Validated stability-indicating methods for the simultaneous determination of amiloride hydrochloride, atenolol, and chlorthalidone using HPTLC and HPLC with photodiode array detector.

Two stability-indicating chromatographic methods are described for simultaneous determination of amiloride hydrochloride (AMI), atenolol (ATE), and chlorthalidone (CHL) in combined dosage forms. The first method was based on HPTLC separation of the three drugs followed by densitometric measurements of their bands at 274 nm. The separation was carried out on Merck HPTLC silica gel 60F254 aluminum sheets using chloroform-methanol-ammonia 27%, w/w (9 + 2 + 0.3, v/v/v) mobile phase. Analysis data was used for the linear regression graph in the range of 0.1-0.5, 0.8-5.0, and 0.3-1.5 microg/band for AMI, ATE, and CHL, respectively. The second method was based on an RP-HPLC separation of the cited drugs performed on an RP stainless steel C18 analytical column (250 x 4.6 mm id) with a gradient elution system of methanol and 0.05 M aqueous phosphate buffer adjusted to pH 4 as the mobile phase, at the flow rate of 1.0 mL/min. Quantitation was achieved with photodiode array detection at 275 nm for AMI and 225 nm for ATE and CHL. The calibration graphs for each drug were rectilinear in the range of 2-50, 25-150, and 2-100 microg/mL for AMI, ATE, and CHL, respectively. The proposed chromatographic methods were successfully applied for determination of the investigated drugs in pharmaceutical preparations. Both methods were validated in compliance with International Conference on Harmonization guidelines in terms of linearity, accuracy, precision, robustness, LOD, and LOQ.

Assay of new combination of tamsulosin and tadalafil using synchronous spectrofluorimetric method coupled with mathematical tools.

A fixed-dose combination therapy of tamsulosin andtadalafilis now available for treatmentof lower urinary tract symptoms associated with benign prostatic hyperplasia. The decrease in sexual performance which is a side-effect of tamsulosin can be compensated by using tadalafil. This study is the first to develop and optimize a synchronous spectrofluorimetric method coupled with derivative and derivative ratio mathematical tools for the determination of tamsulosin andtadalafil in their newly released dosage form. The method successed in quantification of tadalafil by measuring the second-order derivative synchronous spectrofluorimetric amplitude at 278 nm (corresponding to zero-crossing of tamsulosin) using Δƛ=11 nm. On the other hand, first derivative ratio synchronous spectrofluorimetric peak amplitude was measured at 260 nm for determination of tamsulosin, using Δƛ=15 nm and divisor concentration of 0.1 µg mL-1. The method validation was performed using ICH guidelines. The linear responses of tamsulosin andtadalafil were from 0.04 to 0.5 and 0.05-0.5 µg mL-1, respectively. High sensitivity was achieved as represented by slope values of 0.32 and 608.4 for tamsulosin andtadalafil, respectively. The method is sensitive enough to detect concentration as low as 0.024 for both drugs. The proposed integrated spectrofluorimetric method showed good simplicity, selectivity and greenness. It can be successfully applied for analysis of both drugs in dosage form.

Differential pulse cathodic voltammetric determination of floctafenine and metopimazine.

A simple, rapid and sensitive voltammetric method for the determination of floctafenine (FFN) and metopimazine (MPZ) was developed. Well-defined cathodic waves were obtained for both drugs in Britton-Robinson buffer pH 9.0 using the differential-pulse mode at the hanging mercury drop electrode (HMDE). The current-concentration relationship was found to be linear over the ranges 0.4-3.6 and 0.4-2.4 microg ml(-1) for FFN and MPZ, respectively. The quantification of the two drugs in their pharmaceutical formulations was carried out using the proposed voltammetric method and compared with spectrophotometric analysis data. The mechanisms of the electrode reactions for the two drugs were proposed.

High-performance liquid chromatographic determination of proquazone and its m-hydroxy metabolite in spiked human plasma and urine.

A simple and rapid high-performance liquid chromatographic method for the determination of proquazone (PQZ) and its major metabolite, m-hydroxyproquazone, in spiked human plasma and urine was developed. Plasma samples were purified using acetonitrile as a protein precipitant, while urine samples were diluted only with the mobile phase and filtered prior to injection. Samples containing the parent compounds and glafenine (internal standard) were eluted from a reversed-phase C8 column using acetonitrile-0.025 M sodium acetate (60 + 40) adjusted to pH 5 as the mobile phase and detected at 234 nm. Peak area ratios of the analytes versus internal standard were used for calibration. The mean recoveries from plasma and urine samples spiked with PQZ and its m-hydroxy metabolite ranged from 97.87 to 103.88%. The relative standard deviation for the within- and between-day analyses were < 4%. The proposed method was applied for the assay of PQZ in laboratory-made tablets.

Stability indicating HPLC-DAD method for analysis of Ketorolac binary and ternary mixtures in eye drops: Quantitative analysis in rabbit aqueous humor.

Ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) binary mixture (mixture 1) and their ternary mixture with chlorpheniramine maleate (CPM) (mixture 2) were analyzed using a validated HPLC-DAD method. The developed method was suitable for the in vitro as well as quantitative analysis of the targeted mixtures in rabbit aqueous humor. The analysis in dosage form (eye drops) was a stability indicating one at which drugs were separated from possible degradation products arising from different stress conditions (in vitro analysis). For analysis in aqueous humor, Guaifenesin (GUF) was used as internal standard and the method was validated according to FDA regulation for analysis in biological fluids. Agilent 5 HC-C18(2) 150×4.6mm was used as stationary phase with a gradient eluting solvent of 20mM phosphate buffer pH 4.6 containing 0.2% triethylamine and acetonitrile. The drugs were resolved with retention times of 2.41, 5.26, 7.92 and 9.64min for PHE, GUF, KTC and CPM, respectively. The method was sensitive and selective to analyze simultaneously the three drugs in presence of possible forced degradation products and dosage form excipients (in vitro analysis) and also with the internal standard, in presence of aqueous humor interferences (analysis in biological fluid), at a single wavelength (261nm). No extraction procedure was required for analysis in aqueous humor. The simplicity of the method emphasizes its capability to analyze the drugs in vivo (in rabbit aqueous humor) and in vitro (in pharmaceutical formulations).

Development and Validation of a High-Performance Thin-Layer Chromatographic Method for the Simultaneous Determination of Two Binary Mixtures Containing Ketorolac Tromethamine with Phenylephrine Hydrochloride and with Febuxostat.

A validated and highly selective high-performance thin-layer chromatography (HPTLC) method was developed for the determination of ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) (Mixture 1) and with febuxostat (FBX) (Mixture 2) in bulk drug and in combined dosage forms. The proposed method was based on HPTLC separation of the drugs followed by densitometric measurements of their spots at 273 and 320 nm for Mixtures 1 and 2, respectively. The separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using chloroform-methanol-ammonia (7:3:0.1, v/v) and (7.5:2.5:0.1, v/v) as mobile phase for KTC/PHE and KTC/FBX mixtures, respectively. Linear regression lines were obtained over the concentration ranges 0.20-0.60 and 0.60-1.95 µg band(-1)for KTC and PHE (Mixture 1), respectively, and 0.10-1.00 and 0.25-2.50 µg band(-1) for KTC and FBX (Mixture 2), respectively, with correlation coefficients higher than 0.999. The method was successfully applied to the analysis of the two drugs in their synthetic mixtures and in their dosage forms. The mean percentage recoveries were in the range of 98-102%, and the RSD did not exceed 2%. The method was validated according to ICH guidelines and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability.

Spectrophotometric and fluorimetric methods for the determination of meloxicam in dosage forms.

Four simple and accurate methods are presented for the determination of meloxicam in dosage forms. These methods are based on: the direct measurements of the differential spectra at 339.9-384.7 nm (A), the 1D-values at 322-368 nm and 2D-values at 343.2-385.6 nm (B), the formation of an ion-association complex between the drug and safranin T with subsequent absorption measurement at 518 nm (C) and fluorescence measurement at 582 nm (D). All variables were studied to optimize the formation of the ion-association complex. Beer's law was valid over the concentration range 2-10 microg ml(-1) (method A), 1-10 microg ml(-1) (method B), 4.0-12 microg ml(-1) (method C) and 0.4-1.2 microg ml(-1) (method D). The detection limits were 0.11, 0.07, 0.10, 0.33 and 8.74 x 10(-3) microg ml(-1) for methods A, B, C and D, respectively. The proposed methods were successfully applied to the assay of meloxicam in tablets and suppositories. The procedures were rapid, simple and suitable for quality control applications.

Bioavailability study of triamterene and xipamide using urinary pharmacokinetic data following single oral dose of each drug or their combination.

An efficient chromatographic method for the simultaneous determination of triamterene (TRI) and xipamide (XIP) in urine samples, based on high performance liquid chromatography with photodiode array detector (HPLC-DAD) has been developed. The HPLC separation was performed on a RP stainless-steel C-18 analytical column (250 mm × 4.6 mm, 5 µm) with a gradient elution system of 0.05 M phosphate buffer adjusted to pH 4.0 and methanol as the mobile phase. The method was used to determine the urinary excretion profile and to calculate different urinary pharmacokinetic parameters following oral dose of their combination compared with single oral doses of each drug and hence comparing their bioavailability. Quantitation was performed using chlorthalidone as internal standard. The calibration graphs of each drug were rectilinear in the range of 0.2-40 µg/mL urine for TRI and 0.2-15 µg/mL urine for XIP. An HPLC-DAD method was also successfully developed for the simultaneous determination of the investigated drugs in pharmaceutical preparations. The methods were validated in terms of linearity, accuracy, precision, selectivity, limits of detection and quantitation and other aspects of analytical validation.

Enhanced spectrophotometric determination of two antihyperlipidemic mixtures containing ezetimibe in pharmaceutical preparations.

Two spectrophotometric methods are presented for the simultaneous determination of ezetimibe/simvastatin and ezetimibe/atorvastatin binary mixtures in combined pharmaceutical dosage forms without prior separation. The first is the derivative ratio method where the amplitudes of the first derivative of the ratio spectra ((1) DD) at 299.5 and 242.5 nm were found to be linear with ezetimibe and simvastatin concentrations in the ranges 0.5-20 µgml(-1) and 1-40 µgml(-1) , respectively, whereas the amplitudes of the first derivative of the ratio spectra ((1) DD) at 289.5 and 288 nm were selected to determine ezetimibe and atorvastatin in the concentration ranges 5-50 µgml(-1) and 1-40 µgml(-1) , respectively. The second is the H-point standard additions method; absorbances at the two pairs of wavelengths, 228 and 242 nm or 238 and 248 nm, were monitored while adding standard solutions of ezetimibe or simvastatin, respectively. For the analysis of ezetimibe/atorvastatin mixture, absorbance values at 226 and 248 nm or 212 and 272 nm were monitored while adding standard solutions of ezetimibe or atorvastatin, respectively. Moreover, differential spectrophotometry was applied for the determination of ezetimibe in the two mixtures without any interference from the co-existing drug. This was performed by measurement of the difference absorptivities (ΔA) of ezetimibe in 0.07 M 30% methanolic NaOH relative to that of an equimolar solution in 0.07 M 30% methanolic HCl at 246 nm. The described methods are simple, rapid, precise and accurate for the determination of these combinations in synthetic mixtures and dosage forms.

Development of a differential pulse voltammetric method for the determination of Silymarin/Vitamin E acetate mixture in pharmaceuticals.

Differential pulse voltammetric method was developed for determination of Silymarin (SMR)/Vitamin E acetate (VEA) mixture in pharmaceuticals. SMR and VE gave well-resolved diffusion-controlled anodic peaks at +756 and +444mV, respectively (versus Ag/AgCl) in Britton-Robinson buffer at pH 2.8. The solution conditions and instrumental parameters were optimized for their quantitative determination. The linear response was obtained in the range 0.1-4.0mgL(-1) with a detection limit of 0.03mgL(-1) for SMR and 0.05-4.0mgL(-1) with a detection limit of 0.01mgL(-1)for VEA.