Spectrophotometric Methods for Simultaneous Estimation of Tramadol Hydrochloride and Diclofenac Sodium in Combined Dosage Form.

 

Jawed Akhtar_, TY Pasha, Monica Padaria, Bhoomi Shah, Nasimabanu Mansuri, Krishna Prajapati*

Department of Quality Assurance, Parul Institute of Pharmacy and Research, Vadodara, Gujarat, India,

*Corresponding Author E-mail: prajapatikrishna12@yahoo.com

 

A simple, accurate, precise and specific spectrophotometric method has been developed for simultaneous determination of Tramadol Hydrochloride (TRA) and Diclofenac Sodium (DIC) using first order derivative spectrophotometric method by using water as a solvent. The first order derivative method in which absorption at 270.98 nm (zero cross point of TRA) was used for quantification of DIC and 248.38 nm (zero cross point of DIC) for quantification of TRA. This method follows Beer’s linearity in the range of 30-90 μg/ml for TRA and 10-40 μg/ml for DIC. The mean % recoveries were found to be in the range of 99.37-100.83% and 99.61 – 100.06% TRA and DIC respectively for first order derivative method. The proposed method has been validated as per ICH guidelines and successfully applied to the estimation of TRA and DIC in their combined Tablet dosage form.

 

 

INTRODUCTION:

Tramadol Hydrochloride (TRA) is chemically (1R,2R)-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexan-1-ol Hydrochloride (Figure 1a),  synthetic centrally acting analgesic which has been used since 1977 for the relief of moderate to severe acute and chronic pain. TRA can be attributed to norepinephrine and serotonin reuptake blockade in the CNS. Tramadol and its O-desmethyl metabolite (M1) are selective, weak OP3-receptor agonists. TRA produces analgesia in short- and long-term pain states by synergistically combining weak µ-opioid and monoaminergically (noradrenaline and serotonin) mediated mechanisms¹¹. TRA is official in British Pharmacopoeia¹, United States Pharmacopoeia² and Indian Pharmacopoeia³.Chemically DIC is, sodium 2-[(2,6-dichlorophenyl)-amino]phenyl acetate (Figure 1b), used as analgesic and anti-inflammatory drug used in the treatment of rheumatoid arthritis, osteoarthritis gout attacks and in pain management²⁵. Diclofenac sodium is official in British Pharmacopoeia¹, United States Pharmacopoeia² and Indian Pharmacopoeia³.

 

(a)

 

 (b)

Figure 1: Chemical structure of (a) TRA and (b) DIC

 

The review of literature^4-17 revealed that various analytical methods involving spectrophotometry,TLC, HPLC, HPTLC have been reported for TRA in single form and in combination with other drugs. Several analytical methods^18-32 have been reported for DIC in single form and in combination with other drugs including spectrophotometry, HPLC, LC-MS, HPTLC.

 

The present paper describes a simple, accurate and precise method for simultaneous estimation of TRA and DIC in combined tablet dosage form by UV Spectrophotometric first order derivative method³³. The developed methods were validated in accordance with ICH Guidelines³⁴ and successfully employed for the assay of TRA and DIC in their combined dosage form.                   

 

MATERIALS AND METHODS:

Instrument:

Shimadzu UV-1800; UV spectrophotometer with spectral bandwidth of 1.8 nm, wavelength accuracy of 2 nm and matched quartz cells of 10 mm optical path length. Calibrated analytical balance was used for weighing purpose.

 

Drug Sample:

Tramadol hydrochloride and diclofenac sodium were obtained as gift sample from  Sun pharmaceutical industries Ltd., Vadodara, India.

 

Chemicals and Reagents:

In house Distilled water

 

Spectrophotometric condition:

All zero order spectrums (D⁰) were converted to first derivative spectrum (D¹) using delta lambda 1.

Preparation standard stock solutions:

Accurately weighed 100 mg of TRA and DIC standard were transferred to a separate 100 ml volumetric flask and dissolved in 50 ml distilled water. The flasks were shaken and volume was made up to the mark with distilled water to give solutions containing 1000 µg/ml TRA and 1000 µg/ml DIC. From this solution 10 ml was transferred to volumetric flask of 100 ml capacity. Volume was made up to the mark to give a solution containing 100µg/ml of TRA and 100µg/ml DIC.

 

First order Derivative method:

30-90 µg/ml solutions of TRA and 10-40 µg/ml DIC were prepared in distilled water by appropriate dilution and spectrum was recorded between 200-400 nm and first derivative spectrums were obtained using above condition. The overlain first derivative spectrums of TRA and DIC at different concentration were recorded. The zero crossing point (ZCP) of TRA was found to be 270.98 nm and ZCP of DIC was found to be 248.38 nm.

 

Method validation:

The proposed method has been extensively validated in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility. The accuracy was expressed in terms of percent recovery of the known amount of the standard drugs added to the known amount of the pharmaceutical dosage forms. The precision (Coefficient of Variation - C.V.) was expressed with respect to the repeatability, intra-day and inter-day variation in the expected drug concentrations. After validation, the developed methods have been applied to pharmaceutical dosage form.

 

Specificity:
Commonly used excipients (starch, microcrystalline cellulose and magnesium stearate) were spiked into a pre weighed quantity of drugs. The D¹ spectrum (for first order derivative)  were recorded by appropriate dilutions and the quantities of drugs were determined. 

 

Linearity:

Appropriate volume of aliquot from TRA and DIC standard stock solution was transferred to volumetric flask of 10 ml capacity. The volume was adjusted to the mark with distilled water to give solutions containing 30-90 µg/ml TRA and 10-40 µg/ml DIC. All D¹ Spectrum were recorded using above spectrophotometric condition. D¹ absorbance at 248.38 nm and 270.98 nm were recorded for TRA and DIC respectively. Calibration curves were constructed by plotting average absorbance versus concentrations for both drugs. Straight line equations were obtained from these calibration curves.

 

Accuracy:

Accuracy was assessed by determination of the recovery of the method by addition of standard drug to the pre-quantified placebo preparation at 3 different concentration levels 80, 100 and 120 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed 3 times and average recoveries were measured.

 

Precision:

The repeatability was evaluated by assaying 6 times of sample solution prepared for assay determination. The intraday and interday precision study of TRA and DIC was carried out by estimating different concentrations of TRA (20, 30, 40 µg/ml) and DIC (15, 20, 25 µg/ml), 3 times on the same day and on 3 different days (first, second, fifth) and the results are reported in terms of C.V.

 

Detection limit and Quantitation limit:

ICH guideline describes several approaches to determine the detection and quantitation limits. These include visual evaluation, signal-to-noise ratio and the use of standard deviation of the response and the slope of the calibration curve. In the present study, the LOD and LOQ were based on the third approach and were calculated according to the 3.3σ/S and 10σ/S criterions, respectively; where σ is the standard deviation of y-intercepts of regression lines and s is the slope of the calibration curve.

 

Robustness:

The sample solution was prepared and then analyzed with change in the typical analytical conditions like stability of analytical solution.

 

Reproducibility:

The absorbance readings were measured at different laboratory for sample solution using another spectrophotometer by analyst and the values obtained were evaluated using t- test to verify their reproducibility.

 

Determination of TRA and DIC in their Combined Dosage:

A powder quantity equivalent to 50 mg TRA and 75 mg DIC was accurately weighed and transferred to volumetric flask of 100 ml capacity. 60 ml of distilled water was transferred to this volumetric flask and sonicated for 15 min. The above solution was filtered through whatman filter paper (0.45µ).The flask was shaken and volume was made up to the mark with distilled water. From this solution 2 ml was transferred to volumetric flask of 100 ml capacity. Volume was made up to the mark to give a solution containing 8 µg/ml of TRA and 12 µg/ml of DIC. The resulting solution was analysed by proposed methods. The quantitation was carried out by keeping these values to the straight line equation of calibration curve.

 

RESULTS AND DISCUSSION:

In First order derivative method the overlain D¹ spectrum of TRA and DIC at different concentrations revealed that at 248.38 nm (ZCP of DIC) TRA possesses significant D¹ absorbance and at 270.90 nm DIC possesses significant D¹ absorbance. Considering above facts, wavelength 248.38 nm and 270.90 nm were selected for the estimation of TRA and DIC, respectively (Figure 2)

 

Figure 2. Overlain D¹ spectrum of TRA (30-90µg/ml) and DIC (10-40 µg/ml) in distilled water

 

Table 1: Summary of Validation Parameters of first order derivative spectrophotometric method

 

Table 2: Statistical data TRA and DIC by First order derivative method

Parameter	TRA	DIC
Analytical Wavelength	248.38 nm	270.90 nm
Range	30-90 µg/ml	10-40 µg/ml
Slope	0.0001x	0.0003x
Intercept	- 0.00167	- 0.00039
Regression Coefficient (r2)	0.9991	0.9993

 

Linearity was assessed for TRA and DIC by plotting calibration curves of the D¹ absorbance versus the concentration over the concentration range 30-90 µg/ml and 10-40 µg/ml respectively. The correlation coefficients (r²) for TRA and DIC were found to be 0.9991 and 0.9993, respectively (Table 2). The following equations for straight line were obtained for TRA and DIC.

 

Linear equation for TRA, y = 0.0001x-0.00167

Linear equation for DIC, y = 0.00031x - 0.00039

 

The % recoveries were found to be in the range of 99.37-100.83 for TRA and 99.61-100.06% for DIC (Table 3). The precision of method was determined by repeatability, intraday and interday precision and was expressed as the C.V. (Table 4-5), which indicate good method precision.

The Limit of detection for TRA and DIC was found to be 0.502μg/ml and 0.421μg/ml respectively. Limit of quantification for TRA and DIC was found to be 1.506µg/ml and 0.842μg/ml at 248.38 nm and at 270.90 nm respectively (Table 1).

 

The concentration of TRA and DIC in combined dosage form was found to be 7.85 μg/ml and 12.1 μg/ml respectively (Table 6).

 

Both methods were found to be specific, as there was no interference observed when the drugs were estimated in presence of excipients and robust, as there was no significant change in absorbance up to 24 hours of preparation of solution in distilled water. The proposed spectrophotometric method was successfully applied to TRA and DIC combined dosage form.

 

CONCLUSION:

The proposed first order derivative method provide simple, specific, precise, accurate and reproducible quantitative analysis for simultaneous determination of TRA and DIC in combined dosage form. The methods were validated as per ICH guidelines in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility. The proposed methods can be used for routine analysis and quality control assay of TRA and DIC in combined dosage form.

 

 

Table 3: Accuracy data for TRA and DIC by derivative spectrophotometric method

% Level	Amount of drug added (μg/ml)		Amount recovered (μg/ml)		% Recovery
	TRA (μg/ml)	DIC (μg/ml)	TRA (μg/ml)	DIC (μg/ml)	% TRA	% DIC
80 %	8	10.5	7.95	10.46	99.37	99.61
100 %	10	12.5	10.96	12.47	99.84	99.76
120 %	12	14.5	12.1	14.51	100.83	100.06

 

 

Table 4:Precision data for TRA at 248.38 nm

Conc. (μg/ml)	Intraday (Abs. ± S.D)	C.V.	Interday (Abs. ± S.D)	C.V.
30	0.0039 ± 0.00008	1.92	0.0039 ± 0.000075	1.88
40	0.0048 ± 0.000089	1.86	0.0049 ± 0.000075	1.56
50	0.0062 ± 0.00017	1.88	0.0062 ± 0.000103	1.66

(n = 3 determination)

 

 

Table 5: Precision data for DIC at 270.90 nm

Conc. (μg/ml)	Interday (Abs. ± S.D)	C.V.	Interday (Abs. ± S.D)	C.V.
30	0.0051 ± 0.000075	1.48	0.0050± 0.00008	1.50
40	0.0070 ± 0.000137	1.94	0.0068± 0.000121	1.77
50	0.0082 ± 0.000103	1.25	0.0083±0.000143	1.72

(n = 3 determination)

 

 

Table 6: Determination of TRA and DIC in their Combined Dosage

Synthetic Mixture	Drug	Amount Taken (μg/ml)	Amount Found  (μg/ml) (n = 3)	Labeled claim (mg)
TRA + DIC	TRA	8	7.85	50
	DIC	12	12.1	75

ACKNOWLEDGEMENT:

The author wish to extend thanks to Sun Pharma Industries Ltd. Vadodara, India for providing the gift samples.

 

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Received on 04.01.2013       Modified on 13.01.2013

Accepted on 20.01.2013      © RJPT All right reserved