Application of HPTLC, spectrofluorimetry and differential pulse voltammetry for determination of the antifungal drug posaconazole in suspension dosage form.

This work presents the development, validation and application of three simple and direct analytical methods for determination of posaconazole (PSZ) in its pure form and in suspension dosage form. Method I is based on high performance thin layer chromatography (HPTLC) where effective separation of PSZ and the internal standard (itraconazole) was achieved using Merck HPTLC plates (20×10cm aluminium plates with 250µm layer thickness precoated with silicagel 60 F254) and a mobile phase composed of acetone and chloroform (1:2, by volume), followed by densitometric measurement of the drugs' spots at 262nm. Method II involves measurement of the native fluorescence of PSZ in 0.1M H2SO4 at excitation and emission wavelengths of 260 and 365nm, respectively. Method III depends on the voltammetric analysis of PSZ. A well-defined cathodic wave was obtained for PSZ in Britton-Robinson buffer pH 6.5 using the differential-pulse mode at the hanging mercury drop electrode (HMDE). The developed methods were validated according to the International Conference on Harmonization (ICH) guidelines regarding linearity, ranges, accuracy, precision, robustness and limits of detection and quantification. The proposed methods showed good linearity over the concentration ranges 5-50, 0.05-0.3, 0.005-0.05µg/mL PSZ for methods I, II, and III respectively. Intra and inter-day precision were verified by the RSD% values which were less than 2%. The proposed methods were successfully applied for the quantification of PSZ in suspension dosage form with no observable interferences. Assay methods were favorably compared with those obtained by previously reported HPLC method.

Sensitive inexpensive spectrophotometric and spectrofluorimetric analysis of ezogabine, levetiracetam and topiramate in tablet formulations using Hantzsch condensation reaction.

Two highly sensitive, simple and selective spectrophotometric and spectrofluorimetric assays have been investigated for the analysis of ezogabine, levetiracetam and topiramate in their pure and in pharmaceutical dosage forms. The suggested methods depend on the condensation of the primary amino-groups in the three drugs with acetylacetone and formaldehyde according to Hantzsch reaction yielding highly fluorescent yellow colored dihydropyridine derivatives. The reaction products of ezogabine, levetiracetam and topiramate were measured spectrophotometrically at 418, 390 and 380nm or spectrofluorimetrically at λem/ex of 495/425nm, 490/415nm and 488/410nm, respectively. Various experimental conditions have been carefully studied to maximize the reaction yield. At the optimum reaction conditions, the calibration curves were rectilinear over the concentration ranges of 8-25, 60-180 and 80-200µg/mL spectrophotometrically and 0.02-0.2, 0.2-1.2 and 0.2-1.5µg/mL spectrofluorimetrically for ezogabine, levetiracetam and topiramate, respectively with good correlation coefficients. The suggested methods were applied successfully for the analysis of ezogabine, levetiracetam and topiramate in their commercial tablets with high percentage recoveries and negligible interference from various excipients in pharmaceutical dosage forms. The results were statistically analyzed and showed the absence of any significant difference between both developed and published methods. The procedures were validated and evaluated by the ICH guidelines revealing good reproducibility and accuracy. Therefore, the two proposed methods may be considered of high interest for practical and reliable analysis of ezogabine, levetiracetam and topiramate in pharmaceutical dosage forms.

Spectrofluorimetric Determination of Topiramate and Levetiracetam as Single Components in Tablet Formulations and in Human Plasma and Simultaneous Fourth Derivative Synchronous Fluorescence Determination of their Co-Adminstered Mixture in Human Plasma.

Two highly sensitive, rapid, simple, economic and validated spectrofluorimetric methods have been developed for determination of Topiramate and Levetiracetam in pharmaceutical tablets and in human plasma. Topiramate and Levetiracetam were determined separately by derivatization using 4-Chloro-7-nitrobenzofuran-2-oxo-1,3-diazole (NBD-Cl) and measured spectrofluorimetrically. The Relative fluorescence intensities were measured at λem/ex of 547/465 nm and 551/465 nm for Topiramate and Levetiracetam, respectively. While a binary mixture of Topiramate and Levetiracetam were determined by the fourth derivative synchronous fluorescence measurement after their reaction with NBD-Cl. In this method, the fourth derivative synchronous spectra were estimated as peak to peak measurement at 493-497 and 490.5-495 nm corresponding with zero-contribution of Levetiracetam and Topiramate, respectively. Linearity ranges for Topiramate and Levetiracetam in both methods were found to be 0.15-1.2 and 0.2-1.5 µg/mL, respectively. The different experimental parameters affecting the fluorescence of the two drugs were carefully studied and optimized. The proposed methods were validated in terms of linearity, accuracy, precision, limits of detection and quantification and other aspects of analytical validation. The proposed methods were successfully applied for the determination of the investigated drugs in human plasma samples obtained from healthy volunteers after single oral administration of the two drugs.

Caveolin-1 inhibits matrix metalloproteinase-2 activity in the heart.

Apart from its ability to degrade extracellular matrix proteins, matrix metalloproteinase-2 (MMP-2) was recently revealed to have targets and actions within the cardiac myocyte. The localization of MMP-2 in caveolae of endothelial cells suggests that caveolin-1 (Cav-1) may play a role in regulating MMP-2. The caveolin scaffolding domain (CSD) of Cav-1 regulates several proteins including those involved with signaling cascades. Whether Cav-1 is responsible for regulating MMP-2 in the heart is unknown. Hearts from Cav-1(-/-) or Cav-1(+/+) mice were isolated and heart extracts or lipid raft enriched membrane fractions were prepared. MMP-2 activity in Cav-1(-/-) hearts was markedly enhanced when compared with Cav-1(+/+) hearts with no changes in MMP-2 protein levels between groups. In contrast, MMP-2 activity and protein level were greatly reduced in lipid raft enriched fractions of Cav-1(-/-) hearts. Purified CSD inhibited MMP-2 activity in a concentration-dependent manner as assessed using an in vitro degradation assay with a fluorogenic MMP-2 substrate (OmniMMP). These data suggest that Cav-1 plays a role in regulating MMP-2 activity. Cav-1 may thus be a novel mechanism to regulate MMP-2 activity in the heart.