**2.3. On-line SPE**

To meet the ever-growing demands for sensitivity, reliability and speed, the continuous development of more efficient methods for both sample pretreatment and analysis is crucial.

Current Trends in Sample Treatment Techniques for Environmental and Food Analysis 137

**Figure 2.** Typical setup of an on-line SPE-LC system with a 2-position 6-port switching valve; (a) Load

Recent advances in technology have made the Ultra High Performance Liquid Chromatography (UHPLC)/mass spectrometry (MS) system a perfect candidate for combination with on-line SPE. The on-line SPE-UHPLC/MS allows complete separation of

Gosetti et al. [5], applied an automated on-line SPE UHPLC–MS/MS method for the identification and quantification of nine perfluorinated compounds (PFCs) in matrices of environmental, biological and food interest. The SPE protocol was performed by using an anion exchange SPE column (Poros HQ column). The separation of nine PFCs was obtained within 7 min. The limits of detection (LODs) ranged from 3 to 15 ng L−1 whereas the limits of

The same authors performed a simultaneous determination of thirteen polycyclic aromatic hydrocarbons (PAHs) and twelve aldehydes in cooked food by means of an automated online SPE-UHPLC-MS. The resolution allowed the separation of four couples of PAH isomers. SPE treatment was made using Strata C18-E column and the extraction procedure was carefully optimized in order to apply the whole methodology to the analysis of different food matrices as salmon, frankfurter, steak, and pork chop, subjected to different cooking modes (smoked, grilled, cooked in oil or in butter). LODs values ranging from 0.028 to 0.768 µg L−1 for PAHs and from 0.002 to 0.125 µg L−1 for aldehydes were obtained [4].

Previously, PAH, such as naphthalene, biphenyl, acenaphthene, anthracene and pyrene have been determined in natural water by using on-line SPE–HPLC-UV [47]. Fluorocarbon polymer SPE sorbent was used. This application resulted in better extraction selectivity towards PAHs in comparison with several other sorbents and provided no additional peak broadening. Detection limits of method were established as 5 ng L−1 (biphenyl), 7 ng L−<sup>1</sup>

(anthracene), 8 ng L−1 (acenaphthene), 30 ng L−1 (pyrene), 40 ng L−1 (naphthalene).

high number of analytes via a single chromatographic run that takes few minutes.

position and (b) injection position

quantification (LOQs) from 10 to 50 ng L−1.

SPE technique can be easily coupled on-line to high performance liquid chromatography (HPLC) and gaschromatography (GC) systems. On-line systems are beneficial when the amount of sample is limited, or when very high sensitivity is required. In most cases, even though the use of an automated on-line instrument is quite straightforward, experienced personnel are required for method development and eventual trouble-shooting.

The strong differences among the solid phase extraction principle and the gas chromatography analysis made the on-line combination of SPE and GC more complicated.

Nevertheless, the combination with GC has already been successfully applied in environmental analysis [44-46]. In the on-line SPE-GC the analytes are trapped in a short column (10-20 mm×1-4.6 mm i.d.) packed with a suitable stationary phase (typically C8, C18 or styrene-divinylbenzene copolymer). The SPE procedures are essentially the same as the off-line ones. It involves conditioning of the SPE material before loading of the sample. Since water is not a good solvent for GC, primarily because it hydrolyses the siloxane bonds in GC columns causing deterioration of the column performance, the introduction of water directly to GC should be avoided. After trapping, and before elution of the analytes, the SPE column is often dried with a gas flow, or the extract is dried with a separate drying column packed with copper sulphate or silica to remove water, which is placed after the SPE column. The column can also be heated during the drying process, but this increases the risk of losing volatile analytes. The elution of the analytes is performed with a solvent suitable for the GC injector system.

On the other hand, the methods which combine SPE with HPLC are the most frequently used in environmental and food analysis, mainly to determine polar compounds in water solution. Different systems and configurations are available. The most commonly used approach involves the implementation of a small SPE column within the injection loop of a six-port rotary valve (**Figure 2**).

After conditioning, sample application, and eventual clean-up by means of a high-pressure pump, the SPE column is placed in front of an analytical column by switching the valve into the "inject" position. A sample is thus loaded in this SPE column, whereupon the valve is switched in order to elute the analytes out of the sorbent by the LC mobile phase and transfer them into the analytical column [3]. The SPE column is reusable. However, the reusability can cause a progressive deterioration of the column material and thus, lead to a change in their selectivity and capacity. Moreover, the SPE column must be filled with a sorbent compatible with the sorbent of the analytical column which efficiently traps the analytes. The SPE column should be as small as possible in order to prevent band broadening. Usually, the dimension of stainless steel columns is 30 mm length, 2 mm i.d. and 8 mm length, 3 mm i.d.

To meet the ever-growing demands for sensitivity, reliability and speed, the continuous development of more efficient methods for both sample pretreatment and analysis is crucial. SPE technique can be easily coupled on-line to high performance liquid chromatography (HPLC) and gaschromatography (GC) systems. On-line systems are beneficial when the amount of sample is limited, or when very high sensitivity is required. In most cases, even though the use of an automated on-line instrument is quite straightforward, experienced

The strong differences among the solid phase extraction principle and the gas chromatography analysis made the on-line combination of SPE and GC more complicated.

Nevertheless, the combination with GC has already been successfully applied in environmental analysis [44-46]. In the on-line SPE-GC the analytes are trapped in a short column (10-20 mm×1-4.6 mm i.d.) packed with a suitable stationary phase (typically C8, C18 or styrene-divinylbenzene copolymer). The SPE procedures are essentially the same as the off-line ones. It involves conditioning of the SPE material before loading of the sample. Since water is not a good solvent for GC, primarily because it hydrolyses the siloxane bonds in GC columns causing deterioration of the column performance, the introduction of water directly to GC should be avoided. After trapping, and before elution of the analytes, the SPE column is often dried with a gas flow, or the extract is dried with a separate drying column packed with copper sulphate or silica to remove water, which is placed after the SPE column. The column can also be heated during the drying process, but this increases the risk of losing volatile analytes. The elution of the analytes is performed with a solvent suitable for the GC

On the other hand, the methods which combine SPE with HPLC are the most frequently used in environmental and food analysis, mainly to determine polar compounds in water solution. Different systems and configurations are available. The most commonly used approach involves the implementation of a small SPE column within the injection loop of a

After conditioning, sample application, and eventual clean-up by means of a high-pressure pump, the SPE column is placed in front of an analytical column by switching the valve into the "inject" position. A sample is thus loaded in this SPE column, whereupon the valve is switched in order to elute the analytes out of the sorbent by the LC mobile phase and transfer them into the analytical column [3]. The SPE column is reusable. However, the reusability can cause a progressive deterioration of the column material and thus, lead to a change in their selectivity and capacity. Moreover, the SPE column must be filled with a sorbent compatible with the sorbent of the analytical column which efficiently traps the analytes. The SPE column should be as small as possible in order to prevent band broadening. Usually, the dimension of stainless steel columns is 30 mm length, 2 mm i.d.

personnel are required for method development and eventual trouble-shooting.

**2.3. On-line SPE** 

injector system.

six-port rotary valve (**Figure 2**).

and 8 mm length, 3 mm i.d.

**Figure 2.** Typical setup of an on-line SPE-LC system with a 2-position 6-port switching valve; (a) Load position and (b) injection position

Recent advances in technology have made the Ultra High Performance Liquid Chromatography (UHPLC)/mass spectrometry (MS) system a perfect candidate for combination with on-line SPE. The on-line SPE-UHPLC/MS allows complete separation of high number of analytes via a single chromatographic run that takes few minutes.

Gosetti et al. [5], applied an automated on-line SPE UHPLC–MS/MS method for the identification and quantification of nine perfluorinated compounds (PFCs) in matrices of environmental, biological and food interest. The SPE protocol was performed by using an anion exchange SPE column (Poros HQ column). The separation of nine PFCs was obtained within 7 min. The limits of detection (LODs) ranged from 3 to 15 ng L−1 whereas the limits of quantification (LOQs) from 10 to 50 ng L−1.

The same authors performed a simultaneous determination of thirteen polycyclic aromatic hydrocarbons (PAHs) and twelve aldehydes in cooked food by means of an automated online SPE-UHPLC-MS. The resolution allowed the separation of four couples of PAH isomers. SPE treatment was made using Strata C18-E column and the extraction procedure was carefully optimized in order to apply the whole methodology to the analysis of different food matrices as salmon, frankfurter, steak, and pork chop, subjected to different cooking modes (smoked, grilled, cooked in oil or in butter). LODs values ranging from 0.028 to 0.768 µg L−1 for PAHs and from 0.002 to 0.125 µg L−1 for aldehydes were obtained [4].

Previously, PAH, such as naphthalene, biphenyl, acenaphthene, anthracene and pyrene have been determined in natural water by using on-line SPE–HPLC-UV [47]. Fluorocarbon polymer SPE sorbent was used. This application resulted in better extraction selectivity towards PAHs in comparison with several other sorbents and provided no additional peak broadening. Detection limits of method were established as 5 ng L−1 (biphenyl), 7 ng L−<sup>1</sup> (anthracene), 8 ng L−1 (acenaphthene), 30 ng L−1 (pyrene), 40 ng L−1 (naphthalene).

Gallart-Ayala et al. [48], used an automated on-line SPE fast LC–MS/MS method for the simultaneous analysis of bisphenol A (BPA), bisphenol F (BPF), bisphenol E (BPE), bisphenol B (BPB) and bisphenol S (BPS) in canned soft drinks without any previous sample treatment. SPE on-line pre-concentration was performed by using a C18 cartridge. The analysis of all compounds was accomplished in 3 min. Quality parameters of the method were established and the authors obtained a simple, fast, reproducible (RSD values lower than 10%) and accurate (trueness higher than 93%) method for the analysis of bisphenols in canned soft drinks at the ng L−1 level using matrix-matched calibration.

Current Trends in Sample Treatment Techniques for Environmental and Food Analysis 139

**Figure 3.** Schematic representation of non-covalent molecular imprinting procedures: (1) complex formation between the template (methacrylic acid) and the functional monomers (estriol), (2)

Out of all the MIPs applications, the use of MIPs as selective sorbents for solid-phase extraction (MIP-SPE) represents the most important application area in the field of analytical separation sciences [54]. Solid-phase extraction (SPE) is a well-established method routinely used for clean-up and pre-concentration of analytes in a wide range of environmental, pharmaceutical, agricultural and food analysis [1]. Nevertheless, sorbents used in conventional SPE often lack selectivity resulting in co-extraction of interfering matrix components. Therefore, specificity, selectivity and sensitivity together with high extraction efficiency can be obtained using sorbents based on molecularly imprinted polymers

To assess the potential of MIPs in terms of selectivity, we have compared the ability of MIP-SPE for selective extraction of zearalenone from cereal sample extracts with that of a commercial immunoaffinity column (IAC). **Figure 4** shows the similarity of the behavior of these two types of selective sorbents, resulted in high degrees of clean-up. In both cases, very reliable baselines and similar recoveries were obtained, proving that the high selectivity of immunoaffinity sorbents also can be achieved with molecularly imprinted polymers SPE. Furthermore, previous studies have found MIP-SPE to have a similar

selectivity but a higher capacity than commercial IAC columns [49,55].

polymerization, (3) template extraction, (4) analyte rebinding.

**3.1. Applications of MIPs to SPE** 

(MIPs) [8].

Finally, in the current year, Vega-Morales et al. [2] used an on-line SPE-UHPLC-MS/MS to characterize 27 endocrine disrupting compounds (norethindrone, norgestrel, 17-alphaethinyloestradiol, etc.) in sewage samples. SPE treatment was performed by using Oasis HLB columns (mixed-mode sorbent). The complete analysis of each sample required less than 4 min and provided satisfactory recoveries (72–110%) and limits of detection in the order of few nanograms per liter (0.3-2.1 ng L−1).
