HPLC/MS Determination of PFC in Serum

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 1 of 15

Laboratory of Organic Analytical Chemistry
Wadsworth Center
New York State Department of Health

STANDARD OPERATING PROCEDURE
ANALYSIS OF PERFLUOROOCTANE SULFONATE OR OTHER
SIMILAR PERFLUORINATED COMPOUNDS IN SERUM USING
HPLC-ELECTROSPRAY/TANDEM MASS SPECTROMETRY
February 2011

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 2 of 15

APPROVAL PAGE
Revisions to an existing SOP, addition of a SOP change form, or preparation of a new SOP must
be reviewed, approved, and signed by the following:

Authored By:

Qian Wu and Dr. Kurunthachalam Kannan

Supervisor Review By:

______________________________

Date: ____________

Reviewed By:
(QA Coordinator)

______________________________

Date: ____________

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 3 of 15

DEFINITIONS AND ACRONYMS
PFOS

perfluorooctane sulfonate (anion of potassium salt), C8F17SO3-

PFOA

perfluorooctanoic acid, C7F15COO-

PFBS

perfluorobutane sulfonate, C4F9SO3-

PFHxS

perfluorohexane sulfonate, C6F13SO3-

PFDS

perfluorodecane sulfonate, C10F21SO3-

PFHpA

perfluoroheptanoic acid, C6F13COO-

PFNA

perfluorononanoic acid, C8F17 COO-

PFDA

perfluorodecanoic acid, C9F19 COO-

PFUnDA

perfluoroundecanoic acid, C10F21 COO-

PFDoDA

perfluorododecanoic acid, C11F23 COO-

PFOSA

perfluorooctane sulfonylamide, C8F17SO2NH2

HPLC

High Performance Liquid Chromatography

ESI/MS/MS Electrospray/tandem Mass spectrometer
PFCs

Perfluorinated Compounds

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 4 of 15

1.0 PURPOSE
This method describes the procedure for extracting perfluorooctane sulfonate (PFOS) or other
fluorochemical surfactants from serum, or other bodily fluids, using an ion pairing reagent and
methyl-tert-butyl ether (MTBE). In this method, eleven fluorochemicals can be extracted and
analyzed and they are: perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS),
PFOS, perfluorodecane sulfonate (PFDS), perfluoroheptanoic acid (PFHpA), perfluorooctanoic
acid

(PFOA),

perfluorononanoic

acid

(PFNA),

perfluorodecanoic

acid

(PFDA),

perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA) and perfluorooctane
sulfonylamide (PFOSA), and labeled surrogate/internal standards. A strong mineral acid or alkali
is added to the sample to free bound fluorocarbons from the biological matrix and extracted into
a solvent. The sample extracts are analyzed by HPLC equipped with an electrospray (ESI)
tandem mass spectrometry (MS/MS) detector by multiple reaction monitoring (MRM).

2.0 SCOPE AND APPLICATION
This method is for the extraction of PFOS or other fluorochemicals from serum and other bodily
fluids. The extracted fluorochemicals are quantified by HPLC-ES/MS-MS.

3.0 SAFETY CONSIDERATIONS
All safety considerations will be in accordance with Wadsworth-LOAC procedures and with the
requirements of the Wadsworth Center Safety Manual. These requirements include:
Personnel protective equipment (PPE) consisting of lab coats, safety glasses, and latex gloves
will be worn at all times when handling samples.
All personnel using such equipment must be aware of the hazards and must operate equipment
according to the manufacturer’s safety procedures.
Organic solvents are used for rinsing equipment, glassware cleaning, and in the extraction phase
of the method. These solvents represent a potential hazard to personnel in the laboratory.
Care must be taken to minimize exposure in accordance to institutional guidelines.

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 5 of 15

4.0 EQUIPMENT, MATERIALS, AND REAGENTS
Note: Avoid materials, solvents and reagents that contact with teflon. Teflon contains some
polymers that might interfere with PFOS/PFC analysis.
4.1

Equipment and Materials

The following equipment is used while performing this method.
acceptable.
4.1.1

Sample storage and extraction
Balance (sensitivity to 0.100 g)
Freezer @ -20o C (For sample storage)
Alconox detergent
Vortex mixer
Centrifuge
Shaker, Eberbach or equivalent
Nitrogen Evaporator, Organomation
Eppendorf or equivalent pipettes
Timer
15 ml Polypropylene (PP) Centrifuge Tubes
5 ml polypropylene tubes
Oxford Dispenser

4.1.2

Sample Clean-up (if necessary)
Disposable plastic 3cc syringes
0.2 μm, 25 mm nylon filters with leur lock,
Autosampler vials with caps
Syringes (capable of dilivering 5 μl to 50 μl)

Equivalent equipment is

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 6 of 15

Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-Q
water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials.
4.2 Reagents
Milli-Q Water
Sodium Bicarbonate (NaHCO3), J.T.Baker
Sodium Carbonate (Na2CO3), J.T.Baker
Tetrabutylammonium Hydrogen Sulfate (TBAHS), J.T.Baker
Methyl-tert-butyl ether (MTBE), J.T. Baker, glass distilled or HPLC grade
Methanol, J.T. Baker, glass distilled or HPLC grade
Fluorochemical standard solution (stock)
PFBS (3M Specialty Chemical Division), 20 g/mL
PFHxS (3M Specialty Chemical Division), 20 g/mL
PFOS (TCI America), 20 g/mL
PFDS (Wellington), 5 g/mL
PFOSA (3M Specialty Chemical Division), 20 g/mL
PFHpA (Flurochem), 20 g/mL
PFOA (TCI America), 20 g/mL
PFNA (Research Chem Ltd.), 20 g/mL
PFDA (Flurochem), 20 g/mL
PFUnDA (Aldrich), 25 g/mL
PFDoDA (Aldrich), 25 g/mL
Other fluorochemicals, as appropriate

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 7 of 15

4.3 Standards preparation
4.3.1

Secondary standard preparation: mix above standards and methanol into a pp-tube
as listed below, the final solution contains 1

g/mL of each compound listed

above.
PFBS (3M Specialty Chemical Division), 20 g/mL, 200 L
PFHxS (3M Specialty Chemical Division), 20 g/mL, 200 L
PFOS (TCI America), 20 g/mL, 200 L
PFDS (Wellington), 5 g/mL, 800 L
PFOSA (3M Specialty Chemical Division), 20 g/mL, 200 L
PFHpA (Flurochem), 20 g/mL, 200 L
PFOA (TCI America), 20 g/mL, 200 L
PFNA (Research Chem Ltd.), 20 g/mL, 200 L
PFDA (Flurochem), 20 g/mL, 200 L
PFUnDA (Aldrich), 25 g/mL, 160 L
PFDoDA (Aldrich), 25 g/mL, 160 L
MeOH, 1280 L
4.3.2

Working standard preparation: mix 9 mL of methanol and 1 mL of the

secondary dilution standard into a 15 mL pp-tube. Final solution contains 100 ng/mL
of each compound listed above.
4.4 Internal (13C-labeled) standard preparation
Mix 1 mL of 500 ng/mL 13C4-PFOS (Wellington Laboratories, Guelph, Ontario, Canada), 1 mL
of 500 ng/mL 13C4-PFOA (Wellington Laboratories, Guelph, Ontario, Canada), 1 mL of 500
ng/mL 13C2-PFNA (3M Company, St. Paul, MN), 1 mL of 500 ng/mL 13C2-PFDA (3M Company,
St. Paul, MN) and 6 mL of MeOH into a 15 mL pp-tube. The final solution contains 50 ng/mL of
each internal standard listed above.

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 8 of 15

4.5 Reagent preparation
Note: When preparing larger or smaller volumes than what is listed in reagent, standard, or
surrogate preparation, adjust accordingly.
4.5.1 10N sodium hydroxide (NaOH): Weigh 200 g NaOH and transfer into a 1000 mL beaker
containing 500 mL Milli-Q water, mix until all solids are dissolved. Store in a 1 L
Nalgene bottle.
4.5.2 1N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL of 10 N NaOH
solution into a 100 mL volumetric flask and dilute to volume using Milli-Q water. Store
in a 125 mL Nalgene bottle.
4.5.3 0.5 M tetrabutylammonium hydrogen sulfate (TBAHS): Weigh 169 g of TBA into a 1 L
volumetric containing 500 mL Milli-Q water. Adjust to pH 10 using approximately 44 to
54 mL of 10 N NaOH. Dilute to volume with Milli-Q water. Store in a 1 L Nalgene bottle.
Check pH before use. Adjust to pH=10 using 1 N NaOH solution.
4.5.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2CO3/NaHCO3): Weigh 26.5 g
of sodium carbonate and 21 g of sodium bicarbonate into a 1 L volumetric flask and bring
to volume with Milli-Q water. Store in a 1 L Nalgene bottle.
4.6 Standard storage and use conditions
Store spiking solutions at 4 C in sealed polypropylene tubes.
Note: Analysts must allow all spiking solutions to equilibrate to room temperature before use.
Note: Each time a vial containing small volumes of solutions is warmed to room temperature
and opened, a small volume of solvent in the vial headspace evaporates, significantly affecting
concentration.

Solutions should be stored with the smallest possible headspace, and opening

vials should be minimized. Also, the solution must be at room temperature before use.
5.0 SAMPLE HANDLING
In the laboratory, all samples must be kept frozen (-20 C) until grinding and/or sample
extraction. A sample track sheet or laboratory notebook is used to locate samples, processing
date and so on.

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 9 of 15

6.0 QUALITY CONTROLS
For each set of 30 to 40 samples (depending on the sample size and available sample volume and
project goals), there will be a minimum quality control samples that includes a solvent blank,
duplicate method blanks, and duplicate matrix spikes.

6.1 Method Blank
The method blank is extracted with the samples to monitor for any interference that may have
been introduced to the sample during sample preparation. Blanks include:
6.1.1 A 1.0 ml of Milli-Q water is used as a solvent blank.
6.1.2 Extract two 1 mL samples following this procedure and use as matrix blanks. There
should be matrix blanks for each matrix or sample type used in the analysis.
6.2 Surrogate Spike
6.2.1 All samples, blanks and matrix spike samples will be fortified with mass labeled
internal standard before solvent extraction begins.
6.2.2 Surrogate spike samples are needed to monitor the quantitative transfer of the organic
compounds of interest throughout sample preparation to the LC-MS/MS detector.
6.3 Matrix Spike/Matrix Spike Duplicate
6.3.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the
accuracy of the extraction for each matrix that is to be evaluated, especially
interference and ionization suppression due to matrix effects. The spiked compounds
are used to monitor sample matrix effects that could interfere with the analytes of
interest.
6.3.2 Prepare each spike using a sample chosen by the analyst. This is usually a control for
samples received from a laboratory toxicity test.
6.3.3 Expected concentrations for spiking should fall in the mid-range of the initial
calibration curve. Additional spikes may be included and may fall in the low-range
of the initial calibration curve.

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 10 of 15

6.3.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum
of two matrix spikes per batch.
7.0 SAMPLE EXTRACTION
7.1
7.2

Obtain frozen samples and allow thawing at room temperature.
Vortex mix for 15 seconds, then transfer 1.0 mL or appropriate volume to a 15 mL
polypropylene centrifuge tube.

7.3

Return unused samples to freezer after the volume for extraction has been removed.

7.4

Record the initial volume on the lab notebook.

7.5

Label the tube with the study number, sample ID, date and analyst initials.

7.6

Spike all samples, including blanks, ready for extraction with 100 L of surrogate
standards as described in 4.4.

7.7

Matrix spike samples preparation: spike matrix samples with 100

L of native

standards as described in 4.3.2.
7.8

Vortex to mix the samples for 15 seconds.

7.9

To each sample, add 2 mL of 0.25 M sodium carbonate/sodium bicarbonate buffer,
vortex to mix the samples for 15 seconds.

7.10

Check to ensure that 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. To
each sample, add 1 mL of 0.5 M TBA, vortex to mix the samples for 15 seconds.

7.11

Using an Oxford Dispenser, add 5 mL of MTBE.

7.12

Cap each sample and place on an orbital shaker at a setting of 300 rpm, for 40
minutes.

7.13

Centrifuge for 5 minutes at a setting of 3500 rpm, until the liquid layers are well
separated.

7.14

Label a fresh 15 mL pp-tube with the same information as in 7.5.

7.15

Remove 4.5 mL of the organic layer to the clean 15 mL pp-tube.

7.16

Repeat extraction as indicated on 7.11, 7.12 and 7.13 using 3 mL MTBE.

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

7.17

SOP:
Revision: 2
Page 11 of 15

Remove the organic layer to the clean pp-tube as in 7.15, and combine the two
layers.

7.18

Put each sample under nitrogen evaporator until near dryness (takes approximately 2
hours).

7.19

Add exactly 1.0 mL of methanol to each centrifuge tube using a graduated pipette.

7.20

Vortex mix for 60 seconds.

7.21

When necessary, attach a 0.2 m nylon mesh filter to a 3 cc syringe and transfer the
sample extract to this syringe. Filter into a 1.5 mL amber glass autosampler vial.

7.22

Label the autosampler vials with the sample ID, extraction date and initial of the
analyst.

7.23

Cap and store the extracts in the refrigerator until analysis.

7.24

Complete the lab note with sufficient experiment information.

8.0 DATA ANALYSIS AND CALCULATIONS
8.1 Instrumental analysis
Concentrations of 11 PFCs including PFBS, PFHxS, PFOS, PFDS, PFOSA, PFHpA, PFOA,
PFNA, PFDA, PFUnDA, and PFDoDA were detected and quantified on an Agilent 1100 Series
high performance liquid chromatography (HPLC), coupled with an Applied Biosystems API
2000 electrospray triple-quadrupole mass spectrometer (ESI-MS/MS). Aliquots of ten microliters
were injected into a Betasil C18 (100 × 2.1 mm) column with a (20 × 2.1 mm) guard column,
both with a 5 μm particle size (Thermo Electron Corporation, Waltham, MA) at a flow rate of
300 μL min-1. The mobile phase consisted of 2mM ammonium acetate and methanol. The
gradient elution started at 10% methanol and increased to 100% after 10 min; it was held at
100% for 2 min, and then reverted to 10% methanol. The separation of target compounds is
shown in Figure 1.

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 12 of 15

PFBS
RT=8.87 min

PFHpA
RT=11.50 min

PFHxS
RT=11.66 min

PFOA
RT=12.41 min

PFNA
RT=12.73 min
PFOS
RT=12.90 min

PFDS
RT=13.54 min

PFOSA
RT=14.44 min

PFDA
RT=13.08 min

PFUnDA
RT=13.33 min

PFDoDA
RT=13.55 min

Figure 1. Separation of target PFCs using Betasil C18 (100 × 2.1 mm) column.
The MS/MS was operated in a multiple reaction monitoring (MRM) mode; the mass transitions
monitored were: 398.7 > 79.7 for PFHxS, 499 > 99 for PFOS, 503 > 99 for 13C4-PFOS, 599 > 99
for PFDS, 497.7 > 77.7 for PFOSA, 413 > 369 for PFOA, 417 > 372 for 13C4-PFOA, 363 > 319
for PFHpA, 462 > 419 for PFNA, 465 > 420 for 13C2-PFNA, 513 > 469 for PFDA, 515>470 for
13

C2-PFDA, 563>519 for PFUnDA, and 613>569 for PFDoDA.

When possible, multiple

daughter ions were monitored for confirmation, but quantitation was based on a single product
ion. A list of mass transitions of target PFCs is shown in Table 1.
Table 1. Mass transitions and compound specific parameters
Collision
Declustering Collision
Precursor Product
cell exit
Compound
potential
energy
ion
ion
potential
(V)
(eV)
(V)

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

PFBS
PFHxS
PFOS
13
C4-PFOS
PFHpA
PFOA
13
C4-PFOA
PFNA
13
C2-PFNA
PFDA
13
C2-PFDA
PFUnDA
PFDoDA

SOP:
Revision: 2
Page 13 of 15

298.7
398.7
499
503
363
413
417
463
465
513
515
563
613

79.7
79.7
99
99
319
369
372
419
420
469
470
519
569

-30
-60
-60
-60
-10
-10
-10
-10
-10
-15
-15
-15
-22

-50
-80
-80
-80
-15
-15
-15
-15
-15
-10
-10
-7.5
-15

-5.4
-5
-5
-5
-12
-12
-12
-12
-12
-15
-15
-30
-19

8.2. Detection limits
The limit of quantitation (LOQ) is determined based on the linear range of the calibration curve
prepared at a concentration range of 0.1 to 100 ng/mL. Concentrations in samples which are at
least three-fold greater than the lowest acceptable standard concentration are considered to be
valid. A curve point was deemed acceptable if 1) it was back-calculated to be within 30% of
the theoretical value when evaluated versus the 1/x weighted curve, and 2) the peak area of the
standard was at least 3 times greater than that in the blank.

Concentration/dilution factors are

included in the calculation of the LOQ. The LOQ for perfluorochemicals is typically 0.1-0.5
ng/mL.

A list of LOD and LOQ typically detected for PFCs is shown in Table 2.
Table 2. LOD (ng/mL) and LOQ (ng/mL) for Perfluorochemicals

ng/mL
LOD
LOQ

PFBS
0.02
0.1

PFHxS

PFOS

PFDS

0.02
0.1

0.02
0.1

0.05
0.2

PFHp
A
0.02
0.1

PFOA

PFNA

PFDA

0.033
0.1

0.02
0.1

0.033
0.1

PFUn
DA
0.05
0.2

PFDo
DA
0.05
0.2

PFOSA
0.01
0.05

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 14 of 15

8.3. Quality control
Recoveries of labeled internal standards 13C4-PFOS, 13C4-PFOA, 13C2-PFNA, and 13C2-PFDA
should be within 100±30%. If the recoveries do not fall within this range, the sample should be
analyzed again or data should be flagged. Recoveries of native standards spiked into the
sample matrix should be within 100±30%.

If the recoveries do not fall within this range, the

sample should be analyzed again or data should be flagged. Solvents, blood collection tubes,
and method and matrix blanks should be checked for the presence of the perfluorochemicals and
any contamination should be reported as quality control information.

The results are not

corrected for the recoveries of internal standards.

9.0 RECORDS, DOCUMENTATION, AND QC REQUIREMENTS
9.1 Records and Documentation
The primary analyst shall document any anomalies and/or deviation from the specified method in
a bound, serially numbered, laboratory notebook with tear-out carbon copies. All electronic files
and hardcopies will be kept at the LOAC at Wadsworth Center and a duplicate copy will be kept
in the Room D547, Dr. Kurunthachalam Kannan’s office. The primary analyst will sign and date
any forms as the analyst.

9.2 QC Requirements and Data Quality Objectives
9.2.1

Method performance

The method detection limit (MDL) and limit of quantitation (LOQ) is analyte and matrix specific.
The following quality control samples are extracted with each batch of samples to evaluate the
quality of the extraction and analysis
Method blanks and matrix blanks

LOAC
Wadsworth Center, NYSDOH
Albany, NY 12201

SOP:
Revision: 2
Page 15 of 15

Matrix spike and matrix spike duplicate samples to determine accuracy and precision of
the extraction.

9.0 RESPONSIBILITIES
The primary analyst will complete the analysis as specified in this SOP and provide
documentation of raw data and any anomalies and provide data to the data analyst who will
perform data calculations.
The technical reviewer will determine if data quality objectives were met, and to notify the
analyst if any problems were found.

10.0 REFERENCES

Kannan et al. Perfluorooctanesulfonate and Related Fluorochemicals in Human Blood from
Several Countries, Environmental Science and Technology, 2004, 38 (17), 4489-4495

Hansen et al. Compound-Specific, Quantitative Characterization of Organic Fluorochemicals in
Biological Matrices, Environmental Science and Technology, 2001, 35 (4), 766-770