©2018 Waters Corporation 1

Reduction of LC/MS In-Source Fragmentation of HFPO-DA (GenX)Through Mobile Phase Additive Selection:

Experiments to Increase [M-H]- Formation

Lauren Mullin1,2, David Katz3, Nicole Riddell4, Robert Plumb1, Jennifer A. Burgess5 and Ingrid Ericson Jogsten2

1. Waters Corporation, Milford, MA2. MTM Research Centre, Örebro University

3. US EPA –Atlantic Ecology Division, Narragansett, RI4. Wellington Laboratories Inc., Guelph, ON Canada

5. Scientialis Consulting LLC, Hopkinton MA

©2018 Waters Corporation 2

Contents

HFPO-DA introduction and analytical challenge

Mobile phase additive results for HFPO-DA and signal improvement

ADONA and additional PFAS behavior under new mobile phase conditions

©2018 Waters Corporation 3

HFPO-DA Background

2,3,3,3-Tetrafluoro-2-(1,1,2,2,3,3,3,-heptafluoropropoxy)-propanoic acid– pKa = 2.43– Perfluoroalkyl ether carboxylic acid (PFECA)– PFECA developed by DuPont as replacement to existing perfluorinated

compounds– Discharge into waterways has resulted in environmental detection of the

compound– Analyzed by same LC MS/MS ESI- methods for legacy PFAS monitoring

o Ammonium acetate mobile phase additive widely used

Stryner M., Dagnino S., McMahen R. et al. (2015) Environ. Sci. Technol. (49) 11622-11630Hopkins Z., Sun M., DeWitt J. et al. (2018) J. AWWA (110) 13-28Gebbink W., van Asseldonk L., van Leeuwen S. (2017) Environ. Sci. Technol. (51) 11057-11065Sun M., Arevalo E., Strynar M. et al. (2016) Environ. Sci. Technol. Lett.(49)Heydebreck F., Tang J., Xie Z. et al. (2015) Environ. Sci. Technol. (49) 8386-8395

©2018 Waters Corporation 4

Analytical Challenge for HFPO-DA

Stryner et al. 2015 described the identification of HFPO-DA using TOF-MS– Observations in spectra: dimer formation, fragmentation and adduct formation

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Experimental Approach

Goal: to conserve and enhance formation of [M-H]-– MS Source optimization– LC mobile phase additive optimization

o pH of aqueous mobile phase recorded o Hypothesis: Different mobile phase compositions may stabilize the

molecule upon ionization

Mobile Phase Additives Tested

0.1 and 0.5%• formic acid• acetic acid• ammonium hydroxide

2mM and 10mM• ammonium acetate• ammonium formate• ammonium bicarbonate

LC Conditions:• Acquity H-Class UPLC w/ FTN• Column: Acquity BEHC18 100 x• 2.1mm 1.7µm dp• Column Temp: 55ºC• Flow rate: 0.4 mL/min• MPA: aqueous + additive• MPB: methanol + additive• Gradient:

Time (min.) %A %BInitial 45 550.5 45 555 10 90

5.5 10 906 45 558 45 55

MS Conditions:• Xevo TQ-D• MS Scan 50-800Da • ESI- Source Temp.: 150 °C Desolvation Temp.: 350 °C

• Desolvation Flow: 600 L/hr Cone Gas Flow: 50 L/hr

• Cone Voltage: 20 V • Capillary Voltage: 1.0 kV

Standard concentration: 5µg/mLInjection Vol.: 10µL

©2018 Waters Corporation 6

Mobile phase additive experiment: [M-H]-Response

– Result: Ammonium Bicarbonate MP Increased [M-H]-

0

2

4

6

8

10

12

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

[M-H]-

pH of MPA

©2018 Waters Corporation 7

Mobile phase additive experiment: [M-H]- and Adduct Response

0

2

4

6

8

10

12

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

[M-H]-

[M+61]- Adduct

pH of MPA

©2018 Waters Corporation 8

Mobile phase additive experiment: Fragment Response

0

2

4

6

8

10

12

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

8.00E+06

9.00E+06

[M-H]-

[M+61]- Adduct

285 m/z Fragment

169 m/z Fragment

pH of MPA

©2018 Waters Corporation 9

Mobile phase additive experiment: Proportional Comparison of Fragments to Parent Molecule

– Result: decreased proportion of fragmentation

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

169 m/z Fragment

285 m/z Fragment

[M+61]- Adduct

[M-H]-

©2018 Waters Corporation 10

Mobile phase additive experiment: Proportional Comparison of Dimer

– Result: decreased dimer formation with ammonium bicarbonate

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

[M+61] Adduct Response

[M-H]-

Dimer Adduct [2M-2H+Na]-

Dimer [2M-H]-

©2018 Waters Corporation 11

Comparative Spectra: Combined Infusion (5 µg/mL in 50/50 water/methanol + MP)

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Spectral Composition: Combined Infusion (5 µg/mL in 50/50 water/methanol + MP)

HFPO-DA Combined Infusion with2mM ammonium bicarbonate

[M-H]-

285 m/z fragment

169 m/z fragment

Dimer [2M-H]-

Dimer Adduct [2M-2H+Na]-

[M+61]- Adduct

51.5%

HFPO-DA Combined Infusion with2mM ammonium acetate

[M-H]-

285 m/z fragment

169 m/z fragment

Dimer [2M-H]-

Dimer Adduct [2M-2H+Na]-

0.5%

©2018 Waters Corporation 13

Impact of Ammonium Bicarbonate Buffer pH Relative proportions of ions were not impacted by pH, however pH 9 resulted in overall lower signal

response

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

no adjustment pH 8.2 adjusted pH 9.0 adjusted pH 10.0 adjusted pH 11.3 adjusted Ammonium Acetate

%To

tal S

igna

l

Mobile Phase

Dimer adduct

Dimer

[M+HCO3]-

[M-H]-

-CO2 Fragment

CF7 Fragment

©2018 Waters Corporation 14

Impact of Ammonium Bicarbonate Concentration

Concentration of ammonium bicarbonate resulted in increase of adduct formation and decrease in dimerformation

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

0

10

20

30

40

50

60

0.1 0.2 0.5 1 2 5 10 20

% o

f Tot

al S

igna

l

mMolar Concentration Ammonium Bicarbonate

Dimer

[M+61]-

[M-H]-

285 m/z Fragment

169 m/z Fragment

Total Intensity (Absolute)

©2018 Waters Corporation 15

HRMS to confirm adduct identity and fragmentation

HFPO-DA• Theoretical Neutral Mass (Da)= 329.9750• Theoretical [M-H]- (m/z)=328.9677• Observed=328.9679• Mass Error=0.2mDa • Theoretical [M+HCO3]- (m/z)=390.9681• Observed=390.9683• Mass Error=0.2mDa • Theoretical [M+NO3]- (m/z)=391.9634• Mass Error=-995.1mDa X• Theoretical [M-H+NO3]- (m/z)=390.9561• Mass Error=-12.2mDa X

MS Conditions:•Xevo G2-XS Qtof•ESI- Source Temp:120ºC Desolvation Temp: 350ºC

•Desolvation Flow: 1000 L/hr ConeGas Flow: 50 L/hr

•Cone Voltage: 20 V•Capillary Voltage: 1.0 kV

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Identification of [M+61]-

Rannulu et al. 2012: bicarbonate adduct encouraged for steroids through the use of ammonium bicarbonate in ESI- experiments– Overall sensitivity improved for steroids studied using anionic adducts

Complemented [M-H]- formation via formation of [M+HCO3]- for molecules which traditionally had poor [M-H]- yields

Site of adducting: hydroxyl group

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Confirmation with 13C isotope labeled standard

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Initial MRM Results2mM ammonium bicarbonate

Range: 5-5,000 ng/mL 5uL injections RT: 0.97 (%RSD=0.45, n=20) Peak width: ~3.6 sec. R2: 0.995 (Linear fit, 1/x weighting) Ion Ratio (Q/q): Avg. 1.40 (%RSD=4.07, n=20) S/N 5 ng/mL: 14 PtP Note: flow rate adjusted to 0.5mL/min from

original method

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Additional PFAS results for Ammonium Bicarbonate Method

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Behavior of other PFASs: ammonium acetate vs. ammonium bicarbonate

Another PFECA, ADONA, also analyzed across mobile phase experiments

– [M-H]- dominant ion species across all mobile phases

– Ammonium acetate and ammonium bicarbonate response similar, bicarbonate slightly higher

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

2mM ammonium

acetate

10mM ammonium

acetate

2mM ammonium

bicarbonate

10mM ammonium

bicarbonate

ADONA Response

[M-H]- Response

[M+HCO3]-

PFAC-24PAR mix standard analysis in full scan with both ammonium acetate and ammonium bicarbonate

– [M+HCO3]- in carboxylatecompounds with ammonium bicarbonate MP

– Carboxylates also exhibit some in-source fragmentation

– General [M-H]- response and chromatographic peak shape similar between two mobile phase conditions

– FOSA 4x lower– NMe/EtFOSAA and

PFUdA higher

PFAS Formula ClassFragmentation Observed?

( =yes)[M+HCO3]-

( =yes)

PFBA C4HF7O2 Carboxylate

PFPeA C5HF9O2 Carboxylate

PFBS C4HF9SO3 Sulfonate

PFHxA C6HF11O2 Carboxylate

4:2-FTS C6H5F9SO3 Telomer sulfonate

PFPeS C5HF11SO3 Sulfonate

PFHpA C7HF13O2 Carboxylate

PFHxS C6HF13SO3 Sulfonate

PFOA C8HF15O2 Carboxylate

6:2-FTS C8H5F13SO3 Telomer sulfonate

PFHpS C6HF13SO3 Sulfonate

PFNA C9HF17O2 Carboxylate

FOSA C8H2F17NO2S Sulfonamide

PFOS C8HF17SO3 Sulfonate

PFDA C10HF19O2 Carboxylate

8:2-FTS C10H5F17SO3 Telomer sulfonate

PFNS C9HF19SO3 Sulfonate

PFUdA C11HF21O2 Carboxylate

NMeFOSAA C11H6F17NO4S Sulfonamidoacetic acid

NEtFOSAA C12H8F17NO4S Sulfonamidoacetic acid

PFDS C10HF21O2 Sulfonate

PFTrDA C13HF25O2 Carboxylate *

PFDoA C12HF23O2 Carboxylate

PFTeDA C14HF27O2 Carboxylate * **minor

©2018 Waters Corporation 22

Example Comparison: PFOA

©2018 Waters Corporation 23

Conclusions

For HFPO-DA, the use of ammonium bicarbonate mobile phase:– Increased [M-H]-

– Decreased dimer formation– Decreased unintended fragmentation– Increased overall signal

Ammonium bicarbonate mobile phase could also be implemented for analysis of other PFAS, with MRM optimization recommended

Future work: implement method in quantitative analysis of contaminated samples

©2018 Waters Corporation 24

Acknowledgements

Kendon Graham, Kerri Smith, Kari Organtini and Paul Rainville and Adam Ladak (Waters)

My co-authors

Thank you for your attention!