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Journal of Lipid Research, Vol. 48, 242-251, January 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid-phase extraction and HPLC methods

Eva Persson^*, Lars Löfgren, Göran Hansson, Bertil Abrahamsson, Hans Lennernäs^* and Ralf Nilsson^1,

^* Department of Pharmacy, Uppsala University, S-751 23 Uppsala, Sweden
Department of Molecular Pharmacology, AstraZeneca R&D, S-431 83 Mölndal, Sweden
Department of Product Development, AstraZeneca R&D, S-431 83 Mölndal, Sweden

Published, JLR Papers in Press, October 24, 2006.

¹ To whom correspondence should be addressed. e-mail: ralf.nilsson{at}astrazeneca.com

The purpose of the study reported here was to develop a method for the determination of lipid classes in intestinal fluids, including bile acids (BAs). A solid-phase extraction (SPE) method using C18 and silica columns for the separation of BAs, phospholipids (PLs), and neutral lipids (NLs), including free fatty acids, has been developed and validated. Fed-state small intestinal fluid collected from humans was treated with orlistat to inhibit lipolysis and mixed with acetic acid and methanol before SPE to maximize lipid recoveries. BAs, PLs, and NLs were isolated using lipophilic and polar solvents to promote elution from the SPE columns. The different lipid classes were subsequently analyzed using three separately optimized HPLC methods with evaporative light-scattering detectors. High recoveries (>90%) of all lipids evaluated were observed, with low coefficients of variation (<5%). The HPLC methods developed were highly reproducible and allowed baseline separation of nearly all lipid classes investigated. In conclusion, these methods provide a means of lipid class analysis of NLs, PLs, and BAs in human fed-state small intestinal fluid, with potential use in other fluids from the intestinal tract and animals.

Supplementary key words solid-phase extraction • high-performance liquid chromatography • evaporative light scattering • neutral lipids • phospholipids

Abbreviations: BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DAG, diacylglycerol; DCA, deoxycholic acid; ELS, evaporative light scattering; FeSSIF, fed simulated small intestinal fluid; GCA, glycocholic acid; HAc, acetic acid; HIF, human intestinal fluid; LOD, limit of detection; LOQ, limit of quantification; LPC, lysophosphatidylcholine; MAG, monoacylglycerol; MeOH, methanol; MTBE, methyl tert-butyl ether; NL, neutral lipid; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PL, phospholipid; Si, silica; SPE, solid-phase extraction; TAG, triacylglycerol

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