Development and Validation of RP- HPLC Method for Estimation of

Drotaverine Hydrochloride and Nimesulide in Pharmaceutical Dosage Form

G. Tulja Rani¹*, D.Gowri Sankar², P. Kadgapathi³ and B. Satyanarayana⁴

¹Department of Pharmaceutical Analysis, Sarojini Naidu Vanitha Pharmacy Maha Vidyalaya, Exhibition Grounds, Nampally, Hyderabad- 500001, India

²Department of Pharmaceutical Analysis and Quality Assurance, University College of Pharmaceutical sciences, Andhra University, Visakhapatnam- 530003, India

³Hetero Drugs Ltd. Balanagar, Hyderabad- 500055, India

⁴Neosun Biotech (India) Pvt. Ltd., Hyderabad, 500007 India

*Corresponding Author E-mail: tuljapharma@yahoo.com

ABSTRACT:

The present work describes a reverse phase high performance liquid chromatographic method for the simultaneous estimation of drotaverine hydrochloride and nimesulide in combined dosage forms. Chromatography was performed on Phenomenex Luna C18 (250 × 4.6 mm i.d. and particle size 5 µm) column in isocratic mode with mobile phase containing acetonitrile: 0.3% triethylamine aqueous solution (adjusted to pH 3.0 using 1 % ortho phosphoric acid) in the ratio of 75:25v/v. The flow rate was 1.0 mL/min and effluents were monitored at 246 nm. The selected chromatographic conditions were found to be useful in separating drotaverine hydrochloride (run time 2.435 min) and nimesulide (run time 4.019 min). Linearity for drotaverine hydrochloride and nimesulide was found to be in the range of 0.5-100 and 1.0-200 µg/mL, respectively with linearity coefficient of 0.9998 and 0.9993. Percent recovery of the drugs was found to lie between 99.87- 100.13. The proposed method was validated by different parameters. It was found to be accurate, precise, reproducible and specific and hence can be used for simultaneous analysis of these drugs in combined dosage forms.

KEYWORDS: Drotaverine, Nimesulide, Simultaneous, Validation.

INTRODUCTION:

Drotaverine hydrochloride is an antispasmodic drug and it is selective inhibitor of phosphodiesterase 4 and has no anticholinergic effects. Nimesulide¹ is a relatively cox-2-selective non-steroidal antiinflammatory drug (NSAID) with analgesic and antipyretic properties. Chemically drotaverine hydrochloride, is (1Z)-1-[(3, 4-diethoxyphenyl) methylidene]-6, 7-diethoxy-3, 4-dihydro -2-H- isoquinoline and nimesulide is N- (4-Nitro-2 – phenoxyphenyl) -methane sulphonamid.(Figure 1a and 1b) .

Literature survey revealed that there are several analytical and bioanalytical methods were reported for estimation of drotaverine hydrochloride alone and in combination with other drugs in formulations.

Similarly, the methods were also reported for the estimation of nimesulide alone and in combination with other drugs^2-13. However, there is no RP-HPLC method available for simultaneous estimation of drotaverine hydrochloride and nimesulide in combined dosage form. Hence a precise, accurate, specific and sensitive RP-HPLC method was developed for simultaneous estimation of drotaverine hydrochloride and nimesulide in tablet dosage forms and validated.

EXPERIMENTAL:

Chromatographic analysis was performed on Shimadzu HPLC model, equipped with Lc-10At pump, UV-Visible detector SPD-10AVP, rheodyne injector with 20 µL fixed volume loop and Phenomenex C18 column (250 × 4.6 mm I.d. and 5µ particle size). Spinchrome software was used for chromatographic analysis. Spectral measurements were recorded by using ShimadzuUV- 1800 double beam UV-Visible spectrophotometer with 10 mm matched quartz cells and software UV Probe -2.31 version. The pure drug samples drotaverine hydrochloride and nimesulide were obtained as gift samples from Blue Cross Pharmaceuticals Ltd., Nasik and Trident Pharmaceuticals Ltd., Hyderabad (Andhra Pradesh, India) respectively.

Figure1a. Structure of drotaverine hydrochloride

Figure1b. Structure of nimesulide

Tablet formulation A (NOBEL SPAS, Mankind Pharma. Ltd., New Delhi, India) containing labeled amount of 40 mg of drotaverine hydrochloride and 100 mg of nimesulide were procured from local market. Acetonitrile used in the investigation was of HPLC grade, while ortho phosphoric acid and triethylamine were of analytical grade both made by S.D fine chemicals, Mumbai, India. Triple distilled water was used to carry out analysis.

Chromatographic conditions:

A reverse phase C18 column was equilibrated with the mobile phase acetonitrile and 0.3 % triethylamine aqueous solution (adjusted to pH 3.0 using 1 % ortho phosphoric acid) in the ratio of 75:25 v/v. Flow rate of mobile phase was maintained at 1.0 mL/min and effluents were monitored at 246 nm. The sample was injected using a 20 µL fixed loop and the total run time was 10 min. The analysis was carried at room temperature.

Preparation of mobile phase:

Different mobile phases were tried in order to find best possible conditions. A combination of acetonitrile and 0.3 % triethylamine aqueous solution (adjusted to pH 3.0 using 1% orthophosphoric acid) in the ratio of 75:25 v/v was found to be optimum mobile phase. The mobile phase was filtered through 0.45 µm membrane filter and sonicated for 15 min.

Preparation of stock solutions:

Drotaverine hydrochloride and nimesulide were weighed (50 mg each), transferred to separate standard 50 mL volumetric flasks, dissolved and dissolved and made up to the mark with mobile phase to obtain a concentration of 1mg/mL.

Determination of drotaverine hydrochloride and nimesulide in their combined dosage forms:

Twenty tablets were weighed and powdered. Tablet powder equivalent to 20 mg of drotaverine hydrochloride and 50 mg of nimesulide was accurately weighed and transferred to 50 mL volumetric flask. The sample was dissolved by sonicating in 20 mL of the mobile phase for the 5 min. and made up to the mark with mobile phase and filtered through 0.35 µm membrane filter. An aliquot volume was appropriately diluted with mobile phase to obtain solution containing 8 µg/mL of drotaverine hydrochloride and 20 µg/mL of nimesulide. The solution was sonicated for 10 min. and 20 µL of the sample solution was injected into the HPLC system at above chromatographic conditions, chromatogram was recorded and the amount of drotaverine hydrochloride and nimesulide present in table formulation was calculated by comparing the mean the peak areas of sample with that standard. The results were presented in Table -4.

METHOD VALIDATION:

The method was validated for accuracy, precision, specificity, detection limit, quantitation limit and robustness.

Linearity:

Appropriate aliquots of drotaverine hydrochloride and nimesulide stock solutions were taken and diluted with mobile phase to obtain final concentrations of 0.5-100 µg/mL of drotaverine hydrochloride and 1.0-200 µg/mL of nimesulide, respectively. An aliquot of 20 µL of the solution from each flask was injected six times in to the column, then the peak area and retention time were recorded. Calibration curves were constructed by plotting average peak area and concentration. Regression equation for the calibration curve was computed for drotaverine hydrochloride and nimesulide.

Accuracy:

The accuracy of the method was determined by calculating recoveries of drotaverine hydrochloride and nimesulide by standard addition method. A known amount of drotaverine hydrochloride (0, 8, 12, 14 µg/mL), and nimesulide (0, 10,20,30,40 µg/mL) were added to a pre-quantified sample solution and the amount of drotaverine hydrochloride and nimesulide were estimated by measuring peak areas and by fitting these values to the straight line equation of calibration curve.

Precision:

The intra-day and inter- day precision was carried out for 3 different concentrations of drotaverine hydrochloride (60 µg/mL, 80 µg/mL, 100 µg/mL ) and nimesulide (16 µg/mL, 40 µg/mL, 60 µg/mL ) by estimating the corresponding responses six times on the same day and for three different days. The results are reported in terms of % relative standard deviation (% RSD).

Figure 3: calibration curve of drotaverine hydrochloride

Specificity:

Commonly used excipients (starch, micro crystalline cellulose and magnesium stearate) were spiked into a pre-weighed quantity of drugs. The chromatogram was taken by appropriate dilutions and the quantities were determined. No interference of placebo was observed with the principal peaks hence the method was specific for these drugs.

Detection limit and quantitation limit:

The limit of detection and quantification for drotaverine hydrochloride and nimesulide was determined. A signal to noise ratio of 3:1 and 10:1 were calculated respectively for the determination of detection and quatitation limit.

Robustness:

Robustness of the method was determined by making the slight changes in the chromatographic conditions i.e. composition of mobile phase by ± 2 % and pH by ± 0.2 %.

Figure 4: Calibration curve of nimesulide

Stability:

No changes in the assay values were observed after 24 hrs, indicating stability of the drugs in the solvent used during analysis

RESULTS AND DISCUSSION:

Mobile phase was optimized based on resolution, asymmetric factor and the peak areas obtained for both drotaverine hydrochloride and nimesulide. The mobile phase acetonitrile:0.3 % triethylamine aqueous solution pH adjusted to 3.0 with 1 % ortho phosphoric acid (75:25 v/v) was found to be satisfactory and gave two symmetric and well resolved peaks of drotaverine hydrochloride and nimesulide. The resolution between drotaverine hydrochloride and nimesulide was found to be 9.833, which indicates good separation of both the compounds.

Table 1.Column performance test parameters

System suitability parameters

Drotaverine hydrochloride

Nimesulide

Retention time (min) *

Resolution*

Theoretical plates *

Tailing factor*

2.433

……..

4050

1.465

4.017

9.833

8938

1.142

* Each value is the mean of 6 determinations

Table 2 ValidationParameters and Regression Analysis of the Calibration Curves for the Proposed Method

Parameter

Drotaverine hydrochloride

Nimesulide

Linearity

Regression equation

Slope (m)

Intercept (c)

Correlation coefficient

Limit of detection (µg/mL)

Limit of quantification (µg/ml)

Intra-day precision (%RSD)*

Inter-day precision (%RSD)*

0.5-100

42.752x+ 11.569

42.938

11.569

0.9998

0.2

0.58

0.21

0.24

1.0-200

20.106x-10.451

20.022

10.451

0.9993

0.4

1.0

0.32

0.29

* Average of six determinations

The retention time for drotaverine hydrochloride and nimesulide were 2.435 and 4.019 min, respectively [Figure-2]. The asymmetric factor for drotaverine hydrochloride and nimesulide were 1.496 and 1.142, respectively. Overlay spectra of both drotaverine hydrochloride and nimesulide showed that both the drugs absorb appreciably at 246 nm and hence the detection wavelength was fixed at 246 nm. The system suitability test parameters are shown [Table -1]. Data from system suitability studies indicate conformity to compendial requirements. The calibration curves were obtained by plotting the peak areas versus the concentration over the range of 0.5, 1, 2, 4, 6, 8, 12, 14, 16, 18, 20, 40, 50, 60, 80, 100 µg/mL for drotaverine hydrochloride and 1, 2, 8, 16, 20, 40, 50, 60, 80, 120, 160, 200 µg/mL for nimesulide (Figure 3 and 4). The correlation coefficient was found to be 0.9998 and 0.9993 for drotaverine hydrochloride and nimesulide, respectively. The data of regression analysis of calibration curves are shown in [Table-2]. The recoveries of drotaverine hydrochloride and nimesulide were found to be in the range of 99.87 to 100.06 %and 99.90 to 100.3 % respectively. The high percentage recoveries of the drugs indicate that the method is highly accurate. Recovery study data is shown in [Table- 3]. The low coefficients of variation observed in the intra-day and inter-day variation study shows that the proposed HPLC method is highly precise and the results are presented in the Table-2. The absence of additional peaks in the chromatogram indicates non interference of the common excipients used in tablets. The detection limit for drotaverine hydrochloride and nimesulide were 0.2 µg /ml, 0.4 µg/mL and quantification limit were 0.58 µg/mL and 1.0 µg/mL respectively, which suggest that a nanogram quantity of the compound can be estimated accurately. The method exhibits good robustness because the slight changes made in chromatographic conditions did not influence the analytical results. The validation parameters are summarized in [Table -2]. The proposed liquid chromatographic method was applied for the determination of drotaverine hydrochloride and nimesulide in their combined pharmaceutical dosage form. The results for drotaverine hydrochloride and nimesulide were comparable with the corresponding labeled amount [Table -4].

Table 3. Recovery Studies data showing amount of drug recovered from sample solution

Sample amount (mg)

Amount added (mg)

Amount recovered (mg)

% Recovery*

Drotaverine hydrochloride

Nimesulide

7.99

11.99

16.01

19.98

21.99

19.98

30.04

39.98

49.95

59.98

99.87

99.91

100.06

99.90

99.95

99.92

100.13

99.95

99.90

99.96

* Mean of six determinations

CONCLUSION:

The study describes a new RP-HPLC method for the estimation of drotaverine hydrochloride and nimesulide combination in mixture. The method gives good resolution between both the compounds with a short analysis time (< 6 min).The method was validated and found to be simple, sensitive, accurate and precise. Percentage of recovery shows that the method is free from interference of the excipients and hence it can be used for routine analysis of drotaverine hydrochloride and nimesulide in their combined dosage form

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Trident pharmaceuticals, Hyderabad for providing gift sample of nimesulide and M/S Blue Cross Pharmaceuticals Ltd., Nashik for providing gift sample of drotaverine hydrochloride. The authors are also thankful to the principal and management of Sarojini Naidu Vanitha Pharmacy Maha Vidyalaya for providing facilities to carry out research work.