LC/TOFMS and UPLC-MS/MS Methods for the Analysis of Perfluorooctanesulfonate (PFOS) and the Reduction of Matrix Interference in Complex Biological Matrices.

Mark Strynar1, Amy Delinsky1, Andrew Lindstrom1, Shoji Nakayama2, and Jessica Reiner3

Background

Abstract

1U.S. EPA, NERL, Research Triangle Park, NC, USA 2US EPA, NRMRL, Cincinnati, OH, USA 3NIST, Hollings Marine Laboratory, Charleston, SC, USA

Perfluorinated compounds (PFCs) comprise a class of anthropogenic compounds that are regularly detected in biological and environmental samples globally. PFCs are used in a multitude of commercial and industrial processes and products. Perfluorooctane sulfonate (PFOS) is one of the PFCs that are frequently detected in biological and environmental samples. Many studies that have investigated PFOS in marine mammals, avian species, aquatic species, and human serum have been based on LC-MS/MS analysis using a single multiple-reaction-monitoring (MRM) transition (499 m/z → 80 m/z) for quantitation. A recent publication (1) reported a common matrix interferent (taurodeoxycholate) that can complicate PFOS quantitation because it undergoes thesame transition (499 m/z → 80 m/z) and it tends to coelute with PFOS, leading to a positive bias.

The comparison of UPLC/MS/MS and LC/TOFMS for the chromatographic separation and quantitation of PFOS in complex biological matrices is shown. A Waters Aquity UPLC interfaced with a Quattro Premier XE triple quadrupole mass spectrometer and an Agilent 1100 HPLC interfaced with an Agilent Series 6000 TOF mass spectrometer were used for sample analysis. Acquisition files containing quantitation and confirmation ions for each analyte were generated for the triple quadrupole mass spectrometer using values from individual compound optimization. Exact masses and mass defects for each analyte and optimum fragmentation voltage was determined using the TOF instrument. The biological matrices evaluated in this work include whole-fish homogenates, fish livers, avian eggs and human serum.

This work describes an UPLC/MS/MS method for the complete separation of PFOS from known impurities for more accurate quantitation. Most analytical laboratories rely on triple quadrupole MS/MS instruments as the primary means of PFC quantitation. But without careful attention to the appropriate MRMs, PFOS quantitation can be questionable. We have found several common matrix impurities that may co-elute with PFOS which also give the 499 m/z → 80 m/z transition but can be identified with the addition of 2 unique MRMs. This work specifically shows why multiple MRMs are necessary for the proper quantitation and verification of PFOS in complex biological and environmental matrices. To further help avoid this potential problem, an SPE method has been developed for removal of these known matrix impurities from complex biological samples.

LC/TOFMS is another approach for the analysis of PFCs and it is particularly useful for the identification and quantitation of PFOS in complex biological matrices. Accurate mass determination (±10 ppm) provided by TOFMS allows for the separation of PFOS frompotentially co-eluting matrix impurities that have very similar molecular weights and mass transitions. If combined with good chromatographic separation, an LC/TOFMS approach can provide superior selectivity in identification and quantitation of PFOS isolated from complex biological matrices in comparison to standard triple quadrupole MS/MS techniques. Typical MS/MS procedures only provide +/- 0.5 amu accuracy through the first quadrupole when parent → daughter transitions are produced. PFOS (498.65) and a group of common biological matrix impurities (cholic acids at 498.35) are close enough in molecular weight that most triple quadrupole MS/MS systems to fail to distinguish between these two types of compounds. In addition, the primary transition for both is the loss of a sulfonate group (79.90 m/z). However, with TOFMS analysis, the PFOS (498.9302 m/z) is significantly different from the cholic acids (498.2895 m/z) to be easily separated due to molecular weight alone.

Considering the Benskin et al., 2008 (1) report of a cholic acid impurity (taurodeoxycholate –TDCA) with the same 499 m/z → 80 m/z transition as PFOS, many researchers have simply switched to monitoring the 499 m/z → 99 m/z transition exclusively for PFOS. While interfering impurities have not been noted using this approach, it also runs the risk that other coeluting compounds could undergo the same transition, causing inaccuracies in quantitation. If a triple quadrupole instrument is used, a more robust approach is to monitor daughter ion ratios of authentic standards and field samples to provide better assurance of have correct identification and quantitation.

In view of this complexity, we propose a variety of different approaches that could be used to provide for better quantitation of PFOS in biological matrices. The first is to monitor for the impurities known to interfere with PFOS analysis (cholic acids) with unique transitions (m/z 498.35→124.1 and m/z 498.35→106.8) and determine if a matrix interferent actually exists in the test matrix. Secondly, one could enhance chromatographic separation of PFOS from these known impurities and continue to quantitate with multiple transition. Third, we report here on a SPE cleanup method using Supelco ENVI-Carb SPE cartridges to remove the cholic acid interferents from methanolic extracts of various biological matrices. Fourth, one could use LC TOF/MS to obtain exact mass data to distinguish between PFOS (498.9302) and matrix impurities (498.2895) easily. Any of these approaches will help in better quantitation of PFOS in complex biological matrices.

Using a modification of a method by Ye et al., 2008 (2,3) for PFCs in whole-fish homogenates and carp fillets, we isolated PFOS and matrix impurities from a host of complex biological matrices. Not only PFOS but TDCA (and other cholic acids) were found to be common in a variety of complex biological matrices. Given this finding, it is likely that these impurities could have been mistaken for PFOS in some instances due to similar isomeric profiles and retention times as well as transitions (499 m/z →80 m/z). Using the techniques to be described in the following sections, analytical chemists can now use a variety of MS, SPE, and chromatographic techniques to confirm previous PFOS measurements, and improve existing methods.

It should be noted that the presence of these matrix impurities does not necessarily negate quantitation using (499 m/z → 80 m/z) transitions. In some instance of higher relative PFOS concentrations to matrix impurity concentrations, there would likely be little to no effect on quantitation. However, in situations where PFOS is low and matrix impurities are high in concentration it is possible to dramatically overestimate actual PFOS concentrations.

Materials and Methods

Results UPLC/MS/MS

Figure 3. (a) Spiked Method Blank Without ENVI-Carb Cleanup (b) Spiked Method Blank With ENVI-Carb Cleanup.

Extraction15 mL BD Falcon PP tube0.5 g of solid sample or homogenate5.0 mL of 0.1N NaOH in MethanolContaining 18O2-PFOSSonic extraction for 30 minutesCentrifuge 5000 g

Supelco-ENVI-Carb 3 cc(250 mg) SPE

Condition 4 mL MeOHPass 3 mL extract

through SPE

Concentration50 mL BD Falcon PP tube3.0 mL of the extracted supernatantCombine with 27 mL of DI waterLoad onto Waters Oasis WAX SPE cartridgeCartridge pre-conditioned with 4 mL each0.1% NH4OH in waterMethanolDI water

Wash and ElutionWash with 25 mM pH 4 Acetate buffer (4 mL)Wash with methanol (4 mL)

Elute with 7.0 mL of 0.1% NH4OH in Methanol

Oas

is W

AX

Sample split3 mL transferred to new tubeor3 mL passed through ENVI-Carb

No ENVI-Carb3 mL extractEvaporated to 2.0 mLN2 gas 30°C TurboVap

With ENVI-Carb3 mL cleaned up extractEvaporated to 2.0 mLN2 gas 30°C TurboVap

-+Mussel Tissue (SRM 1974b)

-+Cat Serum

-+Lake Michigan fish tissue (SRM 1947)

+-Newborn Calf Serum

+-Whole Chicken Eggs

++Egg Reference #1 (NIST)

++Whale blubber (SRM 1945)

++Whale liver homogenate (II,III,IV)

++Whole fish homogenates (bluegill)

++Bald Eagle Serum

++Osprey Eggs

++Lake Superior fish tissue (SRM 1946)

++Fish liver (bluegill, largemouth bass)

++Human Serum (NIST SRM 1957)

Measurable Cholic Acids

Measurable PFOS

Matrix

PFOS

Cholic Acids

References(1) Benskin et al., 2007 Simultaneous Characterization of Perfluoroalkyl

Carboxylate, Sulfonate, and Sulfonamide Isomers by Liquid Chromatography-Tandem Mass Spectrometry. Anal. Chem. 2007, 79, 6455-6464

(2) Ye et al., 2008 Perfluorinated compounds in whole fish homogenates from the Ohio, Missouri, and Upper Mississippi Rivers, USA. Environmental Pollution 156 (2008) 1227–1232

(3) Ye et al., 2008 Perfluorinated compounds in common carp (Cyprinus carpio) fillets from the Upper Mississippi River. Environment International 34 (2008) 932–938

Acknowledgements

Conclusions

18O2-PFOS

Scan 498-499

PFOS

Cholic acids

(e) Bald Eagle Serum

Cooperative Research & Development Agreement (CRADA) with Waters Corp (CRADA #392-06)CRADA with Agilent Technologies Inc. (CRADA #437-07)Dr. Jennifer Keller at NIST for the kind donation of standard reference materials (SRMs)

Monitoring for transitions for cholic acid matrix impurities (498.35 m/z →124.1 m/z and 498.35 m/z → 106.8 m/z) is suggested to help identify the presence impurities with MS/MS.

Good chromatographic separation of PFOS from these known impurities is possible with standard UPLC and HPLC columns usinggradient methods.

UPLC/MS/MS and LC/TOFMS data suggest that ENVI-Carb cleanup of spiked method blanks and complex biological sample extracts removes cholic acids while not altering PFOS concentrations.

LC/TOFMS appears to be an excellent way to separate PFOS (498.9302) from matrix impurities (498.2895) simply based on themolecular weight of the compounds, and accurate mass determination.

Table 1. Summary of Findings for this Study:

See Delinsky et al. poster TPY 586 For additional information on fish fillets, fish liver and whole-fish homogenates

Figure 2. Picture of the (a) UPLC/MS/MS and (b) LC/TOFMS systemsused

UPLC MS/MS SystemUPLC - Waters Acquity

MS/MS - Micromass Quatro Premier XE

Column - Acquity UPLC BEH C181.7 um 2.1x50 mm

Gradient separation A 2mM Ammonium AcetateB Methanol

(a)

HPLC/TOFMS SystemHPLC - Agilent 1100

TOFMS - Agilent LC/MSD TOF

Column - Agilent Eclipse Plus C183.5 um 3.0x50 mmcustom packed

Gradient separationA 2mM Ammonium AcetateB Methanol

(b)

Figure 1. Molecular weight and MRM transitions for (a) TDCA, (b)TCDCA, (c) TUDCA, and (d) PFOS

NH

S O-

O

OO

CH3

CH3

OH

CH3

H

OH

TDCA

PFOS

S

F

F

F

F

F

F

F

FF

F

F

F

F

FF

F

F

O

O

O-

NH

S O-

O

OO

CH3

CH3

OH OH

CH3

H

TUDCA

CH3

OH OH

CH3

NH

S

O

O-

OO

CH3H

TCDCA

TOFMS MW = 498.2905

MS/MSMRM 1° 498.35-124.1MRM 2° 498.35-106.8MRM 3° 498.35-79.9

TOFMS MW = 498.2905

MS/MSMRM 1° 498.35-124.1MRM 2° 498.35-106.8MRM 3° 498.35-79.9

TOFMS MW = 498.2905

MS/MSMRM 1° 498.35-124.1MRM 2° 498.35-106.8MRM 3° 498.35-79.9

TOFMS MW = 498.9302

MS/MSMRM 1° 498.65-98.8MRM 2° 498.65-79.9

(a)

(c)

(b)

(d)

18O2-PFOS

S

F

F

F

F

F

F

F

FF

F

F

F

F

FF

F

F

O18

O18

O-

PFOS MRM 1°

S

F

F

F

F

F

F

F

FF

F

F

F

F

FF

F

F

O

O

O-

PFOS MRM 2°

S

F

F

F

F

F

F

F

FF

F

F

F

F

FF

F

F

O

O

O-

NH

S O-

O

OO

CH3

CH3

OH OH

CH3

H

TUDCA

NH

S O-

O

OO

CH3

CH3

OH

CH3

H

OH

TDCACH3

OH OH

CH3

NH

S

O

O-

OO

CH3H

TCDCA Cholic Acids MRM 1°

Cholic Acids MRM 3°

Cholic Acids MRM 2°

NH

S O-

O

OO

CH3

CH3

OH OH

CH3

H

TUDCA

NH

S O-

O

OO

CH3

CH3

OH

CH3

H

OH

TDCACH3

OH OH

CH3

NH

S

O

O-

OO

CH3H

TCDCA

18O2-PFOS

S

F

F

F

F

F

F

F

FF

F

F

F

F

FF

F

F

O18

O18

O-

PFOS MRM 1°

S

F

F

F

F

F

F

F

FF

F

F

F

F

FF

F

F

O

O

O-

PFOS MRM 2°

S

F

F

F

F

F

F

F

FF

F

F

F

F

FF

F

F

O

O

O-

Cholic Acids MRM 1°

Cholic Acids MRM 3°Ovelap with PFOS

Cholic Acids MRM 2°

(a)

(b)

No ENVI-Carb With ENVI-Carb

(a) NIST SRM 1957 (Human Serum)

(b) Whole Chicken Egg extract

No ENVI-Carb With ENVI-Carb

(c) Large Mouth Bass Liver

No ENVI-Carb With ENVI-Carb

(d) Bluegill Liver

No ENVI-Carb With ENVI-Carb

Figure 5. UPLC/MS/MS chromatograms for samples with and without ENVI-Carb cleanup for: (a) human serum (b) whole chicken eggs, (c) largemouth bass liver and (d) bluegill liver.

Figure 6. Other matrices without ENVI-Carb cleanup: (a) Lake Superior fish tissue (SRM 1946), (b) bald eagle serum, (c) Lake Michigan fishTissue (SRM 1947), (d) Osprey egg.

(a) SRM 1946

(c) SRM 1947

(b) bald eagle serum

(d) osprey egg

Results LC/TOFMS

(a) Spiked Method Blank4x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14

-ESI EIC(502.9387) Scan Frag=125.0V 042309-002.d

6.0901 1No ENVI-Carb With ENVI-Carb

18O2-PFOS502.9387

Cholic Acids498.2895

PFOS498.9302

18O2-PFOS

18O2-PFOS

Scan 498-499

PFOS

Cholic acids

(d) Osprey Egg

(b) Bluegill Whole-fish Homogenate #1

Scan 498-499

PFOS18O2-PFOS

Cholic acids

(c) Bluegill Whole-fish Homogenate #218O2-PFOS

Cholic acids Scan 498-499

PFOS

Figure 7. LC/TOFMS chromatograms of: (a) spiked method blank, (b) bluegill WFH #1, (c) bluegill WFH #2, (d) osprey egg (e) bald eagle serum (top next column)

Disclaimer: The United States Environmental Protection Agency through its Office of Research and Development funded and managed the research described here. It has been subjected to Agency review and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.