SPECTROFLUOROMETRIC DETERMINATION OF SULPHAMETOXAZOLE IN PHARMACEUTICAL FORMULATION
WIRA WAHYUDI NANDAYASA 1411011009
ABSTRACT
A simple, robust and selective and sensitive spectrofluorometric method has been developed for the determination of sulphamethoxazole in pharmaceutical formulations. The method was based on the scanning of methanolic solution of the drug and methanolic solution of formulation. The fluorescent product formed which was measured after excitation at !"nm. The method showed high sensitivit# with linearit# range from $.%" to %.$ &g'ml. The lower limit of detection ()*+ was found to be .%% x -$ &g'ml and the limit of /uantization ()*0 was determined as the lowest concentration was found to be 1.2" x -$
%
-
&g'ml. The variables that affected the reaction were carefull# studied and optimized. The proposed method was applied successfull# for the determination of sulphamethoxazole in pharmaceutical formulations. The percentage recover# is 3S.+ (n41 were -$$.5 3 $.2!, -$$.$6 3 $.22 and 11.213 $.- for 7harmaceutical formulation.
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Sulfonamides were the first chemotherapeutic agents effective for the treatment of bacterial infections in man and are now available as widel# used pharmaceutical products in medicine and veterinar# practices. Sulphamethoxazole ;9<("meth#lisoxazole #l sulfanilamide= (S>? is the most widel# used sulfonamide for the control of bacterial diseases. The official method is a non selective potentiometric titration with sodium nitrite based on the reaction of the a romatic amine group
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or the development process we used :visible spectrophotometer (7erin@lmer )ambda %", Spectrofluorimeter (7erin@lmer )S"", Sonicater (Branson %"-$, @lectronicbalance (precise 1%sm %$%A. >ethanolD (E7)C grade, water double distilled water, pure sulphamethoxazole has been obtained as gift sample and the drug was used as such for further anal#sis. ormulations were purchased from the local pharmacies and used for anal#sis.
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An accuratel# weighed amount (%"mg of sulphamethoxazole was /uantitativel# transferred into a "$m) calibrated flas, dissolved in methanol and made up the volume. Then $.%ml of the above solution was pipetted out and using micro pipette transferred in to a -$m) standard flas diluted with methanol. The concentration of the woring standard solution was -$&g'ml
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@/uivalent to -%."mg of sulphamethoxazole was weighed accuratel#, from the crushed tablet powder and transferred into a clean %"ml standard flas. -"ml of methanol was added and solicited for "minutes and then made up to the volume with methanol.The above solution was filtered through whatman filter paper and the filtrate was collected. rom the above filtrate $.%ml was pipetted out using micropipette and transferred into a -$ml standard flas, which then was made up the volume with methanol and mixed well. urther dilutions were carried out to get -&g'ml concentration
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>ethod validation regarding reproducibilit# was achieved b# replicate inGection of extracted standard solution at low, medium and high concentration levels, where intensit# of fluorescence was measured in comparison to the intensit# of fluorescence of the standard. 8ntermediate precision stud# was conducted during routine operation of the s#stem over a period of six consecutive da#s. Statistical evaluation revealed relative standard deviations at different values of six replicates. Hithinda# repeatabilit# was studied b# six replicate at three concentration levels. Accurac# was estimated as the deviation to the observed mean concentration from actua l concentration and found to be less than %I for all the concentration. The procedure which was stated in %.! was done in addition of "$I,2"I,-$$I of drug as average along with the tablet powder and further dilution was made to get -.$&g'ml concentration. These solutions were used for further anal#sis to perform recover# studies.
alidation >ethod validation was performed in terms of specificit# and selectivit#, precision and accurac#, linearit# and stabilit#. )inearit# and range Calibration standards of sulphamethoxazole, covering the range $.%"%.$&g'ml were prepared with the suitable dilution made from sulphamethoxazole stoc solution. The calibration curves were obtained b# plotting the intensit# of fluorescence against of concentration of sulphamethoxazole. The slope and intercept of the calibration line were determined b# linear regression using the least s/uares method Specificit# and selectivit# The interference from endogenous compounds was investigated b# the anal#sis of six different blan matrices
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alidation Calibration standards for sulfamethoxazole covering the range of $.%"%.$&g'ml were prepared b# the method mentioned above and the serial dilutions were made with methanol. The calibration curve was obtained b# plotting the intensit# b# fluorescence of the sulfamethoxazole versus anal#te concentration. The slope and intercept of the calibration lie was determined b# li near regression using the least s/uare method. The data was presented in table - and the calibration curve was presented in fig -. Regression anal#sis of the calibration curve showed a linear relationship between the intensit# of fluorescence of sulphamethoxazole and the concentration with corelation coefficient higher than in all the curves assa#ed in pure form.
The precision was carried out as described in method and the results were presented in table %. The values obtained in the repeatabilit# (precision shows that there is no significant difference in the precision values henceJ the developed method can be used to anal#ze the sulfamethoxazole in tablet formulation. The mean of the precision value is -$$.55I. This value was obtained from 11.5- -$-.!.The regression e/uation was found to be # 4 1".--1x 1%.2!5
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8t is evident from the abovementioned results that the proposed methods gave satisfactor# results with sulphamethoxazole in bul. Thus, its tablets were subGected to the anal#sis of their contents from the active ingredient b# the proposed methods and the official (potentiometric titration method. The tablet content, as percentage, was -$$.5 3 $.2!, -$$.$6 3 $.22 and 11.21 3 $.-. These results were compared with those obtained from the official method b# statistical anal#sis with respect to the accurac# and precision. 9o significant differences were found between the calculated and theoretical values.
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A spectrofluorometric method for /uantif#ing sulphamethoxazole in formulation has been developed and validated. The linear range of the proposed spectrofluorometric method was $.%" %.$ &g'ml. The assa# is selective, precise, accurate and linear over the concentration range from $.%" %.$ &g'ml, the concentration of could sulphamethoxazole used for the precision stud# is -.$ &g'ml in formulations be precisel# /uantified and detected was approximated 1.2" x -$- &g'ml and .%% x -$% &g'ml respectivel#. Also, the proposed method involved spectrofluorometric measurements with comparable anal#tical performance devoid from an# potential interference. This gives the advantage of flexibilit# in performing the anal#sis on an# available instrument. urthermore, all the anal#tical reagents are inexpensive, have excellent shelf life, and are available in an# anal#tical laborator#. Therefore, these methods can be recommended for the routine anal#sis of sulphamethoxazole in /ualit# control and clinical laboratories
