Determination of Table 3 Plus Compounds by LC/MS/MS
Chemours Fluoroproducts Analytical Method
Revision date 1/10/2019

Instrument Setup(Note: Trapping column installed on Line A. Delay column installed between mixer andmultisampler.)
Instrument:

Agilent 1290 Infinity II LC with 6470 Triple Quad MS

Analytical Column:

Agilent InfinityLab Poroshell 120 EC-C18(2.1 x 50 mm, 2.7 µm); P/N: 699775-902

Guard Column:

Agilent InfinityLab Poroshell 120 EC-C18(2.1 x 5 mm, 2.7 µm); P/N: 821725-911

Trapping Column:

Restek Ultra Aqueous C18 (4.6 x 50 mm, 5 µm); P/N: 9178555

Delay Column:

Agilent Zorbax Eclipse Plus C18 (4.6 x 50 mm, 3.5 µm); P/N: 959943-902

Consumables(Note: Avoid using glass. All standards, samples, and stock solutions should be prepped and stored
in HDPE or PP containers. Part numbers are provided below, but substitutions of similar products
can be made.)
LC vials:

Microsolv, Cat. No. 9502S-PP-Clear

LC caps:

VWR, P/N: 82028-424

Scintillation vials:

VWR, P/N: 66021-692 (used for sample and standard dilutions)HDPE bottles:

Thermo Scientific, P/N: 2104-0004 (used for calibration stocks)

Luer-Lock syringes:

Microsolv, P/N: 58903-S-K

0.2 um GHP filters:

Pall Acrodisc, P/N: 4554
(or substitute with same dimensions and filter material)

Sample Preparation
All standards and stocks are prepared with a 50:50 methanol:water diluent. A 10-point calibration
curve is made from a 100 ppb mixed stock standard (containing the twenty-four Table 3 Plusanalytes) at the following dilutions: 0.01 ppb, 0.05 ppb, 0.1 ppb, 0.25 ppb, 0.5 ppb, 1 ppb, 5 ppb,10 ppb, 25 ppb, and 50 ppb. Prepped standards should be filtered in the same way as the samples.
Note: The 100 ppb mixed stock standard is prepared in ultrapure water. The stock standard
should be stored in a refrigerator when not in use. After allowing the stock to warm to room
temperature, an aliquot is diluted 2x with methanol to produce a 50 ppb mixed standard in a

50:50 methanol:water matrix; this stock is then used for spiking and to prepare subsequent
dilutions for the calibration curve.
All samples are tested for pH when received to ensure that they are neutral (pH 6-8). If necessary,
dilute solutions of KOH or H2SO4 are used to adjust pH accordingly. Samples are prepared in
duplicate with duplicate spikes at different dilutions depending on the expected concentrationof the analytes. Each sample prep starts with a 2x dilution in methanol to produce a 50:50
methanol:water matrix. Subsequent dilutions are made with a 50:50 methanol:water mix. A 2x
dilution will be appropriate for most groundwater analyses (trace levels expected). If expectedconcentration is unknown, err on side of caution and use large dilution factor initially. If notdetected, reprep with smaller dilution factor. Example preparations are listed below.
2x dilution and spike: 2.5 mL of sample is diluted with 2.5 mL of LC/MS grade methanol in a
scintillation vial, then vortexed and filtered with a 0.2 um GHP syringe filter. To prepare spikes,
2.5 mL of sample is diluted with 2.4 mL of LC/MS grade methanol and 100 µL of 100 ppb spikesolution in a scintillation vial, then vortexed and filtered with a 0.2 um GHP filter. Final expectedconcentration of spike in vial is 2.0 ppb.
100x dilution and spike: 2.5 mL of sample is diluted with 2.5 mL of LC/MS grade methanol in a
scintillation vial, then vortexed to make a 2x prep. 100 µL of the 2x prep is diluted to 5.0 mL using
50:50 methanol:water, then vortexed and filtered with a 0.2 um GHP syringe filter. To prepare
spikes, 100 µL of the 2x prep is diluted with 4.8 mL of 50:50 methanol:water and 100 µL of 100ppb spike solution in a scintillation vial, then vortexed and filtered with a 0.2 um GHP filter. Final
expected concentration of spike in vial is 2.0 ppb.
QC sample: A 5 ppb QC sample is prepared from a stock standard solution separate from the oneused for calibration following the same dilution and filtering protocol as for samples.

Analytical Method
LC Operating Conditions:
Mobile phase A:

2 mM Ammonium Acetate in 5:95 Acetonitrile:Water

Mobile phase B:

Acetonitrile

Needle/seat wash:

50:50 Acetonitrile:Water, Multi-wash option using 2 cycles of seatback flush and needle wash (10 seconds each)Seal wash:

10:90 Isopropanol:Water

Injection volume:

5.00 µL

Flow rate:

0.500 mL/min

Maximum pressure: 600.00 bar
Solvent gradient:
Initial1234
End time:

9.50 mins

Column temp:

50.0°CMS Operating Conditions:
Ion mode:

ESI negative

Scan type:

MRM

Gas temp:

150°C

Gas flow:

8 L/min

Nebulizer:

45 psiSheath gas heater:

200°C

Sheath gas flow:

8 L/min

Capillary:

3500 V

Nozzle voltage:

0VTime0.00 min1.00 min5.00 min6.40 min6.50 min

A
85%
85%
10%
10%
85%

B
15%
15%
90%90%
15%

QQQ Acquisition Parameters:
Compound Name

MRM Transition

RT (mins)

Dwell(msec)

DFSA
MMF
MTP
PPF acid
PFMOAA
R-EVE
Byproduct 4
Byproduct 5PMPA
PFO2HxANVHOSPEPAPFECA B
PFO3OAPESHFPO-DAHFPO-DA_qualifier
PFECA_GPFO4DA
Hydro-EVE Acid
EVE Acid
Byproduct 6
Byproduct 2
Byproduct 1
PFO5DoA

175.0 -> 131.0
139.0 -> 95.0
175.0 -> 97.0
163.0 -> 118.9
179.0 -> 84.9
405.0 -> 217.0
440.9 -> 241.0
439.0 -> 343.0
229.0 -> 184.9
245.0 -> 85.0
297.0 -> 135.0279.0 -> 234.9
295.0 -> 201.0
310.9 -> 85.0
314.9 -> 135.0
329.0 -> 284.9
329.0 -> 169.0
378.9 -> 184.9
376.9 -> 85.0
427.0 -> 282.9
407.0 -> 262.9
397.0 -> 217.0
463.0 -> 262.9
443.0 -> 146.9
442.9 -> 85.0

0.268
0.271
0.370
0.394
0.437
0.420
0.509
0.510
0.662
1.046
1.655
1.702
3.027
3.298
3.388
3.386
3.386
3.702
3.794
3.825
3.825
3.951
4.072
4.084
4.085

50
50
50505050505050
50505050505050505050505050505050

Frag(V)

CE(V)

7293
7858
54100
114
12655
60
12358
55
54
10975
75
87
66
9078
122
160
14366

128128121628
30
4828
48122001216
201282732
30
8

Cell
Accelerator
(V)
2
1
15141
1
5
1
1433541433
21
111

Data Processing
Calibration: The full set of 10 calibration standards should be run at the beginning and end of asequence of samples. All 20 standard injections are used in constructing the calibration curve forquantitation of unknowns. Continuing calibration checks should be performed after every 10
sample injections in the sequence using a mid-level standard; these are not included as points inthe calibration curve. The curve should be linear, not forced through the origin, and weighted
1/x. The acceptable accuracy range is 70%-130%.
Blanks: Three blanks should be run at the beginning of each sequence—a methanol blank takenfrom the vessel used to perform 2x dilutions of aqueous stock standard and samples; a 50:50
methanol:water blank from the vessel used to perform subsequent dilutions; and anenvironmental blank consisting of ultrapure water taken from the same type of container used
for sampling. Target analyte concentrations in all blanks must be less than one-half of the method
LOQ.
Method and/or solvent blanks are also used to monitor for carryover between samples,particularly those with analyte levels detected at high concentrations. A typical sequence consistsof duplicate preps followed by their respective spikes, then a 50:50 methanol:water blank, andrepeating with subsequent sample prep/spike segments. Blanks in between sample segments
should be analyzed to quickly identify carryover contamination and are subject to the same
concentration criteria as described in the paragraph above.
Duplicates: Duplicate preps of the same sample must have a %RPD of ≤25% at mid- and highrange concentrations and ≤50% at the minimum reporting limit (see equation below).
%RPD = (sample result - duplicate result) * 100(sample result + duplicate result)/2Results outside this range require a duplicate reprep. Both results should be reported, along withthe average.
Spikes: Acceptable spike recovery is in the range of 70%-130%. Spike recovery outside this range
does not necessitate reprep, but results should be flagged for further investigation due topossible matrix effects.
QC Sample: A QC sample of known concentration should be prepped from a standard stock
solution separate from the one used for the calibration. Results must be within 70%-130% of thetrue value.

Appendix A: Compound Details
CompoundName

Molecular
FormulaDFSA

C2H2F2O5S

422-67-3

MMF

C3H2F2O4

1514-85-8

MTP

C4H4F4O3

93449-21-9

PPF Acid

C3HF5O2422-64-0

PFMOAAC3HF5O3674-13-5

R-EVE

C8H2F12O5

N/A

Structure

CASRN

Byproduct 4C7H2F12O6S

N/A

Byproduct 5C7H3F11O7S

N/A

PMPA

C4HF7O313140-29-9

PFO2HxA

C4HF7O439492-88-1

NVHOS

C4H2F8O4S

1132933-86-8

PEPA

C5HF9O3267239-61-2

PFECA B

C5HF9O4

151772-58-6

PFO3OAC5HF9O539492-89-2

PES

C4HF9O4S

113507-82-7

HFPO-DAC6HF11O313252-13-6

PFECA GC7HF13O3801212-59-9

PFO4DAC6HF11O639492-90-5

Hydro-EVE
Acid

C8H2F14O4

773804-62-9

EVE Acid

C8HF13O4

69087-46-3

Byproduct 6

C6H2F12O4S

N/A

Byproduct 1

C7HF13O5S

29311-67-9

Byproduct 2

C7H2F14O5S

749836-20-2

PFO5DoA

C7HF13O7

39492-91-6

Appendix B: Recommendations for Instrument Maintenance

Before starting a sequence, run an isopropanol blank and 3 method blanks (50:50
methanol:water) to flush the system and establish a “clean” baseline. At the end of a sequence,
run a “steam clean” line and end with an “idle” line (see screenshot below).

Both of these methods should be set up to utilize lines A2 and B2, which are 95:5 water:methanoland 100% methanol, respectively. This will help to flush out any salts or other buildup that has
accumulated in the system. It is recommended to run the idle method while the system is not
actively being used.
After the idle method has completed and while the source temperatures are still low, open thespray chamber and wipe down all surfaces – especially the spray shield and surrounding area –with a 50:50 mixture of isopropanol and ultrapure water on a Kimwipe. This should be done daily
to prevent buildup.
A 50:50 acetonitrile:water mixture works well as a needle/seat rinse to minimize carryover. Asolution of 90:10 water:isopropanol should be used as the seal wash solvent, which should beprimed if bubbles are observed in the clear tubing between pump heads.
Solvent reservoirs should be periodically rinsed with isopropanol before rinsing and refilling with
the intended solvent. If possible, store solvents in amber bottles to inhibit microbial growth, andprepare fresh solvents if not used within 1-2 weeks.