Sample Preparation and Liquid Chromatography Mass Spectrometry Analysis of Alkylphenolic Compounds and Steroid Sex Hormones in Sediments

A new methodology, based on the use of accelerated solvent extraction (ASE) and highly selective cleanup using restricted access material (RAM) on-line coupled with liquid chromatography–mass spectrometry (LC–MS), is presented for the simultaneous and unequivocal determination of alkylphenol ethoxylates (APEOs), their degradation products and halogenated derivatives, and steroid sex hormones in sediment samples. Using the integrated RAM–LC–MS system, the simultaneous determination of alkylphenolic compounds and sex hormones was achieved, yielding recoveries higher than 60% and producing low MS background noise.


INTRODUCTION
Contaminated sediment, especially sludge produced by sewage treatment plants (STPs), can be considered as one of the most complex matrices to be analyzed. In order to isolate endocrinedisrupting compounds (EDCs) from the solid matrix and to achieve the low limits of detection required, substantial analyte enrichment is necessary. Different extraction protocols, employing Soxhlet extraction, sonication, supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), or microwave-assisted extraction (MAE), have been developed [1]. Because of the complexity of samples, a substantial amount of interfering substances are found in crude extracts. The cleanup step aimed at the reduction of the matrix content is generally based either on solidphase extraction (SPE) or on solid-liquid adsorption chromatography in open columns using a combination of different adsorbents. However, high matrix load affects the performance of the extraction sorbent and gives rise to the partial coextraction of interfering substances.
Among the different sample preparation techniques available, an integrated columnswitching system, in which a precolumn filled with restricted access material (RAM) is coupled to the analytical liquid chromatography (LC) column via a switching valve, offers fully automated on-line enrichment of analytes and sample cleanup[2, 3,4,5]. In RAM-LC integrated systems, the analyte fraction is selectively extracted and enriched at the stationary phase, while the matrix components are flushed into waste. The porous LiChrospher RP-ADS RAM precolumns (Merck, Darmstadt, Germany), consisting of a hydrophilic and an electroneutral external particle surface (alkyl-diol silica) and a hydrophobic reversed-phase internal surface (C4, C8, or C18), are specially designed for the direct and repetitive injection of samples. The bimodal properties allow retention of low molecular analytes at the lipophilic pore surface, while macromolecular constituents are excluded (cut-off 15 kDa). After the enrichment and cleanup step, the analyte fraction is transferred in the back-flush mode from the precolumn onto an analytical column. All transfer and elution processes and the conditioning of both columns are performed with continuously pumping devices, resulting in a fully automated system and high sample throughput.
The multidimensional LC combined with an effective extraction using ASE saves hours of sample preparation in comparison with conventional sample preparation techniques ( Fig. 1) and gives improved results. However, in order to achieve good precision, accuracy, and sensitivity of determination, the operating parameters should be carefully optimized. These include the selection of a sorbent material (C4, C8, or C18), the selection of appropriate mobile phases, for both, the elution of matrix components and for the transfer of analyte onto LC column, and the switching time as defined by the matrix elution profile, breakthrough time of analytes, and analyte elution profile.

Accelerated Solvent Extraction of Solid Samples
Extractions were carried out using a Dionex ASE 200 (Dionex, Idstein, Germany). For extraction, 5.00 g of freeze-dried sediment was mixed thoroughly with Na 2 SO 4 and filled into 11-ml stainless-steel extraction cells. Different solvent mixtures and temperatures were tested, and optimized conditions were as follows: a mixture of acetone/methanol (1:1, v/v) was used as the extraction solvent, with temperature 50 o C, pressure 1500 psi, heating time 5 min, and two 5-min cycles of static extraction. As a final step, the cell was purged with gaseous nitrogen. The total volume of extract was ~20 ml. The extracts were concentrated to an approximate volume of 1 ml using a rotary vacuum evaporator at 30 o C and redissolved in 100 ml of HPLC water. Subsequent cleanup of extracts was performed by SPE using LiChrolute C18 cartridges (Merck, Darmstadt, Germany) as described elsewhere [6]. All experiments were performed with river sediment spiked with 100 ng/g of the composite standard solution of alkylphenolic compounds and steroid sex hormones 72 h before analysis.

RAM-LC Column Switching
The column-switching program is shown in Table 1. The first step was a fractionation step. The sample was applied via an autosampler to the RAM column and flushed using the mobile phase delivered by an HP 1090 pump. During this step, the analytes were retained on the RAM precolumn, while matrix components were flushed into the waste.
Step 2 was the transfer of analytes from the RAM column onto the analytical column using the mobile phase with the stronger elution power, delivered by pump 1. The third step was separation performed in a conventional manner.

RESULTS AND DISCUSSION
In order to optimize the switching time for steps 1 and 2, numerous experiments were performed comparing performances of different ADS RAM precolumns (C4, C8, and C18) and testing different solvents for matrix elution and the analyte transfer. In an ideal situation, the precolumn packing should only retain and enrich the analyte(s) while all other sample components (unwanted matrix) are discharged to waste with the eluent. The best results in terms of recovery of target compounds and selectivity were obtained using the LiChrospher ADS C4 precolumn. It was found that for hydrophilic analytes (APECs), the C8 and C18 precolumns had an advantage with respect to long breakthrough time, but transfer of the most hydrophobic compounds (APs and XAPs) was poor, as was the removal of matrix components. From the other side, using the C4 precolumn, very good fractionation of matrix components and analytes was achieved and good transfer of hydrophobic compounds. However, the breakthrough time for the most polar compounds was short (Table 2), and therefore the fractionation step was limited to a maximum of 5 min. Recoveries (using the ADS C4 precolumn) ranged from 62 to 104% with RSD from 0.87 to 14.5%. Chromatograms showing the separation of alkylphenolic compounds and steroid sex hormones, extracted from the spiked sediments using a RAM-LC integrated system, are shown in Figs. 2 and 3, respectively. When using RAM for cleanup of sediment extracts, the analytes and hydrophobic smaller matrix molecules are adsorbed, while larger matrix molecules, such as    humic substances or proteins, as well as very polar matrix components are eluted in the dead volume of the RAM column and pumped into the waste. The efficiency of the cleanup process is shown in Fig. 4. The elimination of interfering polar matrix components led to more sensitive detection of estradiol. Generally, limits of detection (LODs) achieved using RAM-LC were approximately one order of magnitude lower than those achieved using SPE cleanup.