Development and validation of a rapid and efficient method for simultaneous determination of methylxanthines and their metabolites in urine using monolithic HPLC columns

A new rapid, sensitive and validated HPLC method has been developed for the determination of methylxanthines and their metabolites in asthmatic patients. The method was initiated by using spiked urine samples on a silica monolithic column as a novel packing material. The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer/methanol (87.5:12.5 v/v), at a flow rate 1 mL/min. Detection was set at 274 nm. The LOQ for all the compounds ranged from 14 to 41 ng/mL. Excellent linearity was achieved over the studied range of concentration with correlation coefficients 0.9991–0.9998 (n = 6). The developed method was validated by precision and accuracy with RSD <2.55%. On extraction of the drugs and metabolites from the urine samples high recoveries were achieved ranging from 82.06 to 98.34% w/w on RP18 cartridges and methanol/chloroform (20:80 v/v) as the extraction solvent. This method has advantages over other methods using conventional C18 packings.