DEVELOPMENT OF VALIDATED SPECTROFLUORIMETRIC METHOD FOR T

HE QUANTITATIVE ESTIMATION OF TRAMADOL HYDROCHLORIDE IN BUL

K AND PHARMACEUTICAL DOSAGE FORM
DEEPA.PADMAJA1
*, LEEJA.K1, R.RAJPANDI1, G. BABU2 1,2Department of Pharmaceutical Analysis,Devaki Amma Memorial Co
llege of Pharmacy,Chelembra

Abstract
Tramadol hydrochloride is a synthetic opioid analgesic drug used in the treatment of moderate to
severe pain in adults. Under present study simple, accurate, sensitive and economic spectrofluor
imetric method has been proposed for the determination of Tramadol hydrochloride in pure and
pharmaceutical dosage form. The developed spectrofluorimetric method for the estimation of Tr
amadol hydrochloride is based on the formation of complex between Dragendorffs reagent and
Tramadol hydrochloride, measured at an excitation wavelength of 481nm and emission waveleng
th of 648 nm. The developed method was statistically validated in terms of validation parameter
s as per International Conference on Harmonization (ICH) guidelines .The linearity ranges were
found to be 1.2 2.0 g/ml with the regression coefficient of 0.999. The limit of detection and li
mit of quantification were found to be 0.005 and 0.016 g/ml respectively. Recovery studies wer
e performed in the range 99101 % to confirm the accuracy of the method. The Precision results,
expressed by intraday and interday relative standard deviation values were found to be less than
2% indicating high degree of precision. Excipients used as additives in pharmaceutical
formulations did not interfere in the proposed procedures as shown by the recovery study via sta
ndard addition technique. The analysis results of marketed formulations compiles with their label
ed claim.
Keywords: Dragendorffs reagent, spectrofluorimeter, sulphuric acid, Tramadol
e, Validation

hydrochlorid

MATERIALS AND METHODS

Apparatus:
Fluorescence spectra and intensity measurements were made on an Elico SL 124 PC spectrofluo
rimeter equipped with single quartz cell of 1 cm path length.
Reagents:
Pure Tramadol hydrochloride was obtained from Variety pharmaceuticals Pvt Ltd., shornur, Indi
a. Sulphuric acid and Dragendorffs reagent (Analytical Grade) was purchased from Merck Lab
oratories Pvt Ltd., Mumbai, India. Two commercially
available
tablets
of
tramadol
hydrochloride were selected from the local market. Freshly prepared solutions were always used.

METHOD DEVELOPMENT
Solvent Selection:
To develop a rugged and suitable spectrofluorimetric method for the analysis of Tramadol hydro
chloride in formulations different solvents like distilled water, methanol, phosphate buffer pH 6.
5, 0.1 N H2SO4 and scanned from 200 800 nm to find out the best solvent in which the drug ex
hibit maximum fluorescence. It was found that in, 0.1 N H2SO4 shows a native fluorescence tha
n other solvents but its fluorescence intensity was low. It shows higher fluorescence intensity in t
he presence of Dragendorffs reagent.
Preparation of standard stock solution :
A 1000 mcg/ml standard stock solution of Tramadol hydrochloride was prepared by dissolving 1
00 mg of drug in little quantity of 0.1 N H2SO4 and diluting it to the mark in a 100 ml volume
tric flask with 0.1 N H2SO4.
Preparation of Working standard solution:
From standard stock solution 1 ml was further diluted to 100 ml with same solvent system to
get the solution having concentration 10 g/ml.
Determination of excitation and emission wavelength:
From the above working standard solution, 2 ml was pipette out into a 10 ml volumetric flask. A
bout1ml Dragendorffs reagent was added to the flask. Kept for 5 min at room temperature for c
olor developing and the volume was made up to the mark with 0.1 N H2SO4 to prepare a conce
ntration of 2 g/ml. Then the sample was scanned in Spectrofluorimeter in the range
200
800nm using blank. It was found that the drug exhibit maximum fluorescence at an excitation w
avelength 481 nm and an emission wavelength 648 nm. The excitation and emission spectrum w
as shown in Fig.II.
Selection of sensitivity:
The 2 g/ml of Tramadol hydrochloride was measured at low, medium and high sensitivity. Sens
itivity was fixed as high hence the fluorescent intensities and linearity was satisfactory.
Preparation of calibration curve:
Aliquots of Tramadol hydrochloride ranging from 1.2
2 ml (1ml = 10 mcg/ml) were transferred into a series of 10 ml volumetric flasks. To each flask,
1 ml Dragendorffs reagent was added, allowed to react for 5 min at room temperature. The volu
me was made up to the mark with 0.1 N H2SO4. Fluorescence intensity was measured for each d
ilution at an excitation wavelength 481 nm and an emission wavelength 648 nm. The calibration
curve was prepared by plotting concentration versus fluorescence intensity. Calibration graph w
as shown in Fig III.
Analysis of marketed formulation:

For analysis of Tramadol hydrochloride in tablet dosage form two commercial brands of Tramad
ol (50 mg strength) such as contramal DT and Trambax were procured from local market. 20 tab
lets were taken, weighed accurately and powdered. Powder quantity equivalent to 100 mg of Tr
amadol hydrochloride was taken and transferred to 100 mL volumetric flask. Then 50 ml of 0.1
N H2SO4 was added, and the mixture was sonicated for 10 min, the final volume was made up t
o the mark with same solvent. The mixtures were filtered through whatmann filter paper no1 and
further dilute to get required concentration 10 g/ml .From this pipette out 2 ml into a volumetri
c flask. To each flask,1 mL of Dragendorffs reagent was added to the flask, allowed to react for
5 min at room temperature. The volume was made up to the mark with same solvent to get the
concentration 2 g/ml. Three different mixtures containing different brands of 2 g/ml Tramad
ol hydrochloride were prepared and measure the intensity at an excitation wavelength 481 nm an
d an emission wavelength 648 nm against reagent blank. Similarly prepare the standard solution
as mentioned in above procedure and Fluorescent intensity was noted. The result was indicated
by % recovery given in Table I.
Color stability studies:
The reaction of Tramadol hydrochloride with reagent is complete in 5 min at room temperature.
The stability of the
orange colored product formed from Tramadol hydrochloride
Dragendorffs reagent interaction is more than for 2 hrs. After this time interval, the color inten
sity slowly decreases. However a time interval of 2 hrs was sufficient enough to record the meas
urements. The results were shown in Table II.
METHOD VALIDATION
Validation is a process of establishing documented evidence, which provides a high degree of ass
urance that a specific activity will consistently produce a desired result or product meeting its pr
edetermined specifications and quality characteristics.
The method was validated for diffe
rent parameters like Linearity, Accuracy, Precision, Ruggedness, Limit of Detection (LOD), and
Limit of Quantification (LOQ) [XIII].
Linearity:
Various aliquots were prepared from the working standard solution (10 g/ml) ranging from 1.2
2 g/ml. The samples were scanned in spectrofluorimeter and it was found that the selected drug
showed linearity between the 1.22 g/ml. The result was shown in Table III.
Accuracy:
The accuracy of the method was determined by preparing solutions of different
concentrations (80%,100%,120%) in
which the amount of marketed formulation (Contramal
50 mg) was kept constant(50 mg) and the amount of pure drug was varied i.e,40 mg,50 mg, 60
mg for 80%,100%, and 120% respectively. The solutions were prepared in triplicates and the ac
curacy was indicated by % recovery. The result was shown in Table IV.
Precision
Precision of the method was demonstrated by intra
day, interday and repeatability variation studies. In intraday variation study, six different solutio

ns of same concentration that is 2g/ml were prepared and analyzed in same day. The result was
indicated by % RSD and shown in Table V. In repeatability studies, the fluorescent intensity of t
he same
concentration solution (1.22 g/ml) was measured six times and their fluorescence intensity values were measured at an ex
citation wavelength 481 nm and emission wavelength 648 nm against reagent blank and standard
deviation was calculated.
The results were shown in Table VI. In inter
day variation study, six different solutions of same concentration were prepared and analyzed in
alternate days and the fluorescent intensity values were noted. The result was indicated by % RS
D and was shown in Table VII.
Ruggedness:
Ruggedness of the method was determined by carrying out the analysis by two different analysts
and the respective fluorescent intensity values were noted. The result was indicated by %RSD a
nd was shown in Table VIII.
Limit of Detection:
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sam
ple. The LOD was calculated using the formula involving standard deviation of response and slo
pe of calibration curve as mentioned in Table IX.
LOD= 3.
3 SD/S
Limit of quantification:
The LOQ is the concentration that can be quantified reliably with a specified level of accuracy an
d precision. The LOQ was calculated using the formula involving standard deviation of response
and slope of calibration curve as mentioned in Table IX.
LOQ=10SD/S
RESULTS AND DISCUSSION
It was aimed to develop simple and accurate spectrofluorimetric analytical method for the estima
tion of Tramadol hydrochloride. The reaction between Tramadol hydrochloride and Dragendorff
s reagent produced a complex in acid
media showed fluorescence in the range 1.2
2 g/ml at an excitation wavelength 481nm and emission wavelength 648 nm, linear regression
equation y = 338.7 x
227.2 with a correlation coefficient of 0.999. The excitation and emission spectra were shown i
n Fig II. The developed method was found to be precise as the % RSD values for intraday inter
day were found to be less than 2%. Good recoveries (99.20 % to 100.50 %) of the drug were obt
ained at each added concentration, which indicates that the method was accurate. The LOD and
LOQ were found to be in microgram level, which indicates the sensitivity of the method. The m
ethod was also found to be rugged as indicated by the %RSD values which are less than 2%. Sol
ution stability studies were performed using standard solutions of Tramadol hydrochloride by ke
eping the solutions at room temperature; solution was stable for more than two hours. The assay

results shows that the amount of drug was in good agreement with the labeled claim of the form
ulation as indicated by % recovery.