Validated Simultaneous UV Spectrophotometric Methods for estimation of Tramadol Hydrochloride and ketorolac Tromethamine in bulk and marketed Capsule Formulation

 

V. S. Tambe*¹, M. N. Deodhar², Vijayalakshmi Prakya³

¹J.N.T. University, Hyderabad and PES Modern College of Pharmacy for Ladies, Pune

²J.N.T. University, Hyderabad and S.G.R.S. College of Pharmacy, Pune

³Siddhartha Institute of Pharmacy, J.N.T. University, Hyderabad

 

ABSTRACT:

The present study describes new, simple, rapid and novel spectrophotometric methods for simultaneous estimation of Tramadol Hydrochloride and Ketorolac Tromethamine in bulk and capsule formulation. The combination was analysed by simultaneous equation method (Method A), Absorbance correction method (Method B) and First order derivative spectroscopic method (Method C). Method A involved measurement of absorbance at two wavelengths, 271 nm and 246 nm, λmax of Tramadol hydrochloride and Ketorolac tromethamine respectively. For method B, wavelengths 331 nm (for determination of Ketorolac tromethamine) and 271 nm were used. For method C, wavelengths 339.2 nm (Zero crossing of Tramadol hydrochloride) and 229.4 nm (Zero crossing of Ketorolac tromethamine) were used for determination of Ketorolac tromethamine and Tramadol hydrochloride respectively. Beer’s law was obeyed in concentration range of 5-50 μg/ ml and 2-20 μg/ ml for Tramadol hydrochloride and Ketorolac tromethamine respectively by all the methods. Method C was found to be more simple and sensitive. The proposed methods are recommended for routine analysis of pharmaceutical formulations due to their specificity, rapidity, simplicity and accuracy. These methods were validated for linearity, accuracy and precision as per ICH guidelines.

 

 

 

INTRODUCTION:

Tramadol hydrochloride (TRH, Fig 1), (1RS, 2RS)-2-[(dimethylamine) methyl]-1-(3-methoxyphenyl)-cyclohexanol is an effective centrally acting analgesic used to treat moderate, severe, and chronic pain¹. This drug is a synthetic, centrally acting analgesic, opioid antagonists that binds weakly to μ receptors.

Ketorolac Tromethamine (KTM, Fig 2) is a 5-benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylicacid, 2-(hydroxymethyl)-1,3 propanediol². It is a potent analgesic and with moderate anti-inflammatory activity. It is official in USP.

 

Their combination is available in America and Canada in the form of injections, tablets and capsules. It is prescribed in managements of postoperative pain, migraine, fractures and dental pain.

 

Fig. 1: Tramadol Hydrochloride

Fig. 2: Ketorolac Tromethamine

 

The methods reported for analysis of TRH include UV spectroscopic methods^3-6, HPLC^3,7-10 and GC⁷. KTM has been estimated by UV Spectroscopy¹¹, HPTLC¹² and HPLC^13-17. Gammadol and Sinergix are the solid dosage form containing TRH (25 mg) and KTM (10 mg). Till date, only one method is reported for this combination. The reported method uses HPLC with study on degradation products of both drugs. But, no UV Spectroscopic method is reported for simultaneous estimation of TRH and KTM¹⁸ UV methods are simple, convenient and cost effective. These may be more sensitive due to selection of wavelength of maximum absorption for analysis. Hence, an attempt is made to quantify these drugs in capsule formulation.

 

MATERIALS AND METHODS:

Instrumentation:

A Double beam UV-Visible spectrophotometer (Shimadzu) with 10 mm matched quartz cells was used. All weighing was done on single pan balance (Shimadzu).

 

Reagents and chemicals:

TRH and KTM reference standards were kindly provided by Alkem and Dr. Reddys Pharmaceuticals Pvt. Ltd, Pune. All the reagents were of analytical reagent grade.

 

Selection of solvent:

Drug solubility was tested in distilled water, methanol, 0.1 N NaOH and 0.1 N HCL. This was performed to develop more sensitive method by selecting appropriate solvent. Based on solubility evaluation water was selected as a solvent for further work.

 

Determination of absorptivity values and linearity:

Standard stock solutions of TRH (100 μg/ml) and KTM (100 μg/ml) were prepared in distilled water. For the selection of analytical wavelength, 10 μg/ml solutions of TRH and KTM were prepared separately by appropriate dilution of standard stock solution with distilled water and scanned in the spectrum mode from 200 to 400 nm. From the overlain spectra of these drugs (Figure 3), wavelengths 331 nm, 271 nm and 246 nm were selected for analysis. The calibration curves for TRH (5-50 μg/ml) and KTM (2-20 μg/ml) (Figure 4 and 5) were plotted at the selected wavelengths and absorptivity values were determined. For method C, the zero order spectra were converted in first order spectra for individual drug and zero crossing of both drugs were determined (Figure 6, 7).

 

Fig 3: It shows overlay spectra of TRH (10 μg/ml) and KTM (10 μg/ml)

 

 

(b)

Fig 4: It shows calibration curves for TRH at 271 (a), at 246 (b)

 

 

 

(c)

Fig 5: It shows calibration curves for KTM at 271 (a), at 246 (b), at 331 nm (c)

 

Fig 6: It shows derivative spectra of TRH

 

Fig 7: It shows derivative spectra of KTM

 

Mixed standard stock solution 25 mg of TRH and 10 mg of KTM/25 ml were prepared in distilled water. The working standard solutions were obtained by dilution of the stock solution in water to 5-50 μg/ml and 2-20 μg/ml of TRH and KTM respectively. The zero order spectra of mixed standard was converted into first order spectra (Fig 8) and derivative amplitude was determined at 229.4 nm (zero crossing of KTM) and 339.2 (Zero crossing of TRH)

 

Fig 8: Calibration curve of mixed derivative spectra at 339.2 nm and at 229.4 nm.

 

Formulation analysis:

The content of 20 capsules were mixed and average weight was determined. An accurately weighed sample equivalent to TRH (25 mg) and KTM (10mg) was taken in a 25 ml volumetric flask, 15 ml distilled water was added and solution was sonicated for 15 min. Volume was made up to the mark with distilled water. The solution was then filtered though Whatman filter paper No. 41. The solution was further diluted to obtain concentration of 25 µg/ml of TRH and 10 µg/ml of KTM. The absorbance of resulting solution was measured at selected wavelengths.

 

Method A- Simultaneous Equation Method:

Sample stock solution was appropriately diluted with distilled water to obtain final concentration of 25 μg/ml for TRH and 10 μg/ml for KTM. Absorbance of this solution was measured at 246 and 271 nm. The concentration of drugs was determined by using the Equations 1 and 2. Using absorptivity values following equations were developed for determining concentration of TRH and KTM in Lab mixture.

A₂₄₆ = a _{TRH 246} C _TRH + a _KTM246 C _{KTM -------------}Equation 1

A ₂₇₁= a _{TRH 271} C _TRH + a _{KTM 271} C _{KTM -------------}Equation 2

 

Where a is absorptivity value at specified wavelength.

 

Method B- Absorption Correction method:

A solution containing 25μg/ml for TRH and 10 μg/ml for KTM was evaluated for absorbance at 331 and 271 nm. Following equations were used for determination of both drugs.

 

A₃₃₁ = a _{KTM 331} C _{KTM --------------------------------------------}Equation 3

A ₂₇₁= a _{TRH 271} C _TRH + a _{KTM 271} C _{KTM --------------}Equation 4

 

Method C- First Order Derivative Spectroscopy:

For this method, derivative amplitude of a solution containing 25μg/ml for TRH and 10 μg/ml for KTM was measured at 229.4 nm (zero crossing of KTM) and 339.2 (Zero crossing of TRH). The drug concentrations were determined from calibration curves.

 

Analytical method validation:

All the methods were validated for different parameters like linearity, specificity, accuracy and precision. Linearity was checked by calculating regression coefficient. The accuracy of the method was determined by calculating percentage drug recovery of TRH and KTM at three levels 80%, 100% and 120%. The inter-day and intra-day precision of proposed method was determined. TRH and KTM mixture was prepared and analysed at three different times in a day. The same procedure was followed for three days in order to study inter-day variations. The percent relative standard deviation (% RSD) of prepared concentration was analyzed for precision studies.

 

RESULTS AND DISCUSSION:

Solvent selection: TRH was found to be soluble in distilled water, 0.1 N HCL and in methanol. But in 0.1 N NaOH, it has resulted in globule formation. KTM was found to be insoluble 0.1 N HCL. It is soluble in distilled water and 0.1 N NaOH. Hence distilled water was used as a solvent.

 

The proposed methods for simultaneous estimation of TRH and KTM in a capsule formulation were found to be accurate, simple and rapid which can be well understood from validation data (Table 1 to 4). The % R.S.D. was found to be less than 2, which indicates the validity of methods. Linearity was observed by linear regression equation method for TRH and KTM in different concentration range. The Correlation coefficient of these drugs was found to be close to 1.00, indicating good linearity (Table1).

 

The assay results obtained by proposed methods (Table2) are in fair agreement. In all the cases RSD of percentage drug recovery (± RSD) of TRH and KTM was not more than 2% depicting the accuracy of the developed method (Table 3). Results of precision (Table 4) obtained from intra-day studies and inter-day studies shows % RSD less than 2%. Hence it can be used for routine analysis of two drugs in combined dosage forms. These methods are accurate, simple, rapid, precise, sensitive, reproducible and economic and are validated as per ICH guidelines.

 

 

 

 

Table 1: It shows Linearity data for TRH and KTM for all three methods

Sr. No.	Parameters	TRH			KTM
		Method A	Method B	Method C	Method A	Method B	Method C
1 2	Linearity (μg/ ml) Correlation Coefficient (at specified wavelength)	5-50 0.999(271) 0.995 (246)	5-50 0.999 (271)	5-50 0.999	2-20 0.999(271) 0.999 (246)	2-20 0.998(331) 0.999 (271)	2-20 0.997

N=6

 

Table 2: It shows Assay results for the determination of TRH and KTM in formulation by method A

Drug	Label Claim (μg/ ml)	Amount Found (μg /ml)	% Assay	S. D. (±)
TRH KTM	25 10	24.45 9.81	97.80 98.1	0.96 1.2

N=6

 

Table 3: It shows Assay results for the determination of TRH and KTM in formulation by method B

Drug	Label Claim (μg/ ml)	Amount Found (μg /ml)	% label Claim	S. D.(±)
TRH KTM	25 10	24.88 9.64	99.57 96.4	1.02 1.13

N=6

 

Table 4: It shows Assay results for the determination of TRH and KTM in formulation by method C

Drug	Label Claim (μg/ ml)	Amount Found (μg /ml)	% Assay	S. D. (±)
TRH KTM	25 10	24.32 9.89	97.28 98.9	0.98 0.67

N=6

 

Table 5:  It shows result of recovery studies by the proposed methods

Amount  added (μg/ ml)	Amount recovered (Method A)	% Recovery (Method A) +S.D.	Amount recovered (Method B)	% Recovery (Method B)+S.D.	Amount Recovered (Method C)	% Recovery (Method C) +S.D.
TRH
20	19.733	98.66+ 1.04	19.79	98.66 + 1.24	19.84	101.33+ 1.13
25	24.45	97.80+1.02	24.88	99.0+1.33	24.92	101 + 1.02
30	30.38	101.26+1.07	30.85	98.88+1.55	30.12	100.88 + 0.94
KTM
8	7.73	96.68+1.22	7.7	96.68+0.93	7.8	101.6+ 1.03
10	9.81	97.80+1.24	9.52	96.24+0.95	9.7	100.8+ 0.84
12	12.081	101.26+1.54	11.77	98.14+ 0.86	11.87	100.4+ 0.75

n=6

 

Table 6: It shows intra and interday precision

Intraday Precision
331 nm		246 nm		271 nm		229.4 nm		339.2 nm
S.D.	%RSD	S.D.	%RSD	S.D.	%RSD	S.D.	%RSD	S.D.	%RSD
0.0035	0.6909	0.0033	1.704	0.001	0.423	0.001	2.0	0.0002	1.17
Interday Precision
331 nm		246 nm		271 nm		229.4 nm		339.2 nm
S.D.	%RSD	S.D.	%RSD	S.D.	%RSD	S.D.	%RSD	S.D.	%RSD
0.0045	0.8772	0.0082	0.191	0.001	0.521	0.001	1.9	0.0002	1.2

Measured parameter: Absorbance

 

Table 7: LOD and LOQ

Sr. no	KTM					TRH
	Conc (µg mL-1)	A at 246 nm	A at 271 nm	A at 331 nm	A at 339.2 nm	Conc (µg mL-1)	A at 246 nm	A at 271nm	A at 229.2 nm
LOD		0.173	0.366	0.067	1.544		3.88	0.275	0.516
LOQ		0.521	1.1	0.202	4.63		11.64	0.825	1.53

 

LOD and LOQ at each wavelength was calculated. Method B involving detection at 331 nm and 271nm was found to be more sensitive. (table-7)

 

CONCLUSION:

Three simple UV spectrophotometric methods were developed for the simultaneous determination of Tramadol Hydrochloride and Ketorolac Tromethamine without any interference from the excipients. The methods were applied to the assay of capsule formulation. The results of our study indicate that the proposed UV spectroscopic methods are convenient, rapid, precise and accurate. Statistical analysis proves that, these methods are repeatable and selective for the analysis of TRH and KTM. It can therefore be concluded that use of these methods can save much time and money and they can be with accuracy. Absorption correction method was found to be more sensitive and comparatively simple.

 

ACKNOWLEDGEMENT:

The authors are thankful to management of PES’s Modern College of Pharmacy (for Ladies), Pune for providing necessary facility for the work.

 

REFERENCES:

1.       Tripathi KD. Essentials of Medical Pharmacology. Jaypee Publishers, 6th ed. 460-61.

2.       Goodman and Gilmans, The Pharmacological Basis of Therapeutics, 11th ed. 697-698.

3.       Küçük A and Kadioğlu Y. Determination of tramadol hydrochloride in ampoule dosage forms by using UV spectrophotometric and HPLC-DAD methods in methanol and water media. Farmaco. 2005; Feb 60(2):163-9.

4.       Gharge D and Dhabale P. Simultaneous Estimation of Tramadol Hydrochloride and Paracetamol by UV Spectrophotometric Method from Tablet Formulation. International Journal of Pharm Tech Research. 2(2); 2010: 1119-1123.

5.       Jain V and Sharma R. Simultaneous Spectrophotometric Estimation and Validation of Domperidone, Tramadol hydrochloride and acetaminophen in tablet dosage form. Stamford Journal of Pharmaceutical Sciences. 3(1); 2010: 28-33.

6.       Gogulamudi L and Sujana K. Development and validation of UV spectroscopic method for determination of Tramadol hydrochloride in bulk and formulation. International Journal of Pharmacy and Pharmaceutical Sciences. 4(5); 2012: 275-279.

7.       Tarek B, Tamer A and Randall C. Determination of Paracetamol and Tramadol Hydrochloride in Pharmaceutical Mixture Using HPLC and GC-MS. Journal of Chromatographic Sciences. 47 (10); 2009: 849-854.

8.       Saccomanni G, Del Carloa S, Giorgi M, Manera C and Sabac A. Determination of tramadol and metabolites by HPLC-FL and HPLC–MS/MS in urine of dogs. Journal of pharmaceutical and biomedical analysis. 53; 2010: 194-199.

9.       Mehran J, Mohammad M and Behrouz A. On-line clean-up and determination of tramadol in human plasma and urine samples using molecularly imprinted monolithic column coupling with HPLC. Journal of Chromatography B. 911; 2012: 49– 54.

10.    Vojko K and Robert R. HPLC determination of tramadol in human breast milk. Journal of pharmaceutical and biomedical analysis. 32; 2003: 1061-1066.

11.    Prakash MS and Meena S. Fluorophotometric Determination of Ketororlac Tromethamine.  Indian Drugs. 33;1996: 149-151.

12.    Devarajan PV, Gore SP and Chavan SV. HPTLC Determination of Ketorolac Tromethamine. Journal of pharmaceutical and biomedical analysis. 22; 2000: 679-683.

13.    Irene T, Frances C, Martin K, Marian P and Anne W. HPLC Method for the Determination of Ketorolac in Human Plasma.  Journal of liquid Chromatography and Related Technology. 1996;19(6):957-967.

14.    Chaudhary RS, Gangwal SS, Jindal KC and Khanna S. Reversed-phase high-performance liquid chromatography of ketorolac and its application to bioequivalence studies in human serum. Journal of Chromatogr B: Biomedical Sciences and Applications. 614(1);1993:180-184.

15.    Yang G, He B and Liu S. Determination of the content of ketorolac tromethamine in injection by HPLC. Chinese Journal of Hospital Pharmacy. 12;2003: 12-15.

16.    Reddy BP, Suryanarayana MV, Vemkatraman S, Krupadanam GL and Sastry CS. Purity Evaluation of Ketorolac Tromethamine by HPLC. Indian Drugs. 30;1993:176-179.

17.    Patri S, Patni AK, Iyer SS, Khuroo AH, Tausif M and Rana SA. Validated High-Performance Liquid Chromatography-Tandem Mass Spectrometric (LC-MS/MS) Method for Simultaneous Determination of R(+)-Ketorolac and S(−)-Ketorolac in Human Plasma and its Application to a Bioequivalence Study. Chromatography Research International. 11;2011:1-11.

18.    Tambe VS, Deodhar MN and Prakya V. LC and LC–MS study for simultaneous determination of tramadol hydrochloride and ketorolac tromethamine in bulk and formulation with their major degradation products. Bulletin of Faculty of Pharmacy, Cairo University. 54; 2016: 87–97..

 

 

 

 

Received on 21.06.2017       Accepted on 18.09.2017     

© Asian Pharma Press All Right Reserved