Systematic development of an UPLC-MS/MS method for the determination of tricyclic antidepressants in human urine.

Tricyclic antidepressants have been prescribed for the treatment of depression and other disorders since their discovery in the 1950s but have been replaced in recent decades by newer drugs with more favorable side effect profiles. However, for some patients and conditions, tricyclic antidepressants remain the drug of choice. A fast, sensitive, and robust UPLC-MS/MS method for the monitoring of amitriptyline, nortriptyline, imipramine, doxepin, and desipramine in human urine has been developed using a pre-defined and systematic method development approach. The method was developed using sub-2-µm particle technology, providing a state-of-the-art alternative to older methods. Total cycle time was 2.5min. Human urine samples (200µL) were prepared using an Oasis(®) WCX µElution solid-phase extraction plate, which provided good recovery for all analytes (>92%) and low matrix effects (absolute matrix effects <10%). Standard curves were linear over the range 0.02-250ng/mL with r(2) values>0.994. The method was evaluated against current FDA guidelines and was applied to the analysis of patient samples, including an assessment of incurred sample reanalysis (ISR).

Challenges in developing an ultra-sensitive bioanalytical method for ethinylestradiol in human plasma.

BACKGROUND: Ethinylestradiol (EE) is the active component in most birth control products. It is especially difficult to analyze due to the presence of many closely related endogenous steroids. Endogenous components can coelute with EE making selective extraction and chromatographic separation challenging. Current MS systems are more sensitive to background, contamination and the overall cleanliness of samples and solvents, placing additional emphasis on sample preparation methodology. METHOD: UPLC was combined with a sensitive triple quadrupole MS and a three-step sample preparation method to highlight and resolve method development challenges. RESULTS: EE was adequately resolved using an unendcapped high-strength silica C(18) column. The average matrix factor in six sources of plasma was 1.14 with a %CV of 4.48. Standard curves were linear with 1/x weighting and r(2) value of 0.999 over three orders of magnitude. Average accuracy for standard curves and quality control samples was 96%. LOD of 0.001 ng/ml was achieved.

Sub one minute inhibition assays for the major cytochrome P450 enzymes utilizing ultra-performance liquid chromatography/tandem mass spectrometry.

The measurement of cytochrome P450 (CYP450) isoenzyme inhibition is often done during evaluation of new chemical entities in drug discovery. Typical assay protocol consists of multiple CYP450 probe substrates incubated with selected drug candidates and CYP450. Results of the assay, the amount of probe substrate metabolite formed with respect to control, are used to determine the level of interaction. Liquid chromatography utilizing columns packed with sub-2-micron particles have been shown to provide up to 8X faster analysis time and 3X increases in sensitivity over traditional high-performance liquid chromatography (HPLC). The work presented here shows the development of a high-throughput, sub-2-micron particle LC method coupled with tandem quadrupole mass spectrometry for the rapid analysis of six CYP450 probe substrate metabolites in 30s.