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codeine and lincomycin, yielded quantitative trapping recovery using the EQuan system.
Figure 3. Area response plots for ranitidine demonstrating the pH effect on the preconcentration column.
Effects of varying %Acetontrile The effect on the LC-MS/MS response for the PPCPs was examined as a function of the %ACN in the water samples. Many of the larger, more lipophilic compounds, such as the macrolide antibiotics, showed a significant increase in area response as a function of increasing %ACN in the water sample. For tylosin and roxithromycin, the increased response was most dramatic between 5% and 10% ACN at pH 2.9. The area response increased by a factor of 3 for roxithromycin and a factor of 10 for tylosin when the %ACN was increased from 5% to 10%. The same trend was observed with LC-
MS/MS (5 µL injection) as with the EQuan system indicating that this is a sample solubility effect due to the compounds’ lipophilic nature.
Figure 4. Area response plots showing effect of decreased retention with increasing %ACN.
Although increasing the %ACN in the water sample helped the response of certain PPCPs, it caused a significant decrease in response if the percentage was too high (Figure 4). This effect, observed for ciprofloxacin, trimethoprim, fluoroquinolones, and sulpha drugs, was attributed to a loss of compound retention on the trapping column. Here, compounds have a greater affinity for the solvent than the trapping column stationary phase. This effect is similar to compound “break through” on an SPE cartridge. No reduction
in MS response was observed with a 5 µL injection onto the analytical column.
The effect of decreased analyte retention with increased %ACN in the water sample
was also observed with cotinine using a 5 µL injection on the analytical C18 column. As Figure 5 shows, the LC peak splitting for cotinine was readily observed in acidic (red) and neutral (green) water samples. However, at pH 11.3 the cotinine peak was largely unchanged, even at 20% ACN. This is probably because the basic compound cotinine is uncharged at pH 11.3, which increases its affinity for the C18 stationary phase.
Figure 5. Chromatograms showing the %ACN effect on LC column retention for cotinine.
in response is believed to be attributed to the change in the local partitioning chemistry between ranitidine and the stationary phase of the pre-concentration column; the portioning chemistry was not affected for a
long enough period to change the retention of ranitidine. Nevertheless, under the right sample solution conditions, namely pH 11.3, and 5 - 10% acetonitrile (ACN), ranitidine and other basic PPCPs, such as cimetidine,
As seen with cotinine, the biggest challenge in developing an EQuan method for PPCPs was the small, highly polar organic compounds. Different trapping columns and mobile phases were tested. However, as expected, compromises had to be made to
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