INTRODUCTION:

Analytical Chemistry¹ is the science to analyze morphologies, compositions, and quantities of analytical result have played critical roles from the understanding of basic science to a variety of practical Application, such as biomedical application, environment monitoring, quality control of industrial manufacturing, and forensic science. UV absorption spectroscopy is widely used for quantitative determination of compound that absorbs ultraviolet radiation².

This determination is based on Beer – Lamberts law. The method validation^3,4 can be defined by “Establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specification and quality characteristics⁵. The method validation is an integral part of the method development; it is the process of demonstrating that analytical procedures ^6,7 are suitable for their intended use and that they support the identity, quality, purity, and potency of the drug substances and drug products. Simply, method validation is the process of proving that an analytical method⁸ is acceptable for its intended purpose. The method of Ruggedness is defined as the reproducibility of results when the method is performed under actual use conditions. This includes different analyst, laboratories, column, instruments, source of reagents, chemicals, solvents etc. method ruggedness may not be known when a method is first developed, but insight is obtained during subsequent use of method.

The ornidazole is chemically called as [a-(chloromethyl-2-methyl-5-nitrioimidazoetanol], derivative of nitroimidazole drug known as antiprotozoal and antibacterial agent. Therapeutically used in the treatment of infections due to anaerobic germs such as bacteroides, fragilis, specifically useful in abdominal and gynecological surgery. The Cefixime Trihydrate is chemically knows as Trihydrate of (6R,7R)-7-{[(z)-2-(2-amino-thizol-4-yl)-2-[(carboxymethoxy)imino]acetyl] amino}-3-ethenyl-8-ono-5-thia-1-azabicyclo[4,2,0]oct-2-ene-2-carboxylic acid known as antibacterial antibiotic and antiprotozoal. Therapeutically is used in the treatment of susceptible infections including gonorrhoea, otitis media, pharyngitis, lower respiratory-tract infections.

The pharmaceutical dosage forms of combinational drugs are very much useful in multiple therapies, rather than the use of single drugs formulation because of multiple action, fewer side effects and quicker relief. The present market survey revealed that, day by day new drugs and their combinations with another drugs are being introduced in market as they have more patient compliance than a single drug. Thus it is required to develop methods for analysis with the help of number of analytical techniques ⁹ which are available for the estimation of the drugs in combination.The drug combinations are commonly used clinically and analyst is required to develop suitable method of their analyst. The numbers of techniques like HPLC, HPTLC and Ultraviolet spectroscopy are available from simultaneous estimation of active ingredient in combined dosage formulation. The present market and literature survey revealed that above combination is recently introduced in market and literature survey revealed that few HPLC and HPTLC methods are available for simultaneous estimation of Ornidazole and Cefixime trihydrate in combined dosage form. So, in our study, we have selected Ornidazole, Cefixime trihydrate combined tablet dosage forms by UV simultaneous method for estimation. Since it was thought to develop a precise, accurate, simple, economic, reliable and validated simultaneous UV¹⁰ analytical proper method for these combinations.

MATERIALS AND METHODS:

Instrument

Shimadzu UV 1700 double beam UV- visible spectrophotometer was used along with 1.0 cm path length matched pair of quartz cell for spectrophotometric method.

Experimental:

Standard stock solution:

(a) Ornidazole standard stock solution: An accurately weighed quantity of Ornidazole (100mg) was dissolved in methanolic water (1:1) in 100 ml volumetric flask and volume was made up to mark with methanolic water (1:1). (1000µg/ml)

(b) Cefixime standard stock solution : An accurately weighed quantity of Cefixime (100 mg) was dissolved in methanolic water (1:1) in 100 ml volumetric flask and volume was made up to mark with methanolic water (1:1). (1000µg /ml).

1. Study of Spectra and selection of wavelengths

The aliquot portions of standard stock solutions of Ornidazole and Cefixime were diluted appropriately, with methanolic water (1:1) to obtain a concentration 30µg/ml of Ornidazole and 30µg/ml of Cefixime respectively. The solutions were scanned in the range of 400-200nm in 1.0 cm cell against reagent blank.

From the overlain spectra, the λ _max for Ornidazole and Cefixime are observed at 325nm and 288nm respectively. Hence, the wavelengths 325 nm and 288nm were selected for estimation of Ornidazole and Cefixime respectively. The choice of wavelengths is based on the fact that the contribution of each component to the overall derivative signal is zero at the wavelength at which other component exhibits maximum absorption. Overlain first order derivative spectra of Ornidazole and Cefixime are shown in Fig no.1.

2. Study of Beer-Lambert's Law¹¹

The aliquot portions of standard stock solutions were diluted appropriately with methanol. A linear relationship was observed for Ornidazole (5-30μg/ml) and Cefixime (2-20μg/ml) at 325nm and 288nm.Similarly the aliquot portions of standard stock solution were mixed (5:2) and diluted appropriately with methanol, a linear relationship was observed in range (5-30μg/ml) at 325nm and 288nm at (2- 20μg/ml).The graph plotted as concentration Vs absorbance are shown in Fig No. 2 (a, b, c, d) and Data is given in the Table no.1 (a, b).

3. Estimation of Ornidazole and Cefixime in tablet by proposed method

Standard Preparation:

Accurately weighted the standard drug of Ornidazole and Cefixime Trihydrate 100 mg and 40 mg respectively. Take in separate 100ml volumetric flask add minimum amount of methanolic water dissolve and dilute up to the mark. Aliquot portion of standards solution with methanolic water to get final concentration 20µg/ml of Ornidazole and 8µg/ml of Cefixime. The absorbances were measured at 325nm and 288nm. For the standard solution the absorbance measured were of 0.809 and 0.391 for Ornidazole and Cefixime respectively.

Preparation of sample solution:

Twenty tablets were accurately weighed and crushed to fine powder and mixed thoroughly. An accurately weighed quantity of powder equivalent to about 100 mg of Ornidazole (equivalent to 40 mg of Cefixime ) was taken in 100 ml volumetric flask powder was dissolved in methanolic water (1:1) with vigorous shaking and volume was made up to 100 ml with methanolic water (1:1). The solution was filtered through Whatman filter paper No.41. The aliquot portion of filtrate was diluted with methanol to get final concentration 20μg/ml of Ornidazole and 8μg/ml of Cefixime respectively. The absorbance were measured at 325nm and 288nm.Amount of drugs in the final solution were calculated using same formula as in laboratory mixture.

A_sample Wt. std Avg Weight

% Label claim = ---------- x ------------- x ------------------ x 100 Astd Wt. of sample Label claim

Where

A_sample :Absorbance of sample

Astd :Absorbance of standard

Average weight : 0.8897 g

Results of % estimation of drugs in tablet are shown in Table No.2.

4. Recovery Studies:

Standard solution:

An accurately weighed quantity of Ornidazole (100 mg) and Cefixime (40mg) were mixed and dissolved in methanol in 100 ml volumetric flask. Volume was made up to 100.0 ml with methanolic water (1:1).

Sample Preparation :

An accurately weighed quantity of preanalysed tablet powder equivalent to 100 mg Ornidazole was taken in 100 ml volumetric flask add some methanol to dissolve and volume was adjusted up to the mark with methanolic water (1:1) and the solution was filtered through Whatman filter paper No.41. The aliquot portion of the filtrate was further diluted to get final concentration 20µg/ml and 8 µg/ml for Ornidazole and Cefixime respectively. The absorbance of sample solution was measured at 325nm and 288nm.The total amount of Cx and Cy were calculated by the same formula as under estimation of drugs in laboratory mixture and % label claim using formula given under estimation of drugs in tablet.

The % Recovery was then calculated by using formula;

(A –B)

% Recovery = ----------------- x 100

Where,

A : % Total amount of drug estimated

B : % Amount of drug found on preanalysed basis.

C : % Amount of pure drug added.

Results of recovery study are shown in Table No.3.

RESULT AND DISCUSSION:

Validation¹²parameters

1. Accuracy :

Accuracy of proposed method was ascertained on the basis of recovery study performed by standard addition method. The results of recovery study are as shown in Table No.3

2. Precision :

Precision of an analytical method is expressed as the S.D. and R.S.D. of the series of measurements. It was ascertained by replicate estimation of marketed formulation (five times) and results are as shown in Table No.2.

3. Linearity and range :

According to USP 80% to 120% of test concentration was taken and dilution was done appropriately. Accurately weighed quantity of tablet powder equivalent to 80, 90, 100, 110, 120 mg of Ornidazole were taken into 5 different 100 ml volumetric flasks. Appropriate amount of pure Cefixime was added in same flasks and were dissolved in methanol with vigorous shaking volume was made upto 100 ml with methanolic water (1:1). The solutions were filter through Whatman filter paper No.41 and aliquot portions of filterate were diluted to get final solutions of Ornidazole and CEF. Absorbances were measured at 325nm and 288nm.

Result are shown in Fig.No.3 (a,b) and Tablet No. 4.

4. Ruggedness:

Ruggedness was ascertained by carrying out the analysis for interday variation, intraday variation and different analysts. The results of interday variation, intraday variation and different analyst are shown in Table no.5 (a, b). Validation part are summarized in following Table no.5(c)

Fig no.1: Overlain first order derivative spectra of Ornidazole (ORD) and Cefixime (CEF) (30µg/ml) .

Table no. 1(a): Observation of Calibration Curve of Ornidazole (ORD) and Cefixime (CEF)

Sr. No.	Conc. of Ornidazole (μg/ml)	Absorbance at (nm)	Conc. Of Cefixime (μg/ml )	Absorbance at (nm)
Sr. No.	Conc. of Ornidazole (μg/ml)	325nm	Conc. Of Cefixime (μg/ml )	288nm
1	5	0.198	2	0.106
2	10	0.388	4	0.202
3	15	0.586	6	0.327
4	20	0.805	8	0.391
5	25	1.003	10	0.477
6	30	1.201	12	0.586
7	35	1.471	14	0.677
8	40	1.481	16	0.766
9	45	1.612	18	0.875
10	50	1.688	20	1.001

Fig.no.2(a): Calibration Curve for Ornidazole (ORD)

Fig no. 2(b): Calibration Curve for Cefixime (CEF)

Table no. 1 (b): Observation for the calibration curve of mixture

Sr.No.	Concentration in mixture (μg/ml)		Absorbance at (nm)
Sr.No.	Ornidazole	Cefixime	325nm	288nm
1	5	2	0.203	0.112
2	10	4	0.394	0.211
3	15	6	0.590	0.339
4	20	8	0.812	0.399
5	25	10	1.014	0.485
6	30	12	1.211	0.591
7	35	14	1.465	0.684
8	40	16	1.484	0.775
9	45	18	1.636	0.883
10	50	20	1.665	1.011

Fig no. 2 (c): Calibration graph of mixture at 325 nm Wavelength

Fig no. 2 (d): Calibration graph of mixture at 288 nm Wavelength

Table No.2: Estimation of drugs in tablet dosage form

Sr. No.	Wt. tab. Powder (g)	Absorbance sample (nm)			% label claim
Sr. No.	Wt. tab. Powder (g)	325nm	325nm		ORD	CEF
1.	0.1779	0.807		0.391	99.77	99.25
2.	0.1782	0.809		0.394	99.85	99.56
3.	0.1785	0.812		0.397	100.05	100.19
4.	0.1778	0.805		0.392	99.57	99.29
5.	0.1787	0.815		0.400	100.20	100.80
				Mean	99.88	99.82
				+ S.D.	0.2448	0.6663
				% RSD	0.0024	0.0066

Table No. 3: Result of recovery study

Sr. No.	Weight of tablet Powder (g)	Pure drugs added each (mcg/ml)		Absorbance of sample		% Recovery
Sr. No.	Weight of tablet Powder (g)	ORD	CEF	325 nm	288 nm	ORD	CEF
1	0.1779	5	2	0.810	0.396	100.6	99.6
2	0.1781	10	4	0.816	0.397	100.00	100.5
3	0.1783	15	6	0.819	0.399	100.4	100.86
					Mean	100.33	100.32
					+ S.D.	0.3055	0.6489
					% RSD	0.0030	0.0064

Fig No.3 (b): Linearity and range for Cefixime Trihydrate

Table No.5(a):Ruggdness Interday Variation

Sr.No.	Day	Weight of tablet Powder (g)	Absorbance of sample (nm)			% Label claim*
Sr.No.	Day	Weight of tablet Powder (g)	325		288	ORD	CEF
1	1	0.1780	0.811	0.394		99.96	99.68
2	2	0.1778	0.809	0.392		99.94	100.04
3	3	0.1782	0.811	0.397		100.84	99.82
				Mean		100.25	99.85
				+ S.D.		0.5139	0.1815
				% RSD		0.51	0.18

Table No.5 (b) Ruggedness Intraday Variation

Sr. No.	Time in hour	Weight of tablet Powder (g)	Absorbance of sample (nm)		% Label claim*
Sr. No.	Time in hour	Weight of tablet Powder (g)	325	288	ORD	CEF
1	0	0.1778	0.809	0.398	99.95	100.09
2	2	0.1781	0.812	0.400	99.87	99.91
3	4	0.1783	0.811	0.401	100.78	100.54
				Mean	100.2	100.18
				+ S.D.	0.5038	0.3243
				% RSD	0.50	0.32

Table No.5 (c) Optical and regression characteristics of the proposed method

Parameters	Ornidazole	Cefixime Trihydrate
λ_max	325 nm	288 nm
Linearity Range(µg/ml)	5-30	2-10
Correlation coefficient	1	0.999
Molar extinction coefficient(1mol^-1cm^-1)	8.5 X 10³	2.4 X 10⁴
Sandell’s sensitivity (µg/cm²/0.001 absorbance unit)	0.0257	0.0209
% RSD	0.002	0.006
Standard deviation	0.244	0.666
Slope	0.040	0.048
Intercept	- 0.0121	0.009

CONCLUSION:

The proposed UV Spectrophotometry method for simultaneous estimation of Ornidazole and Cefixime trihydrate in the combined dosage form was to be simple, accurate and reproducible. Beer’s law was obeyed in the concentration range of 5-30 µg/ml and 2-20 µg/ml for Ornidazole and Cefixime trihydrate respectively. Co-efficient of variation was found to be in the range of 0.999 and 0.996 for Ornidazole and Cefixime trihydrate. The percentage recovery studies were found to be 100.33% and 100.32% for Ornidazole and Cefixime trihydrate respectively. So this analytical method that can be employed for routine analysis in quality control laboratory.

REFERENCES:

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Received on 26.07.2016 Accepted on 06.11.2016

Asian J. Pharm. Ana. 2016; 6(4): 246-252.

DOI: 10.5958/2231-5675.2016.00036.3

Sr. No.	% Label claim.	Absorbance
Sr. No.	% Label claim.	325nm	288nm
1	80	0.648	0.313
2	90	0.729	0.352
3	100	0.810	0.391
4	110	0.891	0.430
5	120	0.972	0.469