Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection.

Two analytical methods were developed for quantitative determination of DADLE (H(2)N-Tyr-D-Ala-Gly-Phe-D-Leu-COOH) and its two cyclic prodrugs in rat plasma. For high-performance liquid chromatography with fluorescence detection (LC-FLU), precolumn derivatization of DADLE was accomplished by labeling the N-terminal amino group with the reagent naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA/CN) to form a highly fluorescent 1-cyanobenz[f]isoindole (CBI) derivative. A multi-dimensional LC system was employed to improve selectivity, and solid-phase extraction (SPE) was used for plasma sample preparation. The cyclic prodrugs were converted to DADLE prior to their derivatization. With fluorescence detection after derivatization, the limit of quantitation (LOQ) was 6 ng ml(-1) for the analysis of DADLE, and good linearity was observed up to 6000 ng ml(-1) in rat plasma. Quantitative analysis of DADLE and its cyclic prodrugs was also performed using liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Chromatographic separation was achieved on a C(18) column using gradient elution in a water-acetonitrile system containing 0.1% (v/v) formic acid. The tandem mass spectrometric analysis was performed in the multiple reaction monitoring mode using internal standardization to improve assay precision and accuracy. For plasma sample pretreatment, acetonitrile was added first to precipitate proteins and SPE was used to minimize matrix effects. Using LC-ESI-MS-MS, the LOQ was 0.5 ng ml(-1) for DADLE and 2 to 5 ng ml(-1) for its prodrugs. Good linearity was observed from the LOQ up to 1000 ng ml(-1) for all compounds. For the analysis of DADLE, both analytical methods showed good precision, accuracy and stability. However, for prodrug analysis, LC-FLU showed some sensitivity and accuracy problems, while the LC-ESI-MS-MS method provided consistent and satisfactory results. In conclusion, LC-ESI-MS-MS is the method of choice for the analysis of DADLE and its cyclic prodrugs in rat plasma samples due to its good selectivity, high sensitivity, and fast analysis. Its application was demonstrated through biodisposition and bioconversion studies of the coumarinic acid-based prodrug after intravenous administration in rats.

The effect of enzyme inhibitor and absorption site following [D-ala2, D-leu5]enkephalin oral administration in rats.

The effects of enzyme inhibitor, amastatin, and absorption site following intravenous (i.v.) oral (p.o.), jejunal and ileal administration of [D-ala(2), D-leu(5)]enkephalin (YdAGFdL) were investigated in rats. Model dependent and independent pharmacokinetic parameters were obtained and compared. Linear pharmacokinetics of YdAGFdL were evaluated at 0.28 and 500 microg doses for i.v. and at 1, 500, and 1000 microg for p.o. and ileal routes. Plasma samples were collected and assayed for intact YdAGFdL using a radiometric thin layer chromatography. The clearance (CL) and half lives of the distribution and elimination phases following the 0.28 microg (n=6) i.v. dose were 42.7+/-26.2 (S.D.) ml/min, 0.48+/-0.17 min, and 3.98+/-0.92 min, while those of the 500 microg dose (n=6) were 48.0+/-23.3 ml/min, 0.59+/-0.25, and 6.81+/-3.12 min, respectively, suggesting apparent linear kinetics. The CL values were close to the cardiac output of rats (50 ml/min) indicating very rapid elimination from the body. Mean bioavailability (F) values following p.o. (n=15), jejunal (n=4), and ileal (n=16) administration were 0.40+/-0.24% (S.E.), 1.25+/-0.39, and 1.78+/-0.40, respectively, and were not significantly different (p<0.05) among three doses (1, 1000, 5000 microg). The F value of YdAGFdL following ileal administration in the presence of amastatin was 8.76+/-4.47% (n=6), a 22 fold increase over po administration and a five fold increase over ileal administration without an inhibitor. These results indicate that 'effective' oral delivery of small peptides may be achievable.

Determination of [D-Ala2,D-Leu5]enkephalin and the metabolites containing C-terminal D-leucine by high-performance liquid chromatography.

A high-performance liquid chromatographic procedure has been developed for the determination of [D-Ala2,D-Leu5]enkephalin (DADLE) and the fragments containing D-leucine in rat blood. The procedure was applied to the determination of blood levels of [3H-D-Leu5]DADLE and the C-terminal fragments after intravenous administration of [3H-D-Leu5]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO4 and acetonitrile.