Extraction, Identification, and Functional Characterization of a Bioactive Substance From Automated Compound-Handling Plastic Tips

Abstract

Disposable plastic labware is ubiquitous in contemporary pharmaceutical research laboratories. Plastic labware is routinely used for chemical compound storage and during automated liquid-handling processes that support assay development, high-throughput screening, structure-activity determinations, and liability profiling. However, there is little information available in the literature on the contaminants released from plastic labware upon DMSO exposure and their resultant effects on specific biological assays. The authors report here the extraction, by simple DMSO washing, of a biologically active substance from one particular size of disposable plastic tips used in automated compound handling. The active contaminant was identified as erucamide ((Z)-docos-13-enamide), a long-chain mono-unsaturated fatty acid amide commonly used in plastics manufacturing, by gas chromatography/mass spectroscopy analysis of the DMSO-extracted material. Tip extracts prepared in DMSO, as well as a commercially obtained sample of erucamide, were active in a functional bioassay of a known G-protein-coupled fatty acid receptor. A sample of a different disposable tip product from the same vendor did not release detectable erucamide following solvent extraction, and DMSO extracts prepared from this product were inactive in the receptor functional assay. These results demonstrate that solvent-extractable contaminants from some plastic labware used in the contemporary pharmaceutical research and development (R&D) environment can be introduced into physical and biological assays during routine compound management liquid-handling processes. These contaminants may further possess biological activity and are therefore a potential source of assay-specific confounding artifacts.

Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells

“…Some PFAMs, particularly oleamide and erucamide, are used as slip additives in polyethylenes (51)(52)(53). Thus, background PFAM levels must be determined due to potential slip additive contamination.…”

“…We assumed that the background 18:1 amide reported here is oleamide because oleamide is a common slip additive (51,53).…”

“…We also measured PFAM levels in solvents, glassware, and plasticware used in the extractions to measure background amounts from slip additives (51)(52)(53). PFAM levels in the solvent blanks were low, ranging between 0.5% and 8% of the amounts isolated from the SCP or N 18 TG 2 cells.…”

Mammalian Genotyping Using Acoustic Droplet Ejection for Enhanced Data Reproducibility, Superior Throughput, and Minimized Cross-Contamination

“…Finally, plastic tips have been shown to leach bioactive compounds into the assembled assays, affecting experimental outcomes. [34][35][36] ADE technology has broad applications across the fields of molecular biology, genomics, and drug discovery. For instance, a workflow has been recently described that would apply next-generation sequencing (NGS) techniques to characterizing on-and off-target CRIPSR editing events in a genome.…”

HPLC Biogram Analysis: A Powerful Tool Used for Hit Confirmation in Early Drug Discovery

“…This may be due to irreversible binding of the active component to the HPLC column or, more commonly, to artifactual activity in the original assay, for example, activity due to lipophilic impurities such as plasticizers in the sample. 4,5 In some instances, a "FAIL" is due to no target present in the sample (i.e., sample is badly degraded).…”