Article pubs.acs.org/ac
Cite This: Anal. Chem. 2017, 89, 13365−13373
On-Chip Lipid Extraction Using Superabsorbent Polymers for Mass Spectrometry Geul Bang,†,# Young Hwan Kim,†,‡,§,# Junghyo Yoon,∥ Yeong Jun Yu,†,⊥ Seok Chung,∥,∇ and Jeong Ah Kim*,†,‡ †
Biomedical Omics Group, Korea Basic Science Institute, Chungbuk 28119, Republic of Korea Department of Bio-Analytical Science, University of Science and Technology, Daejeon 34113, Republic of Korea § Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea ∥ School of Mechanical Engineering, Korea University, Seoul 02841, Republic of Korea ⊥ Program in Micro/Nano System, Korea University, Seoul 02841, Republic of Korea ∇ KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Pretreatment of samples is one of the most important steps in analytical methods for efficient and accurate results. Typically, an extraction method used for lipid analysis with mass spectrometry is accompanied by complex liquid−liquid extraction. We have devised a simple, rapid, and efficient lipid extraction method using superabsorbent polymers (SAPs) and developed a high-throughput lipid extraction platform based on a microfluidic system. Since SAPs can rapidly absorb an aqueous solution from a raw sample and convert it into the gel, the lipid extraction process can be remarkably simplified. The hydrophobic lipid components were captured into the fibrous SAP gel and then solubilized and eluted directly into the organic solvent without significant interference by this polymer. The small-scale lipid extraction process minimizes the liquid handling and unnecessary centrifugation steps, thereby enabling the implementation of a SAP-integrated microfluidic lipid extraction platform. The SAP method successfully induced reproducible extraction and high recovery rates (95−100%) compared to the conventional Folch method in several lipid classes. We also demonstrated the feasibility of the SAP method for the analysis of lipids in complex biological samples, such as the brain and liver, as well as Escherichia coli. This small-scale SAP method and its microfluidic platform will open up new possibilities in high-throughput lipidomic research for diagnosing diseases because this new technique saves time, labor, and cost.
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Another difficulty in analyzing lipids is that their concentrations vary over a wide range in various samples. As a result, the less abundant species can easily get lost during inefficient sample pretreatment.10−12 The traditional lipid extraction methods, such as the Folch method and the Bligh−Dyer method, are liquid−liquid extraction (LLE) methods that involve several consecutive steps.13−15 In general, the LLE method is unsuitable for small quantities of samples because it requires several hours for the extraction and generally has a low recovery rate. These are serious considerations because the removal of water is a prerequisite when treating aqueous samples, such as plasma, urine, and other body fluids. Some time ago, a solid-phase extraction (SPE) method that is referred to as the “quick, easy, cheap, effective, rugged, and safe” method (QuEChERS
ipids are one of the major components of biological cell membranes, and they participate in many biochemical functions and metabolic processes, such as intercellular signaling, secretion, and energy storage.1−3 Recent studies have proven that lipids have key roles in biological mechanisms and they are involved in the lipid-related diseases, such as diabetes, Alzheimer’s disease, atherosclerosis, and cancer.4−6 These findings emphasize the importance of research in the field of bioanalytical chemistry that involves comprehensive and quantitative studies of lipids. For this reason, it is essential that a simple and rapid treatment technique for lipid analysis be developed. Despite the highly sophisticated analytical process, successful analysis is determined by the efficient extraction and separation of complex lipid mixtures derived from various biological origins, such as cells, tissues, and body fluids. However, it is very hard to simultaneously analyze all lipid classes with a high recovery rate because such analysis is complicated by the diverse subclasses of lipids, depending on their polar nature and structure of the molecular backbone.7−9 © 2017 American Chemical Society
Received: August 30, 2017 Accepted: November 22, 2017 Published: November 22, 2017 13365
DOI: 10.1021/acs.analchem.7b03547 Anal. Chem. 2017, 89, 13365−13373
Article
Analytical Chemistry
donut-shaped inner well was placed into the lager holes (15 mm in diameter) made of PDMS and bonded onto another PDMS slab as a bottom layer. This assembled microchip consisted of double-layered wells. The overall dimensions of a single device were about 25 × 25 mm2. For a high-throughput device, we used a multiwell plate-based platform. Twenty-four donut-shaped PDMS inner wells were bonded onto the glass bottom of the well plate (well diameter = 13 mm; MatTek Corp., USA). Lipid Extraction Using SAPs. The SAPs were purchased from LG Chem, Ltd., South Korea. The powder was analyzed to measure the size of the individual SAP particles using a scanning electron microscope (JSM-6610LV, JEOL, Japan). Premixed standards, SPLASH, and total lipid extracts, such as porcine brain, bovine liver, and Escherichia coli (E. coli), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and they were dissolved in organic solvents, such as methanol and chloroform, according to the provider’s instruction. The standard mixture includes all of the major lipid classes of human plasma such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), cholesterol ester (Chol Ester), monoacylglycerol (MAG), diacylglycerol (DAG), triacylglycerol (TAG), sphingomyelin (SM), and cholesterol. The standard mixture dissolved in 100% methanol was dried to evaporate the organic solvent. Then, dried lipids were re-dissolved in a one-fourth volume of the mixture (2:1) of methyl tert-butyl ether (MTBE) and methanol (MeOH). This solution was then re-diluted with four volumes of MS-grade water. The final concentration of the standard mixture was equal to the original concentration. The components and the stock concentrations of the standard lipid mixture are listed in Supporting Information Table S1. For preparation of the biological samples, the concentrations of brain, liver, and E. coli extracts were adjusted to 5 mg mL−1 by dissolving the extracts in a 1:4 (v/v) mixture of MTBE and MeOH and re-diluted with MS-grade water to a final concentration of 1 mg mL−1. Plasma (10−100 μL) was used without further dilution. For lipid extraction, 2−15 mg of SAP powder was placed into a microcentrifuge tube or the inner well of a microchip in proportion to the volume of the sample (10− 100 μL). Then, the samples were dropped into SAP powder and left for 30 s to complete the gelation. Four different organic solvents, such as 2:1 (v/v) MTBE:MeOH, 2:1 (v/v) chloroform (CHCl3):MeOH, aceonitrile (ACN), and MeOH, were loaded to the gel and the samples were incubated for 2−3 min. The volume of solvent (40−400 μL) also was proportional to the volumes of the samples. The lipids were solubilized into the organic phase and extracted from the SAP gel. The solvent phase that contained the solubilized lipids was extracted using a pipet. In the case of tests in a microfluidic device, the lipids solubilized in the solvent released out from the inner well to the outer well through the gaps between the pillars that surrounded the inner well. The extracted lipids were collected into the outer well, taken with a pipet. Then, they were analyzed using direct infusion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI/Q-TOF MS) and ultra performance liquid chromatography−mass spectrometry (UPLC/MS) techniques. Modified Folch Method. For comparison with our method, we extracted lipids from the standard mixture using the modified Folch method as described in the previous
method) was introduced for various pretreatments for pesticides, food samples, environmental samples, and lipids.16,17 The QuEChERS method is easy and simple to use, and it reduces the extraction cleanup time (
