Simultaneous determination of caffeine, theophylline and theobromine in human plasma by on-line solid-phase extraction coupled to reversed-phase chromatography.
A reversed-phase liquid chromatographic column switching system was described for the determination of caffeine (CF), theophylline (TH) and theobromine (TB) in human plasma with a direct injection procedure. A short protein-coated mu Bondapak CN silica pre-column (20 x 3 mm, i.d.) was used for enrichment of the drugs and clean up from weakly retained plasma components using phosphate buffer saline pH 7.4. After washing step, the retained drugs were flushed into a reversed-phase column (5 microm TSK gel ODS-80 TM, 150 x 4.6 mm i.d.) with a mobile phase of methanol-0.01 M phosphate buffer, pH 3.5 (30:70, v/v) for the final separation. The eluent was monitored with a UV detector at 275 nm. The resulting chromatograms showed no interference from endogenous plasma components. A linear relationship between the concentration of drug and peak height was confirmed in the range of 0.5-20 microg/mL for all drugs. High extraction recoveries from plasma ranging from 96.12 to 100.32% were achieved. Validation of the method was examined performing intra- and inter-day accuracy and precision and was found to be satisfactory. The coefficients of variation of the three drugs were less than 3% for intra-day and less than 4% for inter-day run assays.
Emara
Samy
S
Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt.
D002021:Buffers; D002110:Caffeine / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D010710:Phosphates; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013805:Theobromine / Q000097:blood*; D013806:Theophylline / Q000097:blood*
D002021:Buffers; D010710:Phosphates; D002110:Caffeine; D013806:Theophylline; D013805:Theobromine; D000432:Methanol