High-throughput bioanalysis of bile acids and their conjugates using UHPLC coupled to HRMS.

BACKGROUND: Quantitative assessment of bile acids in biological matrixes is of growing interest, primarily due to hepatic toxicity resulting from drug interactions with the bile salt export pump. Nevertheless, many bile acids demonstrate poor fragmentation in MS, making conventional MS/MS not a good match for their selective quantitation in biological matrices. RESULTS: The current study was designed to evaluate the feasibility of simultaneous quantitation of 19 bile acids using HRMS coupled to UHPLC separation with minimal instrument optimization. An effective chromatography was developed using an Agilent Zorbax(®) Eclipse XDB-C18 column (1.8 µm, 50 x 2.1 mm internal diameter), achieving separation of 19 compounds in 10 min. Excellent assay reproducibility was demonstrated, with two sets of standard curves, run 42 days apart. CONCLUSIONS: The results show that LC-HRMS is a viable platform for high throughput bioanalysis of bile acids especially in a drug-discovery setting.

In vivo gene transfer using sulfhydryl cross-linked PEG-peptide/glycopeptide DNA co-condensates.

Recent interest in sulfhydryl cross-linked nonviral gene delivery systems, designed to trigger the intracellular release of DNA, has inspired studies to establish their utility in vitro. To determine if this concept can be extrapolated to in vivo gene delivery, sulfhydryl cross-linking peptides (dp 20), derivatized with either an N-glycan or polyethylene glycol (PEG), were used to generate sulfhydryl cross-linked gene formulations. The biodistribution, metabolism, cell-type targeting, and gene expression of sulfhydryl cross-linked PEG-peptide/glycopeptide DNA co-condensates were examined following i.v. dosing in mice. Optimal targeting to hepatocytes was achieved by condensing (125)I-DNA with an add-mixture of 10 mol % triantennary glycopeptide, 5 mol % PEG-peptide, and 85 mol % backbone peptide. Four backbone peptides were substituted into the formulation to examine the influence of peptide metabolism and disulfide bond strength on the rate of DNA metabolism and the level of gene expression in vivo. The half-life of DNA in liver was extended from 1 to 3 h using a backbone peptide composed of d-amino acids, whereas substituting penicillamine for cysteine failed to further increase the metabolic stability of DNA. Optimized gene delivery formulations transiently expressed secreted alkaline phosphatase in mouse serum for 12 days. The results suggest that disulfide bond reduction in liver hepatocytes proceeds rapidly, followed by peptide metabolism, ultimately limiting the metabolic half-life of sulfhydryl cross-linked DNA condensates in vivo.

Application of semi-automated metabolite identification software in the drug discovery process for rapid identification of metabolites and the cytochrome P450 enzymes responsible for their formation.

Rapid identification of metabolites of compound X using data dependent scan function of a quadrupole ion trap mass spectrometer and semi-automated metabolite identification software is described. Compound X is metabolized via monooxygenation and desmethylation. LC-ESI-MS spectra obtained, following incubations of Compound X with microsomes in the presence and absence of chemical inhibitors specific for CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2E1, were processed using semi-automated metabolite identification software to extract information and to identify the cytochrome P450 enzymes responsible for metabolite formation. Chemical inhibition data suggests that the primary cytochrome P450 (CYP450) isozyme responsible for the metabolism of compound X is CYP3A4 with a minor contribution from both CYP2D6 and CYP2E1. Additionally, neither CYP2C9 nor CYP1A2 appears to contribute to the metabolism of compound X.