INTRODUCTION:

Dicyclomine (DICY) is 2-diethylaminoethylbicyclohexyl-1-carboxylate hydrochloride ^{(1, 2)}. Structure of DICY is shown in Figure-1. The empirical formula of DICY is C₁₉H₃₅NO₂ .HCl and molecular weight is 349.5 g/mole. It is anticholinergic drug. It is used for relief of symptoms of Bowel/ irritable Syndrome, anti-inflammatory and renal colic. Dexketoprofen (DEX) is 2-amino-2-(hydroxymethyl)-1,3-propanediol (s)-3-benzoyl-alpha-methylbenzeneacetate^(3,4). Structure of DEX is shown in Figure-2. The empirical formula of DEX is C₁₆H₁₄O₃.C₄H₁₁NO₃ and molecular weight is 375.42 gm/mole. It is a NSAIDs and used for short-term treatment of mild to moderate pain, including dysmenorrhoea.

Many methods have been described in the literature for the determination of Dicyclomine Hydrochloride and Dexketoprofen Trometamol with other drugs, individually and in combination ^(5-19). However there is no UV-Spectroscopic method reported for the simultaneous estimation of these drugs. The present study involves development and validation of UV-Spectroscopic method for estimation of Dicyclomine Hydrochloride and Dexketoprofen Trometamol in bulk and synthetic mixture.

Figure 1: Structure of Dicyclomine

Figure 2: Structure of Dexketoprofen

MATERIALS AND METHODS:

Materials and Reagents:

DICY was obtained as a gift sample from Norris Medicines Limited. DEX was obtained as a gift sample from Lincoln Biopharmaceuticals Ltd. NaOH was obtained from RANKEM, triple distilled water was prepared in the distillation assembly and synthetic mixture was prepared in the laboratory.

Preparation of Standard Stock Solutions:

A standard stock solution of DICY ( 100 μg/mL ) was prepared by dissolving 10 mg of DICY in 100 mL volumetric flask and diluted to 100 mL with distilled water. Working standard solutions were prepared with 0.1 N NaOH: water (50:50) solvent.

A standard stock solution of DEX (100 μg/mL ) was prepared by dissolving 10 mg of DEX in 100 mL volumetric flask and diluted to 100 mL with distilled water. Working standard solutions were prepared with 0.1 N NaOH: water (50:50) solvent.

Selection of analytical wavelength:

Standard solutions of DICY and DEX were scanned over the UV range and overlaid. Two wavelengths selected were 260.0nm (wavelength maxima of DEX) and 311.5nm (Iso-absorptive point). Overlain spectra of both the drugs are shown in figure 3.

Figure 3: Overlain spectra of DICY and DEX

Method Validation:

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

Linearity-Range:

Accurately measured std. working solutions of DICY (1.4, 1.6, 1.8, 2.0, 2.2, 2.4 mL) and DEX (3.5, 4.0. 4.5, 5.0, 5.5, 6.0 mL) were transferred to a series of 10 mL of volumetric flasks and diluted to the mark with 0.1 N NaOH: water (50:50) to prepare the solutions for linearity and range. The calibration curves were plotted over a wide con=centration range and the linear response was observed over a range of 14-24 μg/mL for DICY and 35-60 μg/mL for DEX and the regression equations were calculated.

Precision:

The Intraday Precision was determined by estimating three replicates of three concentrations of DICY (16, 18 and 20μg/mL) and three concentrations of DEX (40, 45 and 50μg/mL) three times a day. Total nine determinations were analyzed at a small interval of time period at same day and results are reported in terms of relative standard deviation.

The Intermediate precision were carried out by estimating the corresponding responses 3 times on three different days for three different concentrations of DICY (16, 18 and 20 μg/mL) and DEX (40, 45 and 50 μg/mL) and the results are reported in terms of relative standard deviation.

Accuracy:

The accuracy of the method was determined by calculating %recovery of DICY and DEX by the standard addition method. Known amounts of standard solutions of DICY (7, 9 and 11 μg/mL) and DEX (17.5, 22.5 and 37.5) were added to prequantified separate sample solutions of DICY (9 μg/mL) and DEX (22.5μg/mL). The amounts of DICY and DEX were estimated by the equation of Absorption Ratio Method.

Cx = (Qm-Qy / Qx-Qy) (A₁/a_x1) ……………… (1)

Cy = (Qm-Qx / Qy-Qx) (A₁/a_y1) ……………… (2)

Where,

C_Xand C_Y = Concentration of X and Y

Q_M = Q absorbance ratio of sample at λ₂ and at λ₁

Q_Xand Q_Y = Ratio of absorptivity of X and Y at λ₂ and λ₁

A₁ = Absorbance of X at λ₁

a_x1anda_Y1 = Absorptivity of X and Y at λ₁

Detection Limit and Quantitation Limit:

The limit of detection (LOD) is deﬁned as the lowest concentration of an analyte that can reliably be differentiated from background levels. Limit of quantification (LOQ) of an individual analytical procedure is the lowest amount of analyte that can be quantitatively determined with suitable precision and accuracy. LOD and LOQ were calculated using following equations as per ICH guidelines:

LOD = 3.3 × (SD/Slope)………………………… (3)

LOQ = 10 × (SD/Slope)…………………………. (4)

Where, SD = Standard deviation of the Y- intercepts of the calibration curves.

Slope = Mean slope of the 5 calibration curves.

Robustness:

Three replicates of three concentrations (16μg/mL, 18μg/mL and 20μg/mL) of standard solution of DICY and three concentrations (40μg/mL, 45μg/mL and 50μg/mL) of standard solution of DEX, total nine determinations were analyzed at three different NaOH concentrations (0.098 N, 0.10 N, 0.102 N). Absorbances of mixture were measured at 260.0 nm and 311.5 nm. %RSD was calculated.

Analysis of Synthetic Mixture:

From the synthetic mixture (injection form) 2 mL was taken in 100 mL volumetric flask and diluted up to the mark with distilled water. From the above solution 1 mL was further taken in 10 mL volumetric flask and diluted with Water: 0.1 N NaOH up to the mark to get final concentrations of DICY (20µg/mL) and DEX (50 µg/mL). The above mixture was analyzed at 225.70 nm and 260.22 nm wavelengths.

Result:

Linearity and Range:

Method is linear within the range, 14-24 µg/ml and 35-60 µg/ml for DICY and DEX respectively, with correlation coefficient more than 0.999. Data are depicted in table 1.

Table 1: Regression analysis of calibration curve

Parameter	DICY	DEX
Linearity	14-24 µg/mL	35-60 µg/mL
Correlation co-efficient	0.9992	0.9992
Slope of regression	y = 0.0077x - 0.0982	y = 0.0433x + 0.1016

Accuracy

Accuracy of the method was determined by recovery study. In the matrix of DICY and DEX, % recovery was found to be 99.025 ± 0.38 and 101.233 ± 0.18 for Dicyclomine Hydrochloride and Dexketoprofen Trometamol respectively. Data are depicted in table 2.

Table 2: Accuracy data of validation parameters

Sr. No.	Level	% Recovery DICY	% Recovery DEX
1	80%	100.6	100.2
	100%	99.7	99.33
	120%	101.05	100.4

Precision

Result of Relative standard deviation (% RSD) for intraday precision and intermediate precision are illustrated in table 3.

Table3: Precision data of validation parameters

%RSD	DICY	DEX
Intraday precision	1.12	0.88
Intermediate precision	1.62	1.13

Robustness:

The method is robust as % RSD for DICY and DEX was found to be less than 2.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

Results of LOD and LOQ for both the drugs are depicted in table 4.

Table 4: Data of LOD and LOQ

Parameter	DICY	DEX
LOD = 3.3 × (SD/Slope) (μg/ml)	1.071	1.153
LOQ = 10 × (SD/Slope) (μg/ml)	3.35	3.49

Analysis of Synthetic Mixture:

Assay of DICY and DEX in synthetic mixture was found to be 99% -101.40%. Data are presented in following table 5.

DISCUSSION:

Absorption Ratio method was developed and validated for simultaneous estimation of Dicyclomine Hydrochloride and Dexketoprofen Trometamol in bulk and Synthetic mixture. R² of the developed method was near to 1; range was found to be 14 - 24 μg/ml for Dicyclomine Hydrochloride and 35 - 60 μg/ml Dexketoprofen Trometamol. % RSD was found to be less than 2 for repeatability and intermediate precision. % Recovery was found 99.025 ± 0.38 and 101.233 ± 0.18 for Dicyclomine Hydrochloride and Dexketoprofen Trometamol respectively. These results indicate that the developed method is accurate, precise and simple. It can be used in the routine quality control of pharmaceutical preparations.

CONCLUSION:

From the validation results, it was concluded that all the developed method is precise, accurate and specific. The method is suitable for QC Laboratories, where economy and time considerations are essential.

Acknowledgement:

The authors are thankful to Norris Medicines Ltd., Ankleswar and Lincoln Biopharmaceuticals Ltd., Ahmadabad, India, for providing a gift sample of DICY and DEX, respectively. The authors are very thankful to staff of Norris Medicines Ltd. for providing necessary facilities to carry out research work.

Table 5: Analysis of Synthetic Mixture of DICY and DEX by Proposed method

SAMPLE NO.	Label Claim (mg/injection)		Amount Found (mg/ injection)		% Label Claim (mg/ injection)
SAMPLE NO.	DICY	DEX	DICY	DEX	DICY	DEX
1	20	50	19.8	50.67	99	101.34
2	20	50	19.73	50.46	98.65	100.92
3	20	50	19.84	50.63	99.2	101.26
4	20	50	19.87	50.69	99.35	101.38
5	20	50	19.7	50.7	98.5	101.4
6	20	50	19.89	50.55	99.45	101.1
Mean			19.805	50.617	99.025	101.233
S.D			0.0766	0.094	0.38	0.18

LIST OF ABBREVIATIONS:

DICY	Dicyclomine Hydrochloride
DEX	Dexketoprofen Trometamol
µg	Microgram (s)
µl	Microliter (s)
µm	Micrometer (s)
AR	Analytical Reagent
HPLC	High Performance Liquid Chromatography
HPTLC	High Performance Thin Layer Chromatography
i.e.	That Is
ICH	International Conference On Harmonization
IP	Indian Pharmacopoeia
LOD	Limit Of Detection
LOQ	Limit Of Quantitation
r²	Correlation Coefficient
UV	Ultra Violate
λmax	Wavelength of Maximum Absorbance

References:

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3. MSDS of Dexketoprofen Trometamol. Available from: URL: http://www.Dexketoprofen Trometamol CAS 156604-79-4 Santa Cruz Biotech.html

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Received on 09.08.2013 Modified on 08.09.2013

Asian J. Research Chem. 6(12): December 2013; Page 1169-1172