An efficient diethyl ether-based soxhlet protocol to quantify faecal sterols from catchment waters

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Abstract

A study was conducted to evaluate the efficiency and reproducibility of a diethyl ether-based soxhlet extraction procedure for faecal sterols occurring from catchment waters. Water samples spiked with a mixture of faecal sterols were filtered and analytes were extracted using the diethyl ether-based soxhlet method and the Bligh and Dyer chloroform extraction process. For diethyl ether-based soxhlet extraction procedure, solvent extracts were saponified with 100 μL of 10% KOH in methanol (100 °C/120 min) and then acidified with 60 μL of 6 M HCl. Lipid contents were extracted by ethanol (0.5 mL) from the saponification products. The lipid extracts were then reacted with 100 μL of bis(trimethyl)trifluoroacetamide (BSTFA) containing 1% trimethyl chlorosilane (100 °C/60 min) to form the trimethylsilyl (TMS) derivatives. The derivatised extracts were then analyzed by gas chromatography–mass spectrometry. For sterol concentrations ranging from 35 to 175 μg mL−1, the soxhlet-based extraction process yielded the following recovery efficiencies for coprostanol (101%), epicoprostanol (97%), cholesterol (97%), dihydrocholesterol (97%) and 5α-cholestane (111%), whereas the Bligh and Dyer process yielded recoveries of 32, 41, 0, 36 and 51%, respectively. The results suggested that the diethyl ether-based soxhlet extraction method was more efficient and reproducible than the Bligh and Dyer chloroform extraction process for the analyses of trace levels of faecal sterols from water samples. Moreover, it was revealed that the diethyl ether-based soxhlet extraction method used less solvent and was logistically easier.

Introduction

Coprostanol has been previously used as a chemical indicator of human sewage contamination [1], [2], [3], [4]. Coprostanol was shown to be produced in high amounts in the human gastrointestinal tract and represented the major faecal sterol comprising approximately 60% of the total sterols in human faeces [5]. It is produced by the metabolism of anaerobic bacteria via the process of biohydrogenation of cholesterol [6], [7]. Coprostanol can also be produced in some animals, but due to differences in diets of herbivores and differences in the gastric microbial flora, their coprostanol levels were observed to be relatively low [8]. In cattle and sheep faeces, 24-ethylcoprostanol was found to be the most prevalent sterol, while in dogs and birds faeces, coprostanol was not detected due to an absence of specific anaerobic bacteria in their digestive tracts that can perform biohydrogenation of cholesterol [8]. Analyses have revealed that human and animal faeces have characteristic faecal sterol “signatures”, which have been proposed as tools to distinguish the sources of faecal pollution. It should be noted that sources of non-faecal coprostanol can arise by the actions of non-intestinal bacteria on cholesterol in environments such as anoxic sediment and hydrophobic particulate matters [9], [10]. In order to minimize these confounding features and maximize interpretative capacity of faecal sterol results, a series of ratio analyses has been compiled by Leeming et al. [11], which allowed an assessment of human contribution to samples, as well as the likely input by herbivores and the presence of aged human faecal components.

This process of source tracking of the faecal pollution required quantitative determination of faecal sterols in water samples by gas chromatography–mass spectrometry (GC–MS). Water samples were first filtered to collect fractions of particulate and faecal sterol components, which were then partitioned by solvent extraction [8], [11]. This solvent extraction process has so far been achieved by following the method of Bligh and Dyer [12]. This method uses high volumes of solvents, which results in large volumes of wastes. No reference has yet been made in the evaluation of solvent recovery steps involving filtration and solvent extraction, which is imperative for accurate and efficient quantitative analyses [13]. This paper reports the evaluation of diethyl ether-based soxhlet protocol for the extraction of faecal sterols from filters (with trapped particulate matter filtered from catchment waters) in comparison with the Bligh and Dyer method for the target sterols—coprostanol, epicoprostanol, cholesterol and dihydrocholesterol and an internal standard, 5α-cholestane.

Section snippets

Reagents

All glassware were soaked in alkaline detergent solution followed by 5% nitric acid solution, washed with deionised water, dried in oven and rinsed with appropriate solvents prior to use for extraction. Diethyl ether was analytical reagent grade (99.7%, v/v) (Merck, Australia) and was distilled prior to its use as a solvent for soxhlet extraction. However, further analyses revealed that distillation of diethyl ether was not necessary. Four standards representative of faecal sterols were used.

Results and discussion

Filters were spiked with standard solutions containing low levels of the faecal sterols and the proposed internal standard, 5α-cholestane to subsequently assess and compare the recovery potentials from water samples using the soxhlet-based and the Bligh and Dyer extraction protocols. The standard solutions were analyzed in triplicate samples by GC–MS at stages before filtration and then after extraction by both methods to determine recovery efficiencies and blank samples (not spiked with sterol

Conclusions

The results of this investigation indicated that the diethyl ether-based soxhlet protocol provided an efficient process for analyzing catchment water samples that yielded better sensitivity, reproducibility and recovery efficiencies than the Bligh and Dyer method. 5α-Cholestane was demonstrated to have appropriate extraction properties and GC–MS characteristics to be utilized as an internal standard for faecal sterol assessment. The use of the soxhlet-based protocol also provided significant

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