Development and Validation of RP-HPLC Method for Simultaneous Estimation of Tramadol Hydrochloride and Diclofenac Sodium in Synthetic Mixture

 

Satish A. Patel, Nilesh B. Chaudhari^*, Pruthviraj K. Chaudhari

Department of Pharmaceutical Analysis, Centre for Health Science Studies, Ganpat University,

Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India.

A simple, accurate, and rapid reversed phase high performance liquid chromatographic method has been developed and validated for the simultaneous estimation of Tramadol Hydrochloride (TRM) and Diclofenac sodium (DIC) in synthetic mixture. The separation was carried out using mobile phase consisting of methanol: phosphate buffer (pH, 6.0) (68:32, v/v). The column used was Phenomenex C18, (250 mm x 4.6 mm i.d., 5 µm) with flow rate 1.2 ml/min using PDA detection at 219 nm. The method was linear over a concentration range 2 - 30 μg/ml for both drugs. The retention time of Tramadol hydrochloride and Diclofenac sodium were found to be 6.30 min and 8.35 min respectively. Results of analysis were validated statistically and by recovery studies. The mean recovery was 99.29 ± 0.54 and 99.03 ± 0.45 for TRM and DIC respectively. The limit of detection (LOD) and the limit of quantification (LOQ) for TRM and DIC were found to be 0.6586 and 1.9956 µg/ml and 0.6169 and 1.8695 µg/ml, respectively. The results of the study showed that the proposed RP-HPLC method was found to be simple, sensitive, precise and accurate and also useful for the routine determination of TRM and DIC in mixture.

 

INTRODUCTION:

Tramadol hydrochloride (TRM) is chemically [2-(dimethylaminomethyl)-1-(3-methoxyphenyl) cyclohexanol] (Figure 1) is a well known μ receptor antagonist analgesic drug ¹. It is official in Indian Pharmacopoeia (IP)²,  British Pharmacopoeia (BP)³. IP and BP describe potentiometry titration method and HPLC method, respectively for its estimation. Literature survey reveals UV^4-8, HPLC^9-12 and HPTLC¹³ methods for estimation of TRM. Diclofenc sodium (DIC) is chemically 2-[2,6dichloroanilino] phenyl acetic acid sodium salt (Figure 2). Diclofenc sodium (DIC) is official in Indian Pharmacopoeia (IP)¹⁴, British Pharmacopoeia (BP)¹⁵ and United States Pharmacopeia (USP)¹⁶. IP describe potentiometry titration method and UV method for its estimation. BP describes potentiometry titration method and HPLC method for its estimation.

 

USP describe potentiometry titration method for its determination. Literature survey reveals UV^17,18, HPLC ^19-21 methods for determination of DIC . The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of TRM and DIC in their combined synthetic mixture. The present communication describes simple, sensitive, rapid, accurate, precise and cost effective RP-HPLC method for simultaneous estimation of both drugs in synthetic mixture.

 

igure 1: Chemical structure of Tramadol hydrochloride (TRM)

 

Figure 2: Chemical structure of Diclofenac sodium (DIC)

 

MATERIALS AND METHODS:

Apparatus:

RP-HPLC instrument (Shimadzu, LC-2010CHT, Japan) equipped with a UV-Visible detector and a photodiode array detector, auto sampler, phenomenex C18 column (250 x 4.6 mm, 5 µ particle size) was used. Chromatograms were automatically obtained by LC-Solution system software. A Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India), Whatman filter paper no. 41 (Millipore, USA) were used in the study.

 

Reagent and materials:

TRM and DIC bulk powder was kindly gifted by Acme Pharmaceuticals Ltd. Ahmedabad, Gujarat, India. Methanol AR Grade was procured from Finar Chemicals Ltd., Ahmedabad, India. Sodium dihydrogen ortho phosphate and disodium hydrogen ortho phosphate (AR grade, Finar Chemicals Limited, Ahmedabad, India) were used in the study.

 

Preparation of phosphate buffer solution:

Phosphate buffer (pH 6.0) was prepared by dissolving 1.56 g sodium dihydrogen ortho-phosphate dihydrate and 0.35 g disodium hydrogen phosphate dihydrate in 1000 ml HPLC-grade water and the pH adjusted to 6.0 by dilute ortho-phosphoric acid.

 

Preparation of standard stock solutions of TRM and DIC (100 μg/ml):

An accurately weighed standard TRM and DIC powder (10 mg) were weighed and transferred to 100 ml separate volumetric flasks and dissolved in methanol. The flasks were shaken and volumes were made up to mark with methanol to give a solution containing 100 μg/ml of each TRM and DIC.

 

Methodology:

To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for TRM and DIC was obtained with a mobile phase methanol: phosphate buffer (pH, 6.0) (68:32, v/v) at a flow rate 1.2 ml/min to get better reproducibility and repeatability. Quantification was carried out at 219 nm based on peak area. Complete resolution of the peaks with clear baseline was obtained (Figure 3). System suitability test parameters for TRM and DIC for the proposed method are reported in Table 1. Overlain UV spectrum showed that both drugs showed good absorbance at 219 nm, hence the wavelength of 219 nm was selected for quantification of TRM and DIC (Figure 4). 

 

Figure 3: Chromatogram of standard solution of TRM and DIC at 219 nm

                                                                                                 

Figure 4: U.V. Spectrum of TRM and DIC.

 

Table 1: System suitability parameters of chromatogram

 

Validation of proposed method:

The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines ²².

 

Calibration Curve (linearity):

Calibration curves were constructed by plotting peak area vs. concentration of TRM and DIC, and regression equation were calculated ( Fig 5 , 6 for TRM and DIC respectively). The calibration curves were plotted over the concentration range of 2 - 30 μg/ml for both drugs. From standard stock solution of mixture of TRM and DIC (0.2, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8 and 3.0 ml) were transferred to a series of 10 ml volumetric flask and diluted to the mark with methanol. Aliquots (20 µl) of each solution were injected under the operating chromatographic condition as described above.

 

Figure 5: Calibration curve of TRM at 219 nm.

 

Figure 6: Calibration curve of DIC at 219 nm

 

Method precision (Repeatability):

The precision of the instrument was checked by repeated injection (n = 6) of standard solutions of TRM (16 µg/ml) and DIC (16 µg/ml) under the same chromatographic condition and measurement of peak area, retention time and tailing factor. The low %RSD values(less than 2%) indicates that proposed method is repeatable.

 

Intermediate precision (Reproducibility):

The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of TRM (4, 8, 12 µg/ml) and DIC (4, 8, 12 µg/ml). The result was reported in terms of relative standard deviation (% RSD).

 

Limit of detection and Limit of quantification:

LOD and LOQ of drugs can be calculated using the following equations designated by International Conference on Harmonization (ICH) guidelines²².

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where, σ = the standard deviation of the response and S = slope of the calibration curve.

Accuracy (Recovery study):

The accuracy of the method was determined by calculating recovery of TRM and DIC by the standard addition method. Known amounts of standard solutions of TRM and DIC (50, 100, 150 %) were added to pre quantified sample solutions of TRM and DIC. The amounts of TRM and DIC were estimated by applying obtained values to the respective regression equation of the calibration curve. The obtained value of recovery indicates that the proposed method is accurate. Results of recovery studies are shown in Table 2.

 

Table 2: Determination of Recovery:

Drug	Level	Amount of sample taken (µg/ml)	Amount of standard   spiked (%)	Mean % Recovery ± RSD
TRM	I	8	50 %	98.21 ± 0.82
	II	8	100 %	100.0 ± 0.40
	III	8	150 %	99.66 ± 0.39
DIC	I	12	50 %	98.51 ± 0.64
	II	12	100 %	99.76 ± 0.29
	III	12	150 %	98.83 ± 0.42

 

Estimation of TRM and DIC from synthetic mixture:

TRM (50 mg) and DIC (75 mg) standard drug powder were accurately weighed and then mixed with commonly used formulation excipients like starch, lactose, magnesium stearate and talc in appropriate proportion. The mixture was then transferred to 100 ml volumetric flask containing 50 ml methanol and sonicated for 20 min. The solution was filtered through Whatman filter paper No. 41 and the volume was adjusted up to the mark with methanol. The above solution (1.0 ml) was transferred to 10 ml volumetric flask and diluted up to mark with methanol to obtain 50 µg/ml TRM and 75 µg/ml DIC. The solution (1.0 ml) was again transferred to 10 ml volumetric flask and diluted up to mark with methanol to obtain final concentration of 5.0 µg/ml TRM and 7.5 µg/ml DIC. Aliquot (20 µl) of sample solution was injected under the operating chromatographic condition as described above and peak area was determined for both drugs (Fig 7). From these area values, the concentrations of TRM and DIC were determined using respective calibration graph. The analysis procedure was repeated six times with synthetic mixture (Table 3).

 

Figure 7: Chromatogram of sample solution of TRM (5.0 µg/ml) and DIC (7.5 µg/ml) at 219 nm

Table 3: Analysis of synthetic mixture of TRM and DIC by proposed method (n = 6)

Sample No.	Label Claim		Amount Found		% Label Claim
	TRM (µg/ml)	DIC (µg/ml)	TRM (µg/ml)	DIC (µg/ml)	TRM (%)	DIC (%)
1	5	7.5	4.91	7.46	98.12	99.53
2	5	7.5	5.06	7.54	101.3	100.6
3	5	7.5	4.90	7.69	98.04	102.5
4	5	7.5	4.82	7.55	96.59	100.7
5	5	7.5	4.85	7.72	96.91	102.9
6	5	7.5	4.89	7.60	97.90	101.4
Mean					98.14	101.2
S.D.					1.67	1.29
RSD%					1.70	1.27

 

RESULTS AND DISCUSSION:

A RP-HPLC method was developed and validated for the determination of TRM and DIC in synthetic mixture on a column (C18, 250 X 4.6 i.d., 5 µm) with variable wavelength detection at 219 nm. The retention time of TRM and DIC was 6.30 min and 8.35 min, respectively. Linear correlation was obtained between area and concentration of TRM and DIC in the concentration range of 2 – 30 µg/ml for both drugs. The low RSD value of interday (1.26 - 1.66 % for TRM and 1.55 - 2.00 % for DIC) and intraday (0.68 - 1.88 % for TRM and 1.61 - 1.82 % for DIC) at 219 nm, reveal that proposed method is precise. The limit of detection (LOD) and limit of quantification (LOQ) for TRM and DIC were found to be 0.6586 and 1.9956 µg/ml and 0.6169 and 1.8695 µg/ml, respectively. These data show that method is sensitive for the determination of TRM and DIC. The recovery experiment was performed by the standard addition method. The mean recoveries were 99.29 ± 0.54 and 99.03 ± 0.45 for TRM and DIC, respectively (Table 2). The results of recovery studies indicate that the proposed method is highly accurate. The proposed validated method was successfully applied to determine TRM and DIC in synthetic mixture. No interference of the excipients with the retention time of drugs appeared; hence the proposed method is applicable for the routine simultaneous estimation of TRM and DIC.

 

Table 4: Regression analysis data and summary of validation parameters for RP-HPLC method

Parameters	RP-HPLC method
	TRM		DIC
Detection wavelength (nm)	219		219
Beer’s law limit	2 - 30		2 - 30
Regression equation y = mx + c	y = 25265x - 8276		y = 67689x + 31245
Slope	25265		67689
Intercept	8276		31245
Correlation coefficient	0.9959		0.9932
Repeatability (% RSD, n = 6)	1.69		1.39
Precision (%RSD)	Intraday (% RSD)	0.68 - 1.88	1.61 - 1.82
	Interday (% RSD)	1.26 - 1.66	1.55 - 2.00
LOD (µg/ml)	0.6586		0.6169
LOQ (µg/ml)	1.9956		1.8695
% Recovery  ±  SD  ( n = 6)	99.29 ± 0.54		99.03 ± 0.45
% Assay ± SD (n = 6)	98.14 ± 1.67		101.2 ± 1.29

 

CONCLUSION:

In this proposed RP-HPLC method, the linearity is observed in the concentration range of 2-30 µg/ml for both drugs with co-efficient of correlation, (r²) = 0.9959 and (r²) = 0.9932 for TRM and DIC, respectively at 219 nm. The results of the analysis of synthetic mixture by the proposed method are highly reproducible and reliable. The method can be used for the routine analysis of the TRM and DIC in mixture without any interference of excipients.

 

ACKNOWLEDGMENTS:

The authors are grateful to Acme Pharmaceuticals Ltd. Ahmedabad, Gujarat, India for providing gift samples of Tramadol Hydrochloride and Diclofenac Sodium and also to Department of Pharmaceutical Analysis, Centre for Health Science Studies, Ganpat University, Mehsana, Gujarat, India for providing the facilities to carry the research work.

 

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Received on 06.03.2013       Modified on 26.03.2013

Accepted on 29.03.2013      © AJRC All right reserved