The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Abstract

This article is available online at http://www.jlr.org implicated in several diverse diseases, such as type 2 diabetes, Alzheimer disease, ( 2 ) and cancer ( 3 ). Lipid analysis has been an important area of research for several decades, and due to technological advances, the fi eld has experienced a renaissance in the last decade. In a modern laboratory, a comprehensive lipid characterization can be performed that generates quantitative data of several hundreds of molecular lipids from several different lipid classes. This kind of analysis, often called lipidomics, is based on HPLC and mass spectrometry (MS) instrumentation. The analysis is performed unattended in the 96-well format and is fully automated.An important component for successful analysis is the quality of the lipid extract. It is important that the lipid extract that is injected on the HPLC or infused into the mass spectrometer is pure; therefore, it is important that interfering substances and particles are removed. Inability in removing these substances might result in a high chemical background, which will have an effect on both the sensitivity and selectivity of the analysis. In contrast to fully automated and high-throughput analysis, lipid extraction is still often performed manually, involving exhaustive and time-consuming pipetting steps and hazardous solvents such as chloroform. Thus, a fully automated, chloroformfree method that can be used with standard 96-well robots would signifi cantly improve sample throughput, as well as reduce the negative impact on health and environment. The aim of this study was to develop that method.Abstract Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profi les of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroformbased reference method. Lipid recoveries were linear from 10-100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by twophase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, highthroughput BUME method can replace chloroform-based methods, saving both human and environment...

A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

“…Over the years some modifi cations to the original solvent system composition have been introduced, tested, and evaluated; although these alterations are not always fully described by the authors, which makes comparison of the lipid content and composition of fl uids in similar pathological conditions somewhat diffi cult. A comparison of the Folch method and the Bligh and Dyer method has shown similar recoveries in the extraction of the predominant phospholipid classes ( 15 ), and similar conclusions were reported in other studies comparing the Folch method with alternative extraction solvent systems (16)(17)(18). Other solvent mixtures that include, for example, hexane ( 19,20 ), butanol ( 18,21 ), ethyl acetate ( 21 ), and more recently tert-butyl methyl ether (TBME) ( 17,(22)(23)(24) are also described in the literature.…”

“…A comparison of the Folch method and the Bligh and Dyer method has shown similar recoveries in the extraction of the predominant phospholipid classes ( 15 ), and similar conclusions were reported in other studies comparing the Folch method with alternative extraction solvent systems (16)(17)(18). Other solvent mixtures that include, for example, hexane ( 19,20 ), butanol ( 18,21 ), ethyl acetate ( 21 ), and more recently tert-butyl methyl ether (TBME) ( 17,(22)(23)(24) are also described in the literature. TBME, which was reported to give comparable results to the Bligh and Dyer method in the profi le of human plasma ( 16 ), has become particularly popular for sphingolipidomic studies and the extraction of lipids in fl uids ( 21,23 ).…”

“…TBME, which was reported to give comparable results to the Bligh and Dyer method in the profi le of human plasma ( 16 ), has become particularly popular for sphingolipidomic studies and the extraction of lipids in fl uids ( 21,23 ). Generally, the alternative solvent systems were not reported to exhibit signifi cant differences in the extraction of predominant lipid classes when compared with the "traditional" solvent systems (15)(16)(17)(18). Rather, the advantage of these extraction systems lies in the avoidance of chlorinated solvents, which are environmentally harmful.…”

Plasma lipid profiling in a large population-based cohort

“…Using the method described, our laboratory can routinely extract 400 samples per day. While robotics has obvious advantages ( 24,25 ), we have demonstrated that our method, even with manual handling and extraction of samples, is robust and applicable to cohorts of 1,000 or more samples.…”

Predator lipids induce paralytic shellfish toxins in bloom-forming algae

“…The mobile phase was 88% (vol/vol) solution A (A = acetonitrile/ methanol/water 40:45:15 with 0.2% formic acid, 0.1% ammonia, and 5 μM phosphoric acid) and 12% (vol/vol) solution B (B = isopropanol with 0.2% formic acid, 0.1% ammonia, and 5 μM phosphoric acid), at 1.0 mL/min. The optimal method for purifying copepodamides G and H differed from that of A-F: freeze-dried Calanus tissue (62.2 g) was extracted with methanol five times and extracts were separated by liquid-liquid partitioning with heptane/methanol (98:2) vs. methanol/water/aqueous ammonium hydroxide (94:5:1) (29). Copepodamides G and H were localized to the aqueous fraction, which was then subjected to reversed phase SPE (ENVI-18, 10 g; Supelco), eluting with a step gradient of aqueous methanol (30-100%).…”