Abstract
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.
Highlights
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)
The BAs adsorbed well to the column when up to 30% MeOH was added to the intestinal fluid; amounts .30% resulted in a decrease of up to 65% in the recovery of BAs
When 30% MeOH was added to the intestinal fluid, the best adsorption of both BAs and neutral lipid (NL) to the column material was obtained
Summary
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. Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid-phase extraction and HPLC methods. HPLC with evaporative light-scattering (ELS) detection provides a simple and affordable detection system for high-concentration samples such as intestinal fluids, bile, 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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