Simultaneous determination of phenytoin, carbamazepine, and 10,11-carbamazepine epoxide in human plasma by high-performance liquid chromatography with ultraviolet detection.
The Bioanalytical Chemistry Department at the Madison facility of Covance Laboratories, has developed and validated a simple and sensitive method for the simultaneous determination of phenytoin (PHT), carbamazepine (CBZ) and 10,11-carbamazepine epoxide (CBZ-E) in human plasma by high-performance liquid chromatography with 10,11 dihydrocarbamazepine as the internal standard. Acetonitrile was added to plasma samples containing PHT, CBZ and CBZ-E to precipitate the plasma proteins. After centrifugation, the acetonitrile supernatant was transferred to a clean tube and evaporated under N2. The dried sample extract was reconstituted in 0.4 ml of mobile phase and injected for analysis by high-performance liquid chromatography. Separation was achieved on a Spherisorb ODS2 analytical column with a mobile phase of 18:18:70 acetonitrile:methanol:potassium phosphate buffer. Detection was at 210 nm using an ultraviolet detector. The mean retention times of CBZ-E, PHT and CBZ were 5.8, 9.9 and 11.8 min, respectively. Peak height ratios were fit to a least squares linear regression algorithm with a 1/(concentration)2 weighting. The method produces acceptable linearity, precision and accuracy to a minimum concentration of 0.050 micrograms ml-1 in human plasma. It is also simple and convenient, with no observable matrix interferences.
Bhatti
M M
MM
Covance Laboratories, Madison, WI 53704, USA. masood.bhatti@covance.com
Hanson
G D
GD
Schultz
L
L
D002220:Carbamazepine / Q000031:analogs & derivatives* / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D010672:Phenytoin / Q000097:blood*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D002220:Carbamazepine; D010672:Phenytoin; C012227:carbamazepine epoxide