A= abc was applied for x (CFN) and y (MND) determination. Working standard solutions of CFN and MND containing 10 mcg/ml conc were separately prepared and used for the method.

A2.ay1 – A1.ay2

Cx = ––––––––––––––––––

ax2.ay1 – ax1.ay2

A1.ax2 – A2.ax1

C_Y = ––––––––––––––––––

ax2.ay1 – ax1.ay2

Where

C _X= conc of CFN in sample solution

A₁and A₂ = absorbance of sample solution at 1 and 2 wavelength

ay₁anday₂= absorptivity of MND at 1 and 2 wavelength of standard solution

ax₁andax₂= absorptivity of CFN at 1 and 2 wavelength of standard solution

C_y= conc of MND in sample solution

Validation of the Method:

Selected critical parameters should meet the performance characteristics of the analytical method so as to attain analytical target profile of the method. An ICH guideline Q2 R1 was applied to study methods performance with critical parameters in order to implement AQbD approach. The method was validated as per ICH guidelines

System suitability:

System suitability is studied to demonstrate the suitability of the developed procedure under consideration for the analytical method. Six replicates of working standard solutions with conc10mcg/ml each of CFN and MND were prepared separately and absorbance was recorded, and SD and %RSD of the response was calculated.

Linearity:

The linearity of an analytical method is its ability to obtain response i.e. absorbance which is directly proportional to the conc of analyte. Series of working standard solutions were prepared in conc. range of 2 - 14μg/ml for CFN and 2-14μg/ml for MND and scanned in 200 to 400nm range in spectrum mode of the spectrophotometer, absorbance of the standard solutions were recorded at their respective wavelength; i.e. 271.5 for CFN and 319nm for MND in spectrum order. Microsoft office excel software tool was used to obtain the standard regression curve and its analysis as slope, intercept, and correlation coefficient.

Assay of formulation:

Assay was carried out by proposed methods and assay was validated by statistical parameters.

Estimation of formulations by simultaneous equation method:

Tablet powder equivalent to 25mg CFN and 15mg MND was weighed and transferred into 50ml volumetric flask. Dissolved into D/W and volume was made to 50ml with solvent. Solution was filtered through what man filter paper and aliquots of solution were further diluted to obtain tablet solution. Solution was scanned in the range of 200 to 400nm to obtain absorbance of tablet solution at 271.5 and 319nm in spectrum order. Obtained absorbance were utilised to estimate unknown conc of formulation; and results were statistically validated to obtain % of nominal conc, standard deviation and % of RSD.

Accuracy and Precision:

The accuracy of an analytical method expresses the closeness of an agreement between test result and true result. Accuracy study was performed by recovery study i.e. standard addition method; diluted standard solutions of CFN and MND were prepared and standard solutions added in 80, 100 and 120% proportionate to the tablet solution. Three replicates at each of these three levels were prepared and measured and % of conc, SD and RSD were calculated.

The precision study was carried out by performing assay of tablet six times; also the reproducibility in result was studied by inter day and intraday precision.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ of CFN and MND by the proposed method were determined using calibration graph method and calculated as 3.3σ/s and 10σ/s for LOD and LOQ respectively; σ is the standard deviation of calibration curve and s is the slope of regression line.

Robustness and Ruggedness:

It is measure of capacity of analytical procedure to remain unaffected by small but deliberate variations in method parameter.

RESULTS AND DISCUSSION:

Method development comprises numerous steps, and of which solvent selection, selection of method for measurement are significant one. Uses of aqueous solvents, eco-friendly solvents like hydrotropic have got remarkable weightage due to low cost, readily available and environmentally sound. Drugs underlying analysis must have appreciable solubility in the selected solvent. Chemical structure of the drug and physico-chemical properties available in the literature guides about use of appropriate solvent in the method.

From UV spectra two wavelengths were selected as 271.5nm (λ_maxof CFN) and 319nm (λ_max of MND) for calculation of both drugs in combined solution shown in Fig No. 7 and 8.

Fig. No. 7: Spectra of MND in D/W

Fig. No. 8: UV-VIS overlain spectrum of CFN and MND in D/W

System Suitability:

The absorbances of six replicates of standard solutions (10mcg/ml) are reported in Table No 2. The SD and % RSD was found for CFN and MND and meets the system suitability requirements indicates method was suitable for analysis.

Table No. 2: System suitability study of CFN and MND

Sr. No.	Conc in mcg/ml	Absorbance of CFN	Absorbance of MND
1	10 mcg/ml	1.083	0.668
2	10 mcg/ml	1.066	0.680
3	10 mcg/ml	1.115	0.690
4	10 mcg/ml	1.068	0.690
5	10 mcg/ml	1.064	0.637
6	10 mcg/ml	1.105	0.638
	SD RSD	0.44551 0.5641	0.0632 0.4963

Linearity:

The calibration curve of both drugs was found to be linear in the conc range of 2-14μg/ml for CFN and 2 – 14μg/ml for MND as shown in Fig No 9. The regression equation of line and its parameters slope, r²value and intercept are tabulated in Table No 3, which proved the linear relationship between conc and obtained response.

Fig. No. 9: Calibration curve of CFN and MND in D/W solvent

Table No. 3: Parameters of regression equation obtained in Microsoft excel

Parameters	CFN	MND
Detection wavelength	271.5	319
Beer’s law limit (μg/ml)	2 – 14 mcg/ml	2 – 14 mcg/ml
Correlation coefficient (r²)	0.9992	0.9901
Regression equation (y = mx + c)	Y = 0.102 X - 0.015	Y = 0.014 X + 0.012

Assay:

The assay was carried out by the proposed method. The spectrum of formulation by method was shown in Fig No 10. The assay of formulation was carried out by proposed method and calculated % of nominal conc and RSD was found within acceptable limits are summarized in Table No 4. The results indicated applicability of the method for estimation of formulation.

Fig. No. 10: Spectra of formulation obtained in the assay

Accuracy and Precision:

The results of accuracy are summarised in Table No 5, the obtained results were within acceptable limit; and methods accuracy was justified by calculating % drug content.

The precision study was carried out by performing assay of solutions; further the reproducibility in result was studied by interday and intraday precision. The values obtained SD and % RSD was shown methods precision and are summarised in Table No 5.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ of CFN and MND by the proposed method were shown in Table No 6.

Robustness and Ruggedness:

Robustness was studied and capacity of analytical procedure to measure analyte was remain unaffected by small but deliberate variations in method parameter. The analytical method was found rugged during development; similarity the result was produced by performing the analysis by different analyst.

Table No. 5: Results of accuracy and precision

S. No.	Parameter	Level of study	Drug Name	S.D.	% RSD
1 2	Precision	Intraday Precision	CFN	0.00246	RSD = 0.2226
		Intraday Precision	MND	0.0057	RSD = 0.8333
		Inter day precision	CFN	0.04803	RSD = 0.0459
		Inter day precision	MND	0.0001	RSD = 0.1572
	Accuracy study of CFN and MND	80%	CFN	1.01725	0.9766
		100%		0.6369	0.6009
		120%		1.3192	0.0134
		80%	MND	0.9429	0.9130
		100%		1.2171	1.2061
		120%		0.5509	0.5117

Table No. 6: Results of LOD and LOQ, robustness

Parameters		CFN	MND
LOD mcg/ml		0.0842	0.0839
LOQ mcg/ml		0.2554	0.2544
Robustness	(conc 2 mcg/ml)	0.213-0.214 (± 2 nm)	0.162 - 0.163 (± 2nm)
Ruggedness	Analyst 1	SD ± 0.06506 RSD ± 0.3031	SD ± 0.00145 RSD ± 0.89506
Ruggedness	Analyst 2	SD ± 0.00246 RSD ± 0.2226	SD = 0.0056 RSD ± 0.8338

CONCLUSION:

Both the drugs were estimated from the combined formulation by simultaneous equation method. Results were found within acceptable limits, statistical data obtained were shown rigidity of the method. The validated method was employed water as solvent thus become economical. The proposed method is precise, accurate, robust and reproducible hence can be routinely used for simultaneous estimation of ciprofloxacin and metronidazole from combined dosage form.

CONFLICT OF INTEREST:

All Authors declared that there is no conflict of interest

ACKNOWLEDGEMENT:

Authors are thankful to Cure Medicines Pvt Ltd Pune for providing Ciprofloxacin hydrochloride; and metronidazole as a gift sample. Authors are thankful to Principal and Management SVPM’S college of Pharmacy Malegaon (BKII) Dist. Pune for providing necessary facilities for research.

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Received on 10.01.2022 Modified on 07.04.2022

Asian J. Pharm. Res. 2022; 12(3):185-191.

DOI: 10.52711/2231-5691.2022.00030