Recent Applications Of Analytical Techniques For .

WSEAS TRANSACTIONS on BIOLOGYand BIOMEDICINERudy Bonfilio, Magali Benjamim De Araujo, Herida Regina Nunes Salgado[13]. This technique is applied for the analysis ofconcentrations in the ng mL-1 range and,sometimes, in the pg mL-1 range in complexmatrices [14]. Thus, measurements of luminescenceintensity have allowed the selective and sensitivequantitative determination of a variety of activepharmaceutical ingredients. However, despite allthe advantages of luminescent methods, onlycertain classes of compounds exhibit luminescencenative, as a consequence of the deactivationprocesses occurring in a molecule. Moreover, theeffects of scattering and absorption limit the use ofluminescent methods when compared to otheranalytical methods such as chromatography andUV/visible spectrophotometry [13]. Luminescencespectroscopy can be applied using three differentprocesses: fluorescence, phosphorescence orchemiluminescence. However, fluorimetry is themost commonly luminescence method used inpharmaceutical analysis. Therefore, below aredescribed relevant examples of fluorimetricmethods applied to analysis of ofluorimetricandspectrophotometricmethods for the determination of pregabalin (PRG).In the spectrofluorimetric method, PRG wasreacted with fluorescamine. The imetric method was applied to thedetermination of the drug in capsules. According tothe authors, no interference could be observed fromthe additives listed to be in capsules. Furthermore,the spectrofluorimetric method was extended to thein-vitro determination of pregabalin in spiked urineand interference from endogenous amino acidscould be eliminated through selective complexationwith copper acetate and co-administered drugs suchas chlordiazepoxide, clonazepam, diazepam,nitrazepam and lamotrigine did not interfere withthe assay. As the method cited in reference number7, since there is no monograph for PGB in anypharmacopoeia, this work is relevant to qualitycontrol and clinical laboratories. Also, theconcentration range was 20-280 ng mL-1 forspectrofluorimetric method and 1-7 µg mL-1 forspectrophotometry. The method proposed byGujral, Haque and Shanker [7] was linear in therange of 0.5–5.0 µg mL-1. These results show thatthe spectrofluorimetric method is more sensitivethan the mentioned spectrophotometric methods fordetermination of this drug.El-Enany and coworkers [16] developed asecond derivative fluorimetric method for thesimultaneous analysis of binary mixture ofchlorzoxazone (CLZ) and ibuprofen (IP). Becausecaffeine and paracetamol (mixture 1) and DR withmetronidazole and diloxanide furoate (mixture 2).The chemometric methods applied were PLS andPCR. These approaches were applied usinginformation included in the UV absorption spectraof appropriate solutions in the wavelength range of210–300 nm with 1 nm intervals. Calibration ofPCR and PLS models was evaluated by internalvalidation, by crossvalidation and by externalvalidation. The authors showed that the proposedmethods were successfully applied for thedetermination of the two ternary combinations inlaboratory-prepared mixtures and commercialtablets. Besides, according to the authors, theresults of PLS and PCR methods were comparedwith the HPLC method, and a good agreement wasfound. All methods show adequate sensitivity forall analytes (detections limits less than 1.99 x 10-2mL-1).Thispaperalsoµgpresents a novelty, because no United States,British, or European compendia analytical methodhas been reported for the simultaneousdetermination of the five drugs in the two studiedternary mixtures.Storlarczyc [10] and coworkers used first andsecond order derivative spectrophotometricmethods for individual determination offluphenazine, pernazine, haloperidol and promazineusing methanol as solvent at two wavelengths. Theauthors concluded that no interference of matrixconstituents was observed and that the developedmethod can be useful for quality control of drugsand it is an option for commonly used expensivechromatographic methods. This method provedsatisfactory, although poor sensitivity for all drugs(detections limits less than 3.23 µg mL-1). Althoughthe authors present this method as a simple optionto chromatographic methods, all drugs are listed inpharmacopoeias. The USP [11] and the Britishpharmacopoeia (BP) [12] describe methods foranalysis of these drugs in tablets as titrimetry,direct and derivative spectrophotometry and HPLC.Therefore, this work does not present a noveltyfrom an analytical point of view.Although the described spectrophotometricmethods show the advantages mentioned by theauthors such as simplicity, low cost and speed ofanalysis, this technique frequently does not allowthe analysis of related substances, which make itdisadvantageous.2.2 Fluorimetric methodsLuminescence spectroscopy is an analytical toolextremely sensitive and has been widely applied insolving problems that require low detection limitsISSN: 1109-9518318Issue 4, Volume 7, October 2010

WSEAS TRANSACTIONS on BIOLOGYand BIOMEDICINERudy Bonfilio, Magali Benjamim De Araujo, Herida Regina Nunes Salgadohydrochloride, diltiazem hydrochloride, nicardipinehydrochloride and flunarizine using water asdiluting solvent. The method was based onoxidation of the studied drugs with ceriumammonium sulphate in acidic medium. Thefluorescence of the produced Ce was measured at365 nm after excitation at 255 nm. The differentexperimental parameters affecting the developmentand stability of the reaction product (Ceconcentration, type of acid and its concentration,heating time, temperature and diluting solvents)were individually optimized. The method wasapplied to the analysis of commercial tablets andthe authors concluded that the proposed method issimple, rapid and inexpensive. Both BP [12] andUSP [11] recommend non-aqueous titration foranalysis of verapamil hydrochloride raw material,the USP describes a HPLC method for analysis intablets and the BP recommends spectrophotometricmethod for its formulations. Regarding diltiazemhydrochloride, the BP recommends HPLC forrelated substances, non-aqueous titration for assayof raw material and the USP recommends HPLCmethod for the raw material and for itsformulations. Nicardipine and flunarizine are notyet listed in Pharmacopoeias, making the proposedmethod more attractive for the later two drugs.Also, the method showed good sensitivity for alldrugs.Marques, da Cunha and Aucélio [20]developed a fluorimetric method to quantifycamptothecin (CPT) in irinotecan (CPT-11) and intopotecan (TPT) based anti-cancer drugs. Forsamples containing TPT, detection was made at368 nm; whereas, in samples containing CPT-11,the detection was made at 267 nm isodifferentialwavelength, using the second derivative of thesynchronized spectrum. The authors concluded thatin practical terms, determinationsusingspectrofluorimetry were made in a faster, cheaper,and simpler way when compared to the ones madeusing HPLC. This work is relevant because no USP[11], BP [12], or European Pharmacopoeia (EP)[18] has been reported a method for thedetermination of these drugs. Besides, the methodwas sensitive, with limits of detection of about 9 ngmL-1, making this method an interesting alternativeto chromatographic techniques.Omar [21] developed spectrophotometric andspectrofluorimetric methods for the determinationof hydrochlorothiazide, indapamide and xipamidebased on ternary complex formation with eosin andlead in the presence of methylcellulose assurfactant. The fluorescence method wasinvestigated for the purpose of enhancing theCLZ and IP exhibit native fluorescence, the methodproposed by the authors was based on measurementof the synchronous fluorescence intensity of thesedrugs in methanol. However, both the excitationand emission spectra of CLZ and IP overlapped.Then, a second derivative fluorescence spectrum ofCLZ and IP was derived from the normalsynchronous spectra. The different experimentalparameters affecting the fluorescence of the twodrugs ( λ selection, pH, type of the dilutingsolvent, stability time and ionic strength) wereoptimized. The authors stated that the highsensitivity attained by the proposed methodallowed the determination of both drugs in their coformulated dosage forms and biological fluids andreal human plasma samples. USP [11] recommendsspectrophotometric method for determination ofCLZ in pure form and HPLC method for itsdetermination in tablets. For IP, BP [12]recommended direct titration for analysis of rawmaterial, while USP [11] recommends an HPLCtechnique. However, a method for simultaneousanalysis of these drugs does not appear inpharmacopoeias, which shows an analyticaladvantage of the fluorimetric method. Besides, thismethod showed a high sensitivity with lowquantitation limits (0.086 and 0.03 µg mL-1 forCLZ and IP, respectively),Walash and coworkers [17] developed twomethods for the determination of rosiglitazonemaleate (ROZ) in pure form, pharmaceuticalpreparations, and biological fluids. Method I was aspectrophotometry method. Method II was basedon the spectrofluorimetric determination of ROZthrough complex formation with Al 3 in acetatebuffer of pH 5. The relative fluorescence of thedrug was measured at 376 nm after excitation at318 nm. Both methods were applied for thedetermination of ROZ in tablets. Furthermore,method II was applied for the determination ofROZ in spiked and real human plasma. Thestability of the formed complexes in both methodswas studied by the authors, and the proposedmethods were found to be stability indicating ones.No United States [11], British [12] or European[18] compendia analytical method has beenreported for analysis of ROZ. Moreover, thequantification limit of the spectrofluorimetricmethod was 0.02 µg ml-1 demonstrating thesensitivity of this technique to human plasmaapplications, making it attractive to laboratorieslacking sophisticated separation techniques.Walash and coworkers [19] published akinetic spectrofluorimetric method for theindividualdeterminationofverapamilISSN: 1109-9518319Issue 4, Volume 7, October 2010

WSEAS TRANSACTIONS on BIOLOGYand BIOMEDICINERudy Bonfilio, Magali Benjamim De Araujo, Herida Regina Nunes Salgadosensitivity of the determination. According to theauthor, both methods have been fully validated andsuccessfully applied for the determination of thestudied drugs in their pharmaceutical tablets.Moreover, common excipients used as additives intablets do not interfere with the proposed methods.The USP [11] describes HPLC method forhydrochlorothiazide raw material and for tablets.BP [12] recommends titration for raw material , both USP and BP recommend a HPLCmethod for raw material and for tablets. No UnitedStates, British or European compendia analyticalmethod has been reported for ximpamide. Thepositives of these methods are the simplicity(prior extraction is not necessary), good sensitivity(quantitation limits less than 0.15 µg mL-1), andpossibility of application to pharmaceutical tablets.Rahman, Siddiqui and Azmi [22] developed aspectrofluorimetric method for the determination ofdoxepin hydrochloride in commercial dosageforms. The method was based on the fluorescention pair complex formation of the drug with eosinin the presence of sodium acetate–acetic acid buffersolution of pH 4.52. The extracted complex showedfluorescence intensity at 567 nm after excitation at464 nm. According to the authors, the method hasbeen successfully applied to the determination ofdoxepin hydrochloride in commercial dosageforms. USP [11] recommends HPLC method fordetermination of doxepin hydrochloride in pureform and capsules. The BP [12] recommendstitration for assay of doxepin hydrochloride rawmaterial and there is no monograph for itsformulatio

Keywords: pharmaceutical analysis; pharmaceutical quality control; analytical techniques. 1 Introduction From an analytical point of view, methods for pharmaceutical analysis are considerably less complex than methods for analysis of drugs and their metabolites in biological samples