INTRODUCTION:

Analytical method development is a standardized laboratory procedure to estimate the concentration or amount of active pharmaceutical ingredients in pharmaceutical dosage form or in pure form^1,2.

Dicyclomine hydrochloride is chemically 2-(Diethylamino) ethyl 1,1’-bis (cyclohexyl)-1-carboxylate³. Dicyclomine hydrochloride has empirical formula C₁₉H₃₅NO₂.HCl with a molecular weight of 345.96⁴. It is an antispasmodic and anticholinergic (antimuscarinic) agent⁵. Dicyclomine hydrochloride helps to relief cramps of intestine, bladder and stomach⁶. The drug is soluble in methanol, ethanol and sparingly soluble in water⁷. The chemical structure of dicyclomine hydrochloride is given in (Fig.1).

Fig. 1: chemical structure of dicyclomine hydrochloride

Numerous HPLC methods has been reported for estimation of dicyclomine hydrochloride with other combinations such as RP-HPLC method for simultaneous estimation of paracetamol, pamabrom and dicyclomine, hydrochloride in tablet dosage form⁷, Paracetamol and Dicyclomine hydrochloride in tablet dosage form⁸, dextropropoxyphene HCL, dicyclomine and paracetamol in capsule formulation⁹, paracetamol and dicyclomine hydrochloride in tablet dosage form¹⁰, dicyclomine and mefanamic acid in tablet dosage form¹¹.

From the literature survey it is evident that there is no RP-HPLC method reported for estimation of dicyclomine hydrochloride in drops form.

MATERIALS AND METHODS:

Instrument:

RP-HPLC Shimadzu LC Prominence SPD 20A model with Spinchrom software were used for method development and validation of dicyclomine hydrochloride. For sonication Kroma Tech (KL-1.5) sonicator was used.

Chemicals and reagents:

Dicyclomine hydrochloride in a pure form was provided by Indoco Remedies Limited, Navi Mumbai, Maharashtra, India. The Marketed formulation of dicyclomine hydrochloride (Cyclopam drops) was purchased from a local market. The chemicals used in the analysis were of HPLC grade obtained from Thermo Fisher Scientific, India Pvt. Ltd., Mumbai.

Chromatographic conditions:

Chromatographic separation of the drug was carried on shimadzu shim-pack GIST C18 (250mmX4.6mm, 5µm) column. Acetonitrile and Buffer pH5.9 in the ratio of 70:30 were used as a mobile phase at 1.0ml/min flow rate. The injection volume was 25µl and detection was carried out at 218nm using UV-visible detector.

Selection of wavelength:

Standard solution of dicyclominehydrochloride (100ppm) was prepared and scanned by a UV spectrophotometer in the range of 200–400nm. Spectra of dicyclomine hydrochloride is shown in (fig.2). Wavelength 218nm was selected as detection wavelength because it shows the maximum absorbance.

Fig. 2: UV Spectra of dicyclomine hydrochloride

Preparation of mobile phase:

A mixture of 70% of acetonitrile of HPLC grade and 30% of buffer pH 5.9 was prepared and it was sonicated for 15 min to degas it.

Preparation of standard stock solution:

100mg of dicyclomine hydrochloride was accurately weighed and transferred it in a 100ml volumetric flask. About 60ml of methanol was added as a diluent and sonicated it for 10 min. Further the volume was made up with methanol. The final concentration of standard obtained was 1000ppm. This solution was used as stock solution.

Preparation of standard solutions:

1 ml of above stock solution was transferred to 10ml of volumetric flask and volume was made up with methanol to obtain the solution of concentration 100ppm. Series of dilutions was made to achieve the concentration in the range of 50-150ppm which was further used to obtain the calibration curve.

Analysis of Marketed Formulations:

For analysis of marketed formulation, 1ml of sample which is equivalent to 10mg of dicyclomine hydrochloride was transferred into 100ml of volumetric flask. 60ml of methanol was added as a diluent and sonicated for 20-30 min and volume was made upto the mark with methanol. This solution was further centrifuged for 5 min at 5000r/min and then by using Whatman filter paper it was filtered to obtain a clear solution. A 25μl volume of above solution was injected into the HPLC system.

RESULTS AND DISCUSSION:

Method development:

A number of trials were carried out using several mobile phases such as water: acetonitrile: methanol (60:20:20), buffer pH 3: acetonitrile (55:45), acetonitrile: phosphate buffer pH 2.3 (70:30) and acetonitrile: phosphate buffer pH 4 (70:30) using various columns such BDS column, Inertsil ODS and prontosil C18 to develop RP-HPLC method for estimation of dicyclomine hydrochloride in marketed formulation. Finally, acetonitrile: buffer pH 5.9 (70:30) was selected as the mobile phase based on better peak shape and peak symmetry using column at 1.0ml/min flow rate and injection volume of 25µl. The run time was 7 min and detection was carried out at 218nm.The retention time for dicyclomine hydrochloride was found to be 4.4 min. The chromatograms of standard and sample of dicyclomine hydrochloride are shown in (Fig.3,4).

The percentage assay for marketed formulation was found to be 99.8% for dicyclomine hydrochloride.

Method validation:

Validation of the developed method for dicyclomine hydrochloride was done by using parameters such as linearity, accuracy, precision, robustness, specificity, system suitability and solution stability as per ICH guidelines^12-15.

Specificity:

To ensure the purity testing, identification and quantification of marker compound from the marketed formulation under analysis specificity is performed. By comparing the UV spectra and retention time of the standard with the component present in the sample specificity is verified. As there was no interference of any other constituents at the retention time obtained for dicyclomine HCL the developed method was found to be specific¹⁶. (Fig.3,4).

Fig. 3: Chromatogram of standard Dicyclomine Hydrochloride

Fig. 4: Chromatogram of sample Dicyclomine Hydrochloride

Linearity:

The linearity of calibration curve was determined at different concentration levels ranging from 50ppm to 150ppm for dicyclomine hydrochloride. By plotting peak area versus concentration, the linearity curve was constructed. The correlation coefficient (r²) was found to be 0.997. Based on the linearity result, the developed method was found to be linear (fig.5). Results are shown in (Table 1).

Fig. 5: Calibration Curve of Dicyclomine Hydrochloride

Table 1: Linearity results for Dicyclomine Hydrochloride

Concentration (µg/ml)

Area

100

125

150

Slope

Intercept

Correlation

183750

260635

347500

429375

431250

3455

5066

0.997

Accuracy (Recovery):

By calculating the percentage recovery of the test sample at three different concentration levels (50%, 100% and 150%) by standard addition method, the accuracy of dicyclomine hydrochloride was performed. Three replicates of each concentration level were injected into the chromatographic system. The percentage recovery for dicyclomine hydrochloride was found within a range of 98-102%. Therefore, from the accuracy results, it was found that the method is accurate. The percentage recovery data is tabulated in (Table 2).

Precision:

The closeness of agreement between various results obtained under the prescribed condition from multiple sampling of the same sample is known as precision¹⁷. Precision includes system precision and method precision. In system precision, six injections of standard dicyclomine hydrochloride were injected and in method precision six injections of sample dicyclomine hydrochloride were injected. The percentage relative standard deviation was found to be less than 2%. Therefore, the method was found to be precise. The system and method precision data are tabulated in (Table 2).

Solution stability:

For evaluating stability of the solution, the sample solution of dicyclomine hydrochloride was injected at different time intervals. The percentage assay was calculated. The percentage assay of sample solution at 24hrs shows that the solution can be used over a period of 24 hours without any degradation. The solution stability data are tabulated in (Table 2).

System suitability:

By evaluating retention time, tailing factor and number of theoretical plates of standard solution, system suitability was checked¹⁸. Six replicates of standard solution was injected into the chromatographic system. All the results was found to be within the limits. The system suitability results are tabulated in (Table 2).

Table 2: Accuracy, Precision, Solution Stability and system Suitability results for Dicyclomine Hydrochloride

Parameter (Units)	Dicyclomine hydrochloride
1. Recovery (%) 50% level 100% level 150% level	99 100.12 100.03
2. Precision (%RSD) System Precision Method Precision	1.06 1.08
3. Solution Stability(%) Initial 6 h 16 h 24 h	100.1 100.24 99.8 100.5
4. Retention time(min)	4.4
5. Tailing factor	1.02
6. Number of theoretical plate	3521

RSD: Relative standard deviation

Table 3: Robustness results for dicyclomine Hydrochloride

Parameters	Dicyclomine hydrochloride (100 µg/ml)
Parameters	RT	% assay
Minus flow (0.8 ml/min)	5.5	99.6
Plus, flow (1.2 ml/min)	3.7	99.2
Minus temperature (26°C)	4.4	101.5
Plus temperature (30°C)	4.4	100.8
Minus wavelength (216 nm)	4.4	100.2
Plus wavelength (220 nm)	4.4	98.3

RT: Retention time

Robustness:

By making small deliberate changes in the optimized method parameters such as flow rate (±0.2 ml), column temperature (±2°C) and wavelength (±2nm), the developed analytical method was evaluated for robustness^19,20. From the results it was found that none of the parameters caused any changes in the peak area. The method was found to be robust as the %RSD value was in the limits. The robustness data is tabulated in (Table 3).

CONCLUSION:

In the current study, the RP-HPLC method was developed and validated for estimation of dicyclomine hydrochloride in bulk as well as in its drops form. Validation of the developed method was done in accordance with ICH Q2 (R1) guidelines. The developed method is simple, precise, accurate, rapid and cost effective for determination of dicyclomine hydrochloride. In this method the run time is short (less than 10 min). This method can be effectively used for routine analysis of dicyclomine hydrochloride in formulations.

ACKNOWLEDGMENT:

The authors are thankful to the Oriental College of Pharmacy for providing necessary facilities and platform to conduct research work. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CONFLICTS OF INTEREST:

There is no conflict to declare.

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Received on 09.03.2020 Modified on 01.05.2020

Research J. Pharm. and Tech. 2021; 14(2):605-609.

DOI: 10.5958/0974-360X.2021.00108.6