Thin-layer chromatographic scanner, spectrophotometric and high-performance liquid chromatographic methods for the determination of colchicine

One high-performance liquid chromatographic (HPLC) and two thin-layer chromatographic (TLC) methods are proposed for the determination of colchicine in crude drugs and pharmaceutical preparations. The TLC scanner method is based on measurement of the absorbance of the separated colchicine spot; alternatively, after scraping the spot from the plate and elution the absorbance can be measured spectrophotometrically. The HPLC assay was carried out isocratically on a reversed-phase column using MeOH-H2O (60 + 40). The recoveries were 99.2 +/- 1.23, 99.1 +/- 1.12 and 99.1 +/- 2.01% for the TLC scanner, spectrophotometric and HPLC methods, respectively. The methods were shown to be sensitive and specific and can be used as an alternative to the pharmacopoeial methods having been applied to the determination of colchicine in corms of Merendera persica and in three pharmaceutical preparations.     

Simultaneous determination of hyoscine butylbromide and ketoprofen in pharmaceutical preparations by spectrophotometric and liquid chromatographic methods

A binary mixture of hyoscine butylbromide and ketoprofen was determined by 4 different methods. The first involved determination of hyoscine butylbromide and ketoprofen using the ratio-spectra first-derivative spectrophotometric technique at 211 and 234 nm over the concentration ranges of 2-14 and 5-45 microg/mL with mean accuracies 99.84 +/-0.92 and 99.98+/- 0.64%, respectively. The second method utilized second-derivative spectrophotometry over the concentration ranges of 2-14 and 5-35 microg/mL with mean accuracies 99.32+/- 1.06 and 99.55+/-1.15%, respectively. The third method was based on the resolution of the 2 components by bivariate calibration depending on a simple and rapid mathematical algorithm and quantitative evaluation of the absorbances at 206 and 254 nm over concentration ranges of 2-16 and 5-35 microg/mL; mean accuracies of 100.21+/-1.30 and 100.19 +/-1.07% were obtained for hyoscine butylbromide and ketoprofen, respectively. The fourth method was reversed-phase liquid chromatography using 0.05 M ammonium dihydrogen phosphate-acetonitrile-methanol (20 + 30 + 6, v/v) as the mobile phase with ultraviolet detection at 220 nm over concentration ranges of 1-90 and 5-70 microg/mL; mean accuracies were 99.92+/-1.02 and 99.61+/- 0.98%, respectively. The suggested procedures were checked using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical preparations. The methods retained their accuracy and precision when the standard addition technique was applied. The results obtained by applying the proposed methods were statistically analyzed and compared with those obtained by the manufacturer's method.     

Simultaneous high-performance liquid chromatographic determination of phenazopyridine and nitrofurantoin in tablets

Summary A reversed-phase, High-performance Liquid chromatographic method for the simultaneous determination of phenazopyridine and nitrofurantoin in tablets is described. An aminopropyl-silica (APS-Hypensil) 5μm column and a mobile phase consisting of Methanol:H₂O: 0.05M sodium dihydrogen phosphate (50∶45∶5) were used. Calibration curves were linear over the concentration range 2–20μg/ml, with minimum detectability of 10ng/ml for both drugs. The method was applied to tablets containing the two species and the results obtained were compared to those given with a published method.     

Determination of eprosartan mesylate and hydrochlorothiazide in tablets by derivative spectrophotometric and high-performance liquid chromatographic methods

Two new simple and selective assay methods have been presented for the analysis of eprosartan mesylate (EPR) and hydrochlorothiazide (HCT) in pharmaceutical formulations. The first method is based on first-derivative ultraviolet spectrophotometry with zero-crossing measurements at 246 and 279 nm for EPR and HCT, respectively. The assay was linear over the concentration ranges 3.0-14.0 μg/mL for EPR and 1.0-12.0 μg/mL for HCT. The quantification limits for EPR and HCT were found to be 1.148 and 0.581 μg/mL, respectively, while the detection limits were 0.344 μg/mL for EPR and 0.175 μg/mL for HCT. The second method involved isocratic reversed-phase liquid chromatography using a mobile phase composed of acetonitrile-10 mM phosphoric acid (pH 2.5) (40:60, v/v). Olmesartan was used as internal standard and the substances were detected at 272 nm. The linearity ranges were found to be 0.5-30 and 0.3-15.0 μg/mL for EPR and HCT, respectively. The limits of detection were found to be 0.121 μg/mL for EPR and 0.045 μg/mL for HCT. The limits of quantification were found to be 0.405 and 0.148 μg/mL for EPR and HCT, respectively. The proposed methods were successfully applied to the determination of commercially available tablets with a high percentage of recovery and good accuracy and precision.     

Simultaneous determination of cefotaxime and desacetylcefotaxime in real urine sample using voltammetric and high-performance liquid chromatographic methods

Two rapid, accurate and sensitive methods are developed and validated for the quantitative simultaneous determination of cefotaxime (CFX) and its active metabolite desacetylcefotaxime (DCFX) in urine.Based on the previous results which showed the four electron reduction of CFX at ≈ −0.5 V, and the new findings that DCFX reduction occurred at more positive potential (−0.23 V), the new adsorptive stripping differential pulse voltammetric (AdSDPV) method was developed for determination of CFX in the presence of DCFX. Linear responses were observed over a wide concentration range (0.07-0.52 μg/ml for CFX and 0.22-1.3 μg/ml for DCFX) in urine.The second assay involves subsequent separation on a reversed-phase HPLC column, with ultraviolet detection at 262 nm. Retention times were 4.057 and 1.960 min for CFX and DCFX, respectively. Linear responses were observed over a wide range, 0.55-6.60 μg/ml for CFX and 1.10-11.00 μg/ml for DCFX, in urine.The statistical evaluation for both methods was examined by means of within-day repeatability (n = 5) and day-to-day precision (n = 3) and was found to be satisfactory with high accuracy and precision.     

Simultaneous high-performance liquid chromatographic determination of pioglitazone and metformin in pharmaceutical-dosage form

A simple, rapid, and precise method is developed for the quantitative simultaneous determination of metformin and pioglitazone in a combined pharmaceutical-dosage form. Separation is achieved with a Zorbax XDB C(18), 15-cm analytical column using buffer-acetonitrile (66:34, v/v) of pH 7.1, adjusted with orthophosphoric acid as the mobile phase. The buffer used in the mobile phase contains 10mM disodium hydrogen phosphate and 5mM sodium dodecyl sulphate in double-distilled water. The instrumental settings are flow rate of 1 mL/min, column temperature at 40 degrees C, and detector wavelength of 226 nm. The internal standard method is used for the quantitation of the ingredients of this combination. Methylparaben is used as an internal standard. The method is validated and shown to be linear for metformin and pioglitazone. The correlation coefficients for metformin and pioglitazone are 0.9991 and 0.9999, respectively. The relative standard deviations for six replicate measurements in two sets of each drug in the tablets are always less than 2%.     

Simultaneous spectrophotometric determination of atrazine and cyanazine by chemometric methods

Three novel ligands containing pyridine-2,6-dicarboxylic acid unit, trans-4 -(4'-methoxystyryl) pyridine-2,6-dicarboxylic acid, trans-4-(4'-(dimethylamino)styryl)pyridine-2,6-dicarboxylic acid, and trans-4-(4'-(diphenylamino)styryl)pyridine-2,6-dicarboxylic acid were synthesized and their complexes with Eu(III), Tb(III) ions were successfully prepared. The ligands and the corresponding metal complexes were characterized by means of MS, elemental analysis, IR, (1)H NMR and TG-DTA. The luminescence spectra of Eu(III) and Tb(III) complexes in solid state were studied. The strong luminescence emitting peaks at 615 nm for Eu(III) and 545 nm for Tb(III) can be observed. The applications in cell imaging of the europium and terbium complexes were investigated.     

Simultaneous high-performance liquid chromatographic determination of antazoline phosphate and tetrahydrozoline hydrochloride in ophthalmic solution

A method for the determination of 2-[(N-phenyl)benzylaminomethyl]-2-imidazoline X H3PO4 (antazoline phosphate) and 2-(1,2,3,4-tetrahydro-1-naphthyl)-2-imidazoline X HCl (tetrahydrozoline hydrochloride) in ophthalmic solution is described. The pharmaceutical preparation is analysed directly by reversed-phase ion-pair high-performance liquid chromatography and the method is very rapid, selective and simple.     

Development and validation of column high-performance liquid chromatographic and ultraviolet spectrophotometric methods for citalopram in tablets

Column high-performance liquid chromatographic (LC) and UV spectrophotometric methods for the quantitative determination of citalopram, a potent and selective serotonin reuptake inhibitor, in tablets were developed. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by the reversed-phase technique on an ACE C18 column with a mobile phase composed of 0.30% triethylamine solution-acetonitrile (55 + 45, v/v) adjusted to pH 6.6 with 10% ortho-phosphoric acid at a flow rate of 1.0 mL/min and 25 degrees C. The UV spectrophotometric method was performed at 239 nm. The linearity of the LC method was in the range of 10.00-70.00 microg/mL, and 2.50-17.50 microg/mL for the UV spectrophotometric method. The interday and intraday assay precision was < 1.5% (relative standard deviation) for the LC and UV spectrophotometric methods. The recoveries were in the range 100.70-101.35% for the LC method and 98.48-98.65% for the UV spectrophotometric method. Statistical analysis by Student's t-test showed no significant difference between the results obtained by the 2 methods. The proposed methods are highly sensitive, precise, and accurate and can be used for the reliable quantitation of citalopram in tablets.     

Stereospecific high-performance liquid chromatographic determination of tocainide

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.     

Normal-phase high-performance liquid chromatographic determination of phenols

Normal-phase high-performance liquid chromatography has been used for separation of phenol and its monoderivatives. Multi-component mixtures of hexane (non-polar component) with butan-1-ol, chloroform, butyl bromide, butyl chloride or diethyl ether (polar additives) were used as selective eluents. Silica gel "Silasorb 600" with specific surface area of about 600 m(2)/g and average particle size of $ 10 mum was used as the sorbent. Phenol and the o-, m-, p-isomers of cresol were concentrated by extraction with n-butyl acetate from aqueous solutions. A method for determination of microamounts of phenols in aqueous solutions in the presence of 160-fold amounts of aromatic hydrocarbons has been developed.     

Purity determination by a combination of spectrophotometric and liquid chromatographic methods: application to 13-cis-retinoic acid

A method is described to determine the purity of 13-cis-retinoic acid. Spectrophotometry is used to determine the non-UV-absorbing impurities, and HPLC is used to determine the UV-absorbing impurities. In combination, these methods permit determination of the purity. An authentic standard is used in developing this method, but it is not needed in the application of the method.     

Simultaneous spectrophotometric determination of diuretics by using multivariate calibration methods

The wavelength range and number of factors used in partial least-squares (PLS) calibration for the resolution of the dihydralazine (DHZ)-hydrochlorothiazide (HCT) binary mixture and the dihydralazine-hydrochlorothiazide-reserpine ternary mixture were optimized in terms of the relative standard error (R.S.E.) and relative mean standard error (R.M.S.E.). Under the optimum conditions thus established, synthetic mixtures of the analytes can be resolved with errors and relative standard deviations (R.S.D) less than 4.5 and 1.0%, respectively. The ensuing method, which was validated by comparison with high performance liquid chromatography (HPLC), also gives good results with real samples (pharmaceutical preparations).     

Rapid ultraviolet spectrophotometric and liquid chromatographic methods for the determination of natamycin in lactoserum matrix

Two rapid and simple methods were developed for the determination of natamycin in lactoserum matrix by ultraviolet (UV) spectrophotometry and liquid chromatography (LC) with diode-array detection. The methods involve protein precipitation with methanol, followed by centrifugation. No cleanup is necessary. The applicable concentrations of natamycin in lactoserum range from 2 to 500 mg/L for samples analyzed by both methods. The detection and quantitation limits are 0.07 and 0.23 mg/L, respectively, for the UV spectrophotometric method and 0.1 and 0.32 mg/L, respectively, for the LC method. The methods were applied satisfactorily to the determination of natamycin in various commercial lactosera. Both methods were validated independently by standard additions and Youden methodologies, which verified their accuracy. Once the 2 proposed methods were validated independently, the validation of one method was carried out with the other.     

Simultaneous high-performance liquid chromatographic determination of catecholamine-related compounds by post-column derivatization involving coulometric oxidation followed by fluorescence reaction

A highly selective and sensitive high-performance liquid chromatographic method for the determination of catecholamines (norepinephrine, epinephrine and dopamine) and related compounds (L-DOPA, normetanephrine, metanephrine, 3-methoxytyramine, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid, 3,4-dihydroxyphenylethylene glycol, 4-hydroxy-3-methoxyphenylethylene glycol and 4-hydroxy-3-methoxyphenylethanol) with a post-column technique involving coulometric oxidation followed by fluorescence derivatization is described. These compounds, 3,4-dihydroxybenzylamine and ferulic acid are separated within 35 min by ion-pair reversed-phase chromatography using acidic buffers (pH 3.1) with methanol-acetonitrile (3:2, v/v) gradient elution, and then oxidized by a commercial coulometric detector to the corresponding o-quinones, which are converted into fluorescent derivatives by reaction with 1,2-diphenylethylenediamine. The detection limits (signal-to-noise ratio = 3) on-column are 1.5-4 pmol for the two mandelic acids, 600 fmol for L-DOPA and 20-70 fmol for the others.     

Isocratic high-performance liquid chromatographic determination of plasma adenosine

Summary Due to manifold physiological and cardioprotective actions of adenosine, the demand for a simple but accurate method to determine its concentration in plasma is increasing. The aim of this study was firstly to develop a simple isocratic method instead of the gradient elution or peak-shifting techniques used earlier and secondly to check conflicting data on the composition of stop-solution, added to the sample in order to prevent changes in adenosine concentration. Isocratic elution improved signal to noise ratio and concentrations of 100 mol L^–1 dipyridamole and 2.5 mol L^–1 erythro-9(2-hydroxy-3-nonyl)adenine in the blood sample effectively prevented both adenosine formation and degradation, even without the use of a 5-ecto-nucleotidase inhibitor. Lowering the concentration of dipyridamole to 25 mol L^–1 caused more than a tenfold increase of adenosine concentration in two out of five cases and even 100 mol L^–1 dipyridamole alone is not sufficient to inhibit adenosine deaminase in blood samples.