Environ. Sci. Technol. 2004, 38, 5522-5528
Analysis of Perfluorinated Acids at Parts-Per-Quadrillion Levels in Seawater Using Liquid Chromatography-Tandem Mass Spectrometry NOBUYOSHI YAMASHITA,† K U R U N T H A C H A L A M K A N N A N , * ,‡ SACHI TANIYASU,† YUICHI HORII,† TSUYOSHI OKAZAWA,† GERT PETRICK,§ AND TOSHITAKA GAMO| National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan, Wadsworth Center, Empire State Plaza, P.O. Box 509, Albany, New York 12201-0509, Department of Environmental Toxicology and Health, State University of New York, Albany, New York 12201-0509, Institute for Marine Research, University of Kiel, Du ¨ sternbrooker Weg 20, D-24105 Kiel, Germany, and Ocean Research Institute, University of Tokyo, Nakano-ku, Tokyo 164-8639, Japan
Perfluorinated acids (PFAs) and their salts have emerged as an important class of global environmental contaminants. Determination of sub-parts-per-trillion or parts-per-quadrillion concentrations of perfluorinated acids in aqueous media has been impeded by relatively high background levels arising from procedural or instrumental blanks. To understand the role of the oceans in the transport and fate of perfluorinated acids, methods to determine ultratrace levels of these compounds in seawater are needed. In this study, sources of procedural and instrumental blank contamination by perfluorinated acids have been identified and eliminated, to reduce background levels in blanks and thereby improve limits of quantitation. The method developed in this study is capable of detecting perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate (PFHS), perfluorobutanesulfonate (PFBS), perfluorooctanoate (PFOA), perfluorononanoate (PFNA), and perfluorooctanesulfonamide (PFOSA) at low pg/L levels in oceanic waters. PFOA is the major perfluorinated compound detected in oceanic waters, followed by PFOS. Further studies are being conducted to elucidate the distribution and fate of perfluorinated acids in oceans.
Introduction Perfluoroalkylated acids, particularly, perfluorooctane sulfonate (PFOS, C8F17SO3-) and perfluorooctanoate (PFOA, C7F15COO-), have recently received considerable attention * Corresponding author phone: (518)474-0015; fax: (518)473-2895; e-mail:
[email protected]. Corresponding author address: Wadsworth Center, New York State Dept of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509. † National Institute of Advanced Industrial Science and Technology (AIST). ‡ Wadsworth Center and State University of New York. § University of Kiel. | University of Tokyo. 5522
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due to their widespread occurrence in the environment (1, 2). The unique physicochemical and biological properties of PFOS and PFOA and their salts contribute to these compounds’ persistence and bioaccumulation. PFOS and PFOA have been detected in the tissues of wildlife and humans (3-5). Because of their persistence and bioaccumulation, 3M, the sole manufacturer of PFOS-related compounds in the United States and the principal manufacturer worldwide, announced phase-out of perfluorooctanyl chemistries in May 2000. In the following years, the United States Environmental Protection Agency (USEPA) identified possible health concerns linked to PFOA and related perfluorinated acids. The USEPA released a preliminary risk assessment on PFOA, which indicated potential exposure of humans to low levels of PFOA in the United States (6). Various international environmental agencies have appealed for more data regarding human and environmental exposures and effects of perfluorinated compounds (PFCs) (7, 8). As a consequence, there has been a rapid increase in the number of publications reporting environmental concentrations of PFCs (9). Given that the findings of these are likely to be used for making regulatory decisions, it is imperative to ensure that the published data are accurate, precise, and reproducible. Nevertheless, the analytical methods for the measurement of perfluorinated acids in environmental matrixes are at various stages of development. The compounds’ unique physicochemical properties render several challenges to analytical chemists endeavoring to measure PFCs at trace levels. In Japan, the National Institute of Advanced Industrial Science and Technology (AIST) initiated a survey of PFCs in environmental samples with financial support from the New Energy and Industrial Technology Development Organization (NEDO) in 2000 (10). The preliminary results of this survey indicated both complexity of sources and the need for monitoring of PFCs in the coastal and oceanic environments in order to evaluate their pathways and mechanisms of transport to remote marine locations. Since the oceans serve as a sink for a variety of environmental pollutants, monitoring PFCs in this ecosystem is crucial. One of the major problems associated with trace level analysis of perfluorinated acids such as PFOS and PFOA is background contamination in the analytical blanks. Because of the contamination in blanks, the limits of detection (LOD) of perfluorochemicals in water samples are high, in the range of several tens to hundreds of ng/L to a few µg/L (11-15). However, concentrations of PFOS in ambient waters are in the lower to upper ng/L range (15-17). At a LOD of 25 ng/L, PFOS and PFOA could not be detected in surface water samples collected from various lakes and rivers in Michigan, U.S.A. There is a need to reduce the contamination in blanks for ultratrace analysis of PFCs in surface waters, particularly the open ocean waters. Contamination sources of PFCs in laboratories have not been well characterized. Two distinct sources of contamination, instrumental and procedural, are expected in PFC analysis. One known source of procedural contamination is fluoropolymers, such as poly(tetrafluoroethylene) and perfluoroalkoxy compounds, which are present in a variety of laboratory products. During the analysis of perfluorinated acids in environmental or biological matrixes, samples or extracts are not allowed to come in contact with such fluoropolymers. However, postinjection contamination, not related to injection-port carryover, has been observed in high performance liquid chromatograph-mass spectrometer (HPLC-MS) analysis of perfluorinated acids. This impedes 10.1021/es0492541 CCC: $27.50
2004 American Chemical Society Published on Web 09/28/2004
TABLE 1. List of Water Samples Collected from Various Seas and Oceans for the Analysis of Perfluorinated Acids n
site ID
depth (m)
date
latitude
longitude
Tokyo Bay
sampling site
3
Mid-Atlantic Ocean
3
Sulu Sea
5
South China Sea
2
Eastern Pacific Ocean
2
Central to Western Pacific Ocean
5
TB1 TB2 TB3 AO1 AO2 AO3 SS1 SS2 SS2 SS3 SS3 SCS1 SCS2 PO1 PO2 PO3 PO3 PO4 PO4 PO5
0-2 0-2 0-2 0-2 0-2 0-2 10 10 3000 10 1000 10 10 0-2 0-2 0-2 4000 0-2 4400 0-2
Mar 2002 Mar 2002 Mar 2002 Oct 2002 Oct 2002 Oct 2002 Dec 2002 Dec 2002 Dec 2002 Dec 2002 Dec 2002 Dec 2002 Dec 2002 Oct 2002 Oct 2002 Jul 2003 Jul 2003 Jul 2003 Jul 2003 Aug 2003
35°23′ N 35°26′ N 35°37′ N 10°00′ N 10°00′ N 10°00′ N 11°28′ N 8°50′ N 8°50′ N 9°17′ N 9°17′ N 13°30′ N 14°30′ N 23°31′ N 26°17′ N 16°30′ N 16°30′ N 15°08′ S 15°08′ S 25°59′ S
139°49′ E 139°54′ E 139°55′ E 43°35′ W 35°11′ W 29°05′ W 121°50′ E 121°48′ E 121°48′ E 120°13′ E 120°13′ E 119°30′ E 118°00′ E 124° 40′ E 128° 31′ E 123°00′ W 123°00′ W 85°49′ W 85°49′ W 140°00′ W
the analysis of sub parts-per-trillion (ppt; ng/L) or partsper-quadrillion (ppq; pg/L) concentrations of perfluorinated acids in ocean waters. In this study, we developed an improved technique for the analysis of parts-per-quadrillion levels of PFOS, perfluorohexanesulfonate (PFHS), perfluorobutanesulfonate (PFBS), PFOA, perfluorononanoate (PFNA), and perfluorooctanesulfonamide (PFOSA) in ocean water. Methods to reduce PFC contamination in instrumental and procedural blanks have been identified, and the sources of contamination have been eliminated. Marine water samples collected from several seas and oceans were analyzed for the presence of PFOS, PFHS, PFBS, PFOA, PFNA, and PFOSA, using the sensitive method developed in this study.
Materials and Methods Chemicals and Standards. Methanol (pesticide grade), distilled water (HPLC-grade), and sodium thiosulfate were purchased from Wako Pure Chemical Industries (Osaka, Japan). Vacuum manifold, solid-phase extraction (SPE) cartridges (Sep-pak tC18, 10 g, 35 cm3; Oasis HLB, 200 mg, 6 cm3) were purchased from Waters (Milford, MA). Nylon syringe filters (13 mm i.d., 0.2 µm) were purchased from Iwaki (Tokyo, Japan), Millipore (Bedford, MA), and Nalgene (Rochester, NY). Milli-Q water and distilled water were obtained from Milli-Q Gradient-A10 (Millipore) and Auto Still WG222 (Yamato, Tokyo, Japan), respectively. Potassium perfluorooctanesulfonate (86.9%), potassium perfluorohexanesulfonate (98.6%), potassium perfluorobutanesulfonate (>98%), and perfluorooctanesulfonamide (>95%) were provided by the 3M Company (St. Paul, MN). Perfluorooctanoic acid (>95%) and perfluorononanoic acid (>95%) were purchased from Avocado Research Chemicals Ltd. (Heysham, Lancashire, U.K.). Tetrahydroperfluorooctanesulfonic acid (THPFOS) was purchased from ICN (Costa Mesa, CA). Sampling. Surface seawater and open ocean water samples were collected during research cruises conducted in the central and eastern Pacific Ocean, South China and Sulu Seas, mid Atlantic Ocean, and Tokyo Bay from 2002 to 2003 (Table 1). Details of each sampling cruise are reported elsewhere (18). Coastal surface seawaters were collected using a clean stainless steel grab sampler with a polypropylene rope and were then transferred into 1-L narrow-mouth polypropylene bottles with screw tops. The bottles were rinsed with methanol and HPLC-grade water at the sampling location, prior to use. Open ocean water samples were
collected by a CTD (conductivity-temperature-depth meter) carousel system equipped with 36 Niskin-X bottles (narrow, cylindrical, 1-m-long plastic tubes capped at both ends) from appropriate depths in the water column. Teflon bottles, Teflon-lined caps, and any suspect fluoropolymer materials were avoided throughout the analysis. The amount of suspended matter was kept to a minimum. To reduce residual chlorine, 200 µL of 250 mg/mL solution (in HPLC-grade water) of sodium thiosulfate was added to each sample. In most cases, samples were extracted within 24 h after collection; otherwise, samples were kept frozen until analysis. Extraction. The analytical procedure for the extraction of water samples was similar to that described elsewhere (12) with some modifications (15). Modifications include the use of 10-g SPE Sep-pak tC18 cartridges instead of 1-g cartridges and extraction of 500 to 1000 mL of water instead of 40 mL of water. Several other modifications and the use of new cartridges are detailed below. Sep-pak tC18 SPE cartridges were preconditioned by passing 100 mL of methanol followed by 50 mL of HPLC-grade water, prior to the passage of samples. A sample aliquot of water (500-1000 mL) was passed through the preconditioned cartridges at a rate of 1 drop/s, and the cartridges were not allowed to dry out at any time during the extraction process. The cartridges were then washed with 20 mL of 40% methanol in water, which was then discarded. The target analytes were eluted with 30 mL of methanol and were collected in a polypropylene tube. The solvent was evaporated under a gentle stream of pure nitrogen gas to 0.2-0.5 mL for ocean waters and 8 mL for Tokyo Bay water samples. Particles that appeared in the final solution of a few of the water samples were removed by filtration using a nylon syringe filter. Recovery of PFBS was low in Sep-pak tC18 cartridges, and these cartridges also introduced some contaminants in blanks. Therefore, in addition to Sep-pak tC18 cartridges, we examined the suitability and performance of Oasis HLB cartridges. Oasis HLB cartridges were preconditioned with 5 mL of methanol and 5 mL of HPLC-grade water. Samples (500 mL to 1 L) were passed, and cartridges were washed with 5 mL of 40% methanol in HPLC-grade water, which was then discarded. The target analytes were eluted with 10 mL of methanol. Instrumental Analysis and Quantification. Analysis of PFOS, PFHS, PFBS, PFOA, PFNA, and PFOSA was performed using a high performance liquid chromatograph-tandem VOL. 38, NO. 21, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. HPLC-Tandem MS Parameters and Ions Monitored for Perfluorochemical Analysisa compound
cone voltage (V)
collision energy (eV)
primary ion (m/z)
product ion (m/z)
PFOS PFHS PFBS PFOA PFNA PFOSA THPFOS
90 70 50 35 35 55 55
35 30 25 10 10 25 22
498.6 398.6 298.6 412.9 462.7 497.6 426.6
98.6 79.7 79.7 368.7 418.8 77.7 406.7
a Different parameters were used in different time segments during chromatography.
FIGURE 1. PFOS, PFOA, PFHS, and PFBS in HPLC-MS/MS instrumental blanks, before and after modifications. mass spectrometer (HPLC-MS/MS), comprising of an Agilent HP1100 liquid chromatograph interfaced with a Micromass (Beverly, MA) Quattro Ultima Pt mass spectrometer operated in the electrospray negative ionization mode. A 5 or 10-µL aliquot of the sample extract was injected into a guard column (XDB-C8, 2.1 mm i.d. × 12.5 mm, 5 µm; Agilent Technologies, Palo Alto, CA) connected sequentially to a Betasil C18 column (2.1 mm i.d. × 50 mm length, 5 µm; Thermo HypersilKeystone, Bellefonte, PA) with 2 mM ammonium acetate aqueous solution (solvent A) and methanol (solvent B) as mobile phases, starting at 10% methanol and increased linearly. At a flow rate of 300 µL/min, the gradient was increased to 30% methanol at 0.1 min, 75% methanol at 7 min, and 100% methanol at 10 min and was kept there until 12 min before reversion to original conditions, at the 20-min time point. The capillary was held at 1.2 kV. Cone-gas and desolvation-gas flows were kept at 60 and 650 L/h, respectively. Source and desolvation temperatures were kept at 120 and 420 °C, respectively. MS/MS parameters were optimized so as to transmit the [M-K]- or [M-H]- ions as shown in Table 2. THPFOS was used as an internal standard, but the quantification was not based on internal standard recoveries. Eight calibration curve points bracketing the concentrations in samples were prepared routinely, to check for linearity. Quantification was based on the response of the external standards that bracketed the concentrations found in samples. For open ocean water samples, a calibration curve containing 1, 5, 10, 20, 100, and 500 ppt standard, injected at 5 µL, was used. For coastal water samples, a calibration curve prepared using 0.1, 0.5, 1, 10, and 20 ppb standard (injected at 5 µL) was used. The limit of detection (LOD) of target chemicals was evaluated for each sample based on the maximum blank concentration, the concentration factors, the sample volume, and a signal-to-noise ratio of 3. The LODs of target chemicals were as follows: 0.8, 0.4, 0.6, 5.2, 1.8, and 1 pg for PFOS, PFHS, PFBS, PFOA, PFNA, and PFOSA, respectively, when 1 L of water sample was used in the analysis. 5524
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FIGURE 2. PFOS, PFHS, PFBS, PFNA, PFOA, and PFOSA in various vial septa and standard (a) snap cap with polyethylene septum, (b) crimp cap with Teflon/silicon/Teflon septum, (c) screw cap with Teflon/silicon/Teflon septum, (d) screw cap with Teflon/rubber septum, (e) crimp cap with Viton septum, and (f) 10 µL of standard (10 ng/L). The numbers of each chromatogram represents intensity relative to 2a. Extreme measures were taken to prevent background levels of PFCs arising from the instrumental analysis. Highly concentrated samples, such as biota, were not analyzed, while the instrument was being used for the analysis of water. Methanol was run between the water samples to prevent carryover effect.
Results and Discussion Instrumental Blanks. Prior to the analysis of samples, several experiments were conducted to reduce contamination by target fluorochemicals in procedural and instrumental blanks. PFOS and PFOA were detected in injections of 10 µL of pesticide-grade methanol taken in an autosampler vial (Figure 1). This contamination was not related to the injection-port carryover arising from the injection of highly concentrated samples or standards, which have led to the appearance of chromatographic peaks at subsequent injections. The abundance of such carryover peaks generally decreases with successive injections of blanks or solvents. The methanol was injected several times, and the abundance of chromatographic peaks was consistent among injections. Furthermore, the abundance did not vary greatly, indepen-
TABLE 3. Contamination Levels (ng/L of Methanol) of Perfluorinated Compounds in Several Materials Used in the Analysis vial septuma A B C D E sodium thiosulfate solution sodium thiosulfate (DW)b sodium thiosulfate (Milli-Q)c polypropylene bottle 1000 mL 500 mL 50 mL 15 mL nylon filter nalgene millipore Iwaki
washed not washed washed not washed washed not washed
PFOS
PFHS
PFBS
PFNA
PFOA
PFOSA
THPFOS
