SPECTROPHOTOMETRIC DETERMINATION OF SOME PHARMACEUTICAL COMPOUNDS USING 2 , 2-DIPHENYL-1-PICRYLHYDRAZYL

A simple, rapid and sensitive spectrophotometric procedure for the assay of some drugs has been developed. The method is based on the reaction of the studied drugs with 2,2-diphenyl-Ipicrylhydrazyl (DPPH). The latter is employed to abstract a hydrogen atom from the drugs thereby promoting a process of radical coupling. This results in a reduction of the violet color of DPPH with the formation of the yellow colored 2,2-diphenyl-Ipicrylhydrazine (DPPH2). The decrease in the intensity of the violet color is used to measure the concentration of the drugs. All measurements are made at h= 520 nm on methanolic solutions of the reagent and drugs. ~ e e r ' s law is obeyed in the ranges of 5-30 pglml (for aceclofenac, diclofenac sodium and thiaprofenic acid), 2-15 pglml (for tenoxicam, furosemide and lansoprazole) and 2-12 pglml (for benoxinate hydrochloride and ritodrine). The validity of the method was tested by carrying out standard addition procedure analyzing the studied drugs in pure form as well as in their pharmaceutical preparations without interference from common additives. Results of the proposed methods are in good agreement with those of the official or reported methods.


INTRODUCTION DPPH
2,2-Diphenyl-I -Picrylhydrazyl (DPPH) is an intense, violetcolored, stable, free radical which reacts as a chromogenic reagentqv2 by abstracting a hydrogen atom from the analyt to form yellow colored N,N-diphenylpicrylhydrazine (DPPH,).This decrease in the intensity of the violet color is used as a measure of the quantity of the tested drugs.The radical DPPH was chosen for the present work because it does not dimerize3 and the problem of cage effect does not arise.In addition, it is deeply colored and its concentration at any time can be estimated by its absorption in the visible range4.

Thiaprofenic acid
Thiaprofenic acid is an anti-inflammatory, antipyretic drug with analgesic properties.Its chemical structure is 2-(5-benzoyl-2-thieny1)propionic acid.Several techniques were used for the determination of this drug such as spectrophotometric5, polarographic6, nuclear magnetic resonance7 , high pressure liquid chroma tog rap hi^^^^ and atomic absorption spectrophotometric methodsq0.

Lansoprazole
Lansoprazole is a substituted benzimidazole, it is one of the first drugs of a new class of orally active anti-ulcer agents.

Benoxinate hydrochloride
Benoxinate hydrochloride is indicated as corneal anaesthesia of short duration.Its chemical structure is 2-(diethylamino)ethyl4-amino-3-n-butoxybenzoate hydrochloride.Spectroph o t 0 m e t t -i ~~~ and pharmacol~gical~~ methods were recommended for the determination of benoxinate hydrochloride.

Furosemide
Furosemide is the most potent diuretic available.It is therapeutically used in cases of acute pulmonary edema, acute renal failure and hypercalecemia.Its chemical structure is 4chloro-N-furfuryl-5-sulfamoyl anthranilic acid24.~harmacological~~, [29][30][31][32][33] c h r ~m -a t o g r a p h i c ~~-~~ and spectrophotometric methods have been reported for the assay of furosemide.

Diclofenac sodium
Diclofenac acid is widely used as an anti-inflammatory agent.Its chemical structure is [2-(2,6-dichloroanilino- phenyllacetic acid.Several techniques have been used for the determination of this drug including chroma tog rap hi^^^'^^ and spectrophotometric methods33t37138.In this work, a simple, rapid and sensitive spectrophotometric method is adopted for the determination of aceclofenac, thiaprofenic acid, tenoxicam, lansoprazole, furosemide, ritodrine, d iclofenac sodium and benoxinate hydrochloride in the pure forms and in pharmaceutical preparations with 2,2-diphenyl-1 -picrylhydrazyl (DPPH).

EXPERIMENTAL Apparatus
A Shimadzu UV1601, UV-visible spectrophotometer (Tokyo, Japan) and Memert type thermostatically controlled water bath (Germany) were used.All volumetric measurements were made with standard glassware.

Materials
All solvents and reagents were of analytical reagent grade.The pharmaceutical grade pure drugs; aceclofenac was obtained from Bristol-Myers Squibb (Squibb, Egypt), furosemide, lansoprazole and thiaprofenic acid were obtained from Hoechst (Hoechst, Egypt), diclofenac sodium and ritodrine were obtained from Pharco (Pharco, Egypt) and benoxinate hydrochloride and tenoxicam were obtained from Eipico (E.I.P. I.Co, Egypt).All compounds were complying with requirements recommended by official or other reported methods and used as such without further purification.A standard stock solution of 50 mg1100 ml of each studied drug was prepared in methanol.

Reagents and solutions
2,2-Diphenyl-I -picrylhydrazyl (Sigma, St. Louis, Mo, USA).A stock solution of 1.5 mgl ml was prepared by dissolving 0.15 g of DPPH in methanol and then diluted to 100 ml with the same solvent.Ten milliliter of this solution were diluted to 100 ml to give 0.1 5 rngl ml (working DPPH solution).The stock and working solutions were kept in a refrigerator and protected from light.The solution was found to be stable for at least one week at 4OC.All chemicals and solvents were of analytical grades.

Pharmaceutical preparations
The commercial dosage forms subjected to analysis were

Assay of pharmaceutical preparations:
Assay of tablets Twenty tablets were accurately weighed and finely powdered.An amount equivalent to about 50 mg of each drug was weighed accurately.The powder was transferred to 100 ml volumetric flask, extracted successively three times with 10 ml of methanol and completed to the mark with the same solvent.The extract is diluted to obtain 50 pglml before carrying out the general procedure.

Assay of capsules
The contents of twenty capsules were weighed and finely powdered.An amount equivalent to 50 mg of each drug was weighed accurately and transferred into a 100 ml volumetric flask.Thirty milliliter of methanol are added to the flask , which was shaken for 10 min.The methanolic supernatant solution is filtered into a 100 ml volumetric flask.The extraction is repeated twice and the content of .thevolumetric flask is diluted to volume with the same solvent.An accurately measured volume of this solution is pipetted into 10 ml volumetric flasks then completed as under general procedure.

Assay of suppositories:
Five suppositories for each drug were accurately weighed and cut into small pieces.The suppository mass was transferred into a porcelain dish and melted on a boiling water bath to complete homogenity.An amount of the suppository mass equivalent to 50 mg of each drug was mixed with 25 ml methanol and shaken for 5 min.The solution was filtered into a 100 mi volumetric flask and completed to volume with methanol and completed as under general procedure.

Assay of ampoules and eye drops
Aliquot of ampoules or eye drops equivalent to 50 mg of each drug was transferred into a 100 ml volumetric flask and completed to volume with methanol and completed as under general procedure.reaction is assumed to proceed via abstraction of hydrogen atoms from the drugs by D P P H ~~. This is accompanied by the change of violet color of DPPH to give the yellow colored DPPH, and the corresponding free radical of the drug ( Scheme 1 ).

Optimization of variables:
Reagent concentration DPPH is added in excess to react with drugs to be analyzed.By measuring the excess reagent, the consumed DPPH would correspond to the amount of the drug.The concentration of the reagent that gives the highest absorption value within the participle sensitivity range of absorbance was found to be 0.15 mglml.Two milliliters of this solution per 10 ml of the reaction mixture was used.

Reaction time and temperature
The reaction time was determined for the interaction of each studied drug with DPPH solution by following the color reduction at ambient temperature (25 + 2 OC), 30, 40, 50 and 60 "C.The optimum temperature and reaction time were recorded in table I.The AA reached a constant level, at once and remained constant for at least 180 minutes.

Molar ratio of the reaction
The stoichiometry of the reaction was assessed by the moleratio method46 and the results have been appeared in Table 1.Such results obtained have been cover the number of abstractable hydrogen present in the drug molecule.

Drug
Heating time Temp.Molar ratio of (min.)

Influence of solvents
The effect of dilution of the reaction product by different solvents namely, methanol, ethanol, n-propanol, isopropanol, nbutanol, acetone was studied.The results indicated that all solvents had no effect on the position of maximum absorption while the reactivity (AA value) was affected.Methanol was found to be the most suitable solvent.

Quantification
Under the optimum parameters, ~e e r ' s law was obeyed for all studied drugs in the range shown in Table 2.The regression analysis of AA value versus concentration were done for all the studied drugs according to the linear regression equation surpmarized in Table 2.

Effect of pH
The effect of pH of the added acetate buffer on color reduction was studied for the investigated drugs.The results revealed that the reaction was independent on pH.Because of this independence of the reaction on pH, further investigations were not carried out to establish whether the constituents or pH range of the buffer solutions have any effect on the interaction of DPPH with the investigated drugs.

Interference
Before proceeding with the analysis of the studied drugs in dosage forms, interference abilities from added common excipients ( such as lactose, sucrose, starch, magnesium stearate and gum acacia) were carried out to explore their effect.Samples were prepared by mixing known amounts of the investigated drugs with various amounts of the common excipients.
The good percentage recoveries of the investigated drugs obtained from those synthetic mixtures show that no interference from these additives takes place with the proposed method.Moreover, accuracy of the suggested procedure was further checked by applying standard addition technique.
The results obtained (Table 3) reveal a high degree of accuracy.

Assay of pharmaceutical dosage forms
It is evident from the results obtained previously that the proposed method gave satisfactory results with the drugs in bulk (Table 4).Thus different pharmaceutical formulations containing the investigated drugs were analyzed for the content of each drug by the proposed procedure as well as the official or reported methods.Data recorded in Table 4 showed no interference by excipients and additives.The results of the analysis recorded in Table 5 indicate the suitability of the method to the assay of the drugs in different pharmaceutical dosage forms.The calculated values of F and t (at 95% confidence level) did not exceed the tabulated (theoretical) ones.This means that there is no significant difference between the proposed and official or reported methods with respect to precision and accuracy.In conclusion, the DPPH method is simple, sensitive, accurate and precise.It also has the advantage of being applicable to pharmaceutical preparations.In comparison to the official or reported methods, the DPPH method is rapid, specific and more sensitive.In addition, the DPPH reagent is stable, doesn't dimerize and is ready for use immediately and its reaction with the selected drugs is rapid and the reaction products are stable for more than two hours.
tenoxicam and ritodrine.Cool and complete to the mark with methanol.Measure the absorbances of the sample and a reagent blank against methanol at h=520 nm.Calculate AA, i.e. the difference between the absorbance values of the blank and sample which corresponds to the drug concentration.

(
Epicotil tablets (labeled to contain 20 mg tenoxicam per tablet), Epicotil suppositories (labeled to contain 20 mg tenoxicam per suppository) and Epicotil vials (labeled to contain 20 mg tenoxicam per vial),E.I.P.I.Co, Egypt; Soral capsules (labeled to contain 20 mg tenoxicam per capsule) and Soral suppositories (labeled to contain 20 mg tenoxicam per suppository), Global Napi Pharmaceuticals (under license of help Ltd-Greece); Benox ophthalmic solution ( labeled to contain 4 mg benoxinate hydrochloride per each ml), E. I.P.I.Co, Egypt; Lanzore capsules (labeled to contain 20 mg lansoprazole per capsule), Hoechst Co., Egypt; Bristaflam tablets (labeled to contain 100 mg aceclofenac per tablet), Squibb Co., Egypt; Surgam tablets (labeled to contain 100 mg thiaprofenic acid per tablet), Surgam suppositories (labeled to contain 100 mg tiaprofenic acid per suppository) and Surgam vials (labeled to contain 300 mg thiaprofenic acid per vial), Hoechst Co., Egypt; Lasix tablets and ampoules were obtained from Hoechst Co., Egypt, labeled to contain 40 mg and 20 mg furosemide per tablet and ampoule, respectively.Declophen tablets and ampoules, Pharco Co., Egypt, labeled to contain 25 mg and 75 mg diclofenac sodium per tablet or ampoule respectively, were used.Yutopar tablets and ampoules (Pharco Co., Egypt), labeled to contain 10 mg of ritodrine hydrochloride per tablet and 1 ml of ampoule, respectively, were used.General procedureTransfer 1 ml of the investigated drug to a 10 ml volumetric flask, add 2 ml of DPPH solution.Mix well for lansoprazole and diclofenac sodium at 25OC, heat in water bath at 60°C for 15 minutes for aceclofenac, thiaprofenic acid, furosemide and benoxinate hydrochloride and at 40°C for 10 minutes for

Table 2 . Spectral characteristics of the studied drugs by the proposed method.
6: Intercept, K: Slope and R: Correlation coefficient.

Table 3 . Assay of the studied drugs in presence of common excipents by the proposed method applying the standard addition technique. Recovery % kS.D.
The amount of excipients added per 50 mg of drug