Investigation of the Effects of Labware Contamination Introduced During Lipid Extractions on Mass Spectrometry-Based Human Serum Lipidome Analysis
Creators: Canez Quijada, Carlos R.
Publisher: name: University of Alberta Library
Year: 2024

<p>In lipidomic workflows, polypropylene microcentrifuge tubes (MCTs) are being increasingly used for lipid extraction and other sample processing steps, over glassware, due to their lower cost and increased convenience for automation, particularly in high-throughput studies. However, the ramifications of utilizing plasticware on mass spectrometry (MS)-based lipidomic investigations remain inadequately explored. The absence of an exhaustive assessment regarding the impact of contamination introduced by plasticware in MS-based lipidomics prompted the initiation of a systematic inquiry aimed at elucidating potential detriments associated with labware contamination in serum lipidomics.Contaminant profiles varied significantly among microcentrifuge tubes from different manufacturers. The most suitable polypropylene tubes evaluated, Eppendorf MCTs, introduced 847 labware-originating contaminant m/z values when three different manufacturing batches were tested for Folch blank extractions. Notably, this included the introduction of 21 primary amide and fatty acid surfactants identical to biological endogenous lipids, 16 of which had not been previously reported as leachables from polypropylene materials. In contrast, the use of borosilicate glassware and polytetrafluoroethylene-lined (PTFE) screw caps introduced 98 different contaminant m/z values across three manufacturing batches of glassware tested for Folch extractions. Despite the astonishing number of labware contaminants identified, existing databases and literature facilitated the identification of only 32 contaminants. To address the dearth of publicly available contaminant information, a comprehensive labware contamination repository is provided, featuring high-resolution m/z values, adductation information, retention times, and a MS/MS spectral repository. This resource is intended to aid researchers in effectively detecting and distinguishing labware-derived contaminants from analytes of interest.During Folch serum lipid extractions, the contamination from Eppendorf MCTs led to severe ion suppression of 40 low abundance serum lipids and induced mild to modest ion suppression across a wide range of higher abundance co-eluting lipids. Alternatively, less compatible polypropylene MCTs affected 75 serum lipids and induced more frequent and pronounced instances of ion suppression. Moreover, by conducting serum lipid extractions with varying initial volumes, it was determined that labware-induced lipid ion suppression is a dynamic phenomenon, dependent on the concentration of both lipids and labware contaminants. Suppressive co-eluting contaminants disproportionately negatively impacted low-abundance lipids. Beyond lipid ion suppression, the identification and quantification of 7 fatty acid endogenous serum lipids were compromised due to the leaching of structurally identical surfactants from MCTs. Polypropylene MCTs artificially introduced 10 additional primary amides extraneous to serum samples. Extreme caution is crucial in data interpretation regarding primary amides and fatty acids when using plastic labware. During biphasic BUME extractions, the use of Eppendorf MCTs resulted in the introduction of 1,010 plasticware contaminant features, comprising 771 distinct m/z values identified via positive ESI and 239 m/z identified via negative ESI, across two different manufacturing batches. Significantly, 210 leachables were uniquely introduced by polypropylene MCTs during BUME extractions, while absent in Folch blank extractions. Additionally, owing to the distinct organic solvents utilized in each extraction protocol, there was a notable disparity in the magnitude of many shared leachables. Conversely, the employment of borosilicate glassware and PTFE-lined screw caps introduced 70 distinct contaminant m/z values when tested for BUME extractions.These labware contaminants caused mild to moderate ion suppression across a broad spectrum of co-eluting lipids. Without strict structural characterization parameters, numerous high resolution m/z values originating from polypropylene leachables could be erroneously mistaken for lipid m/z values. The contaminants from Eppendorf MCTs elicited strong ion suppression in 22 co-eluting lipids, significantly impacting their analysis. The heightened contamination from less compatible polypropylene MCTs profoundly affected 71 co-eluting serum lipids. The presence of structurally identical surfactants leached from MCTs persisted in BUME extractions, compromising the identification and quantification of 6 fatty acid endogenous serum lipids. Furthermore, polypropylene MCTs introduced 8 primary amides, extraneous to the serum samples. By conducting BUME serum lipid extractions with variable initial volumes, the dynamic nature of matrix effects—specifically, the disproportionate impact on low-abundance lipids by co-eluting suppressive contaminants—was further elucidated.These studies aim to raised awareness about the detrimental effects of labware contamination on lipidome analysis. These studies aim to aid researchers in distinguishing contaminants from endogenous analytes of interest and assist in enhancing the data quality of lipidomic workflows.</p>