Improvement in assay sensitivity for plasma dolastatin-10 using capillary electrophoresis at elevated temperatures.

The very potent antimitotic and anticancer agent, dolastatin-10 (DOL-10), currently undergoing testing in a phase II clinical trial, has been quantitated previously in human plasma by high-performance capillary electrophoresis (HPCE). This method provides a lower limit of detection of 25 ng/ml DOL-10 from extracted patient samples. Without changes in preconcentration techniques, we report a significant improvement in the sensitivity of this method using elevated temperatures with conventional UV absorbance detection and liquid-liquid extraction which lowers the detection limit to 3 ng/ml of the drug. An elevated separation temperature of 50 degrees C was critical in achieving this 8x improvement in the detection limit. Partial validation of the method at this temperature gave excellent linearity (0-100 ng/ml; y=0.018x+0.085, r=0.993), limit of quantitation (5 ng/ml), and good overall recovery of the drug (>85%). We have applied this improved method towards the in vivo quantitation of DOL-10 in mice and in a patient receiving the drug in a phase I clinical study. From these analyses we conclude that this method is suitable for clinical studies where plasma levels of DOL-10 are > or = 5 ng/ml.

Quantitation of dolastatin-10 using HPLC/electrospray ionization mass spectrometry: application in a phase I clinical trial.

A highly sensitive and specific assay for the quantitation of the anticancer agent dolastatin-10 (DOL-10) in human plasma is described. The method was based on the use of electrospray ionization-high-performance liquid chromatography/mass spectrometry (ESP-LC/MS). The analytical procedure involved extraction of plasma samples containing DOL-10 and the internal standard (DOL-15) with n-butyl chloride, which was then evaporated under nitrogen. The residue was dissolved in 50 microl mobile phase and 10 microl was subjected to ESP-LC/MS analysis using a C18 microbore column. A linear gradient using water/acetonitrile was used to keep the retention times of the analytes of interest under 5 min. The method exhibited a linear range from 0.005 to 50 ng/ml with a lower limit of quantitation (LLQ) at 0.005 ng/ml. Absolute recoveries of extracted samples in the 85-90% range were obtained. The method's accuracy (< or =5% relative error) and precision (< or =10% CV) were well within industry standards. The analytical procedure was applied to extract DOL-10 metabolites from samples obtained following incubation of the drug with an activated S9 rat liver preparation. Two metabolic products were detected and were tentatively identified as a N-demethyl-DOL-10 and hydroxy-DOL-10. Structural assignments were made based on the fragmentation patterns obtained using the electrospray source to produce collision-induced dissociation (CID). The method was also applied to the measurement of DOL-10 in the plasma of patients treated with this drug. Preliminary investigation of the pharmacokinetics suggested that drug distribution and elimination may be best described by a three-compartment model with t1/2alpha = 0.087 h, t1/2beta = 0.69 h and t1/2gamma = 8.0 h. Plasma clearance was 3.7 l/h per m2.

High-performance capillary electrophoresis measurement of dolastatin-10.

A high-performance capillary electrophoresis (HPCE) assay was used to determine the concentration of a potent cytotoxic agent, dolastatin-10, in human plasma. Following extraction from plasma, using a solid-phase C18 cartridge, capillary zone electrophoresis was used to separate, detect and quantitate dolastatin-10 using the structurally related compound dolastatin-15 as the internal standard. Migration times for both dolastatins are less than 20 min. The recovery of the drug was approximately 90% and was quantified over the assay range of 39 to 5000 ng/ml with good precision and accuracy. The method is linear up to 5000 ng/ml with a lower limit of detection of 25 ng/ml. Data resulting from the use of the assay for the in vitro metabolism of the drug are presented. This is the first report of a validated HPCE assay for determining dolastatin-10 levels in human plasma.