Off-line chromatographic measurements

The first off-line technique employed in this work made use of an Agilent HPLC system equipped with a G1310A isocratic pump, a G1314 variable wavelength detector and a Rheodyne injector (model 7725I). The HPLC measurements were performed on 20 µL samples using a 5 μm C₁₈ Zorbax TM analytical column (25 cm × 0.46 cm) and a mobile phase of 0.01 M sodium-1-heptane sulphonate : acetonitrile (70:30 v/v) at a flow rate of 1.0 mL min⁻¹ and 220 nm UV-detection. The sample and the mobile phase were filtered by 0.22 μm and 0.45 μm Millipore membrane filters, respectively. The mobile phase was then degassed in an ultrasonic bath for 15.0 min immediately before use. Aliquots of DB equivalent to 40.0–320.0 μg were accurately transferred into 10 mL volumetric flasks and the volume was completed with mobile phase. Calibration curves were then constructed for DB by plotting the relative peak areas of DB as a function of respective concentrations.

The second off-line technique selected in this work was densitometry. The device employed was equipped with a UV lamp, a Camag Linomat 5 autosampler with a 100 µL micro-syringe, a TLC scanner-Model 3 S/N 130319 and winCats software for densitometric evaluation (CAMAG, Muttenz, Switzerland). The measurements were obtained using 20 cm × 20 cm TLC plates pre-coated with a 0.25 mm thick layer of silica gel 60 F₂₅₄ (E. Merck, Darmstadt, Germany). The solutions examined were applied as separate spots 20 mm from the bottom of the plates each with a 2 mm band length and developed at 25 ± 2 °C in the absorbance mode at 270 nm running a developing system of acetone:butanone:acetic acid:H₂O:NH₃ (1:1:1:1:0.03 by volume). The drug solutions examined were applied as separate compact spots 15 mm from the bottom of the plates each with a 3 mm band length which were placed in a chromatographic tank saturated with the mobile phase for 30 min prior to development. The normal phase TLC-plates were developed over 8 cm in an ascending manner then left to dry in air and then were scanned at 220 nm. In order to calibrate this method, accurately measured aliquots equivalent to 2.0–12.0 μg DB were spotted on the TLC plates, using the Camag Linomat autosampler using the same TLC chromatographic conditions as stated above. Calibration graphs relating the optical density of each spot to the corresponding concentration of DB were constructed.