INTRODUCTION:

Antibiotic resistance is a prevailing issue globally, particularly among ocular pathogens¹. Quorum sensing (QS) is the communication mechanism of bacteria that led to the development of biofilm, which is the significant reason for antibiotic resistance in ocular infections as it hinders the penetration of antibacterial specialists to treat infections². On the basis of various kinds of literature, it was concluded that the combination of antibacterial agents and Quorum sensing inhibitors (QSIs) will be a remarkable strategy to overcome this problem as QSI will hinder the further development of biofilm, and simultaneously antibacterial agents will cure the infection^3,4. This theory propels to conduct research on the development and validation of a reasonable new analytical technique for the determination of quercetin (QRT), a naturally occurring flavonoid that serves as a QSI⁵, and ciprofloxacin HCl (CIP HCl)⁶, an antibacterial expert of the fluoroquinolone class that is compelling against both gram-positive and negative pathogens and works by repressing the action of enzymes such as DNA gyrase, topoisomerase-II and IV. Technically speaking, CIP HCl is likewise alluded to as 1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-7 (1-piperazinyl)-3-quinoline carboxylic acid hydrochloride. A formulation containing both CIP HCl and QRT must be developed, optimized for their synergistic efficacy, and its drug contents must be examined in light of the aforementioned fact. An examination of the literature revealed that HPTLC^7–9, HPLC^6,10, and UV-spectrophotometric techniques¹¹ can all be used to investigate CIP HCl and QRT when taken alone or in combination with other treatments in various pharmacological frameworks. According to our knowledge, there is currently no technology that can estimate both medications' dosages concurrently. Two simple UV-spectrophotometric methods have been effectively applied in the current study to simultaneously quantify both of these medications. These processes were validated using the ICH recommendations.

MATERIALS AND METHOD:

Instrument-

UV-Vis Spectrophotometer whose scope of frequency is from 190 nm to1100 nm, model -Shimadzu UV-1800, was utilized with sets of Quartz cuvettes of 10 mm path length for investigation.

Chemicals, reagents, and drug samples-CIP HCl and QRT were bought from Himedia Ltd., Mumbai. Any remaining synthetics and reagents utilized for test work were of AR grade.

The procedure of stock solution and calibration curve preparation-

1 mg of each CIP HCl and QRT drug sample was dissolved separately in 10 ml of previously prepared STF to acquire stock solutions of 100 µg/ml concentration of both drug samples. Working dilutions of drugs were prepared in convergences of 1 µg/ml to 10 µg/ml from the stock solution of 100 µg/ml. To decide the particular λmax of both analytes, 5µg/ml dilution of each was examined in the whole UV range of 400 to 200 nm. 271 nm is determined as λmax of CIP HCl and 327 nm of QRT. Apparently from the overlain spectra, 283 nm is the isosbestic point. Calibration curves were plotted at these wavelengths.

Method development-

I- Simultaneous equation method

This method is based on the absorptivity of drugs^12,13, CIP HCl and QRT at their absorption maxima, and from overlain spectra, it is evident that the λmax of one as well as other drugs was at 271nm (λ₁) and 327 nm (λ₂) respectively. The absorbance of each dilution of concentration 1-10 µg/ml was recorded and absorptivity of the same was determined by dividing absorbance by concentration and taking an average of ten estimations for both analytes. For quantitative estimation, the absorptivity, ax1and ax2 for CIP HCl were found to be 0.188 and 0.070 respectively and ay1 and ay2 for QRT were 0.111 and 0.119 respectively at absorption maxima of each other (λ₁ and λ₂). Further equations 1and2 were utilized to estimate drug concentration Cx= CIP HCl and Cy= QRT in their combination, where A₁ and A₂ are the absorbances of sample solutions at 271nm and 327nm respectively.

Cx= A₂(0.111)-A₁(0.119)/-0.0146 (1)

Cy= A₁(0.070)-A₂(0.188)/-0.0146 (2)

II- Absorbance ratio method

This strategy for synchronous assessment of two drugs, which is based on the fact that at any two wavelengths, the ratio of absorbances was found to be constant, without being dependent on concentration, is utilized^12,14. In this technique, two wavelengths were chosen for utilizing this strategy for the development of equations 3 and 4 known as Q absorbance equations in which one is an isosbestic point, at 283 nm as evident in figure 05 and the other is the λmax of one of the two components, i.e. 271 nm, λmax of CIP HCl. The drug concentration of CIP HCl and QRT (Cxand Cy respectively) were estimated by substituting the values of absorbances of both analytes at λmax 271 nm and 283 nm in equations5and6 in which the determining absorptivity values for CIP HCl, ax1and ax2 were 0.188 and 0.088 respectively and for QRT, ay1 and ay2 were 0.111 and 0.109 respectively.

Cx= (Q_M – Q_Y) / (Q_X - Q_Y) × A₁/ ax1 (3)

Cy= (Q_M – Q_X) / (Q_Y – Q_X) × A₁/ ay1 (4)

Where,

Q_M = A₂ / A₁; A₁ and A₂were the absorbances of the analyte at 271nm and 283nm respectively.

Q_X = ax2 / ax1= 0.468; Q_Y = ay2/ay1= 0.981

Substituting these values in equations 3and 4, we get equations 5 and 6:

Cx= (Q_M – 0.981) / (-0.513) × A₁/ 0.188 (5)

Cy= (Q_M – 0.468) / (0.513) × A₁/ 0.111 (6)

Method validation-

Q2AandB ICH guidelines^15,16are the basis of validation of two developed methods, I and II i.e. simultaneous equation method and absorbance ratio method¹⁷. The methods are validated according to the following parameters:

(i) Specificity and Selectivity: Solutions containing CIP HCl and QRT were scanned between the range of 400-200 nm and the spectrum was obtained. A spectrum of blank solvent was obtained and compared for any interference at the maximum wavelength of absorbance of analytes.

(ii) Linearity:As per the developed methods, appropriate dilutions of standard stock solutions of CIP HCl and QRT were assayed for their linearity data as compiled in table 01. The Beer- Lambert’s concentration range was found to be 1-10 µg/ml.

(iii) Limit of Detection (LOD) and Quantification (LOQ):The LOD and LOQ of CIP HCl and QRT were calculated by using the formula, LOD = 3.3s/ S; LOQ = 10s/ S, and reported in table 01, where, s is the standard deviations of the y-intercept and S is the Slope of the calibration curve of both analytes (figure 4, 5, 6 and 7).

(iv) Precision:To determine how precise the developed method is, the assay of dilutions of CIP HCl and QRT on different days (inter-day) and on the same day at different time spans (intra- day) was conducted (table 03, 04,). These precision studies followed by the calculation of % RSD are compiled in table 01.

(v) Accuracy:The accuracy as per ICH guidelines Q2(R1), of the recommended methods, was examined by the assay of the investigating solutions of the CIP HCl and QRT at levels of 80, 100, and 120 % after the addition of the known amount of standard solution and then determines the recovery percentage of the analyte reported in table 02 by the following formula-

% recovery= amount found/ amount added × 100

RESULT AND DISCUSSION:

Method development:In order to determine CIP HCl and QRT in their pure powder or combination delivery framework, a simultaneous equation method and absorbance ratio method based on UV Spectrophotometric methodology were developed. STF was used as the solvent system. λmax of CIP HCl was found to be at 271nm and QRT at 327nm.

Method Validation:All the statistical parameters of method validation are displayed in table 01.

Table 01: The statistical parameters of method validation for simultaneous estimation of CIP HCl and QRT

S. No.	PARAMETERS		Method I		Method II
S. No.	PARAMETERS		CIP HCl	QRT	CIP HCl	QRT
1.	Absorption maxima (λmax)		271 nm	327 nm	Isosbestic point at 283 nm
2.	Linearity Range (µg/ml)		1-10	1-10	1-10	1-10
3.	Correlation coefficient (R²)		0.9992	0.9973	0.9976	0.9976
4.	Regression equation (y)		Y= 0.1861x+ 0.0066	Y= 0.1166x+ 0.0096	Y= 0.0842x+ 0.0159	Y= 0.097x+ 0.0425
5.	Slope value (m)		0.1861	0.1166	0.0842	0.097
6.	Standard deviation of Intercept (c)		0.0066	0.0096	0.0159	0.0425
7.	Accuracy (%Recovery)		98.62-101.15	99.34-100.94	99.63-104.62	101.23-102.64
8.	Precision (%RSD)	Intra-day	0.91-1.63	0.83-1.39	0.88-1.72	0.73-1.02
8.	Precision (%RSD)	Inter-day	0.63-1.01	0.8-1.62	0.7-1.91	1.04-1.23
9.	Limit of Detection (LoD) (µg/ml)		0.9	1.73	1.65	1.5
10.	Limit of Quantification (LoQ) (µg/ml)		2.842	5.248	5	4.7

(i) Specificity and Selectivity:When CIP HCl and QRT were measured at their maximum wavelengths, the solvent's spectrum revealed no interference of absorbance, and both analytes exclusively displayed their maximum wavelengths at 271 nm and 327nm, respectively. As a result, the approach was found to be selective and focused. Figures 1 and 2 depict the UV spectrums of the drugs CIP HCl and QRT, respectively. The isosbestic UV-Spectrum of the two analytes is depicted in Figure 3.

Figure 01: UV Spectrum of CIP HCl in STF Figure 02: UV Spectrum of QRT in STF

Figure 03: Overlain spectra of CIP HCl and QRT

(ii) Linearity: Utilizing the concentration vs. absorbance data generated by each linear dilution of both analytes, thecalibration curve was plotted. Each concentration displayed a linear absorbance range between 1-10 µg/mL, with a correlation coefficient for CIP HCl and QRT, respectively, of 0.9992 and 0.9973 (Figs. 4 and 5). Figures 6 and 7 displayed the calibration curves of the CIP HCl and QRT linearity spectra at 283 nm (isosbestic point) withcorrelation coefficients of 0.9976 and 0.9978 respectively.

Figure 04: Calibration curve of CIP HCl in STF at 271 nm Figure 05: Calibration curve of QRT in STF at 327 nm

Figure 06: Calibration curve of CIP HCl in STF Figure 07: Calibration curve of QRT in STF

at 283 nm (isosbestic point) at 283 nm (isosbestic point)

(iii) Limit of Detection (LOD) and Quantification (LOQ): CIP HCl and QRT have LOD and LOQ values of 0.194 and 0.352 µg/ml and 0.832 and 2.386 µg/ml, respectively by a method I, and 0.742, 0.486, and 1.48, 3.27 µg/ml, respectively, by method II are displayed in Table 01.

(iv) Precision: Studying repeatability and intermediate precision allowed for the determination of precision. The method was determined to be accurate because, as shown in table 01, the %RSD calculated for five replicates of solutions for both analytes at each precision level was discovered to be less than 2%. The% RSD for CIP HCl and QRT was computed. Tables 2 and 3 list the outcomes of experiments conducted by methods I and II using both analytes over a short period of time. Tables 4 and 5 contain data from intermediate precision research that expresses laboratory variance on various days. Studies on intra- and inter-day precision for both approaches show that good repeatability and intermediate accuracy are indicated when the RSD is less than 2.0%.

Table 02: Intraday precision data of CIP HCl and QRT (Method-I)

Concentration (µg/ml)	1^st hour		2^nd hour		3^rd hour
Concentration (µg/ml)	CIP HCl	QRT	CIP HCl	QRT	CIP HCl	QRT
5	4.924234	4.866209	5.020956	4.385935	4.918861	4.394511
5	4.951102	4.789022	5.037077	4.445969	4.913487	4.454545
5	5.04245	4.763293	5.128426	4.428816	4.74691	4.497427
5	5.101558	4.797599	5.112305	4.497427	4.800645	4.403087
5	5.047824	4.831904	5.074691	4.463122	4.908114	4.540309
%RSD	1.47	0.83	0.91	0.93	1.63	1.39

Table 03: Intraday precision data of CIP HCl and QRT (Method-II)

Concentration (µg/ml)	1^st hour		2^nd hour		3^rd hour
Concentration (µg/ml)	CIP HCl	QRT	CIP HCl	QRT	CIP HCl	QRT
5	5.155582	4.93617	5.286223	4.976697	5.06057	4.956434
5	5.191211	4.895643	5.26247	5.057751	5.143705	5.027356
5	5.226841	4.915907	5.167458	4.946302	5.167458	4.946302
5	5.2981	4.905775	5.309976	4.956434	5.0962	5.007092
5	5.381235	4.986829	5.191211	4.926039	5.155582	5.027356
%RSD	1.72	0.73	1.17	1.02	0.88	0.78

Table 04: Interday precision data of CIP HCl and QRT (Method-I)

Concentration (µg/ml)	Day 1		Day 2		Day 3
Concentration (µg/ml)	CIP HCl	QRT	CIP HCl	QRT	CIP HCl	QRT
4	4	4.025729	4.166577	4.085763	4.225685	4.180103
4	4.037614	3.965695	4.268673	4.120069	4.268673	4.077187
4	4.059108	4.085763	4.263299	4.16295	4.29554	4.137221
4	4.075228	4.128645	4.204191	4.09434	4.252552	4.111492
4	4.026867	4.102916	4.225685	4.085763	4.236432	4.128645
%RSD	0.72	1.62	1.01	0.8	0.63	0.91

Table 05: Interday precision data of CIP HCl and QRT (Method-II)

Concentration (µg/ml)	Day 1		Day 2		Day 3
Concentration (µg/ml)	CIP HCl	QRT	CIP HCl	QRT	CIP HCl	QRT
4	4.027316	4.225386	4.300475	4.367734	4.312352	4.782918
4	3.991686	4.177936	4.336105	4.403321	4.336105	4.652432
4	4.039192	4.284698	4.371734	4.462633	4.395487	4.711744
4	3.991686	4.260973	4.312352	4.415184	4.359857	4.759193
4	3.849169	4.189798	4.300475	4.344009	4.21734	4.794781
%RSD	1.91	1.08	0.7	1.04	1.55	1.23

(v) Accuracy: In the recovery study level, recovery experiments of the test concentration were carried out three times at each site to assess the accuracy of the recommended procedures. The outcomes of the recovery studies are listed in Table 6. Based on the mean percent drug estimated, it was decided that the suggested analytical method's accuracy was sufficient (Table 1).

Table 06: Result of recovery study

Drug	Level of recovery (%)	Method-I				Method- II
Drug	Level of recovery (%)	Amount of drug added (µg/ml)	Recovery (%)	SD	RSD (%)	Amount of drug added (µg/ml)	Recovery (%)	SD	RSD (%)
CIP HCl	80	2	98.62	0.37	0.38	2	99.63	1.59	1.60
	100	4	101.15	0.46	0.46	4	104.62	1.41	1.36
	120	6	100.46	0.70	0.71	6	102.53	1.22	1.19
QRT	80	2	99.34	1.26	1.27	2	102.64	0.99	0.97
	100	4	99.93	1.56	1.57	4	102.07	1.64	1.62
	120	6	100.94	0.66	0.66	6	101.23	1.07	1.06

%Recovery is the mean of three estimations.

CONCLUSION:

In the present research work, because it was discovered to be fundamental, quick, specific, selective, linear, precise, and accurate in its unadulterated and consolidated dose structure, the developed UV Spectrophotometric approach based on absorptivity measurements can be used for in vitro depiction and concurrent assessment of CIP HCl and QRT.

ACKNOWLEDGEMENT:

The authors acknowledge the Department of Science and Technology, New Delhi, India (DST /INSPIRE Fellowship/2019/IF190329 and DST/ NM/NT/2018/20) for providing financial assistance for the successful completion of this work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest, financial or otherwise.

REFERENCES:

1. Stewart PS. Mechanisms of antibiotic resistance in bacterial biofilms. Int J Med Microbiol. 2002;292(2):107-113. doi:10.1078/1438-4221-00196

2. Diriba K, Kassa T, Alemu Y, Bekele S. In Vitro Biofilm Formation and Antibiotic Susceptibility Patterns of Bacteria from Suspected External Eye Infected Patients Attending Ophthalmology Clinic, Southwest Ethiopia. Int J Microbiol. 2020;2020. doi:10.1155/2020/8472395

3. Vasudevan S, Swamy SS, Kaur G, Princy SA, Balamurugan P. Synergism between quorum sensing inhibitors and antibiotics: Combating the antibiotic resistance crisis. Biotechnol Appl Quor Sens Inhib. Published online 2018:209-225. doi:10.1007/978-981-10-9026-4_10

4. Singh N, Romero M, Travanut A, et al. Dual bioresponsive antibiotic and quorum sensing inhibitor combination nanoparticles for treatment of: Pseudomonas aeruginosa biofilms in vitro and ex vivo. Biomater Sci. 2019;7(10):4099-4111. doi:10.1039/c9bm00773c

5. Ouyang J, Sun F, Feng W, et al. Quercetin is an effective inhibitor of quorum sensing, biofilm formation and virulence factors in Pseudomonas aeruginosa. J Appl Microbiol. 2016;120(4):966-974. doi:10.1111/jam.13073

6. Singh R, Maithani M, Saraf SK, Saraf S, Gupta RC. Simultaneous Estimation of Ciprofloxacin Hydrochloride, Ofloxacin, Tinidazole and Ornidazole by Reverse Phase – High Performance Liquid Chromatography. Eurasian J Anal Chem. 2009;4(2):161-167. http://eurasianjournals.com/index.php/ejac/article/view/21

7. Baghel US, Nagar A, Pannu MS, Singh D, Yadav R. HPLC and HPTLC methods for simultaneous estimation of quercetin and curcumin in polyherbal formulation. Indian J Pharm Sci. 2017;79(2):197-203. doi:10.4172/pharmaceutical-sciences.1000217

8. Fazil Ahmed M, Srinivasa Rao A. Simultaneous estimation of quercetin and rutin in ethanolic extract of Melia azedarach. Linn leaves by HPTLC method. Asian J Biomed Pharm Sci. 2013;3(21):56-59. http://www.jbiopharm.com

9. Jain A, Lodhi S, Singhai AK. Simultaneous estimation of quercetin and rutin in Tephrosia purpurea Pers by high performance thin- layer chromatography. Asian J Tradit Med. 2009;4(3):104-109.

10. Patel KB, Thula KC, Maheshwari DG. Original Research Paper stability indicating HPLC method for simultaneous Estimation of Ciprofloxacin and Phenylephrine in Pharmaceutical dosage form. Pharmacophore. 2014;5(2):262. http://www.pharmacophorejournal.com/

11. Baldi A, Upendra S, Ashish B. Simultaneous Estimation of Quercetin and Silymarin: Method Development and Validation. Int J Pharm Biol Arch. 2013;4(3):527-531. www.ijpba.info

12. Mohamed EH, Lotfy HM, Hegazy MA, Mowaka S. Different applications of isosbestic points, normalized spectra and dual wavelength as powerful tools for resolution of multicomponent mixtures with severely overlapping spectra. Chem Cent J. 2017;11(1):1-15. doi:10.1186/s13065-017-0270-8

13. Sawant RL, Hadawale SD, Dhikale GK, Bansode CA, Tajane PS. Spectrophotometric methods for simultaneous estimation of rabeprazole sodium and aceclofenac from the combined capsule dosage form. Pharm Methods. 2011;2(3):193-197. doi:10.4103/2229-4708.90362

14. Dhumal DM, Shirkhedkar AA, Nerkar PP, Surana SJ. Simultaneous estimation of moxifloxacin hydrochloride and dexamethasone sodium phosphate in bulk and in ophthalmic solution by RP-HPLC. J Chil Chem Soc. 2012;57(4):1344-1347. doi:10.4067/S0717-97072012000400003

15. Bellah SF, Ullah S, Harris KK. IOSR Journal of Pharmacy paper , research paper publishing , where to publish ... IOSR J Pharm. 2012;6(3):44-48.

16. ICH Q2B. ICH Q2B Guideline Validation of Analytical Procedures Methodology Comments for its application. Methodology.:71-76.

17. Sireesha D, Monika ML, Bakshi V. Development and Validation of UV Spectrophotometric Method for The Simultaneous Estimation of Rosuvastatin and Ezetimibe in Pharmaceutical Dosage Form. Asian J Pharm Anal. 2017;7(3):135. doi:10.5958/2231-5675.2017.00021.7

Received on 08.11.2023 Accepted on 01.12.2023

Int. J. Tech. 2023; 13(2):101-107.

DOI: 10.52711/2231-3915.2023.00013