Spectrophotometric Determination of Aciclovir , Cefepirne Hydrochloride , Etamsylate and Metoclopramide Hydrochloride Usina 1 . 1 0 Phenanttrroline-Fe ( lll ) Reaqent

Analytical Chemistry Department, Faculty of PharmacyZagazig University Zagazig, Ey9pt Abstract A simple, rapid,sensitive and accurate method for the determination of aciclovir,cefepime HCI, etamsylate and metoclopramide HCI in pure form and in pharmaceutical formulations is developed.The method is based on the fomabon of tns(o-phenanthroline) iron(ll) complex (Ferroin) upon the reaction of the ated drugs wrth iron(l l l )-ophenanthroline mixture.The ferroin complex is colorimetncally measured at &, 51 0 nm against a reagent blank.0ptimization of the experimental conditions is described.Beerls law is obeyed in the concentration range from 0.2530 pg ml-' with molar absorp&vWs (E) ranging from 4.796 x 1 o3 -9.51 2 x 1 o4 L.mol'.ae' and Sandell sensitivities (S) of 2.129 x 34.5 x 10" pg cmhe developed method is applied successfully for the determination of the cited drugs in pure forms and in the corresponding pharmaceutical formulations wtthout any interferences from common excipients.


INTROOUCTlON
Aciclovir is an anti-viral agent that is used currently for treatment of Herps Cefepime hydrochloride is a fourth generation cephalosporin that's widely used as anti-bacterial and administred via injection"'.lthas been determined by several procedures e.g.spectrophotometry~g~,diffuse reflectance IRB X-ray diff~acbon''~) and HPLC'").

m ) , ~~~( Z B ' and HPLC'~).
The aim of the present work is to apply 1.10-phenanthroline -~e ~' reagent to react with the studied drugs yielding a colored ferroin complex and presenting a simple and rapid assay procedure for the studied drugs in pure form and in pharmaceutical forrnulations.This work describes a spectrophotometnc method that can be used in laboratories where modem and expensive apparatus such as that required for GLC or HPLC is not available.

Apparahrs:
Shimadzu UV 260 recording spectrophotometer with matched 10 mm quartz cells was used for all absorbance measurements.

-1s
and Reagents : All chemicals used were of analybcal grade and all solutions were freshly prepared in doubly distilled water.

Procedure:
For pure phnnaceufjcak : Aliquots of the standard drug solutions ranging from 2.5-300 pg ml-' (Tab.2) were pipetted into test tubes,followed by 1.5-2.5 ml Fe(ll1)-o-phenanthroline reagent,the tubes were then heated on boiling water bath for the specrfied times (Tab.l).After completion of heat treatment the solution was coded to room temperature,transferred carefully and quantitatively to 10 ml volumetric flasks and the volume was made up to the mark with distilled water.The developed colored complex was measured at 510 nm against a reagent blank similarly treated (drug is omrtted).The concentration is then calculated from regression e q M o n s or calibmon graphs.

For dosage fonns:
Fortabh?ts:-Ten tablets were finely gr0und.A portion of powder equivalent to 100 mg of the active ingredient was extracted with 3 x 25 ml portions of distilled water,the porhons were filtered, the filter washed with distilled water and the salmon diluted to 100 ml wrth distilled water.The drug content in the obtained extract was determined as described under the general procedure.
For capsules : The contents of ten capsules were emptied.A portion equivalent to 100 rng of the adve ingredient was extracted with 3 x 25 ml portions distilled water by aid of heating for 2 min.in boiling water bath and the portions were filtered into 100 ml calibrated flask,cornplete as described under tablets and follow the general procedure.

For vials :
The contents of 2 vials were mixed and an amount of aciclovir Na equivalent to 100 mg of aciclovir was weighed and dissolved in 100 ml distilled water,complete following the general procedure.

For ampules:
The requisite volume equivalent to 100 mg drug was transferred into 100 ml volumetric flask and the volume was made up to the mark with water then complete as before following the general procedure.The proposed method is based on the formation of tns(o-phenanthroline) iron(ll) chelate upon the readon of ctted drugs wtth an iron(ll1)-o-phenanthroline reagent.The reaction proceeds through reduction of iron(l l I) ions to iron(ll) and subsequent formation of intensive orangered coloration of the complex.
The absorption spectra of the colored species in the proposed method shows a characteristic A ,at 510 nm where the orange-red color was completely developed.The experimental conditions were established by varying each parameter individua~ly'~' in order to establish the favorable experimental conditions.
For the proposed method,the cited drugs were allowed to react with Fe(lll) in the presence of 1,lO-phenanthroline and factors as temperature,readion time and reagent concentration were studied.
Samples prepared as described under the general procedures were left at different sets of temperatures ranging from 25 -100°C for a fixed time (Tab. 1 ),(Fig. 1 ),maximum absorbance and sensitivity were obtained by heating the reactants in boiling water bath.On the other hand,heating times on boiling water bath were varied and the absorbance was measured.ltwas found that 20 minutes were sufficient for metoclopramide hydrochloride and etamsylate,25 minutes for cefepime hydrochloride while aciclovir required 30 minutes for complete color development (Tab.1 ).Further heating causes no change in the color while measurements taken before the recommended times render inaccurate results so heating in boiling water bath for the recommended times was adopted for all subsequent measurements.
Another study on the volume of iron(ll1)-o-phenanthroline reagent was done where various volumes ranging from 0.5 -3.5 ml were used.It was found that 1.5 -2.5 mi were adequate for maximum absorbance and sensitivity (Tab.l).Smaller volumes gave incomplete complex forrnation,while larger volumes had no effect on the complex formation, although absorbance increased slightly due to background of the reagent used.The d o r obtained was stable for at least 24 hours at room temperature (Tab. 1 ).
The effects of common additives and excepients were studled and it was found that there were no interferences,~ the examined drugs can be assayed directly in their dosage forms by the suggested method without prior extraaon or separation.

Calibration graphs
Calibration graphs were constructed by plotttng the concentration of the drug against the corresponding absorbance values.The resulting graphs were linear up to 30 pg ml-' aciclovir , 4.2 pg ml-' cefepime hydrochloride, 2.75 pg ml" etamsylate and 1 1 pg ml-' metdopramide hydrochloride.
The linearity of calibration graphs is apparent from the correlation coefficient,r, and the intercepts which are nearly close to zero.All calibration data are calculated and listed in (Tab.2).

Sensitivity, accuracy and precision:
The mean molar absorpitivity (E) and Sandell's sensitivity (S) as calculated from Beer's law are presented in (Tab.2).The %S.D. and % range of S.E. at 95% confidence limits are given in (Tab.3&4).
The utilrty of this method was venfied by means of replicate measurements of pharmaceutical formulations and recovery experiments.Recoveries were determined either by standard addition method or by calibration method.
The performance of this method was assessed by calculation of "tn and Fvalues compared with the reported methods for determination of these drugs.Results showed that no significant difference in accuracy and precision between the proposed and the reported methods (Tab.3&4).

Conclusion
The proposed method is advantageous when compared to many of the reported spectrophotometnc methods in having higher sensitivrty .The data given before reveal mat proposed method is simple, sensitive with good accuracy and preusion.It doesn't require expensive toxic chemicals or sophisticated expenmental setup.ltcan be used directly to determine the examined drugs &out prior extramon as common additives don't interfere.
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Tab. 1 .
Anatytical data for the determination of examined drugs Ta b.2.Optical characteristics for the reaction of examined drugs with Iron(b = slope c = concentration (pg ml-') A = absorbance unit phenanthroline is an organic base which contains iron(ll) spectfic group'29' .
Fig.l.Effect of readat!t m p m t u m(1)30pgImlacickvir(2)3pgImlcek@meHCI (3) 2 pg Iml ebmrrylate (4) 7 pg Iml mdockpnmide HCI of the proposed and the reported method for the tested drugs in

(2.365) 5.66 (5.79) Tab.4. Comparative analytical resutts of the proposed and reported method for the tested drugs in some pharmaceutical formulations.
umber of experiments S.D. = Standard Deviation.S.E.= Standard Error.
t = "t" test of unpaired data.F = V a r i n c e t e s t .