INTRODUCTION:

Ofloxacin (OFLOX) is (RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3,-de]-1,4 benzoazeine-6-carboxylic Figure 1(a) is a well-known antibiotic of second generation fluoroquinoline that inhibits the supercoiling activity of bacterial DNA gyrase, halting DNA replication and is used to treat bacterial infection¹. Tinidazole (TZ) is chemically 1-(2-ethylsulfonylethyl)-2-methyl-5-nitro-imidazoleFigure 1(b). Tinidazole is an anti-parasitic drug used against protozoan infections. It is widely known throughout Europe and the developing world for the treatment for a variety of amoebic and parasitic infections².Several methods are reported for quantitative determination of OFLOX and TZ in single and in combination such as UV^{3,4,5,6,7,8,9,10,11,12,13}, HPTLC^14,15 and RP-HPLC^{16,17,18,19,20,21}.

Chemometric is the science of extracting information from chemical systems. Multivariate calibration method e.g. multiple linear regression (MLR), principle component regression (PCR) and partial least squares (PLS) utilizing spectrophotometric data are the important chemometric approach for determination of mixtures including drugs combination.^{22,23,24,25,26,27,28} As there are no reports on chemometric analysis of these drugs, this work was undertaken which presents simple, accurate and reproducible multivariate spectrophotometric methods for simultaneous determination of OFLOX and TINI in tablet dosage form. HPLC method was also developed and validated for the simultaneous determination of OFLOX and TZ. The sample (tablet) was assayed with the optimized chemometric-assisted spectrophotometric methods and HPLC method for comparison.

Structure of Ofloxacin (OFLOX),

Fig. 1(a): Fig. 1(b): Structure of Tinidazole (TZ)

MATERIALS AND METHOD:

Reagents:

Reference standard of OFLOX and TZ were obtained from Cipla Ltd, Mumbai as gift samples and Methanol used was of AR grade (LOBA Chemie, India). OfloxTZ tablets manufactured by Cipla Ltd containing Ofloxacin IP 200 mg and Tinidazole IP 600 mg were procured from local pharmacy shop.HPLC grade water was obtained by ELGA LABWATERpurification system (PURELAB UHQ-11, United Kingdom). Methanol used for HPLC was of HPLC grade and that used for spectrophotometry was of AR grade (LOBA Chemie, Mumbai, MH, and India).

Instrumentation:

Double beam UV- Vis spectrophotometer (Jasco V-730) with matched pair of 1 cm quartz cells were used to record spectra of all solutions. The spectra were recorded in the range 280 to 320 at spectral band width of 2.0 nm, scanning speed 100 nm/min, data pitch 0.5 nm. Unscrambler X (10.3) (64-bit) trial version and Microsoft Excel 2013 were used for model generation and application of chemometric.

The RP-HPLC was carried on JASCO HPLC (PU 2080 Plus, Japan) equipped with Jasco UV detector (PU 2010 Plus, Japan). Samples were injected through Rheodyne sample injection port (20 µl). THERMOSIL C18 Column (250 x 4.5mm, i.e. 5 μm) was used. Data acquisition and integration was performed usingBorwin software (version 1.5).

Chromatographic Conditions:

The mobile phase was prepared by mixing Acetonitrile and 10mM Phosphate Buffer in ratio (85:15 % v/v). The flow rate was 1 ml/min. Quantitation based on peak area was achieved using UV detector at 300nm. All determinations were performed at ambient temperature.

Preparation of standard stock solution:

Stock solution of OFLOX and TZ were prepared by dissolving accurately weighed 10mg of standard drug in 10ml of methanol, separately. The concentration of OFLOX and TZ were 1000μg/ml from which further 5 ml was pipetted and diluted to 50ml to achieve final concentration of 100μg/ml of OFLOX and TZ, respectively.

One component calibration:

To find linear concentration of each drug, one component calibration was performed. Linear dynamic ranges were studied in the concentration range of 2.0-12.0μg/ml for OFLOX and 5.0-30.0μg/ml for TZ. Absorbance values were recorded at λ_max of each drug (295 nm for OFLOX and 311nm for TZ) against methanol as blank. Linear dynamic range for each compound was determined by least-square linear regression of concentration and the corresponding absorbance. Fig 2 represents overlain spectra of OFLOX and TZ and their mixture.

Fig.2: Overlain spectra of OFLOX and TZ (10 µg/ml each)

Working solutions:

Working standard solutions were prepared from standard stock solution of 100μg/ml by appropriate dilution to obtain final concentration of 2.0-12.0μg/ml for OFLOX and 5.0-30.0μg/ml for TZ for chemometric analysis as well as HPLC method.

Experimental Procedures:

Construction of calibration and validation set for UV method:

A total set of 36 mixtures were prepared by combining working standard of OFLOX and TZ in their linear concentration range of 2.0-12.0μg/ml and 5.0-30.0 μg/ml, respectively (Table 1). From these 24 mixtures were used for calibration set and 12 mixtures were used for validation set. The validation set was randomly selected. The absorbance spectra were recorded in range of 280- 320nm with 0.5nm interval. The spectra were saved as ASCII (.txt) format which were further extracted in MS-Excel as required by Unscrambler software for model generation. The PCR and PLS models were developed utilizing absorption data using Unscrambler software.

Table 1: Composition of calibration and validation sets.

Mixt No.		OFLOX (μg/ml)		TZ (μg/ml)	Mixt No.	OFLOX (μg/ml)	TZ (μg/ml)
1	2		5		19	10	15
2	2		10		20	10	30
3	2		15		21	12	5
4	2		20		22	12	10
5	4		5		23	12	15
6	4		10		24	12	30
7	4		15		25	2	25
8	4		25		26	2	30
9	6		10		27	4	30
10	6		15		28	4	20
11	6		25		29	6	5
12	6		30		30	6	20
13	8		10		31	8	5
14	8		15		32	8	25
15	8		20		33	10	20
16	8		30		34	10	25
17	10		5		35	12	20
18	10		10		36	12	25

*Mix no. 1-24 calibration set

*Mix no. 25-36 validation set

After the PCR and PLS models have been constructed, it was found that the optimum number of LVs were two factors for both PCR and PLS. For validation of generated models, concentration in validation set was predicted by using proposed PCR and PLS models (Table 2).

Table 2: Predicted results for validation set by PLS and PCR method.

METHOD		PLS				PCR
*OFLOX*	*TINI*	*OFLOXACIN*		*TINIDAZOLE*		*OFLOXACIN*		*TINIDAZOLE*
Actual (μg/ml)		Predicted	% R*	Predicted	% R*	Predicted	% R*	Predicted	% R*
2	25	1.9802	99.01	24.7174	98.87	1.9753	98.77	24.8412	99.36
2	30	1.9812	99.06	29.8414	99.47	1.9901	99.51	29.5813	98.60
4	30	3.9853	99.63	29.9679	99.89	3.9454	98.64	29.7676	99.23
4	20	4.0824	102.06	19.7588	98.79	4.1001	102.50	19.7599	98.80
6	5	6.1387	102.31	4.9835	99.67	6.1255	102.09	4.9438	98.88
6	20	6.1105	101.84	20.2707	101.35	6.0801	101.34	20.2845	101.42
8	5	7.8987	98.73	4.9273	98.55	7.9156	98.95	4.9877	99.75
8	25	7.9421	99.28	24.7173	98.87	7.8919	98.65	24.6875	98.75
10	20	10.0324	100.32	20.3664	101.83	10.1304	101.30	20.1664	100.83
10	25	9.8849	98.85	24.8934	99.57	9.885	98.85	24.593	98.37
12	20	11.8547	98.79	19.7123	98.56	11.9545	99.62	19.7119	98.56
12	25	12.2138	101.78	25.0878	100.35	12.1137	100.95	24.9871	99.95

Table 4: Assay result by PLS and PCR methods for TZ and OFLOX in tablet (OfloxTZ)

METHOD		PLS				PCR
TZ	OFLOX	TZ		OFLOX		TZ		OFLOX
Actual (μg/ml)		Predicted (μg/ml)	% R	Predicted (μg/ml)	% R	Predicted (μg/ml)	% R	Predicted (μg/ml)	% R
10	3.33	9.986	99.86	3.356	100.78	9.998	99.98	3.285	98.65
10	3.33	10.119	101.19	3.281	98.53	10.1345	101.35	3.261	97.93
10	3.33	10.086	100.86	3.261	97.93	10.129	101.29	3.273	98.29
10	3.3	10.144	101.44	3.321	99.73	10.051	100.51	3.328	99.94
10	3.33	9.861	98.61	3.301	99.13	10.2245	102.25	3.352	100.66
10	3.33	10.192	101.92	3.351	100.63	9.894	98.94	3.285	98.65
MEAN		10.065	100.647	3.312	99.454	10.072	100.718	3.297	99.019
SD		0.121	1.213	0.038	1.141	0.117	1.166	0.035	1.053

Validation by HPLC method:

For validation of the developed method, the ICH Q2 (R1) guidelines were followed.

a. Specificity: Specificity was checked by injecting blank, placebo and comparing the peaks observed in sample solution with standard solution. No interference was observed. Observed peaks in sample solution matched standard peak of OFOX and TINI showed that, the method was specific.

b. System suitability: System suitability performance was evaluated by system suitability parameters such as retention time, theoretical plates, asymmetric factor and resolution and the method indicated good performance of the system as depicted inTable 5.

Table 5: HPLC method system suitability parameters for OFLOX and TZ (n=3)

Drugs

Conc.

(μg/ml)

(min)

Area

Plates

Asymmetry

3.308

882176.5

2871.08

1.09

OFLOX

8.508

2234216.964

2170.43

1.52

c. Linearity: Linearity study of HPLC detector response for determination of OFLOX and TZ was evaluated by analyzing a series of standard solutions of five different concentrations of each compound. Calibration curves constructed were linear over the concentration range of 2.0-12.0μg/ml and 5.0-30.0μg/ml for OFLOX and TZ respectively. Regression analysis has been carried out with coefficient of determination (r2) 0.9987 and 0.9937 for OFLOX and TZ respectively.

d. Precision: The precision of the method was studied. Intra-day studies by injecting three concentrations of each drug at three levels. The study was carried at 4, 6 and 8 μg/ml for OFLOX while 10, 15 and 20μg/ml for TZ. % RSD were calculated and were well within limit of less than 2

e. Accuracy: To check an accuracy of the method, recovery studies were carried out by spiking the standard drug solution to the sample injection solution, at three different levels of 50, 100 and 150 % of assay concentration. The % recovery and % RSD were calculated.

f. Assay: 20 tablets of OFLOX-TZ® were accurately weighed and finely powdered. Tablet powder equivalent to 10 mg of TINI (3.33 mg of OFOX) was taken and transferred to 10 ml volumetric flask and was diluted to 10 ml with methanol. The solution was sonicated for 10 minutes. This solution was then filtered with help of whatmanfilter paper no. 41. 1 ml of filtrate solution was diluted to 10 ml with mobile phase. Further 1.0 ml of this solution was diluted to 10 ml with methanol to get final concentration of 10 μg/ml and 3.33 μg/ml respectively of TINI and OFOX. The procedure was repeated 6 times for tablet formulation. The assay results are presented in Table 6.

Table 6: Assay results of OFLOX-TZ tablet along with % recovery by HPLC

	TZ				OFLOX
Sr. No.	Actual amount	Peak area	Amount Recovered (µg/ml)	% Recovery	Actual amount	Peak area	Amount Recovered (µg/ml)	% Recovery
1	10	868094.75	10.032	100.320	3.33	760158.82	3.327	99.896
2	10	854060.75	9.867	98.667	3.33	762546.65	3.337	100.216
3	10	878832.25	10.159	101.585	3.33	772385.33	3.381	101.532
4	10	869656.43	10.050	100.504	3.33	756269.9	3.309	99.376
5	10	872891.5	10.089	100.885	3.33	767325.88	3.358	100.855
6	10	882749	10.205	102.047	3.33	758598.27	3.320	99.688
Mean 100.668					100.260
SD 1.178					0.802
%RSD 1.170					0.800

g. Robustness:The robustness of the method was evaluated by deliberately varying the chromatographic conditions i.e. altering mobile phase concentration by ± 2 ml and change in wavelength of detection by ± 2nm and flow rate by ± 0.1 ml. At these altered conditions the standard solutions were injected and effects were noted and % RSD was calculated. The method was found to be robust

h. Limit of detection (LOD) and Limit of quantitation (LOQ): LOD and LOQ were calculated as 3.3 σ/S and 10 σ/S, respectively; where σ is the standard deviation of the y-intercept and S is the slope of the calibration plot. (Table7).

Table 7: Summary of validation parameters by proposed HPLC method

Sr. No.	Validation Parameter	OFLOX	TZ
1	Regression Equation	y = 224531x + 13245 R² = 0.9987	Y=84900x + 16374 4² = 0.9937
2	Range	2.0-12 µg/ml	5.0-30 µg/ml
3	Intraday precision (% RSD)	1.511	1.507
4	LOD (μg/ml)	0.2465	0.4726
5	LOQ (μg/ml)	0.7471	1.4324
6	Accuracy (% recovery)	101.018	98.813
7	Robustness (% RSD)	<2	<2
8	Specificity	Specific	Specific

RESULT AND DISCUSSION:

Chemometric method:

The first step in multivariate method involves constructing the calibration matrix. The wavelength range used was 280-320 nm. Eighty-one spectral points at 0.5 nm intervals were selected within this range. The composition of the calibration mixtures was randomly designed in order to collect maximum information from the spectra of these mixtures. The quality of multicomponent analysis is dependent on the wavelength range and spectral mode used.

The PCR and PLS models were developed by the Unscrambler® software. Model development was performed by using 24 mixtures as calibration standards. Leave-one-out cross-validation (LOO-CV) was used to validate the PCR and PLS models in model development and to obtain optimum latent variables (number of factors). The predicted concentrations of the components in each sample were compared with the actual concentrations and the root mean square error of cross validation (RMSECV) was calculated for each method. The RMSECV was used as a diagnostic test for examining the error in the predicted concentrations.The resulting models were also validated by prediction of the concentration of analytes in a separate validation set shown in Table 2. Statistical parameters of the chemometric methods are represented in Table3.

HPLC method:

The validity of the analytical procedure as well as the resolution between the peaks of interest was ensured by the system suitability test (Table 5). All critical parameters tested meet the acceptance criteria. As shown in the chromatogram (Figure 3), the two analytes are eluted by forming symmetrical single peaks well-separated from each other. OFOX and TZ showed a good correlation coefficient in the concentration range of 2.0-12.0μg/ml and 5.0-30.0μg/ml. The linear regression analysis obtained by plotting the peak areas of analytes versus concentration showed excellent coefficient of determination (r2 greater than 0.9987). The proposed method afforded high recoveries of almost 100% for OFLOX and TZ in synthetic formulation, indicating that this assay procedure can be used for the routine quality control analysis of the pharmaceutical dosage form.

The system precision is a measure of the method variability that can be expected for a given analyst performing the analysis and was determined by performing six repeats. The %RSD for OFOX and TZ response was found to be less than 2.0 (values in limit). The summary of HPLC results are depicted in Table 7.

Comparison of the Chemometric method with the HPLC Method:

In order to compare the results of the proposed PCR and PLS models for the determination of OFLOX and TZ in mixture, the HPLC method was also employed. The same sample solutions used for the PCR and PLS models were analyzed by the HPLC method. The assay results of PCR, PLS, and HPLC methods are presented in Table 8. The data were expressed in terms of percent labeled amount. The results showed that the average percent labeled amount obtained from the PCR and PLS models were not significantly different from those obtained from the HPLC method with the confidence limit of 95%.

Average of six determinations

CONCLUSION:

RP-HPLC techniques are generally used for separation and determination of components in pharmaceutical formulations and are considered superior with regard to identification and specificity. However, the chemometric methods are less expensive by comparison and do not require sophisticated instrumentation nor any prior separation step. The proposed chemometric-assisted spectrophotometric methods are applicable and specific for the simultaneous determination of OFLOX and TZ in their synthetic mixtures. The results obtained were compared with the proposed RP-HPLC method and good coincidence in the means of percentage recovery was observed as there was no significant difference between the methods compared. The three proposed methods were accurate, precise with good reproducibility and sensitivity; hence can be used for the routine analysis of these in their combined pharmaceuticals.

ACKNOWLEDGEMENT:

Authors are thankful to the Principal, AISSMS College of Pharmacy for providing necessary facilities to carry out the experiment. Authors are also thankful to Cipla Pharmaceuticals Ltd, Mumbai for providing a working standard of Ofloxacin and Tinidazole.

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Received on 06.08.2020 Modified on 31.12.2020

Research J. Pharm. and Tech 2021; 14(11):5713-5718.

DOI: 10.52711/0974-360X.2021.00993

Statistical Parameters	OFLOXACIN		TINIDAZOLE
Statistical Parameters	PCR	PLS	PCR	PLS
Range (μg/ml)	2.0-12.0		5.0-30.0
Offset	0.0035	0.0035	0.0292	0.0292
Regression Coefficient (R²)	0.9994	0.9994	0.9981	0.9981
RMSE	0.0770	0.0770	0.3599	0.3598
PC/Factors	02		02

DRUG	OFLOX*					TZ*
Actual amount	3.3μg/ml					10 μg/ml
Method	HPLC	PCR	PLS	HPLC	PCR	PLS
Amount found	3.339	3.297	3.312	10.067	10.072	10.065
% Amount found	100.260	99.919	100.359	100.668	100.718	100.647
T test value	0.2118					0.0283
P value	98.11%					99.22%