An Efficient Ethanol-Based Analytical Protocol to Quantify Faecal Steroids in Marine Sediments

[摘要] Steroids are widely distributed in the marine environment as a result of numerous biological syntheses, diagenetic and degradation transformations, and anthropogenic inputs of organic matter. The marine biogeochemistry of steroids has been extensively studied since the mid-1970s.1 The behaviour of anthropogenic organic matter in marine environment plays an important role in our understanding of the effects of marine pollution. A group of steroids, derived from cholesterol in the digestive tract of higher animals, has often been used as biomarkers of sewage contamination in marine waters and sediments.2-5 Despite all the investigations of steroids in the marine environment, the current analytical methodologies for steroids in marine sediments are laborious and time-consuming, involve multi-step procedures and the use of large volumes of solvents, which consequently produces large quantities of wastes. Evaluation and optimisation of methods are essential for both economical and analytical results. Environmental benefits resulting from reduced amounts of hazardous wastes should also be considered. In general, the analytical methods comprise the following procedures: 1) extraction of the steroids from the matrix; 2) fractionation (also called clean up) of the extract by adsorption column chromatography; 3) derivatisation of the steroids into their trimethylsilyl ethers; 4) determination of the steroid by gas chromatography coupled to flame ionization detector, and 5) confirmation of the compounds with mass spectrometry. All these steps have to be adapted to meet the needs of specific applications. One must choose the most suitable procedures depending on the type and number of samples, type of analysis (only steroids or steroids and a series of other compounds, such as saturated and polycyclic aromatic hydrocarbons, chlorinated pesticides, polychlorinated biphenyls, fatty acids), and equipment available. In this work, a method for analysis of steroids commonly used in investigations of sewage contamination (coprostanol, epicoprostanol, cholesterol, cholestanol, 5a-coprostanone and 5b-coprostanone) has been developed. The method based on ethanol extraction has been compared to the others tested in terms of recoveries of standards and solvent facilities (i.e. toxicity, storage, costs). A rapid clean up has also been tested. The analytical performance of the method was assessed through analysis of reference material.  Experimental Reagents and glasswareAll glassware were soaked in solution of alkaline detergent, washed with distilled water and ethanol, dried in oven, and rinsed with organic solvents prior to use. The adsorbents alumina, Al2O3, (70-230 mesh) and silica gel, SiO2, (70-230 mesh) were heated overnight to 400 oC for activation and, after cooling in dissicator, 5% deactivated with pre-extracted water on a w/w basis as described in Aceves et al.6 Sodium sulphate was also heated to 400 oC and stored in dissicator. All solvents were pesticide grade except for ethanol, analytical grade, which was distilled. Authentic standards of steroids (Sigma) were dissolved in methylene chloride and diluted in concentrations ranging from 0.25 to 20.0 mg L-1. A synthetic matrix was prepared for the extraction tests with sodium sulphate (Na2SO4) spiked with a mixture of the steroid standards comprising coprostanol (5b-cholestan-5b-ol), epicoprostanol (5b-cholestan-3a-ol), cholesterol (cholest-5-en-3b-ol), 5b-coprostanone (5b-cholestan-3-one), 5a-coprostanone (5a-cholestan-3-one), cholestanol (5a-cholestan-3b-ol), and 5a-cholestane in the concentration range of 10 mg L-1.Instrumental analysisThe instrumental analysis was performed with a Hewlett-Packard 5890 series II gas chromatograph equipped with flame ionization detector and a splitless injector. The capillary column was of 25 m x 0.32 mm I.D. coated with Ultra-2 (5% phenyl-methyl silicone). The programmed temperature was 40 to 240 oC (1 min) at 20 oC min-1, 240 oC to 260 oC (1 min) at 1 oC min-1, and 260 oC to 310 oC at 50 oC min-1. The injector and detector temperatures were 300 and 325 oC respectively. Hydrogen was used as the carrier gas (2.0 mL s-1) and the injection volume was 1.0 mL. Calibration of the peak area to concentration was done with the steroid standards in the derivatized form within the range of 0.25 to 20.0 mg L-1, linear response for coprostanol was 0.992 (Figure 1). Standards of 5a-cholestane and 5a-androstanol (steroids frequently used as internal standards) and standards of petroleum and polycyclic aromatic hydrocarbons were also injected in the established chromatographic conditions. Data were acquired and processed on an HP Chemstation data system.   Detection limits (DL), defined as three times the standard deviations of the lowest steroid concentrations detected7 were in the range of 1 ng g-1 for the steroids.Extraction of steroidsTo check recoveries of steroids, extractions with different solvents were performed in triplicates using the synthetic matrix previously described (25.0 g). In test 1, the extraction was carried out with 80 mL of methanol (MERCK) reflux for 5 h (adapted from LeBlanc et al.8). Both tests 2 and 3 were performed for 6 h in a Soxhlet apparatus. Extraction of test 2 was done with 80 mL of a mixture of 50% acetone (J.BAKER) in methylene chloride (MERCK) and test 3 was extracted with 80 mL of distilled ethanol (Cia Nacional de Álcool). Test 4 (adapted from Mudge & Bebianno9) was done with 90 mL of 0.5 mol L-1 KOH in ethanol under reflux for 3 h (saponification and extraction simultaneously). Extracts of test 4 were treated via aqueous/organic liquid-liquid partitioning (1 mL of n-hexane/10 mL of pre-extracted water) in a separation funnel. The organic phase was reduced by rotary evaporation with the same following procedures of the previous tests. The resulting extracts of all tests were taken to the derivatisation procedure, described further on, and then analysed by gas chromatography.Fractionation proceduresTwo clean up procedures were assessed in glass columns of 38 x 0.7 cm I.D. The first test consisted of the glass column packed with 1.0 g of alumina and 2.0 g of silica gel (both 5% deactivated), in the top and bottom of the column respectively. It was introduced 1.0 mL of the steroid standard solution (10 mg L-1). The fractionation was done with the following increasing polarity gradient: 15.0 mL of n-hexane; 15.0 mL of 20% methylene chloride in n-hexane; and 15.0 mL of 50% acetone in methylene chloride, and provided three fractions with the last one comprising the steroids. The second clean up test was carried out with 2.0 g of alumina and elution with 15.0 mL of distilled ethanol, providing one fraction.Derivatisation of steroidsThe fractions obtained from the extraction and cleanup tests, after reduced by vacuum evaporation and transferred to 1.0 mL vials, were taken to dryness under nitrogen blow and submitted to derivatisation with the commercial mixture N,O,-bis(trimethylsilyl)trifluoracetamide and trimethylchlorosilane, BSTFA/TMCS 99:1 (Supelco), 40 mL, 65 oC/1.5 h (adapted from Green et al.10). After reaction, the final residues were redissolved to 1.0 mL with methylene chloride/n-hexane (1:1) and stored in glass ampoules until gas chromatographic analysis.Analysis of reference materialReference material (lyophilised marine sediment) provided by the International Atomic Energy Agency (IAEA-383) was also analysed for steroids. The solvent of choice for the extraction was ethanol and the clean up procedure consisted of alumina column/ethanol elution. A summary of the analytical protocol is shown in Figure 2.  Results and Discussion Recovery of steroidsDue to the low concentrations of total and individual steroids in the water column and sediments, these compounds are typically analysed by gas chromatography after forming their trimethylsilyl ether derivatives. In order to perform gas chromatographic analysis, steroids should be transformed in more volatile compounds. In the

[学科分类] 化学（综合）