Determination of Fecal Sterols Following a Diet with and without Plant Sterols

Abstract

The aim of this study was to develop a method for neutral fecal sterols determination in subjects receiving a normal diet with or without a plant sterols-enriched beverage using gas chromatography-mass spectrometry (GC/MS). Sample preparation conditions (homogenization of lyophilized feces with water) were evaluated. Sterol determination required direct hot saponification, unsaponifiable extraction with hexane, and the formation of trimethylsilyl (TMS) ether derivatives. The method allows the quantification of cholesterol, plant sterols and their metabolites (coprostanol, coprostanone, cholestanol, cholestanone, methylcoprostanol, methylcoprostanone, ethylcoprostenol, stigmastenol, ethylcoprostanol and ethylcoprostanone). Good linearity was obtained (r > 0.96) and interference was only observed for coprostanone, where the standard addition method proved necessary for quantification. The limits of detection (LOD) ranged from 0.10 to 3.88 µg/g dry feces and the limits of quantitation (LOQ) from 0.34 to 12.94 µg/g dry feces. Intra- and inter-assay precision (RSD %) were 0.9-9.2 and 2.1-11.3, respectively. Accuracy, expressed as percentage recovery (80-119%) was obtained for all determined sterols.

Relationship Between Dietary Sterols and Gut Microbiota: A Review

“…Fresh feces were initially used for fecal sterol determination. However, freeze‐dry feces are currently the most widely used option, since these samples offer several advantages in terms of conservation, safety, and sampling . Generally, both fresh and freeze‐dry feces are homogenized with organic solvents (e.g., chloroform (Cl 3 CH): methanol (MetOH)) (Table ).…”

“…Besides, due to the presence of particles in suspension that hindered pipetting, the influence of filtration was evaluated. We concluded that subjecting the samples to ultrasound (20 min) and keeping the dispersion (feces + water) at room temperature for 2 h was better than the filtration process, which caused a low precision (between 1.2 and 47.5 RSD%) . Other authors have attempted to improve feces homogenization with water by applying only agitation, or using an Omni‐Mixer …”

“…On the other hand, nitrogen was the most commonly used carrier gas in GC‐FID when a packed column was used, versus hydrogen and helium in the case of capillary columns. Helium was used with GC‐MS, though in some cases hydrogen has also been used . Table summarizes the validation analytical parameters for sterols and their metabolites in feces.…”

“…In the 1970s and 1980s, some authors reported incomplete validation parameters in the determination of sterols and their metabolites in feces. At present, few studies carry out complete validation (selectivity and linearity, precision, accuracy and detection and quantitation limits) …”

“…However, freeze-dry feces are currently the most widely used option, since these samples offer several advantages in terms of conservation, www.advancedsciencenews.com www.ejlst.com safety, and sampling. [78] Generally, both fresh and freeze-dry feces are homogenized with organic solvents (e.g., chloroform (Cl 3 CH): methanol (MetOH)) ( Table 3). In a previous study carried out by our group, freeze-dry feces were homogenized with water to obtain a more environmentally friendly method.…”