Environmental Engineering Reference
In-Depth Information
0.1 M aqueous acetic acid (pH 4) and methanol] in a 20mL borosilicate glass vial
(Wheaton; VWR Scientific, Rockwood, TN), and the mixture was homogenized
using a Tissuemiser (Fisher Scientific) at 30,000 rpm. Five surrogates (100.0
μ
g/
mL in acetonitrile) were added to each sample: acetaminophen-
d
4
(454 ng), fluox-
etine-
d
6
(636 ng), diphenhydramine-
d
3
(8.9 ng), carbamazepine-
d
10
(38.5 ng),
and ibuprofen-
13
C
3
(789 ng). Samples were shaken vigorously and mixed on a
rotary extractor for 5 min. Following extraction, samples were rinsed into 50-mL
polypropylene copolymer round-bottomed centrifuge tubes (Nalge Co.; Nalgene
Brand Products, Rochester, New York) using 1 mL of extraction solvent and cen-
trifuged at 16,000 rpm for 40 min at 4 °C. The supernatant was decanted into
18-mL disposable borosilicate glass culture tubes (VWR Scientific), and the
solvent was evaporated to dryness under a stream of nitrogen at 45 °C using a
Zymark Turbovap LC concentration workstation (Zymark Corp., Hopkinton,
MA). Samples were reconstituted in 1 mL of mobile phase, and a constant amount
of the internal standards 7-aminolunitrazapam-
d
7
(100 ng) and meclofenamic
acid (1000 ng) was added. Prior to analysis, samples were sonicated for 1 min and
filtered using Pall Acrodisc hydrophobic Teflon Supor membrane syringe filters
(13 mm diameter; 0.2
μ
m pore size; VWR Scientific, Suwanee, GA).
LC
-
MS/MS detection
(Ramirez et al.
2007
): A Varian ProStar model 210
binary pump equipped with a model 410 autosampler was used to detect the ana-
lytes, which were separated on a 15 cm
×
2.1 mm (5
μ
m, 80 Å) Extend-C18 col-
umn (Agilent Technologies, Palo Alto, CA) connected with an Extend-C18 guard
cartridge 12.5 mm
×
2.1 mm (5
μ
m, 80 Å) (Agilent Technologies). A binary
gradient consisting of 0.1 % (v/v) formic acid in water and 100 % methanol was
employed to achieve chromatographic separation, whereas the time-scheduled
elution program was as follows (min): 0, 2, 7, 12, 21 28, 34, 45, 50, 51, 65. The
mobile-phase composition for 0.1 % formic acid was 93, 93, 85, 85, 52, 52, 41, 2,
2, 93, 93 and for methanol was 7, 7, 15, 15, 48, 48, 59, 98, 98, 7, 7, respectively.
Additional chromatographic parameters were as follows: injection volume, 10
μ
L;
column temperature, 30 °C; flow rate, 350
μ
L min
−
1
. Eluted analytes were moni-
tored by MS/MS using a Varian model 1200L triple-quadrupole mass analyzer
equipped with an electrospray interface (ESI).
Each compound was infused individually into the mass spectrometer at a con-
centration of 1
μ
g mL
−
1
in aqueous 0.1 % (v/v) formic acid at a flow rate of 10
μ
L min
−
1
for determining the best ionization mode (ESI
+
or
−
) and optimal
MS/MS transitions for target analytes. All analytes were initially tested using both
positive and negative ionization modes while the first quadrupole was scanned
from
m
/
z
50 to [M
+
100]. This can enable identification of the optimal source
polarity and the most intense precursor ion for each compound. Once these param-
eters have been defined, the energy at the collision cell was varied, while the third
quadrupole was scanned to identify and optimize the intensity of product ions for
each compound. Additional instrumental parameters held constant for all analytes
were as follows: nebulizing gas, N
2
at 60 psi; drying gas, N
2
at 19 psi; tempera-
ture, 300 °C; needle voltage, 5000 V ESI
+
, 4500 V ESI-; declustering potential,
40 V; collision gas, argon at 2.0 mTorr.
