Comparison of HPLC, capillary electrophoretic and direct spectrofluorimetric methods for the determination of temoporfin–poly(ethylene glycol) conjugates in plasma†

Abstract

High-performance liquid chromatographic (HPLC), capillary electrophoretic (CE) and direct spectrofluorimetric methods for the determination of temoporfin–poly(ethylene glycol) 2000 conjugate (m-THPC–PEG 2000) in plasma are described and compared. m-THPC–PEG 2000 in plasma was quantitatively 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 method was that it gave a broad and split peak even under gradient elution 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 drug. m-THPC–PEG 2000 gave a single and relatively sharp peak when separated by CE with sodium tetraborate buffer (pH 9.45) in the presence of sodium dodecyl sulfate as the running buffer. However, this method lacks the necessary sensitivity for detecting the drug in plasma extract because of the limited sample volume that can be injected. Direct spectrofluorimetry is the method of choice because of its simplicity, specificity and sensitivity. Using an excitation wavelength of 423 nm and the specific emission maximum of 657 nm, the fluorescence intensity could be sensitively measured. The calibration curve constructed by plotting 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 quantification (S/N = 10) was 2.24 ng ml^–1. The precision and reproducibility were assessed by repeated analysis (n = 24) of spiked plasma samples at 350.8 and 699.3 ng ml^–1. The RSD was 4.5% and 1.6%, respectively.