Development and validation of an HPLC method to be used for simultaneous detection and quantification of different statins after ex vivo skin diffusion studies.

A novel high performance liquid chromatography (HPLC) method was developed and validated to simultaneously analyse all statins currently available globally (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin). A Venusil XBP C18(2) reverse phase column (150 x 4.6 mm) with a 5 µm particle size was used. The gradient conditions started at 25% acetonitrile, which linearly increased to 90% after 1.0 min, held at 90% until 6.5 min, and lastly, re-equilibrated to starting conditions. The mobile phase consisted of acetonitrile/water and 0.005 M (0.2%) octane sulphonic acid-Na (pH 3.5). The flow rate was set at 1.0 ml/min with a 10 µl injection volume. The HPLC method indicated linearity (R² =0.9999) within the concentration range of 0.2-206.4 µg/ml. The limit of detection (LOD) and limit of quantification (LOQ) values were found to be within the permissible criteria of ≤15% and ≤20%, respectively. Following an appropriate investigation of all the parameters for method validation, it was confirmed that the HPLC method was successfully validated and proven to be accurate to simultaneously quantify statins even in combination with other excipients used during the formulation of nano-emulsions and nano-emulgels.

Development and validation of a HPLC electrochemical detection method to measure COMT activity as a tool in drug development.

The determination of catechol-O-methyltransferase (COMT) activity is considered valuable for various pharmaceutical and biomedical research projects. A specific high performance liquid chromatography-coulometric electrochemical detection method, for the assay of COMT activity was developed by measuring the formation of normetanephrine from norepinephrine. The chromatographic separation was achieved on a C18 reversed phase column with a mobile phase consisting of 10 mM sodium dihydrogen phosphate buffer, 4 mM sodium 1-octanesulfonate, 0.17 mM ethylenediaminetetra-acetic acid disodium salt, 6 % methanol and 4 % acetonitrile (pH ± 4.0). The detection of normetanephrine was achieved through electrochemical detection, with a coulometric cell potential setting of +450 mV. The flow rate was at 1 ml/min and the total run time was 45 min. The method was validated according to validation guidelines (Shabir 2006; European Medicines Agency 2011; US FDA 2018). The method was found to be linear (R² > 0.99) over the analytical range (100 to 2500 ng/ml) for all the analytes. All the other validation parameters (sensitivity, precision, accuracy, recovery and stability) were acceptable and within range. The method was applied for the determination of COMT activity in rat liver homogenate test samples. The known selective COMT inhibitor entacapone was used as test inhibitor. The results confirmed the ability of entacapone to inhibit COMT activity by decreasing the production of all the metabolites of norepinephrine.

An HPLC-DAD validated method for the detection and quantification of cortisol, corticosterone and melatonin in plasma samples of two different animal species.

The monitoring of endogenous hormone plasma levels could be valuable in biomedical, veterinary and pharmaceutical research. A specific high performance liquid chromatography method with diode array detection, for the assay of cortisol, corticosterone and melatonin in animal plasma was developed and validated. The chromatographic separation was achieved on a C8 reversed phase column with a mobile phase consisting of HPLC-grade water and 35% v/v acetonitrile (pH ± 3.36). The detection was achieved through diode array detection, with two set wavelengths; 245 and 275 nm. The flow rate was at 1 ml/min and the total run time was 50 min. The method was validated according to validation guidelines (Shabir, 2006; US FDA, 2013). The method was found to be linear (R² > 0.99) over the analytical range (10 to 500 ng/ml) for all three analytes. All the other validation parameters were acceptable and within range. The method was applied to plasma samples from Sprague-Dawley rats and white rhinoceros.

Development and validation of the simultaneous determination of artemisone, clofazimine and decoquinate with HPLC.

The aim of this study was to develop and validate a novel HPLC method for the simultaneous analysis of artemisone, clofazimine and decoquinate. Detection was obtained at two wavelengths; 284 nm (clofazimine) and 210 nm (artemisone and decoquinate). Gradient elution was used with mobile phase A (A) consisting of 0.005 M sodium octanesulphonic-acid (pH 3.5) and mobile phase B (B) of HPLC grade acetonitrile. The flow rate was set to 1.0 ml/min with (A) at 35% and (B) at 65% for 2 min, followed by a gradient shift of 10/90% ((A)/(B)) over a duration of 4 min. After 10 min, the initial gradient conditions were readjusted to 35/65% ((A)/(B)). Distinctive peaks were identified for clofazimine, artemisone and decoquinate, respectively. The proposed HPLC assay method was validated and found to be reliable, reproducible and accurate for simultaneous analysis of the three compounds.