INTRODUCTION:

The 5-nitroimidazole derivative ornidazole can be employed in a variety of applications such as to treat intestinal amoebiasis and urogenital infections as an antiprotozoal and antimicrobial agent. IUPAC nomenclature of ornidazole is α – (chloro-methyl)-2-methyl-5-nitroimidazole-1 ethanol. Ofloxacin is a fluroquinolone derivative used as antibacterial agent. IUPAC nomenclature of Ofloxacin is (±)-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. Dissolution¹, UV², RP-HPLC⁴ and HPTLC⁵, Stability Indicating RP-HPLC⁶ are some of the analytical methods described in the literature to quantitatively estimate Ofloxacin and Ornidazole in single and combined forms. However, chemometric-based multivariate methods to simultaneously estimate these molecules in a binary mixture have not been reported.

This work demonstrates accurate, precise, and robust multivariate methods to simultaneously determine Ofloxacin and Ornidazole in binary mixture.

Chemometrics is the science of obtaining information from chemical systems. Partial Least Square Regression (PLS) and Principal Component Analysis (PCR) are the two most widely employed multivariate calibration methods to determine a specific compound qualitatively and quantitatively. A relationship is developed from the instrumental data and mathematical model is developed to predict the composition of analyte of interest from the sample. Theoretical details of these methods and applications can be found in the literature.^8-10.

MATERIAL AND METHODS:

UV-VIS spectrophotometer (Lab India UV 3200) was used for the measurement of absorbance of ofloxacin and ornidazole within the range of 200-400nm with an interval of 1nm and slit width of 1 nm was used to collect the spectra. All spectral measurement were performed using quartz sample cell and methanol as a reference. Solo 9.0 (Eigenvector) with was used to perform Partial Least Square and Partial Component Regression techniques.

Chemicals and Reagents:

Highly reputed pharmaceutical companies provided Ofloxacin and Ornidazole as a gift sample, accompanied with a certificate of analysis. Solvents and reagents of spectroscopic grade were used. Pharmaceutical tablet formulation were purchased from local pharmacy shop.

Preparation of standard solution:

The standard stock solutions of Ofloxacin and Ornidazole were prepared by weighing 10mg of each drug and dissolving it in enough methanol to make up the volume up to 100ml. This standard stock solution was diluted to a concentration range of 2-12mcg/ml for Ofloxacin and 5-60mcg/ml for Ornidazole. The resulting solutions were scanned in the UV spectrophotometer in the range of 200 nm to 400nm.

Chemometric Method:

PCR and PLS are the tools which are used for factor analysis using multivariate analysis. Initial step in both methods involve preparation of calibration set with known concentration of both drugs in a binary mixture. All the solutions with varying concentrations of ofloxacin and ornidazole were scanned between 200 nm to 400 nm. Second step involve preparation of validation set with known concentration of analyte. Finally, a model will be developed and used for determination of concentration from the sample. Sample solutions were scanned within given range of wavelength and further processed with the PLS toolbox. Mean centering preprocessing was used for smoothing the raw data. In case of PLS two latent variables were used and in case of PCR two principal components were selected based on PRESS value. Venetian blinds method was used as a cross validation tool. Table 1 shows the calibration set that was used to develop the model.

Table 1. The composition of calibration set

Sample number		Ofloxacin (ug/ml)	Ornidazole (ug/ml)
1		0.5	1.25
2		1	2.5
3		1.5	3.75
4		2	5
5		2.5	6.25
6		3	7.5
7		3.5	8.75
8		4	10
9		4.5	11.25
10		5	12.5
11		5.5	13.75
12		66	15
13		6.5	16.25
14		7	17.5
15		7.5	18.75
16		8	20
	17	8.5	21.25
	18	9	22.5
	19	9.5	23.75
	20	10	25
	21	10.5	26.5
	22	11	27.5
	23	11.5	28.75
	24	12	30

RESULTS AND DISCUSSION:

The PRESS value (Prediction Error Sum of Square) was computed, and the number of optimum components was chosen based on this value. It is shown in fig 1 and 2 for Ofloxacin and Ornidazole respectively.

Figure 1. PRESS PCR Plot

Figure 2. PRESS plot for PLS

With the values obtained by the Standard Error of Calibration (SEC) and the Root Mean Standard Error of Prediction (RMSEP) the prediction capability of these methods were evaluated. The developed PCR and PLS models were validated with the help of a validation set of 12 random samples was prepared developed model was applied and concentration of both analytes was determined with the results of amount recovered, percentage recovery and standard deviation is shown in table 2.

Table 2. Predicted validation using PCR and PLS methods

Method		PLS				PCR
OFX	ORNI	OFX		ORNI		OFX		ORNI
Actual ug/ml		predicted	%R*	predicted	%R*	Predicted	%R*	predicted	%R*
2	5	1.98	99	4.99	99.8	2.01	100.5	5.04	100.8
3	7.5	2.96	98.66	7.48	99.73	3.04	101.3333	7.51	100.1333
4	10	3.97	99.25	10.04	100.4	3.96	99	10.09	100.9
5	12.5	4.96	99.2	12.47	99.76	4.99	99.8	12.505	100.04
6	15	6.02	100.33	15.03	100.2	5.98	99.66667	14.98	99.86667
7	17.5	7.04	100.57	17.51	100.05	7.02	100.2857	17.47	99.82857
8	20	7.95	99.37	19.98	99.9	7.98	99.75	20.05	100.25
9	22.5	8.96	99.55	22.46	99.82	9.03	100.3333	22.513	100.0578
10	25	10.06	100.6	25.06	100.24	10.06	100.6	25.06	100.24
11	27.5	11.02	100.18	27.5	100	11.04	100.3636	27.52	100.0727
12	30	11.98	99.83	29.98	99.93	12.05	100.4167	30.11	100.3667
		Mean	99.68		99.98	Mean	100.1863		100.2323
		SD	0.6599		0.2175	SD	0.609768		0.344371
		%RSD	0.6620		0.2176	%RSD	0.608634		0.343573

Method Validation:

Validation of the following validation parameters precision, accuracy, limit of detection, and limit of quantitation, linearity was done with the help of the developed Partial Least Square (PLS) and Principal Component Regression (PCR) chemometric techniques. Linearity of the method was studied using calibration curve which shows linearity from 2-12 mcg/ml and 5-60 mcg/ml. The USP guidelines were used to calculate the Limits of Detection (LOD) and Limits of Quantitation (LOQ), results of regression analysis is shown in table 3 and calibration curves are shown in fig 3 and 4.

Table 3. Results of the regression analysis

Parameter	PLS		PCR
Parameter	OFX	ORNI	OFX	ORNI
Linearity	2-12	5-60	2-12	5-60
Correlation Coefficient	0.9978	0.9992	0.9983	0.9988
RMSEP	0.5398	0.7233	0.9523	0.6311
RMSECV	0.9251	0.8933	0.8203	0.7194
LOD	0.527	0.976	0.487	0.874
LOQ	1.487	1.917	1.197	1.744

Figure 3. Calibration curve for Ofloxacin

Figure 4. Calibration curve for Ornidazole

Accuracy studies were performed using recovery studies at three levels of 80%, 100% and 120% with standard addition method as per regulatory guidelines. Standard solutions of Ofloxacin and Ornidazole were spiked into sample solutions that were pre analyzed, and the resulting solutions were scanned in the 200-400nm range. Tables 4 and 5 demonstrate the results by using the developed PCR and PLS models to estimate the concentrations of both compounds.

Interday and Intraday precision was calculated using mid concentration and it was done in triplicate. Standard deviation and relative standard deviation were used to evaluate the precision of the method. Both methods showed good precision below 2 which is within the specified range of regulatory guidelines, results are shown in table 6 and 7.

Table 4 Validation parameter (Accuracy) data- Ofloxacin (using PCR and PLS techniques)

Level	Sample conc. ug/ml	Standard added ug/ml		Total conc. ug/ml	Recovered conc. ug/ml		% Recovery
					PLS	PCR	PLS	PCR
80%	4	3.2	7.2		7.17	7.25	99.58	100.69
	4	3.2	7.2		7.19	7.28	99.86	101.11
	4	3.2	7.2		7.21	7.32	100.13	101.66
100%	4	4	8		7.98	8.12	99.75	101.5
	4	4	8		7.96	8.1	99.5	101.25
	4	4	8		8.02	8.09	100.25	101.12
120%	4	4.8	8.8		8.82	8.9	100.22	101.13
	4	4.8	8.8		8.81	8.93	100.11	101.47
	4	4.8	8.8		8.79	8.86	99.88	100.68
Mean							99.92	101.18
SD							0.2765	0.3402
%RSD							0.2767	0.3362

Table 5 Validation parameter (Accuracy) data- Ornidazole (using PCR and PLS techniques)

Level	Sample conc. ug/ml	Standard added ug/ml	Total conc. ug/ml	Recovered conc. ug/ml		% Recovery
				PLS	PCR	PLS	PCR
80%	10	8	18	17.98	18.29	99.88	101.61
	10	8	18	17.95	18.25	99.72	101.38
	10	8	18	18.08	18.23	100.44	101.27
100%	10	10	20	20.1	20.27	100.5	101.35
	10	10	20	19.94	20.34	99.7	101.7
	10	10	20	19.86	20.29	99.3	101.45
120%	10	12	22	21.93	22.27	99.68	101.22
	10	12	22	21.98	22.38	99.90	101.72
	10	12	22	22.03	21.89	100.13	99.5
Mean						99.92	101.24
SD						0.3853	0.6794
%RSD						0.3856	0.6711

Table 6 Inter-Day Precision

Amount Taken μg/ml		Predicted Conc.(μg/ml)				% Recovery
		PCR		PLS		PCR		PLS
OFX	ORNI	OFX	ORNI	OFX	ORNI	OFX	ORNI	OFX	ORNI
4	10	4.01	10.11	4.06	10.08	101.5	101.1	101.5	100.8
4	10	4.08	10.07	4.04	10.12	101	100.7	101	101.2
4	10	4.05	10.04	3.93	10.07	98.25	100.4	98.25	100.7
4	10	4.11	10.03	4.06	10.14	101.5	100.3	101.5	101.4
4	10	4.08	9.97	3.99	10.19	99.75	99.7	99.75	101.9
4	10	4.13	9.94	4.07	10.05	101.75	99.4	101.75	100.5
Mean				4.025	10.1083	100.625	100.267	100.625	101.083
SD				0.05468	0.05193	1.36702	0.62823	1.36702	0.51929
%RSD				1.35853	0.51373	1.35853	0.62655	1.35853	0.51373

Table 7. Assay results-Actual values and Predicted values using PLS and PCR techniques (Ofloxacin, Ornidazole)

Method		PLS				PCR
OFX	ORNI	OFX		ORNI		OFX		ORNI
Actual ug/ml		predicted	%R*	predicted	%R*	Predicted	%R*	predicted	%R*
4	10	3.99	99.75	10.11	101.1	4.1	102.5	10.13	101.3
4	10	4.02	100.5	10.12	101.2	4.06	101.5	10.15	101.5
4	10	4.04	101	10.03	100.3	4.02	100.5	10.11	101.1
4	10	3.99	99.75	10.06	100.6	4.01	100.25	10.07	100.7
4	10	3.96	99	9.96	99.6	4.08	102	10.04	100.4
4	10	4.06	101.5	9.98	99.8	4.05	101.25	10.17	101.7
Mean			100.25		100.43		101.33		101.11
SD			0.9219		0.6592		0.8612		0.4915
%RSD			0.9196		0.6564		0.8498		0.4861