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Effects of %ACN
The effect on the LC-MS/MS response for the PPCPs was
examined as a function of the % ACN in the water sam-
ples. 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 ef-
fect due to the compounds’ lipophilic nature.
Although increasing the %ACN in the water sample
helped the response of certain PPCPs, it caused a signifi-
cant decrease in response in others if the percentage was
too high (Figure 5). This effect, observed for ciprofloxacin,
trimethoprim, fluoroquinolones, and sulfa drugs, was at-
tributed to a loss of compound retention on the trapping
column, where 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 fall-off in MS response was observed with a
5 µL injection onto the analytical column.
The effect of decreased analyte retention with increas-
ing %ACN in the water sample was also observed with
cotinine using a 5 µL injection on the analytical C18
column. As Figure 6 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 virtually unchanged, even at 20% ACN. This is
likely due to the fact that the basic compound cotinine is
uncharged at pH 11.3, which increases its affinity for the
C18 stationary phase.
As seen with cotinine, the biggest challenge in develop-
ing an EQuan method for PPCPs was the small, highly-
polar organic compounds. Different trapping columns and
mobile phases were tested, but as expected, compromises
had to be made to allow the largest breadth of PPCPs in
one LC-MS/MS run. Metformin was the clearest example.
Despite many approaches, no satisfactory reverse-phase
LC method could be discovered because of its very high
polarity. Hence, as described in EPA Method 1694,
hydrophilic interaction liquid chromatography (HILIC)
was used for the successful LC separation of metformin in
water. Again, pH had a dramatic effect on the response of
metformin (and other Group 4 PPCPs). The best response
for metformin was with the water sample adjusted to pH
11.3 prior to injection on the reverse-phase EQuan trap-
ping column.
EQuan Method Summary
Despite all of the challenges in the development of one
single LC/MS method for this diverse group of compo-
nents, a balance was found that allowed the measurement
of the 67 PPCPs in water by the EQuan system, with a
large majority being quantified at or below 10 ng/L using
a 0.5 mL injection volume with detection on the TSQ
Vantage mass spectrometer.
The best compromise for the online sample prepara-
tion method was to run an acidified and a basified water
sample containing 10% ACN. Figure 7 shows example
chromatograms for the PPCPs in water at the ng/L level us-
ing this approach. The red chromatograms were the water
samples at pH 2.9, and the blue chromatograms were the
water samples at pH 11.3. In general, basic conditions
were preferable for analyzing the smaller, more polar com-
pounds, and acidic conditions were preferable for analyz-
ing the larger, more lipophilic compounds.