DOI: 10.36347/sasjm

Pages: 72-75

The developed HPLC method was validated in the identification and quantification of diazepam, oxazepam, clonazepam, flunitrazepam and nitrazepam (benzodiazepines). These analyses were performed using an HPLC system with a diode array detector (λ =254nm), a Lichrospher RP 18 column having a 5µm internal diameter, a 10 mM potassium phosphate buffer (pH 2.5), drop-wise addition of phosphoric acid as well as using methanol-acetonitrile (10:27, v/v) as mobile phase having a flow rate of 1ml min-1 and an injection volume of 20 µl. Confirmation of using the instrument was done with identification of caffeine prepared standards which showed good linearity hence validating the method and proving the instrument was suitable for the carrying out the project. In analyzing these benzodiazepines, the isocratic elution was used which gave good peaks but took longer retention times to elute especially the diazepam which eluted further away taking the longest retention time. The gradient elution was introduced where changing the mobile phase to 33 after 15 minutes and then to 60 gave best peak resolution amongst the four close benzodiazepines as well as making the diazepam take a shorter retention time to elute hence giving the best peak separation and identification of these benzodiazepines. The elements of validation were performed on two selected benzodiazepines (nitrazepam and diazepam) where their standard preparations gave correlation values of 0.99626 and 0.99978 respectively indicating a good linearity of all the points hence confirm that the method is valid with their LOD being 8ppm and 2 ppm as well as LOQ being 26ppm and 6 ppm respectively. It can be conclude that, this method proved to give good separation of peaks, shorter retention times for peak elution, accurate, precise results as well as good linearity and so, it is recommended for the identification and quantification of most drugs.