© 2021 NSF NSF/ANSI 53 – 2021

Informative Annex 5
Test method for PFOA (perfluorooctanoic acid) and PFOS (perfluorooctanesulfonate) in general
test water by LC/MS/MS in electrospray negative ionization mode

The information contained in this annex is not part of this American National Standard (ANS) and
has not been processed in accordance with ANSI’s requirements for an ANS. Therefore, this annex
may contain material that has not been subjected to public review or a consensus process.
In addition, it does not contain requirements necessary for conformance to this standard.

I-5.1 Summary of method

Test waters to be analyzed for perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) are
directly injected and then analyzed by liquid chromatography triple quadrupole mass spectroscopy
(LC/MS/MS) in electrospray negative mode. Method sensitivity is 10 ng/L.

I-5.2 Definitions
I-5.2.1 stock standard solution (SS): A concentrated solution containing one or more method analytes

NOT FOR
prepared in the laboratory using assayed reference materials or purchased from a reputable commercial
source.

I-5.2.2 working calibration standard (WCS): A solution prepared from the stock standard solution. The
calibration solutions are used to calibrate the instrument response with respect to analyte concentration.

I-5.2.3

DISTRIBUTION
matrix spike (MS): A quality control sample composed of a known amount of standard is added
to a sample before the analysis to access the effect the matrix has on the performance of measurement
process.

OR SALE
I-5.2.4 matrix spike duplicate (MSD): A quality control sample composed of a known amount of
standard is added to a sample, analyzed with a matrix spike to access the effect the matrix has on the
precision of the measurement process.

I-5.2.5 continuing calibration verification (CCV): An analytical standard prepared from the same
source as the calibration curve that is analyzed periodically after the sample to verify the accuracy of the
existing calibration of the analytes.

I-5.3 Standards for PFOA/PFOS analysis

I-5.3.1 SS shall be prepared from reference PFOA and PFOS chemicals purchased commercially which
have optimal purity as a trace analytical standard. A second source of the standards are used in quality
control (QC). Internal standards shall be prepared from commercially available C13 labeled PFOA and
PFOS chemicals. Dilutions from the SS serve as the working standards in this method.

a) Prepare 5,000 µg/mL SS from purchased reference standards.

b) Add about 50 mg of the reference standard [PFOA and about 110 mg PFOS (40.7%)] to a separate
40-mL VOC vials, recording actual mass.

c) Adjust mass based upon purity and salt content.

d) Add 10.0 mL of methanol and vortex or sonicate as necessary to dissolve.

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e) Calculate the concentration by the following equation:

mass[mg] 1,000 µg purity[%] FW of free base or acid

concentration [µg/mL] = ( )( )( )( )
10 mL 1 mg 100 FW of salt

Alternately, many SS are available as a 1.0 mg/mL solution from commercial vendors. The analyst has
the option of using them if they choose.

f) Prepare secondary SS in methanol as needed using the following equation:

(C1 )(V1 )= (C2 )(V2 )

where:

C1 = concentration of SS (ng/mL)
V1 = volume of SS (mL)
C2 = concentration of working standard (ng/mL)
V2 = volume of working standard (mL)

I-5.3.2 Preparation of 20 µg/L PFOA and PFOS working solutions (WS) in 95/5 (v/v) water /
methanol:

NOT FOR
a) Prepare a working solution by making dilutions of the 5000 µg/mL individual primary stock solutions
to the 20 ug/mL solutions. The analyst has the option of preparing the 20 µg/mL SS mixture using other
analytical dilution protocols.

DISTRIBUTION
b) Add 38 mL of water and 2 mL of methanol to a 40 mL VOC vial.

c) Prepare mixed standard solution by adding 40 µL aliquot volume for each standard to the 40-mL
VOC vial.

OR SALE
d) Label with contents and preparation date. Solution is viable for 6 mo.

e) Store solution in 40 mL VOC vial at 4 °C (7.2 °F).

I-5.3.3 Preparation of 1,000 µg/mL individual internal standard stock solutions:

a) Stock internal standards are made from pure reference standards

b) Add about 10 mg of the internal standard to a 40-mL VOC vial, recording actual mass.

c) Adjust mass based upon purity and salt content.

d) Add 10.0 mL of methanol and vortex or sonicate as necessary to dissolve.

e) Calculate the concentration by the following the equation in Section I-5.3.1. Dilute the 1,000 µg/mL
individual internal standard stock solutions to a concentration of 1,000 ng/mL to be used in
Section I-5.3.4.

I-5.3.4 Preparation of 10 µg/L internal standard mixture in 50/50 (v/v) water / methanol:

a) Prepare the internal standard mixture by adding appropriate aliquot volume for each individual
internal standard PFOA-13C8 and PFOS-13C8 to a 40-mL VOC vial.

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b) Add adequate volume of diluent 50/50 (v/v) water/methanol to give a final volume of 40 mL and a
final concentration of 10 µg/L. Stir to mix.

c) Label with contents and preparation date. The internal standard solution is viable until a noticeable
decrease in response is observed.

d) Store solution at 4 °C (7.2 °F).

I-5.3.5 Preparation of multiple PFOA and PFOS WCS in the range of 10 ng/L to 1,000 ng/L in 95/5
(v/v) water / methanol:

a) Use the PFOA and PFOS SS prepared in Section I-5.3.2 to prepare WCS.

b) Prepare each WCS by adding the appropriate aliquot volume and diluent for each standard to a
40-mL VOC vial for a final volume of 40 mL. Calculate the concentration based on the water portion of
the resulting solution excluding the 5% methanol. Methanol will be added to all samples, blanks and
QC preparations to result in equivalent proportions of water, methanol and other additions for
subsequent instrumental analysis.

c) Label with contents and preparation date. Solution is viable for 1 mo.

NOT FOR
d) Store Solution in 40-mL VOC vial at 4 °C (7.2 °F).

I-5.4 Preparation for PFOA/PFOS instrumental analysis

DISTRIBUTION
General test water samples must be preserved with 6 to 10 mg of sodium thiosulfate per 125 mL of sample
at time of sampling. Samples are stored at 4 °C (7.2 °F).

I-5.4.1 Preparation of samples:

OR SALE
a) Pipette 90 µL of methanol into 2-mL autosampler vials to be used for blanks and samples.
b) Pipette 20 µL of the 10 µg/L internal standard solution to each of the 2-mL autosampler vials.
c) Pipette 1,800 µL of blank water or sample into the 2-mL autosampler vial.
d) Pipette 9 µL of concentrated formic acid into each vial.
e) Vortex all blanks, and samples for 15 s.

I-5.4.2 Preparation of the standards for analysis:

a) Pipette 20 µL of the 10 µg/L internal standard solution to 2-mL autosampler vials for the calibration
standard solutions.

b) Pipette 1,890 µL of each WCS solution into the 2-mL autosampler vial.

c) Pipette 9 µL of concentrated formic acid into each vial.

d) Vortex all standards for 15 s.

I-5.4.3 Preparation of matrix spike samples:

a) Follow steps below to prepare MS and MSD in one of the effluent sample waters to a concentration
of 350 ng/L or other mid-range concentration using the 20 µg/L working standard (WS) prepared above
in Section I-5.3.2.

b) Pipette 90 µL of methanol to each of the MS and MSD into two 2-mL autosampler vials.

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c) Pipette 20 µL of the 10 µg/L internal standard solution to each of the MS and MSD.

d) Pipette 1,800 µL of effluent sample water into a 2-mL autosampler vial for each MS and MSD.

e) Pipette appropriate aliquot of the 20 µg/L PFOA and PFOS working solution standard (WS) into the
2-mL autosampler vial for each MS and MSD.

NOTE — Calculation is to be based on the volume of the water sample to obtain the resulting spike
concentration. All other additions are adjusted to be equivalent between the samples, blanks and
calibration standards and therefore these volumes are not included in the calculation

f) Pipette 9 µL of concentrated formic acid into each vial using separate pipette tips.

g) Vortex MS and MSD for 15 s.

I-5.5 Apparatus and conditions for PFOA/PFOS analysis

The following apparatus shall be used for PFOA/PFOS analysis:

— ultra-high pressure liquid chromatography system with autosampler and triple quadrupole mass

NOT FOR
spectrometer (LCMS);

— columns: C18 5 µm (3.0 × 150 mm) scrubber column and C18 5 µm (3.0 × 50 mm) analytical
column; and

DISTRIBUTION
— LCMS conditions:

— analysis with 500 µL loop in sample injector;

— injection volume: 500 µL; and
— mobile phase solutions:

OR SALE
— mobile phase A: 10 mm ammonium acetate in LCMS grade water; and
— mobile phase B: LCMS grade methanol.

Flow
Start Seconds Loop Grad %A %B
(mL/min)
0.00 25 out 0.20 step 100 0
0.42 400 out 0.20 ramp 20 80
7.08 60 out 0.20 ramp 10 90
8.08 60 out 0.20 step 10 90
9.08 260 in 0.20 step 5 95
13.42 240 out 0.20 step 100 0

— ion source settings, negative mode:

— spray voltage: 2500 V;

— vaporizer temperature: 400 °C (720 °F);
— sheath gas pressure: 30 units or as applicable to the LCMS system being used;
— aux gas pressure: 20 units or as applicable to the LCMS system being used;
— ion transfer tube capillary temperature: 350 °C (630 °F);
— declustering voltage: 0; and
— polarity: ES-.

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MRM Collision
Compound CAS # Dwell (s) S-lens
transition energy
PFOA 335-67-1 413.02 > 369.11 0.05 10 52
PFOA – 13C8 — 421.05 > 376.11 0.05 10 55
PFOS 1763-23-1 498.99 > 80.22 0.05 46 160
PFOS – 13C8 — 507.03 > 80.22 0.05 46 160

I-5.6 Sample analysis

— analyze four control blanks to condition the system, then the calibration standards, reagent blank,
laboratory fortified blank, second source, a reagent blank, the samples, a MS and MSD, and CCV at a
minimum for every ten samples being analyzed;

— the sample concentrations are calculated using internal standard (least squares linear regression)
with a 1/X weighting; and

— report limit determination:

NOT FOR
— the report limit is determined by the following equation.

I-5.7 Quality control

ng ng

DISTRIBUTION
report limit [ ] = low standard concentration [ ] × dilution
L L

— calibration criteria for quantitative analysis:

OR SALE
— each sample batch run will contain a minimum of three calibration standards. If the lowest
standard is excluded, the reporting limits (RLs) will be adjusted accordingly;

— the calibration curve correlation coefficient 0.995 or better, or R2 ≥ 0.99; and

— a method blank will be run after calibration standards. The area ratio for any peak present in
the retention window of a target analyte should be less than 80% of the area ratio of the lowest
standard.

— acceptance criteria for QC samples:

— percent recoveries for MS and MSD are calculated. Average percent recoveries should fall
within established control limits or ± 30% if control limits have not been established;

— the relative percent difference (RPD) for MS and MSD are calculated. RPDs should fall within
established control limits or ± 30% if control limits have not been established;

(MS - MSD)
RPD = | | × 20
(MS + MSD)

— a bracketing standard (CCV) prepared at a midrange concentration will be run after every ten
samples. The percent recovery for the bracketing standard should be within 80% to 120% of the
standard concentration value;

— all positive results must be within the calibration range; and

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