COMPARISON OF HPLC, CAPILLARY ELECTROPHORETIC AND DIRECT SPECTROFLUOROMETRIC METHODS FOR THE DETERMINATION OF TEMOPORFIN POLY(ETHYLENE GLYCOL) CONJUGATES IN PLASMA

High-performance liquid chromatographic (HPLC), capillary electrophore tic (CE) and direct spectrofluorimetric methods for the determination of temoporfin-poly(ethylene glycol) 2000 conjugate (m-THPC-PEG 2000) i n plasma are described and compared. m-THPC-PEG 2000 in plasma was qua ntitatively extracted (recovery 101-107%) with CH3OH-DMSO (4 + 1 v/v). The supernatant after centrifugation was used for HPLC, CE or direct spectrofluorimetric determination. The major drawback of the HPLC meth od was that it grave a broad and split peak even under gradient elutio n conditions, resulting in difficulty in detection and quantification. This is because m-THPC-PEG 2000 consists of a group of compounds with an average molecular mass of approximately 8680 Da owing to the wide molecular mass distribution of PEG 2000 used in the synthesis of the d rug. m-THPC-PEG 2000 gave a single and relatively sharp peak when sepa rated by CE with sodium tetraborate buffer (pH 9.45) in the presence o f sodium dodecyl sulfate as the running buffer. However, this method l acks the necessary sensitivity for detecting the drug in plasma extrac t because of the limited sample volume that can be injected. Direct sp ectrofluorimetry is the method of choice because of its simplicity, sp ecificity and sensitivity. Using an excitation wavelength of 423 nm an d the specific emission maximum of 657 nm, the fluorescence intensity could be sensitively measured. The calibration curve constructed by pl otting fluorescence intensity against concentration was linear within the range 1.32-1056 ng ml(-1). The detection limit (S/N = 3) was 1.32 ng ml(-1) and the limit of yuantification (S/N = 10) was 2.24 ng ml(-1 ). The precision and reproducibility were assessed by repeated analysi s (n = 24) of spiked plasma samples at 350.8 and 699.3 ng ml(-1). The RSD was 4.5% and 1.6%, respectively.