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2
Experimental
Materials and Reagents
Certified PAH and isotopically labeled PAH standard
mixtures, along with additional single PAH standards,
were used.
18
Standard reference materials (SRM 2260a
and SRM 1491a) were obtained from NIST
(Gaithersburg, MD). Stock solutions were stored at
−20 °C until needed. Chlorobenzene dopant (extra dry,
99.8% pure) is available from Thermo Fisher Scientific.
Artificial seawater (3.5% w/v) was prepared using the
commercially available Instant Ocean
®
salt.
Chromatographic studies were performed using
Fisher Chemical
™
Optima
™
LC/MS-grade
acetonitrile, methanol, and water.
Sample Collection
All glassware used to collect and store samples was
cleaned by heating to 450 °C for at least 6 h before use.
Field samples were collected using 60 mL amber glass
vials rinsed once with surface water, filled, and capped
with PTFE-lined plastic caps being careful to eliminate
trapped air. Vials were then placed in plastic bags and
transported on ice to the laboratory. A sampling blank,
consisting of a 60 mL vial filled with artificial seawater,
was placed on ice and transported during sampling.
Seawater samples were collected in a single trip during
August 2012 around Northern Biscayne Bay, adjacent to
the metropolitan area of Miami, Florida. Two reclaimed
water samples were collected from the North District
Wastewater Treatment Plant in the Miami-Dade County
during August and September 2012.
Rainwater runoff samples were collected during a heavy
rain event in June 2013 from drainage openings in two
parking lots at the Florida International University (FIU)
Biscayne Bay campus and at the parking lot of a nearby
residential complex. A reference rainwater sample was
collected during the same event using a 1 L amber glass
bottle and a glass funnel. All samples were stored at 4 °C.
Seawater samples were analyzed no more than 14 days
after collection. Rainwater, rainwater runoff, and
reclaimed waters were analyzed within 24 h of collection.
Sample Preparation
Environmental water
Working solutions of all PAHs were prepared each
analysis day in methanol from stock solutions or certified
standards. Refrigerated samples were allowed to reach
room temperature before preparation. Vials were
vigorously shaken for at least 20 s. Then 10 mL aliquots
of raw water samples were transferred using disposable
glass graduated pipettes directly from the sampling
containers into 10 mL LC vials containing 0.55 mL of a
methanol solution of isotopically labeled PAHs and
0.45 mL of water. The resulting solutions contained
5% methanol and 95% water with 100 ng/L of each
isotopically labeled PAH. Solutions were capped,
thoroughly mixed, and loaded into the online SPE
system without further treatment.
Calibration solutions
Matrix-matched calibration solutions (5–500 ng/L) were
prepared using the same procedure, using artificial
seawater and working solutions containing analytes and
internal standards in methanol. A seven-point set of
calibration solutions was freshly prepared for each
analysis batch.
Liquid Chromatography
Online preconcentration was performed using an EQuan
online SPE system consisting of an HTC-PAL
™
autosampler system (CTC Analytics, Zwingen,
Switzerland) equipped with a 5 mL glass syringe, a
Thermo Scientific
™
Accela
™
1000 LC pump as an
analytical HPLC pump, and an Accela 600 LC pump
as an SPE loading pump. The online SPE column was a
Thermo Scientific
™
Hypersil GOLD aQ
™
column
(20 × 2.1 mm, 12 μm particle size). Analytical separations
were carried out using a Hypersil Green PAH column
(150 × 2.1 mm, 3 μm particle size), protected by a
Hypersil Green PAH guard column (10 × 2.1 mm,
3 μm particle size). Stainless steel tubing was used
throughout the SPE-LC-MS/MS system. Dopant to
assist photo-ionization was delivered using the combined
output of two programmable syringe pumps.
The samples, quality controls, and calibration solutions
were loaded onto the 10 mL stainless steel loop (rotary
valve A, Figure 1). The SPE column was placed in a
second rotary valve (valve B, Figure 1), allowing
connection with either the loading pump or the analytical
pump. Analysis steps, determined by valve turning events,
are graphically presented in Figure 1. Ten milliliters of
sample were passed through the SPE column within
5 min, followed by 2 mL of 1% methanol in water to
remove inorganic species. Then, 0.5 mL of a short
gradient to 60% methanol and 0.5 mL of 60% methanol
were passed to prepare the SPE column for connection
with the organic-rich analytical stream (Step 1). The SPE
column was connected to the analytical column and
gradient separation was started, while the sample loop
was completely filled with methanol from the SPE LC
pump (Step 2). At 15 min, valve A turned and the
methanol-filled sample loop was connected with the
injection port. The autosampler sequentially injected
5 mL of methanol, 5 mL of water, and two 5 mL portions
of the next sample in the queue while the chromato-
graphic separation continued (Step 3). Finally, at 24 min,
valve B turned again (Step 4) and connected the SPE LC
pump to the SPE column, which was then cleaned with
1 mL of acetonitrile and progressively taken to the highly
aqueous initial conditions. These steps added to a total
run time of 28 min per sample.