2 Affinity-Based Methods for the Analysis of Emerging …
43
within an SPE cartridge or disposable syringe (Hage 1998). This support is then used
in a sample application and elution scheme like the one shown in Fig. 2.1 to capture
and concentrate the desired target and to wash away non-bound sample components.
The target analyte is then collected by passing an elution buffer through the support
(Ferguson et al. 2001; Nelson and Hage 2006; Qiao et al. 2009; Ryu et al. 2015;
Laranjeiro et al. 2018). This target can be used directly or placed into an alternative
solvent for analysis by LC, GC, MS, or capillary electrophoresis (Hage 1998; Hage
and Nelson 2001). This technique avoids the laborious extraction and derivatization
steps that are often needed in trace analysis with more traditional sample pretreatment methods. Off-line immunoextraction does typically require manual steps for
sample handling and solution transfer, which can limit the precision and accuracy of
this method. In addition, some off-line immunoextraction techniques, such as those
used with GC, may require solvent evaporation and target resuspension in an organic
solvent, thereby increasing the cost and overall analysis time (Ferguson et al. 2001;
Nelson and Hage 2006; Qiao et al. 2009; Ryu et al. 2015; Laranjeiro et al. 2018).
A number of reports have described the use of off-line immunoextraction with
RPLC for environmental analysis (Hage and Nelson 2001). For example, this combination has been used to determine 17β-estradiol, estrone, and 17α-ethynylestradiol
in wastewater (Ferguson et al. 2001). In this approach, monoclonal antibodies that
could specifically bind 17β-estradiol or estrone were immobilized to controlled-pore
glass beads. Samples of wastewater effluent were filtered and spiked with deuteriumlabeled estrogens. The estrogens in these samples were extracted by using commercial supports in a layered-bed SPE system that contained both an ethylvinylbenzenedivinylbenzene polymer and a C18 adsorbent. The extracts were collected, evaporated
to dryness, and resuspended in a solution of 5% methanol in water. The resuspended
extracts were then passed through the immunosorbent and eluted with 70% methanol
in water. The recovery was greater than 90% for both 17β-estradiol and estrone. Separation of estrogens in the eluted fraction was carried out on an analytical RPLC
column, and these compounds were detected by MS using selected ion monitoring
and electrospray ionization (ESI). Figure 2.3 shows some chromatograms that were
obtained with and without the use of immunoextraction for wastewater effluents from
sewage plants. The chromatograms generated when using off-line immunoextraction
resulted in well-defined peaks for each of the target steroids, with detection limits
for these analytes in the low ng L
−1 range (Ferguson et al. 2001).
Several other reports have utilized off-line immunoextraction for the analysis of
trace contaminants in wastewater and related samples. One study used this method
to measure levonorgestrel (Qiao et al. 2009) by employing polyclonal antibodies
for this analyte that were immobilized to activated Sepharose. Levonorgestrel was
measured at levels in the ng L
−1 range in wastewater (Qiao et al. 2009). Immunoextraction followed by LC-MS/MS has been used to measure zearalenone in surface
waters that were collected upstream from a water treatment plant (Laranjeiro et al.
2018). This work used a commercial wide bore immunoaffinity column that contained monoclonal antibodies specific for this mycotoxin. This immunosorbent was
combined with a C18 RPLC column and LC-MS/MS to detect zearalenone in the
low-to-mid ng L
−1 range (Laranjeiro et al. 2018).
43
within an SPE cartridge or disposable syringe (Hage 1998). This support is then used
in a sample application and elution scheme like the one shown in Fig. 2.1 to capture
and concentrate the desired target and to wash away non-bound sample components.
The target analyte is then collected by passing an elution buffer through the support
(Ferguson et al. 2001; Nelson and Hage 2006; Qiao et al. 2009; Ryu et al. 2015;
Laranjeiro et al. 2018). This target can be used directly or placed into an alternative
solvent for analysis by LC, GC, MS, or capillary electrophoresis (Hage 1998; Hage
and Nelson 2001). This technique avoids the laborious extraction and derivatization
steps that are often needed in trace analysis with more traditional sample pretreatment methods. Off-line immunoextraction does typically require manual steps for
sample handling and solution transfer, which can limit the precision and accuracy of
this method. In addition, some off-line immunoextraction techniques, such as those
used with GC, may require solvent evaporation and target resuspension in an organic
solvent, thereby increasing the cost and overall analysis time (Ferguson et al. 2001;
Nelson and Hage 2006; Qiao et al. 2009; Ryu et al. 2015; Laranjeiro et al. 2018).
A number of reports have described the use of off-line immunoextraction with
RPLC for environmental analysis (Hage and Nelson 2001). For example, this combination has been used to determine 17β-estradiol, estrone, and 17α-ethynylestradiol
in wastewater (Ferguson et al. 2001). In this approach, monoclonal antibodies that
could specifically bind 17β-estradiol or estrone were immobilized to controlled-pore
glass beads. Samples of wastewater effluent were filtered and spiked with deuteriumlabeled estrogens. The estrogens in these samples were extracted by using commercial supports in a layered-bed SPE system that contained both an ethylvinylbenzenedivinylbenzene polymer and a C18 adsorbent. The extracts were collected, evaporated
to dryness, and resuspended in a solution of 5% methanol in water. The resuspended
extracts were then passed through the immunosorbent and eluted with 70% methanol
in water. The recovery was greater than 90% for both 17β-estradiol and estrone. Separation of estrogens in the eluted fraction was carried out on an analytical RPLC
column, and these compounds were detected by MS using selected ion monitoring
and electrospray ionization (ESI). Figure 2.3 shows some chromatograms that were
obtained with and without the use of immunoextraction for wastewater effluents from
sewage plants. The chromatograms generated when using off-line immunoextraction
resulted in well-defined peaks for each of the target steroids, with detection limits
for these analytes in the low ng L
−1 range (Ferguson et al. 2001).
Several other reports have utilized off-line immunoextraction for the analysis of
trace contaminants in wastewater and related samples. One study used this method
to measure levonorgestrel (Qiao et al. 2009) by employing polyclonal antibodies
for this analyte that were immobilized to activated Sepharose. Levonorgestrel was
measured at levels in the ng L
−1 range in wastewater (Qiao et al. 2009). Immunoextraction followed by LC-MS/MS has been used to measure zearalenone in surface
waters that were collected upstream from a water treatment plant (Laranjeiro et al.
2018). This work used a commercial wide bore immunoaffinity column that contained monoclonal antibodies specific for this mycotoxin. This immunosorbent was
combined with a C18 RPLC column and LC-MS/MS to detect zearalenone in the
low-to-mid ng L
−1 range (Laranjeiro et al. 2018).