Injection-port Silylation Using N-O-bis-(trimethylsilyl)-trifluoroacetamide for the Determination of the Fecal Sterols by Gas Chromatography-Mass Spectrometry

摘要

Fecal coliform bacteria are widely used as indicators of sewage contamination in surface water. However, there are some disadvantages in these microbial techniques such as long analysis time needed (18-48h), lacking specificity, etc. Therefore, it is necessary to seek another, more specific indicator of human sanitary waste. Certain fecal sterols, metabolites of cholesterol, may be useful for this purpose. One of fecal sterols, coprostanol is formed during catabolism of cholesterol by indigenous bacteria present in the gut of humans and higher animals and is the primary sterols detected in domestic water. Unaffected by physical factors like temperature and salinity, fecal sterols have been used to monitor sewage and to detect fecal pollution in live-aboard marinas. A calculated concentration ratio of coprostanol (representing sewage concontamination) versus the sum of cholesterol and dihydrocholesterol (representing a rough estimate of sew sewage and non-sewage sources) has been utilized to measure the source of fecal contamination. Cononventinal analytical method for fecal sterols relied on the time consuming off-line derivatization before instrumental analysis. In this work, a novel analytical method based on solid-phase extraction (SPE) and injection-port silylation for the determination of five types of fecal sterols (coprostanol, cholestanol, epicholestanol, epicoprostanol, cholesterol) with gas chromatography-mass spectrometry (GC-MS) is presented. In this method, silylation of fecal sterols were performed at the GC injection-port with N-O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA). The factors influential to this technique such as injection-port temperature, purge time, derivatization reagent (BSTFA) volume, and the type of organic solvent were investigated. In addition, the conditions of SPE (including the type of SPE cartridge, the type of elution organic solvent) were also studied. After SPE followed by injection-port silylation by GC-MS, typical limits of detection (LODs) of fecal sterols were in the range of low ng/L in environmental water samples. Compared with traditional silylation of fecal sterols (performed with water bath (60℃, 30min)), this method is simpler and more convenient.