INTRODUCTION:

Ornidazole¹is a 5-nitroimidazole derivative used as an anti-infective agent mainly in the treatment of amoebiasis and other protozoal diseases. It is chemically 1-chloro-3-(2-methyl-5-nitro-1H-imidazol-1-yl) propan-2-ol (Figure 1). In general RP-HPLC estimation is usually performed with the help of organic mobile phase, which are harmful, volatile and expensive. In the present study, hydrotropic solution was employed as solubilising agent and mobile phase for the estimation of poorly aqueous soluble drug Ornidazole by RP-HPLC. Only few methods were reported till date for the estimation of drugs in RP-HPLC by using hydrotropic solution as mobile phase.^2-4 Literature survey revealed that a few spectrophotometric^5-7 HPLC^8-11and HPTLC^12-14 methods were reported for the estimation of Ornidazole in pharmaceutical dosage forms. In all reported methods expensive, volatile and toxic organic solvents are used as mobile phase. It was observed that various poorly water-soluble drugs were analysed spectrophotometrically^15-19 using various hydrotropic agents. Also, hydrotropy had applications in TLC^{20, 21} and HPTLC²². Hydrotropy²³ refers to the ability of a concentrated solution of a chemical compound to increase the aqueous solubility of another compound (usually a sparingly soluble organic compound). Each hydrotropic agent is effective in increasing the water solubility of selected hydrophobic drugs. Examples for hydrotropic agents include Sodium acetate, Sodium caprylate, Sodium salicylate, Sodium citrate, Sodium benzoate, Nicotinamide, Urea etc. Hence, the present study is aiming to develop and validate accurate, simple, sensitive, eco-friendly, cost effective method for the estimation of Ornidazole by using hydrotropic solution (5% urea solution) as mobile phase.

Fig.1: Structure of Ornidazole

MATERIALS AND METHODS:

Reagents and chemicals:

· Ornidazole standard from M/S Parenteral Drug India Ltd., Indore.

· HPLC grade water from Merck specialities Pvt limited, Mumbai.

· Urea Analytical grade from Loba Chemie, Mumbai.

· Ornida tablets 500mg, manufactured by Aristo Pharmaceuticals Ltd., India.

Instrumentation:

The HPLC system used for this method was Shimadzu LC6AD dual pump with PDA Detector SPD-M20A and Rheodyne injector (20μl loop). Analytical column used was Shimadzu shim pack C₁₈ Column with 5μm particle size and dimension of 4.6 x250 mm. Apart from this, Shimadzu analytical balance was used for weighing standards and samples. Membrane filter of pore size 0.45μm from Merck Millipore was used for mobile phase filtration and PVDF syringe filters 0.22μm pore size from Agilent technologies were used for sample filtration.

Methodology adopted:

1. Determination of Solubility

2. Selection of Hydrotropic Agent

3. Preparation of standard drug solution

4. Method development and optimization

5. Preparation of Working Standard Solution

6. Preparation of calibration curve

7. Analysis of Marketed Formulation

8. Estimation of amount of Ornidazole in tablet dosage form.

9. Validation of the proposed method.

Determination of Solubility:

Preliminary Solubility Studies:

Solubility of Ornidazole was determined at 28±1 °C. An excess amount of drug was added to screw capped 30ml glass vials containing different aqueous systems viz. distilled water, 5% urea solution and other hydrotropic agents. The vials were shaken mechanically for12 hours at 28±1°C in a mechanical shaker. These solutions were allowed to equilibrate for next 24 hours and then centrifuged for 5min at 2000rpm. The supernatant liquid was taken for appropriate dilution after filtration through Whatman filter paper # 41 and analysed spectrophotometrically against corresponding solvent blank. After analysis, it was found that there was tremendous increase in solubility of Ornidazole in 5% urea solution as compared to solubility studies in other solvents.

Selection of hydrotropic agent:

Ornidazole was scanned in various solutions of hydrotropic agents in the spectrum mode over the UV range (200-400nm) and was found to be most appropriate in 5% urea solution because Ornidazole was soluble and stable in urea solution. Also, Urea solution has no interference with the λ-max of Ornidazole (320nm). The spectra of Ornidazole in urea was shown in Figure 2.

Fig. 2: Spectra of Ornidazole in Urea

Preparation of standard drug solution:

Stock solution of Ornidazole:

Weighed accurately about 100mg of Ornidazole RS and transferred to a 100mL standard flask. 40ml of 5% Urea solution was added and flask was shaken for 10-15 minutes. The final volume was made up to 100ml with HPLC grade water to get a concentration of 1000μg/ml (solution A).

Method Development and Optimization:

The mobile phase influences resolution, selectivity and efficiency of separation. In reverse phase chromatography, usually the mobile phase consists of toxic, volatile and costlier organic solvents. But here an attempt was made to find out an eco-friendly, cost effective, non-volatile mobile phase for the estimation of poorly water-soluble drug Ornidazole. Selection of mobile phase was carried out by trial and error method. Different concentrations of Urea solution were tried as mobile phase; among them 5%Urea solution gave sharp peak for ornidazole. pH of Urea solution also changed, but no significant change occurred on the shape of peaks. Finally, the mobile phase was optimized as 5%Urea solution. All the stock solutions are prepared by using mobile phase and further dilutions are made by HPLC grade water.

HPLC operating conditions:

Column:	Shimadzu shim pack C₁₈ (4.6 x250 mm with 5μm particle size)
Detector:	PDA Detector (SPD-M20A)
Injection volume:	20μl
Flow rate:	1.0ml per minute
Temperature:	Ambient
Run time:	10 Minutes
Mobile phase:	5% Urea solution
Wave length:	320nm

Preparation of Working Standard Solution:

From the standard stock solution of Ornidazole (1000μg/ml, solution A), accurately pipetted out 10mL and transferred into a 100ml standard flask and made up the volume using HPLC grade water. The resulting solution contains 100μg/ml of Ornidazole and further diluted with HPLC grade water to obtain final concentrations ranging from 10-50μg/ml.

30μg/ml concentration was used for the preparation of standard chromatogram for assay.

Preparation of Calibration Curve:

The standard solutions were prepared in a range of 10-50μg/ml. Five replicates of each concentration were injected and chromatograms were recorded. Chromatograms showing linearity was given in Fig.3. Calibration curve was plotted which is shown in Fig. 4 and data is presented in Table 1.

Fig. 3: Integrated chromatograms showing linearity

Fig. 4: Calibration curve of Ornidazole

Table 1: Results of linearity study

SI No	Concentration (μg/mL)	Peak area of Ornidazole
1	10	513741
2	20	1015825
3	30	1527427
4	40	2039079
5	50	2468150
Slope		49321
Intercept		33223
Correlation Coefficient		0.9990

Analysis of Marketed Formulation:

Weighed accurately twenty Ornida tablets and average weight was calculated and then finely powdered. Then powder equivalent to 100mg of Ornidazole was weighed and transferred to a stoppered flask. The powder was extracted initially using 20mL of 5% urea solution (5%urea in HPLC grade water) by vigorous shaking for 15 minutes. Then solution was transferred to a 100ml standard flask through Whatman No.41 filter paper. The residue was further extracted twice with 10mL of urea solution and transferred to the standard flask through the same filter paper and then washed out the filter paper using HPLC grade water. The volume was finally made up to 100ml with HPLC grade water to attain a concentration of 1000μg/ml of Ornidazole. From the above solution, accurately pipetted out 10ml and transferred into a 100ml standard flask and made up the volume using HPLC grade water. The resulting solution contains 100μg/ml of Ornidazole and further diluted with HPLC grade water to obtain a final concentration of 30μg/ml. Typical chromatogram for the standard and sample were recorded at 320nm and presented in Figure 5. The results are shown in Table 2.

Fig. 5: Ornidazole standard and sample chromatogram

Table 2: Assay result of formulation

Formulation	Label claim (mg)	Amount found (mg)*	% Label claim
Tablet – Ornida	500	496.8	99.36

*Average of six determination

Validation of the Proposed Method:

The proposed method was validated as per ICH guidelines²⁴. The parameters studied for validation were system suitability, accuracy, precision, linearity, specificity, robustness, ruggedness, limit of detection (LOD) and limit of quantitation (LOQ).

1. System Suitability:

System suitability was performed by injecting six replicate injections of Ornidazole standard. The obtained results showed that all the parameters tested were within the acceptable range. Ornidazole was repeatedly retained and well separated at 3.996 min. The tailing factor for Ornidazole peak never exceeded 1.3 indicating good peak symmetry (acceptance limit is < 2) and the number of theoretical plates were always >2000 in all chromatographic runs to ensure good column efficacy throughout the developed separation process.

2. Accuracy:

Accuracy of the proposed method was determined by recovery studies. To the formulation (pre-analysed sample), the reference standard of the drug was added at the level of 80%, 100%, 120%. The recovery studies were carried out three times and the percentage recovery were calculated. The statistically evaluated results are shown in Table 3.

3. PRECISION:

Precision was determined in two levels

· Repeatability

· Intermediate precision

Repeatability (System stability study):

The repeatability of the method was studied using 100% test concentration of Ornidazole prepared separately, and the peak area was determined six times at 320nm. The Result was presented in Table 4.

Intermediate/Inter - day precision:

The inter-day precision study of Ornidazole was carried out by estimating the corresponding responses for three days. The Results were presented in Table 5.

5. LINEARITY:

Linearity was determined by using five different concentration of standard Ornidazole solution. Suitable dilutions were made from the standard stock solution containing 1000μg/ml of Ornidazole (solution A) to prepare standard solutions of five different concentrations ranging from 10-50μg/ml.

Table 3: Accuracy: statistical validation

Spike level	Amount recovered (mg/mL)	% Recovery	Average % recovery	Standard deviation	% RSD	Coefficient of variation
80%	79.864	99.83	99.81	0.016996	0.017290	0.000170
	79.856	99.82
	79.832	99.79
100%	99.300	99.30	99.28	0.016329	0.016448	0.000164
	99.280	99.28
	99.260	99.26
120%	118.62	98.85	98.85	0.004714	0.004768	0.000476
	118.63	98.86
	118.64	98.85

Table 4: Result of repeatability

Sl. No	Concentration (μg/mL)	Peak area	%Label claim
1	30	1514580	99.58
2	30	1509878	99.28
3	30	1509932	99.29
4	30	1511010	99.36
5	30	1512411	99.45
6	30	1508929	99.22
Mean	99.36
Standard deviation	0.120646
% RSD	0.121423
Coefficient of variation	0.001214

Table 5: Results of inter-day precision

Parameter	Peak area	% Label claim	Average % Label claim	Standard deviation	% RSD	Coefficient of variation
DAY 1	1514013	99.12	99.08	0.028674	0.028946	0.00028940
	1512986	99.05
	1513643	99.09
DAY 2	1514580	99.15	98.95	0.148847	0.150426	0.00150426
	1508929	98.79
	1511010	98.92
DAY 3	1512410	99.01	98.90	0.077888	0.078755	0.00078755
	1509936	98.85
	1509875	98.84

Table 6: Results of Robustness

Parameters	Adjusted to	Peak area	Average Peak area	Standard deviation	% RSD	Coefficient of variation
Flow rate	0.8mL /minute	1509875	1511976	1568.59	0.103744	0.00103744
		1513643
		1512410
	1 mL /minute	1514580	1511506	2333.55	0.1543857	0.0015438
		1511010
		1508929
	1.2 mL /minute	1509936	1507402	1798.94	0.11934042	0.0011934
		1506331
		1505939
Mobile phase composition	3% Urea	1508396	1508524	127.96	0.0084824	0.0000848
		1508478
		1508699
	4%Urea	1508781	1508859	55.427	0.0036764	0.0000367
		1508896
		1508901
	5% Urea	1514578	1514042	654.35	0.0432187	0.0004321
		1513121
		1514428

Table 7: Results of Ruggedness

Parameter	Peak area	% Label claim	Average % Label claim	Standard deviation	% RSD	Coefficient of variation
Analyst I	1509878	99.28	99.32	0.0974109	0.098077	0.00098077
	1508929	99.22
	1512411	99.45
Analyst II	1512410	99.01	98.93	0.0654896	0.0661979	0.00066197
	1509936	98.85
	1511010	98.92

6. RUGGEDNESS:

Interday variations were performed by using six replicate injections of sample solutions which were prepared and analysed by different analyst on three different days over a period of one week. Ruggedness also expressed in terms of %RSD and statistical analysis showed no significant difference between results obtained employing different analyst which is presented in Table 7.

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

The LOD and LOQ were estimated from the set of six calibration curves used to determine the linearity of the developed method. Six calibration curves were drawn for the drugs that come across within its linearity range. From each calibration curve y-intercept and slope were determined and are substituted in the corresponding equation for finding the LOD and LOQ.

LOD = 3.3 (σ/S)

LOQ =10 (σ/S)

Where σ is the standard deviation of the response and ‘S’ is the slope of the linearity curve.

The LOD and LOQ of Ornidazole were 0.015771μg/ml and 0.047793μg/ml respectively.

RESULTS AND DISCUSSION:

The present study was aimed to develop a simple, eco-friendly, cost effective, precise and accurate RP-HPLC method for the estimation of Ornidazole from bulk and tablet dosage forms by using an eco-friendly novel hydrotropic mobile phase. C₁₈ analytical column was chosen as the stationary phase for the separation and determination of Ornidazole. After solubility studies different concentrations of Urea solution were tested as mobile phase. The choice of the optimum composition is based on the chromatographic response factor, a good peak shape with minimum tailing. 5% solution of Urea as hydrotropic mobile phase was proved to be most suitable since the chromatographic peak obtained was well defined and almost free from tailing. The retention time of Ornidazole was found to be 3.996 min (Figure 5). The system suitability studies were carried out to check various parameters such as number of theoretical plates and tailing factor. The tailing factor for Ornidazole peak was 1.3, indicating good peak symmetry (acceptance limit is < 2) and the number of theoretical plates was 4731.7 shows good column efficacy. The linearity was found satisfactory for the drug in the range 10-50μg/ml (Table1). The regression equation of the linearity curve of Ornidazole was found to be Y=49321X+33223 (where Y is the peak area and X is the concentration of Ornidazole in μg/ml). Precision of the method was studied by repeatability (Table 4) and inter-day precision (Table 5) and the results showed lower %RSD values, indicate that the method is quite precise. The percent recoveries of the drug solutions were studied at three different concentration levels. The percentage individual recovery and the %RSD at each level were within the acceptable limits (Table 3). This indicates that the method is accurate. The absence of additional peaks in the chromatogram indicates non-interference of the commonly used excipients in the tablets and hence the method is specific. The deliberate changes in the method have not much affected the peak tailing, number of theoretical plates and the percent assay. This indicates that the present method is robust (Table 6). The result of analysis of tablet was found 99.36% (Table 2). The LOD and LOQ of Ornidazole were 0.015771μg/ml and 0.047793μg/ml respectively. The lowest values of LOD and LOQ as obtained by the proposed method indicate that the method is sensitive. The summary of all the results was presented in Table 8.

Table 8: Summary of Results

PARAMETERS	ORNIDAZOLE
^{Absorption maxima(nm)}	320
Retention Time(min)	3.996
^{Concentration range(}^μ^g/ml)	10-50
^{Regression equation (y= mx+c)}	Y=49321X+33223
^{Correlation coefficient}	^0.9990
^Specificity	^{No interference of any peaks}
^{Tailing Factor}	^1.3
^{Number of Theoretical plates}	^4731.7
^{Accuracy(%RSD)} ^80% ^100% ^120%	0.01702 0.01644 0.00476
^{LOD (}^μ^g/ml)	0.015771
^LOQ(^μ^g/ml)	0.047793
^{Intra-day Precision (%RSD)}	0.121423
^{Intermediate Precision} ^{Day 1} ^{Day 2} ^{Day 3}	0.02894 0.15042 0.07875
^{Percentage recovery}	99.36
^{Robustness(%RSD)} ^{Flow rate} ^{Mobile phase composition}	^˂2 ^˂2
^{Ruggedness(%RSD)} ^{Analyst 1} ^{Analyst 2}	0.098077 0.066197

CONCLUSION:

The proposed method describes a novel eco-friendly RP-HPLC method for the determination of Ornidazole employing a special mobile phase comprising of 5% Urea solution was found to be satisfactory and give sharp peak for Ornidazole. By proper choice of hydrotropic agents, the use of organic solvents in analysis may be discouraged to a large extent. Hydrotropic solubilisation technique and hydrotropic solution as mobile phase will be surely utilized in large scale for the method development of poorly-water soluble drugs by RP-HPLC in future. So, it can be concluded that the developed method is novel, simple, precise, cost-effective, eco-friendly, safe and can be successfully employed for the routine analysis of Ornidazole in bulk and pharmaceutical dosage forms.

ACKNOWLEDGEMENT:

The authors are thankful to State Board of Medical Research A2/ (SBMR. 2019-2020)/17842/2019/MCT for providing fund and IUCGGT, University of Kerala, Karyavattom for providing necessary facilities to carry out this research work.

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Received on 06.07.2020 Modified on 10.08.2020

Asian J. Res. Pharm. Sci. 2021; 11(1):15-21.

DOI: 10.5958/2231-5659.2021.00003.5