In the Laboratory
W
An Undergraduate Experiment for the Measurement of Perfluorinated Surfactants in Fish Liver by Liquid Chromatography–Tandem Mass Spectrometry Naomi L. Stock, Jonathan W. Martin, Yun Ye, and Scott A. Mabury* Department of Chemistry, University of Toronto, Toronto ON, M5S 3H6, Canada; *
[email protected] In the past decade, advances in liquid chromatography– tandem mass spectrometry (LC–MS兾MS) techniques have permitted the analysis of trace environmental contaminants in complex matrices and, in some cases, have allowed for the detection of compounds that were previously unmeasurable (1, 2). One such class of contaminants currently receiving considerable attention are the perfluorinated surfactants (3). Perfluorinated surfactants are a specialty class of chemicals employed in many industrial and commercial applications including soil- and stain-resistant coatings for fabrics and carpets, oil- and grease-resistant coatings for paper products, fire-fighting foams, lubricants, paints, and cosmetics (3). Two important perfluorinated surfactants are perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) (Figure 1). Both PFOS and PFOA have recently been detected in biological and environmental matrices (4, 5) and are of concern owing to potential toxicity, persistence, and bioaccumulation. As such, in May 2000, the 3M Company, a major manufacturer of perfluorinated surfactants globally, announced that it would be discontinuing the production of both PFOS and PFOA (3). The primary objectives of this laboratory experiment are to provide undergraduate students with the opportunity to learn the fundamentals of LC–MS兾MS and employ this technique for the analysis of perfluorinated surfactants. This laboratory experiment provides an excellent hands-on introduction to the specific techniques of LC–MS兾MS and electrospray ionization and also allows students to practice the analytical principles of sample extraction, detection, quantification, and quality control using a fresh fish sample. Materials and Methods
Sampling Students were directed to obtain a whole, fresh fish from a local fish market or grocery store and to note the species,
size, and location where caught (if known). The liver was isolated and removed in the laboratory with the assistance of the teaching assistant. Only liver tissues were used for the analyses, as previous findings indicate that perfluorinated surfactants, such as PFOS and PFOA, accumulate primarily in the liver tissue (3).
Extraction A subsample of the liver (approximately 500 mg) was homogenized with 4 mL of sodium carbonate (NaCO3, 0.25 M), 1 mL of tetrabutylammonium hydrogen sulfate (TBAS, 0.5 M, adjusted to pH 10), and 50 µL of the internal standard solution (perfluoroheptanoic acid (PFHpA),100 ppb) in a 15-mL polypropylene centrifuge tube. The homogenate was vigorously shaken for 5 minutes with a 5-mL aliquot of methyl tert-butyl ether (MTBE), followed by centrifugation to isolate the organic phase. The extraction process was repeated and the MTBE extracts combined in a separate centrifuge tube. The organic solvent was evaporated under a gentle stream of nitrogen, reconstituted in 0.5 mL of methanol, filtered (0.2-µm nylon filters), and placed in polypropylene autosampler vials. This was an optimized version of the method of Hansen et al. (6). Analysis The filtered extracts were analyzed by LC–MS兾MS employing the conditions described by Moody et al. (7). Samples (20-µL injection volume) were separated using a Genesis C8 column (2.1-mm, 4-µm; C18 Security Guard) and a flow rate of 200 µL兾minute (Waters 600 pump). The solvent gradient was operated from 40 to 95% eluent B for two minutes, then held at 95% eluent B for 12 minutes, where eluent A was 10:90 methanol:water and eluent B was 100% methanol. Analytes were detected using a Micromass Quattro LC triple quadrupole mass spectrometer employing negative electrospray ionization and operating in multiple reaction moni-
Table 1. Student-Optimized MS/MS Parameters for Three Perfluorinated Surfactants Parent Ion/(m/z)
Daughter Ion/(m/z)
Collision Energy/eV
Cone Voltage/V
PFOS
499
099a
40–50
50–60
PFOA
413
369
11–13
13–15
PFHpA
363
319
11–13
13–15
Analyte
The most intense daughter ion was m/z = 80; however, owing to a possible interferrence with m/z = 80 (6), students were instructed to select m/z = 99. a
Figure 1. Perfluorooctane sulfonate (PFOS) (top) and perfluorooctanoate (PFOA) (bottom).
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Journal of Chemical Education
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Vol. 84 No. 2 February 2007
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www.JCE.DivCHED.org
In the Laboratory Table 2. Perfluorooctane sulfonate (PFOS) and Perfluorooctanoate (PFOA) Concentrations in Fish Collected from Markets and Grocery Stores in Toronto Ontario Size/cm
Wgt/g
PFOS/ (ng/g wet wgt)
PFOA/ (ng/g wet wgt)
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134
