Reversed Phase High Pressure Liquid Chromatography Technique for Determination of Oxolamine Citrate from Pharmaceutical Formulation

 

Rele R.V.* and Patil S.P.

D.G.  Ruparel College, Matunga, Mumbai - 400 016.

*Corresponding Author E-mail: spp30@rediffmail.com

ABSTRACT:

A simple, rapid and accurate high performance liquid chromatography method is described for determination of oxolamine citrate from active pharmaceutical ingredients. The separation of drug was achieved on kromasil C18 (250 X 4.6 mm) 5µ column. The mobile phase consisted a mixture of buffer and methanol (60:40 v/v). The buffer was a mixture of 0.05 M potassium dihydrogen phosphate monobasic and triethylamine. The pH of buffer was adjusted to 4.1 by dilute ortho-phosphoric acid. The detection was carried out at wavelength 230 nm. The method was validated for system suitability, linearity, accuracy, precision, robustness, stability of sample solution. The method has been successfully used to analyze oxalamine citrate from pharmaceutical formulation.

 

 

INTRODUCTION:

In this communication the present work proposes a new HPLC method for assay of oxolamine citrate from active pharmaceutical ingredients. Its chemical name is 3-phenyl-5(diethylaminoethyl)-1, 2, 4-oxadiazole citrate. Oxolamine citrate is an anti- inflammatory drug. The drug is official in Chemical Abstracts Service Registry Number¹. Literature survey reveals no official method for assay of this drug. A new, simple, rapid and reliable HPLC method is developed for the determination of oxolamine citrate. This method can be used for the routine analysis. In the proposed work optimization and validation of this method are reported.

 

EXPRIMENTAL:

MATERIALS AND METHODS:

Chemical and reagents:

Reference standard of oxolamine citrate was obtained from reputed firm with certificate of analysis. Potassium dihydrogen phosphate monobasic, triethylamine were used of analytical grade. The HPLC grade water was used from Merck. Standard and sample solutions were prepared in diluent i.e. buffer containing 0.05 M potassium dihydrogen phosphate monobasic and triethylamine. The pH of buffer was adjusted to 4.1 by dilute ortho-phosphoric acid.

 

Instrumentation:

The HPLC system, Water Alliance (2695) HPLC system equipped with separation module and DAD detector (2996), was used. The chromatogram was recorded and peak quantified by mean of PC based Empower software.

A SHIMADZU analytical balance was used.

 

Preparation of Standard preparation:

Standard solution:

About 25 mg standard oxolamine citrate was weighted accurately and transferred in 25 ml volumetric flask. About 15 ml of diluent was added and sonicated for 5 minutes. The volume was adjusted up to the mark with diluent to give concentration as 1000 μg /ml. The working standard solution was prepared by diluting 5 ml of 1000 µg /ml solution to 100 ml with diluent to get concentration 50 µg /ml.

 

Sample preparation:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 25 mg of oxolamine citrate sample was weighted accurately and transferred in 25 ml volumetric flask. About 15 ml of diluent was added and sonicated for 5 minutes. The volume was adjusted up to the mark with diluent to give concentration as 1000 μg /ml. The sample solution was prepared by diluting 5 ml of 1000 µg/ml solution to 100 ml with diluent to get concentration 50 µg /ml.

 

Chromatographic condition:

Chromatographic separation was performed at 40°C temperature on a reverse phase kromasil C₁₈ (250 X 4.6 mm) 5µ column. The mobile phase consisted a mixture of buffer and methanol (60:40 v/v). The buffer was a mixture of 0.05 M potassium dihydrogen phosphate monobasic and triethylamine. The pH of buffer was adjusted to 4.1 by dilute ortho-phosphoric acid. The flow rate of the mobile phase was adjusted to 1.0 ml /min. The detection was carried out at wavelength 230 nm figure 1. The injection volume of the standard and sample solution was set at 20 µl.

 

Figure 1: UV spectra of oxolamine citrate

 

Method Development:

A reverse phase approach was used for estimation of oxolamine citrate by HPLC. Different columns like Techno chrome C18 (250 X 4.6 mm, 5 µ), Unishere C18 (250 X 4.6 mm, µ) were tried to estimating oxolamine citrate. Finally Kromasil C18 (250 X 4.6 mm, 5 µ) offered more advantage over the other columns. It was found that a good symmetrical peak observed at about 7.8 minutes for standard solution of oxolamine citrate. The typical chromatogram of standard and sample assayed are given in the figure 2 and 3 respectively.

 

Figure 2: Typical chromatogram of oxolamine citrate (standard)

 

Figure 3: Typical chromatogram of oxolamine citrate (sample)

 

Method validation:

System suitability:

System performances of developed HPLC method were determined by injecting standard solutions. Parameter such as theoretical plates (N), tailing factor, and relative standard deviation were determined. The results are shown in table 1 which indicates good performance of the system.

 

 

Table 1: System suitability parameters evaluated on standard solution of oxolamine citrate

Retention Time	Area	Area %	USP Plate Count	USP Tailing
7.82 minutes	1128096	100.0	7920	1.28

 

Specificity:

Specificity is the ability of the method to resolve the active ingredients. Hence blank (diluent), benzamidoxime, oxolamine citrate were injected to prove specificity. The typical chromatogram of the standard and sample assayed are given in figure 2 and 3 respectively.

 

Linearity:

Under the experimental conditions described above, linear calibration curve were obtained throughout the concentration range studied. Regression analysis was done on the peak area (y) v/s concentration (x). The regression analysis data obtained is tabulated in table no. 2.

 

Table 2: Statistical evaluation of the data subjected to regression analysis

Parameters	Oxalamine citrate
Correlation Coefficient (r)	1.0000
% Intercept (y)	-303.350
Slope (m)	11332.613

 

 

Accuracy:

The accuracy method was determined by applying proposed method to synthetic mixture containing known amount of drug corresponding to 80 %, 100 % and 120 %. The accuracy was then calculated as the percentage of analyte recovered by the assay. The results of the recovery analysis are enclosed under table no.3.

Table 3: Statistical evaluation of the data subjected to accuracy of oxolamine  citrate

Injection	Level (%)	Conc. of spiked oxalamine citrate (ppm)	Area	Amount found in ppm	% Recovery
01	80 %	80	904811	80.48	100.2
02			903074	80.33	100.0
01	100 %	100	1129291	100.45	100.4
02			1130880	100.59	100.6
01	120 %	120	1364887	120.40	101.2
02			1364549	120.37	101.1
				Mean	100.6
				Standard Deviation	0.556776
				% RSD	0.55

 

 

Precision:

The method precision was established by carrying out the analysis of oxalamine citrate. The assay was carried out of the drug using analytical method in six replicates. The value of relative standard deviation lies well with the limits (0.10 %). The results of the same are tabulated in the table no. 4.

 

Table 4: Statistical evaluation of the data subjected to method precision of oxolamine citrate

Sr. No.	Sample name	Assay (%)
01	Test solution – 1	99.5
02	Test solution – 2	99.5
03	Test solution – 3	99.1
04	Test solution – 4	99.7
05	Test solution – 5	99.4
06	Test solution – 6	99.4
Mean		99.4
Standard Deviation		0.20110
% RSD		0.20

 

 

Robustness:

The robustness of the method was determined to check the reliability of an analysis with respect to deliberate variations in method parameters.

The typical variations were given below:

Variation in the flow rate by + 0.2 ml /min

Variation in wavelength by + 5 nm

Variation in concentration of mobile phase by + 2 %

The results of the analysis of the samples under the conditions of the above variation indicated the nature of robustness of the method.

 

Stability of solution:

The stability of the solutions under study was established by keeping the solutions at room temperature for 24 hours. The results indicated no significance change in assay results of the solutions. It confirmed the stability of the drug in the solvents used for the analysis.

 

Method application:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 25 mg of oxolamine citrate sample was weighted accurately and transferred in 25 ml volumetric flask. About 15 ml diluent was added and sonicated for 5 min to dissolve it. Further volume was made up to the mark with the diluent to give 1000 µg /ml. Further the 5 ml of this solution was diluted to 100 ml with diluent to give 50 µg /ml of oxolamine citrate. From this solution 20 µl was injected specific conditions. The analyte peak was identified by comparison with that of respective standard. The (%) assay results were expressed in table no. 4. It indicates the amount of oxalamine citrate in the product meets the requirement.

 

RESULT AND CONCLUSION:

The reproducibility, repeatability and accuracy of the proposed method were found to be satisfactory which is evidenced by low values of standard deviation and percent relative standard deviation. The accuracy and reproducibility of the proposed method was confirmed by recovery experiments, performed by adding known amount of the drug to the pre-analyzed active pharmaceutical ingredient and reanalyzing the mixture by proposed method. Thus the proposed RP-HPLC method is novel method for the estimation of oxolamine citrate from active pharmaceutical ingredient. It is more precise, accurate, linear, robust, simple and rapid method. Hence the proposed RP-HPLC method is strongly recommended for the quality control of the raw material, active pharmaceutical ingredient and pharmaceutical formulation.

 

ACKNOWLEGMENTS:

Authors express sincere thanks to the principal and head of chemistry department of D.G. Ruparel College, Mumbai for guidance, encouragement and providing laboratory facilities.

 

REFERENCES:

1.       Chemical Abstract Service Register Number 1949-20-8

 

 

 

Received on 28.12.2010       Modified on 19.01.2011

Accepted on 23.01.2011      © RJPT All right reserved