Comparison of pressurized solvent and reflux extraction methods for thedetermination of perfluorooctanoic acid in polytetrafluoroethylenepolymers using LC-MS-MS

Barbara S. Larsen,a Mary A. Kaiser,a Miguel Botelho,a Gilbert R. Woolera and L. William Buxtonb

Received 16th August 2004, Accepted 4th October 2004

First published as an Advance Article on the web 24th November 2004

DOI: 10.1039/b412609b

Both pressurized solvent extraction (PSE) and reflux extraction in various solvents were used to

select the most efficient system for the determination of the quantity of perfluorooctanoic acid

(PFOA) present in polytetrafluoroethylene polymers. After evaporating the solvent, PFOA was

determined using liquid chromatography tandem mass spectrometry. Ethanol, water and

methanol gave comparable results and were shown to be good solvents for this extraction.

Acetonitrile was a reasonable solvent using the reflux extraction method, but not with PSE.

Chloroform resulted in poor recovery for both extraction methods. PSE proved to be the more

efficient extraction method.

Introduction

In 1999 Moody et al. reported the presence of perfluorinated

compounds in groundwater impacted by fire-fighting

activities, even though the activities had concluded many

years before the analyses were conducted.1 Several investiga-

tors have reported that human sera contained low concentra-

tions (viz., ppb) of perfluorinated organic molecules [e.g.,

perfluorooctane sulfonate (PFOS), perfluorooctanoic acid

(PFOA)] in the general population.2–5 Other reports showed

that low levels of these and similar compounds could be found

in wildlife and in the environment.6–8

Organofluorine compounds have unusual properties that

make them difficult to measure.9 In addition, perfluorocar-

boxylates are used in the production of many fluoropolymers10

as a processing aid.11,12 Fluoropolymers are important inert

components commonly used in laboratory apparatus and

analytical laboratory instrumentation. If residual perfluoro-

carboxylates are present in analytical systems, their levels and

ease of extraction will be important in deciding the low levels

of quantitation needed, especially for exposure, environmental,

and toxicological studies.

In this study, we compare solvents and extraction methods

used for the determination of perfluorooctanoic acid (PFOA),

a common fluoropolymer processing aid, in two polytetra-

fluoroethylene (PTFE) polymers.

Based on the work of Vandenburg et al.,13 we selected

pressurized solvent extraction and boiling under reflux to

determine optimal conditions for these extraction measure-

ments. Since PFOA is quite soluble in water14 and since water

is a good solvent used to ascertain potential exposure to PFOA

from perfluoropolymers, water was selected for this study.

Ethanol has been used for studies involving food contact in

FDA studies.15 Acetonitrile and methanol are common

solvents. Chloroform was evaluated since it is a common

halogenated solvent.

Experimental

Apparatus

Reflux extractions were done with a 500-mL round-bottom

flask with glass magnetic stirring bar and a glass water

condenser. The pressurized solvent extraction was done with a

Dionex (Sunnyvale, CA) accelerated solvent extractor (ASEH

series 200).

Materials and reagents

Polytetrafluoroethylene fluoropolymer resins were obtained

from a commercial lot and an intermediate of a commercial lot

(average particle size approximately 500 mm). Liquid chroma-

tographic grade or analytical grade water, methanol, acetoni-

trile, chloroform and standard Ottawa sand were obtained

from EMD Chemicals, Inc. (Darmstadt, Germany). Ethanol

(100%) was obtained from Sigma–Aldrich (Milwaukee, WI). A

dual 13C enriched perfluorooctanoic acid was synthesized

in house.

Standards for the seven-point calibration curve were pre-

pared by dilution with methanol from a 1000 ppb (mg L21)

standard solution to concentrations of 0.5, 1, 5, 10, 25, 50, and

100 ppb.

Reflux extraction procedure

Five reflux extraction systems were run concurrently. One

hundred mL of solvent was added to each 500-mL round-

bottom flask with a glass stirring bar and reflux water

condenser. The solvent was refluxed for 20 min and the

solvent discarded. Then, 250 mL of the solvent of interest was

added to each flask. To one of the flasks was added 1.0 mL of

the recovery check standard that was subsequently used to

determine percent recovery of PFOA. Approximately 0.75 g of

each polymer was weighed (¡ 0.1 mg) and added to three of

the remaining flasks. The fifth flask had only solvent, which

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served as the solvent blank. The solvents in the prepared flasks

were refluxed for two hours, cooled and filtered through a

no. 40 filter paper (Whatman, Clifton, NJ). The solvents were

then evaporated on a Buchi rotary evaporator (Brinkmann,

Westbury, NY) to remove most of the solvent (approximately

10 mL remaining). The remaining solvent was then transferred

to either a 30 mL or 60 mL disposable collection vial and

evaporated to dryness under nitrogen on a temperature-

controlled heating block (ambient for chloroform, 45 uC for

methanol, 55 uC for ethanol and acetonitrile, and 85 uC for

water).

Pressurized solvent extraction procedure

Five pressurized solvent extractions were performed concur-

rently. The instrument’s solvent reservoir was filled with the

appropriate solvent and the lines rinsed four times with

approximately 5 mL of solvent. The cells were filled with

Ottawa sand to approximately 3 mm from the top. Each cell

was closed and loaded onto the instrument. The cells with sand

were preconditioned. The preconditioning cycle was set to

1500 psi (10.3 kPa), 150 uC, 7 min heating time, 100% volume

flush, 240 second purge for one cycle. The solvents were

then discarded and new collection vessels set in place.

Approximately 5/6 of the sand was removed from three of

the vessels and a weighed quantity of polymer added to each

vessel. Each cell was refilled to 3 mm from the top with the

sand and the lid secured. To the fourth sand-filled cell 1.0 mL

of recovery check standard was added on top of the sand and

the lid secured. The fifth sand filled flask served as the solvent

blank. The extraction conditions were set to 1600 psi

(11.0 kPa), 150 uC, 7 min heating time, 100% volume flush,

240 s purge for 4 cycles. The collection vials were removed

when the extraction was complete and placed in a heating

block where they were evaporated to dryness under nitrogen

(ambient for chloroform, 45 uC for methanol, 55 uC for

ethanol and acetonitrile and 85 uC for water).

Analytical method

The concentration of PFOA in the reconstituted extracts was

determined using high performance liquid chromatography

coupled with negative ion electrospray tandem mass spectro-

metry (LC-MS-MS) (Micromass Quatro Ultima, Beverly,

MA). The extracts were reconstituted by adding 1 mL of

100% methanol to the vial and shaking for 30 min on a wrist-

action shaker. The methanol was then placed into a 5-mL

volumetric flask, and brought to volume with the LC mobile

phase A, as described below. The dual 13C-enriched standard

(final concentration 50 ppb) was added as an internal standard

to all the reconstituted samples.

The analyte was separated using an Agilent 1100 liquid chro-

matograph (Wilmington, DE) modified with low dead-volume

internal tubing. A guard column, Hypersil C18 2 6 50 mm

(Thermo Keystone, Bellefonte, PA), was installed between

the mixer and the autoinjector. Twenty-five microliters of

the reconstituted extract were injected onto a Hypersil ODS

2.1 6 200 mm (Thermo Keystone, Bellefonte, PA) at a flow

rate of 0.3 mL min21 and maintained at 60 uC. Duplicate

injections were made for all samples.

Initial gradient conditions were 15% mobile phase B, where

mobile phase A is 2 mM ammonium acetate–1% methanol and

B is 100% methanol. A linear gradient was used from 15–67%

B over 16 min. The conditions were returned to 15% B for two

additional minutes. Typical elution time for PFOA was

approximately 16.5 min. Fig. 1 shows a typical total ion

chromatogram of the internal standard and the PFOA analyte.

In negative ion mode, PFOA is observed as an anion at

413 [CF3(CF2)6COO2]. The internal standard is observed at

415 [CF3(CF2)513CF2

13COO2].

Quantitative analysis was performed using selected ion

monitoring for the transition of 413 . 369 (loss of CO2) for

the analyte and 415 . 370 (loss of 13CO2) for the internal

standard. Any samples that fell outside the calibration were

diluted appropriately and reanalyzed. A seven-point linear

calibration curve was prepared (not including zero) for

external calibration. Each calibrant concentration set was

run in duplicate and bracketed the samples. A typical

calibration curve consisted of all fourteen calibration points.

With no weighting, the acceptance criterion for the calibration

curve required a correlation coefficient, R, ¢0.992.

A methanol blank was run after each 100 ppb standard. The

acceptance criterion was set so that the lowest level PFOA

standard’s area was at least five-times greater than the area of

the methanol (solvent) blank. (No quantifiable area was

observed in the blank.)

The limit of quantitation (LOQ) was set at 0.5 ppb, the

concentration of the lowest calibration standard. A signal

between the blank background and the LOQ was defined as

not quantifiable (NQ).

Results and discussion

In the extraction of the PTFE polymers with the various

solvents, a recovery check standard was run. The recovery

check standard vessel contained just the solvent with

500 ng mL21 of PFOA added in order to determine the

recovery of PFOA in the solvent. Table 1 summarizes the

results of the recovery check standard for each solvent used in

this study. The recoveries of PFOA for methanol, water and

ethanol for both extraction methods were acceptable (80–

120%). With acetonitrile acceptable recovery was observed

using reflux extraction: however, using PSE as the extraction

system, acetonitrile had no detectable recovery of PFOA. The

results indicated that there was no recovery of PFOA using

chloroform by either the reflux extraction or pressurized

solvent extraction.

The initial extraction results with chloroform stabilized with

1% hexane were unexpected and additional trials using

different stabilizers were performed. The chloroform modified

with 1% or 5% ethanol did not extract PFOA quantitatively or

reproducibly. The initial extraction in the PSE was followed by

a re-extraction with methanol, showing an improvement in the

recovery to 57%. This indicates that the chloroform extraction

was ineffective. Recoveries for the water and alcohols ranged

from 89% to 104%.

To ensure that the low sample recoveries observed in

chloroform were not a result of loss during evaporation of the

solvent, a sample of PFOA (500 ng mL21) in chloroform was

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dried overnight in the hood. An identical sample was

evaporated to dryness with nitrogen and heat. These samples

were reconstituted and analyzed by the analytical method. The

observed recoveries were similar, 103% and 94%, respectively.

This indicates that the low recoveries in chloroform extracts

are not the result of losses during the evaporation or

reconstitution process, but are due to inefficient extraction of

PFOA in chloroform stabilized with either 1% hexane or 1%

ethanol. The experiment with chloroform containing 5%

ethanol showed modest recovery, indicating that the ethanol

plays a role in the extraction efficiency.

Tables 2 and 3 show data for the spike recovery and the

extraction results for the two different fluoropolymers (PTFE)

comparing reflux extraction with pressurized solvent extrac-

tion. The results in Table 2 are comparable to those shown in

Table 1. For water and alcohols under these conditions, PSE

percent recoveries were generally the same as the reflux

extraction method. The amount of analyte extracted and

detected using the reflux extraction method appeared to be the

same level for the water and alcohols (Table 3). The level of

analyte detected using the water and alcohols was greater

under the same PSE conditions than that with acetonitrile and

chloroform. PSE is the more efficient extraction method for

determining PFOA content in PTFE polymers (Table 3). The

PTFE sample labeled polymer I is from a commercial lot and

polymer II is the intermediate of a commercial lot, showing

that the finishing steps significantly reduce the PFOA

concentration in the commercial polymer. When the chloro-

form extraction was followed by methanol extraction and the

two quantities added together (Polymer II, Table 3), the

combined solvent results agreed with the water and alcohols

extraction results under the same PSE conditions (1580 ppb).

Table 1 Check standard percent recovery

Solventa Refluxb PSEb,c

Methanol 97 95Acetonitrile 89 4.1 (0.31)Ethanol 104 99Water 103 96Chloroform–1% hexane 0.9 2.8 (0.06)Chloroform–1% ethanol 60* 0 (58)Chloroform–5% ethanol 42* 45 (11)a Chloroform was purchased with either 1% hexane or 1% ethanol asthe stabilizer and the % ethanol was increased by volume to 5%.b Results are an average of two measurments and * denotes anaverage of three measurements. c A second solvent extraction withmethanol was made with the extraction efficiency indicated in theparenthesis

Table 2 Check standard percent recovery from PTFE samples

SolventRefluxtemperature/uC

Polymer I Polymer II

Reflux PSE Reflux PSE

Methanol 65 93.1 88.4 92.9 89.2Acetonitrile 82 91.3 0 86.4 7.9Ethanol 78 99.1 85.5 104 92.5Water 100 104 104 104 106Chloroform 61 3.1a 0a 36.1b 59.5b

a Chloroform stabilized with 1% hexane was used for theseextractions. b The results reported is the sum of the initial extractionusing chloroform stabilized with 1% ethanol followed by a secondextraction with methanol.

Table 3 Concentration of PFOA (ppb) extracted from PTFEpolymers

SolventRefluxtemperature/uC

Polymer I Polymer II

Reflux PSE Reflux PSE

Methanol 65 42 140 485 1420Acetonitrile 82 46 NQa 412 241Ethanol 78 46 135 535 1100Water 100 46 59 493 1040Chloroform 61 NQa,b NQa,b 59c 1580c

a NQ is nonquantifiable, indicating that the observed signal was lessthan the lowest calibrant. b Chloroform stabilized with 1% hexanewas used for these extractions. c The result reported is the sum ofthe initial extraction using chloroform stabilized with 1% ethanolfollowed by a second extraction with methanol.

Fig. 1 LC–MS–MS of a PTFE extract showing in the upper trace the internal standard (415 . 370), lower trace the PFOA analyte (413 . 369).

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Conclusions

Ethanol, methanol and water efficiently extracted PFOA from

PTFE polymers. PSE was able to extract more analyte in a

shorter time period than reflux extraction with water,

methanol and ethanol. Acetonitrile as the solvent gave

reasonable recoveries with reflux extraction but not with

PSE. Chloroform gave unacceptable results by either extrac-

tion method. This result supports the use of water, ethanol, or

methanol for extracting PFOA from PTFE matrices.

Acknowledgements

The authors thank Charles F. Koerting, Donald A. Wilson

Jr. and Gregory A. Urove for their advice and assistance.

Barbara S. Larsen,a Mary A. Kaiser,a Miguel Botelho,a

Gilbert R. Woolera and L. William Buxtonb

aDuPont Corporate Center for Analytical Sciences, ExperimentalStation 402/5321, P.O. Box 80402, Wilmington, DE 19880-0402, USA.E-mail: mary.a.kaiser@usa.dupont.com; Fax: 302 695-1053;Tel: 302 695-8435bDuPont Fluoroproducts, P.O. Box 80713, Wilmington, DE 19880-0713,USA

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