Development of a Chromatographic Technique for Quantification of Drotaverine Hydrochloride and Diclofenac Potassium

Introduction

The benzyl isoquiniline derivative Drotaverine Hydrochloride (DROTA) has parasymptholytic action. It may relieve pain without causing drowsiness or loss of awareness. The spasmolytic activity of this drug act by inhibiting the phosphodiesterase-4. It shows clinical importance in various disease associated with smooth muscle spam ^1-3. The phenylacetic acid derivative, Diclofenac Potasium (DICLO) is classified as a non-steroidal anti-inflammatory medication acts by inhibiting cyclooxygenase enzyme, which is the key enzyme responsible for the synthesis of prostaglandins (PGs). PGs play a role not only in the inflammatory response but also in the transmission of pain signals. ^4-6.

Based on the reported articles DROTA can be determined in pharmaceutical formulations using a number of different UV and HPLC procedures, both alone and in combination with other medications ^7-11. Several UV and chromatographic approaches have been developed for estimating DICLO in pharmaceutical formulations in conjunction with other medicines ^12-16. An RP-HPLC technique was used to evaluate DROTA and DICLO in human plasma simultaneously. ¹⁷. Various spectrophotometric methods like 1^st order Spectra, absorption corrected method and area under curve; and HPTLC had been developed for combination of DROTA and DICLO ^18-20. From the literature survey, it is concluded that simultaneous analysis of DROTA and DICLO in drug formulation by RP-HPLC is not available. Hence, an attempt was made in this study for simultaneously estimation of dosage form by RP-HPLC .

Figure 1: Structural Formula of A. Drotaverine Hydrochloride B. Diclofenac Potassium Click here to View Figure

Materials and Methods

Ingredients Used

Active pharmaceutical ingredients of Drotaverine Hydrochloride and Diclofenac Potassium were purchased from Yarrow Chemicals, Mumbai, India. Throughout the course of the research, AR grade distilled water, methanol, and orthophosphoric acid were all utilized.

Instrumentation and Chromatographic conditions

A highly sensitive Adventurer-Pro, AVG264C electronic balance, the HPLC (shimadzu) outfitted with LC-20 AT binary pump, SPD-20A PDA detector and LC solution as software for the investigation. For the separation, a Phenomenex C18 column (250 mm × 4.6 mm i.d., 5 μm) was used, and the injector volume was 20 μL. In order to carry out the chromatographic separation, a mobile phase that consisted of methanol and water that had been adjusted to a pH of 3 using orthophosphoric acid (80:20) was employed at a flow rate of 1.0 ml/min. The peak area of the effluent was measured using a PDA detector, while it was being observed at 280 nm.

Preparation of Mobile Phase

Mix Methanol and Water in the proportion of 80:20 (v/v), and modify the pH to 3 with orthophosphoric acid. Sonicate it for 30 minutes before passing it through a 0.2 µ membrane filter. The mobile phase is used throughout the process as diluents.

Stock solution preparation for Standard

Weigh 40 mg DROTA, add 30 ml diluent, sonicate for 20 min & make up the volume to 50 ml with diluent to produce standard stock solution of 800 µg/ml. Weigh 25 mg DICLO, add 30 ml diluent, sonicate for 20 min & make up the volume to 50 ml with diluent to produce standard stock solution of 500 µg/ml. Withdraw 10 ml of standard stock solution of DROTA and DICLO, transfer into 100 ml volumetric flask and dilute upto mark with diluent. It gives 80 μg/ml of DROTA and 50 μg/ml of DICLO.

Preparation of Calibration Curve

The linearity of the HPLC method for analysis of DROTA and DICLO were assessed and evaluated by analyzing a series of different concentrations of DROTA and DICLO standard solutions. In which the reasonable linearity was achieved in the range of 16-48 μg/ml for DROTA and 10-30 µg/ml for DICLO. Peak areas of these solutions were measured at 280 nm.

Stock solution preparation for Formulation sample

Weigh accurately 20 tablets and calculate the average weight. Triturate them in glass mortar. Powder equivalent to 80 mg of DROTA and 50 mg of DICLO was weighed and transferred into the 100 ml of volumetric flask, add 60 ml diluent and sonicate it for 30 minutes. Filter the solution through 0.2μ membrane filter and dilute upto mark with diluent. The solution was further diluted to obtain the conc. 80 μg/ml of DROTA and 50 μg/ml of DICLO.

Method validation ^21-23

Linearity and Range

When calculating the linearity of the response, the concentration limits of 16-48 µg/ml for DROTA and 10-30 µg/ml for DICLO were employed. After visualizing peak regions against concentration on the calibration curve, we were able to determine the correlation coefficient for DROTA and DICLO as well as the equation for the regression line. The linearity was quantified using the correlation coefficient of linear regression line.

Precision

Repeatability

The repeatability of the synthetic combination was evaluated based on the findings of six separate experiments by taking a solution containing 16 µg/ml of DROTA and 10 µg/ml of DICLO from working standard solution.

Intra-day precision

Triplicate samples of solutions containing 24:15, 32:20 and 40:25 µg/ml of DROTA and DICLO were analyzed on the same day to determine the intraday precision.

Interday precision

Triplicate samples of solutions containing 24:15, 32:20 and 40:25 µg/ml of DROTA and DICLO were analyzed on the three separate days to determine the interday precision.

Accuracy

The recovery study was carried out to test the reliability of the procedure. Increasing aliquots of standard stock solution (0, 0.16, 0.20, and 0.24 ml of 800 μg/ml of DROTA and 0, 0.16, 0.20, and 0.24 ml of 500 μg/ml of DICLO) were introduced simultaneously to a constant amount of preanalyzed sample (2.0 ml) in a 10ml vol. flask. The vol. was adjusted with mobile phase. The measured concentration and the actual concentration were compared.

LOD and LOQ

The LOD and LOQ were estimated by

LOD = 3.3× SD/Slope

LOQ = 10× SD/Slope

Estimation of Marketed formulation

2 ml was drawn from the sample stock solution and volume was adjusted up to 10 ml with mobile phase. The peak area was measured at 280 nm.

Results and Discussion

Mobile phase optimisation

Different solvent systems were tried for separation of DROTA and DICLO. Separation was achived in mobile phase, Methanol and Water in the proportion of 80:20 (v/v), and modifies the pH to 3 with orthophosphoric acid. The Retention times were found to be 2.003 min. for DROTA and 6.820 for DICLO. Chromatograms of DROTA and DICLO were shown in Figure 2.

Figure 2: A. Chromatogram of Drotaverine Hydrochloride B. Chromatogram of Diclofenac Potassium C. Chromatogram of Drotaverine Hydrochloride (RT: 2.003) and Diclofenac Potassium (RT: 6.820). Click here to View Figure

Method Validation

Linearity was determined between 16-48 µg/ml of DROTA and 10-30 µg/ml of DICLO (Table 1).  Correlation coefficients of 0.999 were found for both the DROTA and DICLO. The calibration curve of DROTA and DICLO were shown in Figure 3. The regression line equation was y = 13.96x + 82.68 for DROTA and y = 48.16x + 164.6 for DICLO. The linearity chromatogram of DROTA and DICLO were shown in Figure 4.

Table 1: Calibration data for Drotaverine Hydrochloride and Diclofenac Potassium

Drotaverine Hydrochloride		Diclofenac Potassium
Conc. (µg/ml )	Peak area (n=3)	Conc. (µg/ml )	Peak area (n=3)
16	306.981 ± 4.45	10	649.938 ± 05.59
24	418.969 ± 4.52	15	887.818 ± 07.68
32	523.979 ± 4.67	20	1129.575 ± 13.51
40	646.361 ± 9.35	25	1349.474 ± 07.03
48	752.009 ± 8.03	30	1623.268 ± 10.08

n= number of determinations

Figure 3: Calibration curve of (A) Drotaverine Hydrochloride and (B) Diclofenac Potassium Click here to View Figure

Figure 4: Linearity chromatogram of Drotaverine Hydrochloride  and Diclofenac Potassium Click here to View Figure

Precision

The repeatability, intraday and interday pecisions of the suggested procedure were all taken into consideration while evaluating its accuracy. The fact that the percentage RSD is less than 2% at each level, as evidenced by the findings, makes it abundantly evident that the suggested approach is accurate enough for the analysis of the drug (Table 2). 

LOD and LOQ

The LOD and LOQ for DROTA were calculated to be 1.08 µg/ml and 3.28 µg/ml respectively whereas The LOD and LOQ for DICLO were esimated to be 0.25 µg/ml and 0.76 µg/ml respectively (Table 2).

Accuracy

It was determined that the analytical process was accurate by conducting a percentage recovery analysis. The accuracy of the suggested approach for the analysis of the medication is demonstrated by the recovery values, which fall within the range of 98–102% (Table 2).

Formulation Assay

The applicability of the suggested procedure was investigated by examining the Tablet formulation of Verin D that is available for purchase commercially. Their findings are presented in Table 3.

Table 2: Brief Description of Method Validation

Parameters	Drotaverine Hydrochloride	Diclofenac Potassium
Range of Linearity (µg/ml )	16-48	10-30
Correlation coefficient	0.999	0.999
Regression equation	y = 13.96x + 82.68	y = 48.16x + 164.6
LOD (μg/ml)	1.08	0.25
LOQ (μg/ml)	3.28	0.76
Precision (%RSD)
Repeatability (n=6)	0.60	0.20
Intraday (n=3)	0.80	0.50
Interday (n=3)	1.10	0.80
Accuracy
80% (n=3)	100.94	101.02
100% (n=3)	100.52	100.61
120% (n=3)	100.57	100.92

n= number of determinations

Table 3: Assay of Formulation (Verin D)

Drugs	Labeled Claim (mg)	Amount found (mg)	Amount found (%)*	% RSD
Drotaverine Hydrochloride	80	79.32	99.15 ± 0.813	0.820
Diclofenac Potassium	50	49.46	98.92 ± 0.741	0.749

* mean ± SD

Conclusion

The newly developed RP-HPLC method for the detection of Drotaverine hydrochloride and Diclofenac potassium in a combination formulation shows accurate sensitive, rapid, reproducible results than other established methods. The method had a good resolution for both of the medications despite having an analysis time that was less than ten minutes, which allowed the quick quantification of a large number of samples during routine and quality control examination of tablet formulations. The developed method has been proven to work. It was discovered to be straightforward, specific, and accurate. The fact that the percentage of recovered drug in tablets is high shows that the excipients that are included in the dosage forms do not interfere with the estimation. The procedure that was just described is one that is suitable for use in the regular examination of mixed dose forms of Drotaverine hydrochoride and Diclofenac potassium. Additionally, it has applications in the quality control of large-scale manufacturing.

Acknowledgement

Authors are thankful to Ms. Puja Bhimani for technical support for completing the research work.

Conflict of Interest

Authors do not have any conflict of interest.

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