Simultaneous Determination of Drotaverine and Omeprazole in Combine Dosage Form by RP-HPLC Method

Analytical Chemistry is a measurement of science consisting of a set of powerful ideas and methods that are useful in all fields of science and medicine. It seeks ever-improved means of measuring the chemical composition of natural and artificial materials. This branch of chemistry, which is both theoretical and practical science, is practiced in many laboratories in many diverse ways while the analytical method is a specific application of a technique to solve an analytical problem. Methods of analysis are routinely developed, improved, validated, collaboratively studied, and applied. The discipline of analytical chemistry consists of qualitative and quantitative analysis.1

CHROMATOGRAPHY:²

The term ‘Chromatography’ covers those processes aimed at the separation of the various species of a mixture based on their distribution characteristics between a stationary and a mobile phase.

This may be regarded as an analytical technique employed for the purification and separation of organic and inorganic substances. There are various advanced chromatographic techniques, widely used for the estimation of multi component drugs in their formulations. The various chromatographic techniques are.

• High Performance Liquid Chromatography
• High-Performance Thin Layer Chromatography
• Gas Chromatography

DRUG PROFILE

Drotaverine:²⁷

Omeprazole:³⁸

• Solubility of Drotaverine: The solubility of the drug sample was determined according to I.P.1996.
• Solubility of Omeprazole: The solubility of the drug sample was determined according to I.P. 1996.

RESULT AND DISCUSSION:

Drotaverine was found to be insoluble in water and soluble in acetonitrile & methanol. Omeprazole was found to be soluble in water and freely frsoluble in methanol & acetonitrile.

Method Development and Its Validation for Simultaneous Estimation of Drotaverine & Omeprazole by Rp-HPLC In Combination Tablet Dosage Form

• Selection of wavelength

The λmax of the two ingredients i.e. Drotaverine & Omeprazole, were found to be 277 nm and 307 nm respectively in methanol as solvent system. The isosbestic point for the drugs were found at 284 nm.

• Preparation of standard solution of Drotaverine:

10 mg of Drotaverine was weighed accurately and transferred into a 100 ml volumetric flask. About 10 ml of HPLC- grade methanol was added and sonicated to dissolve. The volume was made up to the mark with the same solvent. The final solution contained about 100 μg/ml of Drotaverine.

• Preparation of standard solution of Omeprazole:

10 mg of Omeprazole was weighed accurately and transferred into a 100 ml volumetric flask. About 10 ml of HPLC- grade methanol was added and sonicated to dissolve. The volume was made up to the mark with the same solvent. The final solution contained about 100 μg/ml of Omeprazole.

Fig: 3 Drotaverine

Fig: 4 Omeprazole

Preparation of mix Standard solution of Drotaverine & Omeprazole:

Accurately weighed 100 mg of Drotaverine and 100 mg of Omeprazole were transferred to 100 ml volumetric flask. About 40 ml of mobile phase was added and sonicated to dissolve. The volume was made up to mark with same solvent. Then 2 ml of the above solution was diluted to 100 ml with the solvent system. The resultant solution was filtered through a 0.45 ?m membrane filter and degassed under an ultrasonic bath before use. Several working standard solutions are prepared from the above standard solution by serial dilution technique.

Initialization of the instrument:

The HPLC instrument was switched on. The column was washed with HPLC water for45 minutes. The column was then saturated with mobile phase for 45 min. The mobile phase was run to find the peaks. After 20 minutes the standard drug solution was injected in HPLC.

Different chromatographic conditions used and their Optimizations.

The different HPLC chromatographic conditions were used to find out the optimum chromatographic condition for best elution of drugs.

Trial -1

Table: 7 Chromatographic conditions1 (Trial-1)

Trial-2

No peaks were found. Hence chromatogram was not acceptable.

Table: 8 Chromatographic conditions 2 (Trial-2)

Trial-3

Only one peak was observed, resolution were not there. Hence chromatogram was not acceptable.

Table: 9 Chromatographic conditions 3 (Trial-3)

Trial-4

Peak separation was not good, and a third peak was found. Hence chromatogram was not acceptable.

Table: 10 Chromatographic conditions 4 (Trial-4)

The chromatogram obtained after condition 4, Typical chromatogram of DROTAVERINE (RT=2.67 min) and OMEPRAZOLE (RT= 4.95 min).

RESULT AND DISCUSSION:

Here resolution was good, theoretical plate count and symmetry was appropriate. Also, no unwanted little peaks were seen between two peaks. Hence it was acceptable.

Optimized Method:

The selected and optimized mobile phase was acetonitrile : potassium dihydrogen phosphate buffer (0.02M, pH 3.0) (55:45v/v) and conditions optimized were : flow rate (1.0 ml/minute), wavelength (273 nm), Run time was 20 min. Here the peaks were separated and showed better resolution, theoretical plate count and symmetry. The proposed chromatographic conditions were found appropriate for the quantitative determination of the drugs.

Preparation of mobile phase:

Mobile phase was prepared by taking acetonitrile: potassium dihydrogen phosphate buffer (0.02M, pH 3.0) (55:45v/v). Mobile phase was filtered through 0.45 ?m membrane filter and degassed under ultrasonic bath prior to use. The mobile phase was pumped through the column at a flow rate of 1.0 ml/min.

Running the standard solution of Drotaverine:

2 ml of stock solution prepared as mentioned under section 4.5.2was pipette out into a 10 ml volumetric flask. The volume was made up to the mark with mobile phase. The solution was filtered through the 0.45 ?m membrane filter and degassed under ultrasonic bath prior to use. The solution was injected into the HPLC system.

Fig: 6 Chromatogram of Drotaverine

Result & Discussion: Retention time was found to be 2.67 min.

Running the standard solution of Omeprazole:

4 ml of stock solution prepared as mentioned under section 4.5.3 was pipetted into a 10 ml volumetric flask. The volume was made up to the mark with methanol. The solution was filtered through the 0.45 ?m membrane filter and degassed under ultrasonic bath prior to use. The solution was injected into the HPLC system. The chromatogram obtained is shown in figure 7.

Fig:7 Chromatogram of Omeprazole

Result & Discussion: Retention time was found to be 4.97 min.

Result & Discussion: The HPLC system was set with the optimized chromatographic conditions to run the standard solution of Drotaverine and Omeprazole for 10 min. The retention time were found to be2.69 min and 4.97 min respectively.

• Linearity and Range

Result & Discussion: Linearity range was found to be 0-140 µg/ml for Drotaverine. The correlation coefficient was found to be 0.995, the slope was found to be 8031 and intercept was found to be 10243 for Drotaverine. 

Linearity range was found to be 5-100 µg/ml for Omeprazole. The correlation coefficient was found to be 0.998, the slope was found to be 38873 and intercept was found to be 13606 for Omeprrazole.

• Accuracy:

Result & Discussion: The mean recoveries were found to be 98.976, 99.54, 99.57 % for Omeprazole and 98.94, 99.76, 99.31% for Drotaverine The limit for mean % recovery is 95-105% and as both the values are within the limit, hence it can be said that the proposed method was accurate.

• Precision:

Result & Discussion: The repeatability study which was conducted on the solution having the concentration of about 20mg/ml for Drotaverine and 40mg/ml for Omeprazole (n =5) showed a RSD of 0.07684% for Drotaverine and 1.203503% for Omeprazole. It was concluded that the analytical technique showed good repeatability.

• Intermediate precision

Result and discussion: Intraday and interday studies show that the mean RSD (%) was found to be within acceptance limit (≤2%), so it was concluded that there was no significant difference for the assay, which was tested within day and between days. Hence, method at selected wavelength was found to be precise.

Limit of detection and limit of quantification

The detection limit (LOD) and quantitation limit (LOQ) may be expressed as:

L.O.D. = 3.3(SD/S).

L.O.Q. = 10(SD/S)

Where, SD = Standard deviation of the response

             S = Slope of the calibration curve

Result & Discussion

The LOD was found to be 0.32mg/ml and 1.44mg/ml and LOQ was found to be 0.96mg/ml and 4.32mg/ml for Drotaverine & Omeprazole respectively which represents that sensitivity of the method is high.

ASSAY OF DROTAVERINE & OMEPRAZOLE INDOSAGE FORM:

Assay was performed as described in previous chapter. Results obtained are tabulated below:

Table :17 Assay ofDrotaverine &Omeprazole Tablets