Analysis of some antifungal drugs by spectrophotometric and spectrofluorimetric methods in different pharmaceutical dosage forms

Simple spectrophotometric and spectrofluorimetric methods are suggested for the determination of antifungal drugs; clotrimazole, econazole nitrate, ketoconazole, miconazole and tolnaftate. Spectrophotometric one depends on the interaction between imidazole antifungal drugs as n-electron donor with the pi acceptor 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in methanol or with p-chloranilic acid (p-CA) in acetonitrile. The produced chromogens obey Beer's law at lambda(max) 460, and 520 nm in the concentration range 22.5-200 and 7.9-280 microg ml(-1) for DDQ, and p-CA, respectively. Spectrofluorimetric method is based on the measurement of the native fluorescence of ketoconazole at 375 nm with excitation at 288 nm and or the induced fluorescence after alkaline hydrolysis of tolnaftate with 5 M NaOH solution at 420 nm with excitation at 344 nm. Fluorescence intensity versus concentration is linear for ketoconazole at 49.7-800 ng ml(-1) while for tolnaftate, it is in the range of 20.4-400 ng ml(-1). The proposed methods were applied successfully for the determination of all the studied drugs in their pharmaceutical formulations.     

Application of first-derivative, ratio derivative spectrophotometry, TLC-densitometry and spectrofluorimetry for the simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical dosage forms and plasma

Four sensitive methods are described for the direct determination of telmisartan (TELM) and hydrochlorothiazide (HCT) in combined dosage forms without prior separation. The first method is a first derivative spectophotometry (1D) using a zero- crossing technique of measurement at 241.6 and 227.6 nm for TELM and HCT, respectively. The second method is the first derivative of ratio spectrophotometry (1DD) where the amplitudes were measured at 242.7 nm for TELM and 274.9 nm for HCT. The third method is based on TLC separation of the two drugs followed by the densitometric measurements of their spots at 295 and 225 nm for TELM and HCT, respectively. The separation was carried out on silica gel 60 F254 using butanol: ammonia 25% (8:2 v/v) as mobile phase. The fourth method is spectrofluorimetric determination of TELM, depending on measuring the native fluorescence of the drug in 1 M sodium hydroxide at lambda excitation 230 nm and emission at 365 nm. The proposed methods were applied successfully for the determination of the two drugs in bulk powder and in pharmaceutical formulations. The spectrofluorimetric method was utilized for the analysis of TELM in human plasma.     

Simultaneous spectrofluorimetric and spectrophotometric determination of melatonin and pyridoxine in pharmaceutical preparations by multivariate calibration methods

Partial least-squares (PLS) calibration and principal component regression (PCR) methods were utilized for the simultaneous spectrofluorimetric and spectrophotometric determination of pyridoxine (PY) and melatonin (MT). Since emission and adsorption spectra of these drugs overlap, PY and MT cannot be directly determined by fluorimetric nor by spectrophotometric methods. Full-spectrum multivariate calibration PLS and PCR methods were developed for both fluorimetry and spectrophotometry. The conditions were optimized for fluorimetric as well as for spectrophotometric determination of both drugs. The simultaneous determination of PY and MT was carried out in mixtures by recording the emission fluorescence spectrum between 324 and 500 nm (lambda(ex) 285 nm) for fluorimetry, and by recording the absorption spectrum between 250 and 350 nm for spectrophotometry (lambda(max(PY)) 310 nm, lambda(max(MT)) 278 nm). The experimental calibration matrixes were designed orthogonally. At the optimum conditions, dynamic ranges were 0.04-1.3 and 0.1-4 microg ml(-1) for fluorimetry and 1-22 and 1-24 microg ml(-1) for spectrophotometry for MT and PY, respectively. The calibration concentrations were prepared in the dynamic ranges. The parameters of the chemometrics procedure for the simultaneous determination of MT and PY were optimized, and the proposed methods were validated with prediction set. Finally the procedures were successfully applied to simultaneous spectrofluorimetric and spectrophotometric determination of PY and MT in synthetic mixtures and in a pharmaceutical formulation.     

Spectrofluorimetric determination of vigabatrin and gabapentin in urine and dosage forms through derivatization with fluorescamine.

A stability-indicating, sensitive, simple and selective spectrofluorimetric method was developed for the determination of vigabatrin (VG) and gabapentin (GB). The method is based on the reaction between the two drugs and fluorescamine in borate buffer of pH 8.2 to give highly fluorescent derivatives that are measured at 472 nm using an excitation wavelength of 390 nm for both drugs. The optimum conditions were ascertained and the method was applied for the determination of VG and GB over the concentration range of 0.20-4.00 and 0.1-1.0 microg/ml, respectively with detection limits of 0.05 microg/ml (2.9 x 10(-7) M) and 0.06 microg/ml (2.3 x 10(-7) M) for VG and GB, respectively. The suggested method was applied, without any interference from the excipients, to the determination of the two drugs in their pharmaceutical formulations. Furthermore, the method was extended to the in-vitro determination of both drugs in spiked human urine. Interference from endogenous amino acids could be eliminated through selective complexation with copper acetate, the % recovery (n=4) is 98.0 +/- 7.05. Co-administered drugs such as lamotrigine, phenobarbitone, valproic acid, clopazam, carbamazepine, clonazepam and cimitidine did not interfere with the assay. The method is also stability-indicating; as the degradation product of vigabatrin: 5-vinylpyrrolidin-2-one, produced no interference with its analysis.     

Spectrofluorimetric and micelle-enhanced spectrofluorimetric methods for the determination of gemfibrozil in pharmaceutical preparations

A spectrofluorimetric method for the determination of antihyperlipoproteinemic gemfibrozil was developed based on its native fluorescence. This method allows the determination of 0.10-6 microg ml(-1) gemfibrozil in aqueous solution (without using any buffer solution) with excitation and emission wavelengths of 276 and 304 nm, respectively. Detection and quantification limits were 0.03 and 0.10 microg ml(-1), respectively. The fluorescence properties of gemfibrozil in micellar media were also studied. It was shown that in the presence of 0.4% Brij-35 surfactant (pH 4.0, acetic acid-acetate buffer) about 2.4-fold enhancement can be achieved in the fluorescence of this drug. Based on the obtained results, a micelle-enhanced fluorescence method was also developed that is more sensitive than aqueous fluorescence method and has lower detection limit (0.02 microg ml(-1)). Both methods were applied satisfactorily to the determination of gemfibrozil in a commercial pharmaceutical formulation.     

Stability-indicating spectrophotometric and spectrofluorimetric methods for determination of alfuzosin hydrochloride in the presence of its degradation products

Validated stability-indicating spectrophotometric and spectrofluorimetric assays (SIAMs) were developed for the determination of alfuzosin hydrochloride (ALF) in the presence of its oxidative, acid, and alkaline degradation products. Three spectrophotometric methods were suggested for the determination of ALF in the presence of its oxidative degradation product; these included the use of zero order (0D), first order (1D), and third order (3D) spectra. The absorbance was measured at 330.8 nm for (0D) method, while the amplitude of first derivative (1D) method and that of third derivative (3D) method were measured at 354.0 and 241.2 nm, respectively. The linearity ranges were 1.0-40.0 microg/ml for (0D) and (1D) methods, and 1.0-10.0 microg/ml for (3D) method. Two spectrofluorimetric methods were developed, one for determination of ALF in the presence of its oxidative degradation product and the other for its determination in the presence of its acid or alkaline degradation products. The first method was based on measuring the native fluorescence of ALF in deionized water using lamda(excitation) 325.0 nm and lamda(emission) 390.0 nm. The linearity range was 50.0-750.0 ng/ml. This method was also used to determine ALF in human plasma with the aid of a suggested solid phase extraction method. The second method was used for determination of ALF via its acid degradation product. The method was based on the reaction of fluorescamine with the primary aliphatic amine group produced on the degradation product moiety. The reaction product was determined spectrofluorimetrically using lamda(excitation) 380.0 nm and lamda(emission) 465.0 nm. The linearity range was 100.0-900.0 ng/ml. All methods were validated according to the International Conference on Harmonization (ICH) guidelines, and applied to bulk powder and pharmaceutical formulations.     

Determination of aceclofenac in bulk and pharmaceutical formulations.

Three sensitive and reproducible methods for quantitative determination of aceclofenac (AC) in pure form and in pharmaceutical formulation are presented. The first method is based on the reaction between the drug via its secondary aromatic amino group and p-dimethylaminocinnamaldehyde (PDAC) in acidified methanol to give a stable coloured complex after heating at 75 degrees C for 20 min. Absorption measurements were carried out at 665.5 nm. Beer's law is obeyed over concentration range 20-100 microg ml(-1) with mean recovery 100.33 +/- 0.84. The other two methods are high performance liquid chromatography (HPLC) and densitometric methods by which the drug was determined in the presence of its degradation products over concentration range of 20-70 microg ml(-1) and 1-10 microg per spot and mean recoveries are 99.59 +/- 0.90 and 99.45 +/- 1.09, respectively.     

A validated spectrofluorimetric method for determination of some psychoactive drugs

Five psychoactive drugs namely, chlorpromazine HCl, thioridazine HCl, clomipramine HCl, imipramine HCl and desipramine HCl were analyzed by a simple spectrofluorimetric method. The method is based on oxidation of the studied drugs using cerium(IV) in presence of sulphuric acid and monitoring the fluorescence of the formed cerium(III) at lambda(ex.) = 254 nm and lambda(em.) = 355 nm. All variables affecting the reaction conditions such as; cerium(IV) concentration, sulphuric acid concentration, heating time, temperature and dilution solvents were carefully studied. The effect of potential interference due to common ingredients as glucose, sucrose, lactose, citric acid and propylene glycol were investigated. A validation study of the proposed method was carried out according to USP 2002. Beer's law was obeyed for all the studied drugs in the concentration range of 0.05-1.3 microg/ml. Limits of detection range was 0.035-0.038 microg/ml and limits of quantitation of 0.116-0.125 microg/ml were obtained. The method was successfully applied for the assay of the studied drugs in pure form and in pharmaceutical dosage forms. Results were compared with official methods. The t- and F-values were calculated and compared with the theoretical values, which indicate high accuracy and good precision of the proposed method.     

Spectrofluorimetric determination of manzamine A in spiked human urine and plasma

The native fluorescence of manzamine A (a biologically active beta-carboline marine-derived alkaloid) has been studied under different conditions. The highest fluorescence intensity was obtained in methanol. Two wavelength settings were found to be suitable for excitation, 280 nm and 340 nm; while lambdamax emission was constant in both cases at 387 nm. The fluorescence intensity at 340/387 nm setting was 1.6 greater than that obtained at 280/387 nm settings. The calibration curves were rectilinear over the range 0.1-2.0 and 0.5-2.5 microg/ml for the two settings, respectively. The detection limits were 0.05 microg/ml (9.1 x 10(-9) M) and 0.1 microg/ml (1.82 x 10(-8) M) at 340/387 nm and 280/387 nm, respectively. The proposed method was applied to the determination of manzamine A in spiked human urine and plasma samples adopting the 340/387 nm wavelength setting, the % recoveries (n = 6) were 99.61 +/- 0.90 and 100.25 +/- 1.63, respectively.     

Spectrofluorimetric and spectrophotometric methods for the determination of vigabatrin in tablets

Two sensitive and selective spectrofluorimetric and spectrophotometric methods have been developed for the determination of vigabatrin in tablets. The methods are based on derivatization with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The product showed an absorption maximum at 460 nm and a fluorescence emission peak at 520 nm in ethyl acetate. The color was found to be stable for at least 48 h in this solvent. The optimum conditions of the reaction were investigated and it was found that the reaction proceeds quantitatively at pH 10.0, 70 degrees C in 50 min when the mole ratio of the reagent to drug was 30. The reaction obeys Beer's law over the ranges of 2-10 and 0.05-1.00 microg ml(-1) for the spectrophotometric and spectrofluorimetric measurements, respectively. The detection limits were found to be 0.50 and 0.01 microg ml(-1) for the spectrophotometric and spectrofluorimetric methods, respectively. The proposed methods were applied to the assay of vigabatrin in tablets. The results were compared statistically with those obtained by the modified spectrofluorimetric method reported in the literature.     

Colorimetric and fluorimetric methods for determination of panthenol in cosmetic and pharmaceutical formulation

Two methods are suggested for determination of panthenol. The first is colorimetric method where panthenol is subjected to alkaline hydrolysis; the resulting beta-alanol is allowed to react with vanillin (Duquenois reagent) in presence of McIlvain buffer pH 7.5. The color developed is measured at 406 nm. The linearity range was found to be 50-500 microg/ml while the lower limit of detection was about 10 microg/ml. The second is a sensitive and reliable modified fluorimetric method is also suggested, whereas panthenol, after alkaline hydrolysis is treated with ninhydrin. The fluorescent product was found to have excitation lambda(max) at 385 nm and emission lambda(max) at 465 nm. The method showed high sensitivity with linearity range from 0.01 to 3 microg/ml. The lower limit of detection (LOQ) reached 0.005 microg/ml. Validation of both methods was carried out and the two methods were applied for determination of panthenol in some cosmetic and pharmaceutical formulations.     

Voltammetric, spectrofluorimetric and spectrophotometric methods to determine flufenamic acid

Simple, rapid and sensitive voltammetric, spectrofluorimetric and spectrophotometric methods for determination of flufenamic acid (FF) in bulk powder and capsule dosage form are presented. The methods are based on the cyclisation reaction of FF with concentrated sulphuric acid to produce the corresponding acridone derivative. The voltammetric method is based on the adsorptive stripping differential pulse (DP) technique. The acridone derivative is determined over the concentration range of 8-60 ng ml(-1) using adsorptive preconcentration at the hanging mercury drop electrode (HMDE). The lower detection limit was found to be 1.02 ng ml(-1). The fluorimetric and spectrophotometric methods are based on the measurement of the fluorescence intensity at 450 nm (lambda(ex) = 400 nm)and peak-to-peak measurements of the first- (D1) and second-derivative (D2) curves, respectively. Beer's law is obeyed over the concentration ranges of 2-20 ng ml(-1) and 0.2-8.0 microg ml(-1) for the fluorimetric and spectrophotometric measurements, respectively. The three methods were proved to be accurate and reproducible as indicated by a relative standard deviation of <2%.     

Spectrofluorometric determination of montelukast in dosage forms and spiked human plasma

The native fluorescence of montelukast has been studied under different experimental conditions. The highest fluorescence intensity was obtained in methanol at 390 nm using 340 nm for excitation. Surfactants and sensitizers had either a negative or a slightly positive effect on its fluorescence intensity. The fluorescence intensity-concentration plot was rectilinear over the range 0.125 to 5 microg/ml with a lower detection limit of 0.02 microg/ml (3.4 x 10(-8) M). Interference likely to be introduced from co-formulated drugs (such as loratadine) or co-administered drugs (such as verapamil, carbazepam, propranolol) or other common drugs, was studied. The method was successfully applied to the determination of the drug in tablets (pediatric tablets, chewable tablets and adult tablets). The mean % recoveries were in agreement with those provided by the manufacturer. The method was further applied to the in vitro determination of montelukast in spiked human plasma, the mean % recovery (n = 5) was 100.08 +/- 1.40.     

Spectrofluorimetric and micelle-enhanced spectrofluorimetric determination of gatifloxacin in human urine and serum

A spectrofluorimetric method to determine gatifloxacin has been developed and applied to the quantification of this fluoroquinolone in spiked human urine and serum. The native fluorescence of gatifloxacin allow the determination of 0.040-0.700 micro gmL(-1) of this molecule in aqueous solution containing acetic acid-sodium acetate buffer (pH 3.5), with lambda(exc)=292 nm and lambda(em)=484 nm. Micelle-enhanced fluorescence led to 75% higher analytical signals in presence of 12 mM sodium dodecyl sulphate, which allow the determination of 0.020-0.450 microg mL(-1) fluoroquinolone with lambda(exc)=292 nm and lambda(em)=470 nm. Both methods were successfully applied to gatifloxacin determination in spiked human urine and serum.     

Use of mixed anhydrides for the determination of terfenadine in dosage forms and spiked human plasma

Terfenadine reacts with mixed anhydrides (malonic and acetic anhydrides) producing a yellow-coloured product with intense fluorescence. Based on this fact, a spectrophotometric method was developed for the determination of terfenadine in dosage forms. The relation between the absorbance at 395 nm and the concentration is rectilinear over the range 0.5-5 microg ml(-1) (molar absorptivity is 1.405 x 10(5) l mol(-1) cm (-1)). The reaction product was also measured spectrofluorimetrically at 435 nm after excitation at 395 nm. The fluorescence intensity was directly proportional to the concentration over the range 0.5-4 ng ml(-1) with minimum detectability (S/N = 2) of 0.07 microg ml(-1) (approximately 1.5 x 10(-10) M). The different parameters affecting the development and stability of the reaction product were carefully studied and incorporated into the procedure. The proposed spectrophotometric method was successfully applied to the determination of terfenadine in tablets and suspensions; the percentage recoveries were 99.83 +/- 0.75 and 99.65 +/- 0.83, respectively. The proposed spectrofluorimetric method was applied to the determination of terfenadine in spiked human plasma. The percentage recovery was 99.35 +/- 2.19. The method is highly sensitive and specific. No interference was noticed from co-formulated drugs, such as pseudoephedrine and ibuprofen.     

Spectrofluorimetric determination of velnacrine in human serum and urine

A simple, sensitive and rapid spectrofluorimetric method to determine velnacrine, a cholinesterase inhibitor, has been developed and validated. The influence of pH, temperature, ionic strength, presence of excipients, metal ions and surfactants on the fluorescence intensity has been studied. The proposed method allows the determination of 5-100 ng ml(-1) of velnacrine in aqueous solution containing sodium acetate buffer (pH 5.6; 0.04 M) with lambda(excitation) 242 nm and lambda(emission) 359 nm. The detection and quantitation limits were 1.7 and 4.5 ng ml(-1) respectively. The method was successfully applied to the determination of velnacrine in human serum and urine.     

Stability-indicating methods for the determination of mosapride citrate in the presence of its degradation products according to ICH guidelines

In the present work, different spectrophotometric methods and one spectrofluorimetric method have been developed and validated for the determination of mosapride citrate in the presence of its acid-induced degradation products. The drug was subjected to stress stability study including acid, alkali, oxidative, photolytic, and thermal stress degradation. The developed spectrophotometric methods included the use of first order derivative ((1)D), derivative of ratio spectra ((1)DD), mean centring of ratio spectra (MC) and H-point standard additions (HPSAM) spectrophotometric methods. For (1)D method, the peaks amplitudes at 282.8 and 319.6 nm were measured, while for (1)DD method those at 308 nm and 323 nm were measured. Mean centring of ratio spectra method used the values at 317 nm for calibration, while for HPSAM the absorbance at 273 and 288.6 nm were used. These methods were successfully applied for determination of mosapride in the concentration range of 5-70 μg.ml(-1). The spectrofluorimetric method was based on measuring the native fluorescence of mosapride in 0.1 M NaOH using λ(excitation) 276 nm and λ(emission) 344 nm and 684 nm with linearity ranges of 50-3000 ng.ml(-1) and 50-9000 ng.ml(-1), respectively. All the developed methods were validated according to the International Conference on Harmonization (ICH) guidelines and were applied for bulk powder and dosage form. The results obtained were statistically compared to each other using one-way ANOVA testing.     

Use of 7-fluoro-4-nitrobenzo-2-oxo-1,3-diazole (NBD-F) for the determination of ramipril in tablets and spiked human plasma.

Ramipril, as a secondary amine compound, reacts with 7-fluoro-4-nitrobenzo-2-oxo-1,3-diazole (NBD-F) producing the corresponding fluorescent NBD-ramipril. According to this fact, spectrophotometric and fluorimetric methods for the determination of ramipril were developed. The effect of these parameters on the reaction product were carefully studied to optimize reaction conditions. The relationship between the absorbance at 465 nm and the concentration was found to be linear over the range 1-10 microg/ml. Moreover, the fluorescence intensity was also found to be directly proportional at the concentration over the range of 20-100 ng/ml at 530 nm after excitation at 465 nm. The proposed procedure was successfully applied to the determination of ramipril in both tablet dosage form and in plasma. Spectrophotometric determination of ramipril tablets yielded a percentage recovery of 98.66+/-0.38, while the percentage recovery of spectrofluorimetric determination of ramipril in spiked human plasma was 99.08+/-1.11%. The results obtained are in good agreement with those obtained by the reference method. No interference could be observed from the co-administered drug (hydrochlorothiazines).     

Selective spectrofluorimetric determination of phenolic beta-lactam antibiotics through the formation of their coumarin derivatives

A simple, sensitive and selective spectrofluorimetric procedure was developed for the determination of amoxycillin, cefadroxil and cefoperazone. The method is based on the reaction between these drugs and ethyl acetoacetate, in acidic medium, to give yellow fluorescent products with excitation wavelengths ranging from 401 to 467 nm and emission wavelengths ranging from 465 to 503 nm. The reaction conditions were studied and optimized. The reaction obeyed Beer's law over the range of 10.0-20.0, 1.5-1.0 and 50.0-100.0 microg ml(-1) for amoxycillin, cefadroxil and cefoperazone, respectively. Interference's from other antibiotics, drugs and dosage forms additives, in capsules and vials dosage forms, were investigated. The proposed method was applied to the analysis of pharmaceutical formulations (capsules and vials) containing the above antibiotics, either alone or in combination with other antibiotics or drugs. The validity of the method was tested by the recovery studies of standard addition which were found to be satisfactory. The results of the proposed method demonstrated that the method is equally accurate, precise and reproducible as the official methods (USP XXIII) and those published for the non-official binary mixtures.     

Spectrofluorometric determination of some beta-blockers in tablets and human plasma using 9,10-dimethoxyanthracene-2-sodium sulfonate

A simple and sensitive spectrofluorometric method was developed for the quantitative determination of some beta-blockers, namely arotinolol, atenolol and labetalol as hydrochloride salts. The method is based on the reaction of these drugs as n-electron donors with the fluorogenic reagent 9,10-dimethoxy-2-anthracene sulfonate (DMAS) as pi-acceptor in acidic medium. The obtained ion-pairs were extracted into chloroform and measured spectrofluorometrically at 452 nm after excitation at 385 nm. The fluorescence intensity-concentration plots are rectilinear over the ranges of 0.5-5 microg x ml(1), 1.0-11.0 microg x ml(1) and 0.6-6.4 microg x ml(1) for labetalol, atenolol and arotinolol, respectively. The different parameters affecting the reaction pathway were thoroughly studied and optimized. No interference was observed from the common pharmaceutical excipients. The proposed method was successfully applied to the analysis of tablets and the results were statistically compared with those obtained by reference methods. The method was further extended to the in vitro determination of the drugs in spiked human plasma, the % recoveries (n = 3) ranged from 96.98 +/- 1.55 to 98.28 +/- 2.19. A proposal of the reaction pathway was postulated.