Validation of a HPLC method for quantification of midazolam in rat plasma: Application during a Maytenus ilicifolia-drug interaction study.

Midazolam (MDZ) is routinely employed as a marker compound of cytochrome P450 3A (CYP3A) activity. Despite the many HPLC-UV methods described to quantify MDZ in plasma, all of them use acetonitrile (ACN) or a mixture of methanol-isopropanol as organic solvent of the mobile phase. Since the ACN shortage in 2008, efforts have been made to replace this solvent during HPLC analysis. A simple, sensitive, accurate and repeatable HPLC-UV method (220 nm) was developed and validated to quantify MDZ in rat plasma using methanol instead. The method was applied during a herb-drug interaction study involving Maytenus ilicifolia, a Brazilian folk medicine used to treat gastric disorders. Plasma samples were alkalinized and MDZ plus alprazolam (internal standard) were extracted with diethyl ether. After solvent removal, the residue was reconstituted with methanol-water (1:1). The analyte was eluted throughout a C18 column using sodium acetate buffer (10 mm, pH 7.4)-methanol (40:60, v/v). The precision at the lower limit of quantification never exceeded 19.40%, and 13.86% at the higher levels of quality control standards, whereas the accuracy ranged from -19.81 to 14.33%. The analytical curve was linear from 50 to 2,000 ng/ml. The activity of the hepatic CYP3A enzymes was not affected by the extract.

Development of spectrophotometric method for iron determination in fortified wheat and maize flours.

The determination of iron in fortified foods is mandatory by many global regulatory agencies. However, the spectroscopic techniques require elevated investments limiting their applicability especially in developing countries. Therefore, simple, viable and analytical methods with sufficient sensitivity can become an alternative. In this work, a sensitive, simple and viable spectrophotometry method to determine iron in wheat and maize flours was developed following a cloud point extraction (CPE) procedure. The analyte was first complexed with 2-(5-Bromine-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) in the presence of the surfactant octylphenoxypolyethoxyethanol (Triton X-114). For the CPE optimization the variables: pH of the medium, stoichiometry of the complex, surfactant, and salt concentrations were evaluated. Linearity in the analytical blank was obtained by using the square root of absorbance (Abs) in order to adjust the residues of the curve. The precision was lower than 5% and the accuracy ranged from 97 to 101%. The limits of detection and quantification were 0.004µgmL-1 and 0.01µgmL-1, respectively. The method was applied to investigate the content of iron in 14 brands of fortified flours. The concentrations of iron varied from 0.435 to 3.62mg/100g and 0.570 to 3.15mg/100g in wheat and maize flour, respectively. The content of iron in all brands investigated in this study was approximately 10-fold lower than the value required by (ANVISA). The amount of iron in fortified foods was satisfactorily determined by using a simple, sensitive, and low cost spectrophotometric method.

Determination of fexofenadine in Hank's balanced salt solution by high-performance liquid chromatography with ultraviolet detection: application to Caco-2 cell permeability studies.

The drug-transporting proteins can affect the pharmacokinetics and pharmacodymanics of many drugs, resulting in an erratic and unpredictable pharmacological response. The Caco-2 monolayer is routinely applied to investigate the carrier-mediated transport of drugs. Therefore, the selection of a marker compound able to characterize the activity of such transporters is crucial. Fexofenadine (FEX), a P-gp/OATP substrate, can be considered a suitable probe. However, in order to use be used as a marker compound, it is mandatory to develop an analytical method able to quantify this drug during the in vitro permeability assay. An HPLC method with ultraviolet detection was developed; the mobile phase consisted of phosphate buffer (pH 3.2) containing 10 m m of sodium octanosulphonate and acetonitrile (60:40) and the flow rate was set at 1.2 mL/min. Fexofenadine was eluted at 40°C, the retention time was about 4.6 min. The LOD and LOQ values were 1.9 and 6.2 ng/mL, respectively. Verapamil and ketoconazole, the most common P-gp inhibitors, were eluted as distinct peaks of that corresponding to fexofenadine The method was successfully applied to quantify the amount of FEX transported across the Caco-2 monolayer and could be an additional tool for those investigating the role of membrane transporters on drug absorption.