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.

HPLC electrochemical detection and quantification of monoamines and their metabolites in rat brain tissue samples.

The monitoring of monoamines and their metabolites in CNS samples can be very valuable in pharmaceutical and biomedical research. A specific high performance liquid chromatography, coupled to a coulometric electrochemical detection method, for the assay of monoamines (dopamine, norepinephrine, epinephrine and serotonin) and their metabolites in rat brain tissue samples was developed. The chromatographic separation was achieved on a C8 reversed phase column with a mobile phase consisting of 0.1 M sodium formate buffer, 5 mM sodium 1-heptanesulfonate, 0.17 mM ethylenediaminetetraacetic acid disodium salt and 5% v/v acetonitrile (pH ±4.0). The detection was achieved through electrochemical detection, with a coulometric cell potential setting of +650 mV. The flow-rate was at 1 ml/min and the total run time was 50 min. The method was validated according to validation guidelines. The method was found to be linear (R2 > 0.99) over the analytical range (5 to 200 ng/ml) for all monoamines and their metabolites. All the other validation parameters were acceptable and within range. The method was applied to three rat brain areas (pre-frontal cortex, hippocampus and striatum), where the monoamines (except for epinephrine) and their metabolites were easily detected.

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.

A novel RP-HPLC method for the detection and quantification of roxithromycin in topical delivery studies.

A novel HPLC method with UV detection for the identification and quantification of roxithromycin (ROX) during in vitro skin penetration studies has been developed and validated. The method proved to be simple and rapid with isocratic elution (flow rate: 1.0 mL/min) of ROX, using a C18 column and UV detection at 205 nm. The mobile phase consisted of 0.06 M potassium di-hydrogen orthophosphate buffer (pH adjusted to 7.4 with sodium hydroxide) and acetonitrile in a 50:50 (v/v) ratio. This method showed linearity across the concentration range of 5 - 1000 µg/mL with a correlation coefficient of 0.9999. An average recovery of 101.78% was obtained. Limit of detection (LOD) and lower limit of quantification (LLOQ) values proved that ROX can still be detected at a concentration level of 0.3 µg/mL and accurately quantified at a concentration of 0.5 µg/mL. The specificity testing during method validation proved that this method is suitable for the accurate detection and quantification of ROX even when combined with different compounds typically used during the formulation of topical delivery systems.

A novel reversed-phase LC method for quantitative detection of azithromycin in bulk drug and tablet formulations in various aqueous media.

A novel reversed-phase HPLC method with UV-detection for separation, identification and quantification of azithromycin (AZH) was developed. The method was validated for the purpose of quantifying AZH in stability, dissolution and solubility studies. The method was validated at two optimum wavelengths where linear regression at both 205 and 210 nm, resulted in correlation coefficients of r2 = 0.9999. This HPLC method proved to be superior to other published methods due to the specificity, resulting in less peak interference and tailing. The pH of the mobile phase, as well as the ratio of buffer to organic component included in the mobile phase, proved to be critical factors in the improved detection of AZH.