Measurement of catecholamines in rat and mini-pig plasma and urine by liquid chromatography-tandem mass spectrometry coupled with solid phase extraction.

A tandem mass spectrometry method combined with an ion-pair chromatographic separation after weak cation exchange solid phase sample extraction for epinephrine (E), norepinephrine (NE) and dopamine (DA) has been developed. Two surrogate matrixes for plasma and urine as well as stable isotope labeled internal standards were utilized for quantitation. The observed dynamic range of E, NE and DA was 0.

Understanding and reducing the experimental variability of in vitro plasma protein binding measurements.

The experimental measurement of plasma protein binding is a useful in vitro Absorption Distribution Metabolism and Excretion(ADME) assay currently conducted in both screening and definitive early development candidate modes. The fraction unbound is utilized to calculate important pharmacokinetic (PK) parameters such as unbound clearance and unbound volume of distribution in animals that can be used to make human PK and dose predictions and estimate clinically relevant drug-drug interaction potential. Although these types of assays have been executed for decades, a rigorous statistical analysis of sources of variability has not been conducted because of the tedious nature of the manual experiment.

Quantification of intact and truncated stromal cell-derived factor-1α in circulation by immunoaffinity enrichment and tandem mass spectrometry.

Stromal cell-derived factor 1α (SDF-1α) or CXCL12 is a small pro-inflammatory chemoattractant cytokine and a substrate of dipeptidyl peptidase IV (DPP-IV). Proteolytic cleavage by DPP-IV inactivates SDF-1α and attenuates its interaction with CXCR4, its cell surface receptor. To enable investigation of suppression of such inactivation with pharmacologic inhibition of DPP-IV, we developed quantitative mass spectrometric methods that differentiate intact SDF-1α from its inactive form.

Ask the experts: automation: part I.

Bioanalysis invited a selection of leading researchers to express their views on automation in the bioanalytical laboratory. The topics discussed include the challenges that the modern bioanalyst faces when integrating automation into existing drug-development processes, the impact of automation and how they envision the modern bioanalytical laboratory changing in the near future. Their enlightening responses provide a valuable insight into the impact of automation and the future of the constantly evolving bioanalytical laboratory.

Standardized workflows for increasing efficiency and productivity in discovery stage bioanalysis.

Merck consolidated discovery stage bioanalytical functions into the Department of Pharmacokinetics, Pharmacodynamics & Drug Metabolism in 2007. Since then procedures and equipment used to provide important quantitative data to project teams have been harmonized and in many cases standardized. This approach has enabled movement of work across the network of laboratories and has resulted in a lean, flexible and efficient organization.

Flexible automated approach for quantitative liquid handling of complex biological samples.

A fully automated protein precipitation technique for biological sample preparation has been developed for the quantitation of drugs in various biological matrixes. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and controls from a Watson laboratory information management system generated work list, shaking of 96-well plates, and vacuum application. Processing time is less than 30 s per sample or approximately 45 min per 96-well plate, which is then immediately ready for injection onto an LC-MS/MS system.

Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery.

The use of a cassette incubation of probe substrates with human liver microsomes (HLM) - also known as the 'cocktail' approach - is becoming a widely accepted approach to determine the interaction of new chemical entities (NCEs) with cytochrome P450 enzymes (CYP450) in early drug discovery. This article describes two LC-MS/MS-based analytical methods used at the high-throughput (HT) stage and late discovery (LD) stage for analysis of 'cocktail' incubates to analyze the probe metabolites 1'-hydroxymidazolam (CYP3A4), 4'-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), 1'-hydroxytacrine (CYP1A2) and 4'-hydroxymephenytoin (CYP2C19). The analytical methods are advantageous over currently reported methods due to their sensitivity, shorter analyses times (<2 min/sample for the HT method and 4 min/sample for the LD method) and their ability to monitor a unique set of clinically relevant probe metabolites from a biological incubate containing low microsomal protein (0.

Evaluation of online extraction/mass spectrometry for in vivo cassette analysis.

An online extraction/mass spectrometry technique was evaluated for direct analysis of plasma samples. A simple user-friendly online extraction system that consists of two pumps, an autosampler, a six-port switching valve and a mass spectrometer is described. The system was controlled by the LC-MS software (Masslynx 3.

Bergamottin contribution to the grapefruit juice-felodipine interaction and disposition in humans.

Objectives: Our objectives were to evaluate the contribution of bergamottin to the grapefruit juice-felodipine interaction and to characterize bergamottin disposition.

Methods: In this study 250 mL grapefruit juice; 2-, 6-, or 12-mg capsules of bergamottin plus water; or water was administered with 5 mg extended-release felodipine to 11 volunteers in a partially randomized, 5-way crossover study. Plasma concentrations of felodipine, its primary metabolite (dehydrofelodipine), bergamottin, and 6',7'-dihydroxybergamottin were determined.

Enzymatic tissue digestion as an alternative sample preparation approach for quantitative analysis using liquid chromatography-tandem mass spectrometry.

Compound extraction from biological tissue often presents a challenge for the bioanalytical chemist. Labor-intensive homogenization or sonication of whole or powdered tissue is performed before compounds can be extracted and analyzed. Enzymatic digestion is commonly used for tissue dissociation and cell harvesting and offers the advantages of unattended sample preparation, potential automation, and low cost.

Quantitation of endogenous analytes in biofluid without a true blank matrix.

A method is presented that describes a reliable and practical procedure for quantitation of an analyte present at relatively high background levels in blank (untreated) biological matrixes. Using a "surrogate analyte" approach, an endogenous analyte was quantitated in a variety of biological matrixes containing both very low (<10 ng/mL) and high (>2000 ng/mL) background levels of the desired analyte. This quantitative "surrogate analyte" approach was applied during the development of an HPLC/MS method for alpha-ketoisocaproic acid (KIC), which was identified as a potential biomarker for branched chain amino acid transferase inhibitor activity.

Investigation of EDTA anticoagulant in plasma to improve the throughput of liquid chromatography/tandem mass spectrometric assays.

In this study, EDTA and heparin are compared as anticoagulants with respect to their efficiency in preventing clot formation in plasma samples that were subsequently analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A pilot in vivo pharmacokinetic study for the drug chlorpheniramine was conducted in which both EDTA and heparin plasma samples were collected simultaneously. All conditions except the anticoagulant were held constant during the pharmacokinetic study.

Small molecule analysis by MALDI mass spectrometry.

This review focuses on the application of matrix assisted laser desorption/ionization (MALDI) mass spectrometry to the characterization of molecules in the low mass range (<1500 Da). Despite its reputation to the contrary, MALDI is a powerful technique to provide both qualitative and quantitative determination of low molecular weight compounds. Several approaches to minimize interference via sample preparation and matrix selection are discussed, as well as coupling of MALDI to liquid and planar chromatographic techniques to extend its range of applicability.

Evaluation of the human serum albumin column as a discovery screening tool for plasma protein binding.

A total of 69 compounds with a variety of chemical structures were assayed using a human serum albumin column in combination with UV and mass spectrometric detection. A moderate correlation, R(2)=0.661, between the plasma protein binding, determined by traditional techniques of equilibrium dialysis or ultrafiltration, and chromatographic retention factor (k'/k'+1) was observed.