Quantitation of PFAS using MALDI-TOF MSBrooke Dilmetz

Per and polyfluoroalkyl substances• PFOS and PFOA manufactured from early 1950’s – present1

• Highly persistent and bioaccumulative

University of Adelaide 2

PFOS (Perfluorooctanesulfonate)

PFOA (Perfluorooctanoate)

1 Oliaei et al. (2013). Environ Sci Pollut Res

Per and polyfluoroalkyl substances• PFOS and PFOA manufactured from early 1950’s – present1

• Germany, Italy and China• Highly persistent and bioaccumulative

University of Adelaide 3

PFOS (Perfluorooctanesulfonate)

PFOA (Perfluorooctanoate)

- Evidence that PFAS:• Disrupt normal endocrine activity;• Reduce immune function;• Cause adverse effects on multiple organs, including the liver and

pancreas; • Cause developmental problems in rodent offspring exposed in the

womb.2,3

2Lindstrom AB, Strynar MJ, Libelo EL. (2011). Environ Sci Technol

3White SS, Fenton SE, Hines EP. (2011). J Steroid Biochem

Measuring PFAS: LCMS • Liquid chromatography coupled tandem mass spectrometry

University of Adelaide 4

1. Zheo et al. (2011). Mircochem.2. Shimadzu, Application note C813. Adelaide Proteomics Centre, 20174. Cao et al. (2011). Talanta5. Food standards Australia, 2017

Drinking water quality guideline5

PFOS PFOA

0.07µg/L 0.56µg/L

Advantages• Sensitive/Selective • Low detection (LOD 0.03 – 3.0 ng/L1,2)

Disadvantages• Cost ($200.00-400.00/sample3)• Time - column conditioning,

- carry-over• Contamination of fittings and tubing4

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• Typical analytes are macromolecules (proteins, polymers)• Limited for the detection of small molecules (M.W. <500 Da)• Analyte co-deposited with matrix

PFAS analysis by MALDI-TOF MS

Advantages• High throughput MS analysis:

– typically 2-10 sec/spot

• Able to analyse multiple samples:– 1536 spot plate format (800µm)

• No carry-over:– Straight-forward target washing

• Low Cost ($5-10 per sample)

University of Adelaide 6Technical Note # TN-41, Bruker Daltonics GmbH

Disadvantages: small molecule analysis

• Quantitation is difficult, due to:o Matrix interference for small

molecules o Spot-spot variability

PFAS analysis by MALDI-TOF MS

University of Adelaide 7

PFAS analysis by MALDI-TOF MS

Project Aims

To create a screening platform for the detection of PFAS by MALDI-TOF MS

University of Adelaide 8

1. Determine suitable matrices for PFAS detection 2. Determine LODs/ LOQs for various PFAS

3. Quantify of PFAS spiked into water samples4. Apply methodology to contaminated water sample

MALDI Matrix Properties

Name Other Names Applications

α-Cyano-4-hydroxycinnamic acid CHCA Peptides and proteins

2,5-Dihydroxybenzoic acid 2,5-DHB Peptides, phosphoprotein digests, glycoprotein digests, proteins, glycans

2,5-Dihydroxyacetophenone 2,5-DHAP Proteins

Sinnapinic acid SA Proteins, lipids

3-Hydroxypicolinic acid 3-HPA Oligonucleotides

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• Strong UV light absorption at the wavelength of the laser (355nm)• Typically low molecular weight acid• Different matrices for different analyte classes – ionisation threshold

Matrices for PFAS Detection

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Cao et al. (2011) Paper

TMGN Replicate 1

TMGN Replicate 2

TMGN Replicate 3

TMGN Replicate 4

TMGN Replicate 5

Matrices for PFAS Detection

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Cao et al. (2011) Paper

Matrices used for detection of PFAS

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Matrix Advantages Disadvantages

CHCA Homogenouscrystallisation

Interfering matrix ion peaks

Novel Proton Sponge

Less interfering

matrix peaks

Heterogeneous crystallisation

TMGN Homogenouscrystallisation

Interfering matrix ion peaks

• Matrix-free applications• NALDI• Monoliths

CHCA for PFOS 

Novel Matrix for PFOA Novel Matrix for PFHxS

Quantitation of PFAS by MALDI-TOF MSPFOS -Matrix: CHCA

y = 5.1488x ‐ 0.4286R² = 0.99980

20

40

60

0 2 4 6 8 10 12

Average Intensity

 Ra

tio

Concentration PFOS (ng/uL)

PFOA -Matrix: Novel proton sponge

y = 2.3366x ‐ 0.4994R² = 0.988

0

5

10

15

20

25

30

0 2 4 6 8 10Average Intensity

 Ratio

Concentration of PFOA (ng/µL)

PFHxS -Matrix: Novel proton sponge

y = 0.7298x ‐ 0.0295R² = 0.9997

0

2

4

6

8

10

0 2 4 6 8 10Average Intensity

 Ratio 

Concentration PFHxS (ng/µL)

• Above detection limits of drinking water

• All 3 compounds show linearity – (0.1-10ng/µL)

MC

Slide 13

MC2 You need to have the ccalculated concentration hereMark Condina, 7/09/2017

Workflow for extraction of samples

University of Adelaide 14

SPE of contaminated water sample

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• 10 replicates per concentration• No heavy labelled standard

PFOS Evaluation

SPEContaminated

Water Concentration

(µg/L)

LC-MSContaminated

Water Concentration

(µg/L)

% Difference

1278.69 1170 8.5

y = 16357x + 9423.6R² = 0.9862

0

50000

100000

150000

200000

250000

0 2 4 6 8 10 12

Average Intensity

Concentration of PFOS (ng/uL)

PFOS Calibration Contaminated Water (After SPE)

Matrix free: NALDI application• Nanostructured laser desorption ionisation • Disposable target • Low mass organic molecules (e.g. 100-2000 m.w.) ion target• Surface deposited layer of inorganic nanostructures

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Daniels, H., Dikler, S. Technical Note 22, Bruker Daltonics, and NanoSys Inc. 

Quantitation of PFOS by NALDI-TOF

• Able to achieve linearity for same concentration range as with matrix• Matrix: NALDI target

y = 1.3584x + 0.0997R² = 0.9912

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

0 2 4 6 8 10 12

Average Intensity

 Ratio

Concentration of PFOS (ng/µL)

MC

Slide 17

MC2 You need to have the ccalculated concentration hereMark Condina, 7/09/2017

PFC evaluation by NALDI-TOF MS: screening• PFOS Threshold limit: 0.07µg/L

University of Adelaide 18

‐Matrix free allows lower levels of detection for PFOS-Will be used to determine LOQ

498.915

0

500

1000

1500

2000

2500

Inte

ns. [

a.u.

]

498.5 498.6 498.7 498.8 498.9 499.0 499.1 499.2m/z

Conclusions• Quantitate using MALDI-TOF MS

– Possible to achieve quant without labelled standard (higher variance)– Able to detect down to required thresholds using matrix free platform

• MALDI-TOF MS offers high-throughput assessment of samples after SPE enrichment– Able to screen 1000’s samples low cost

• More comprehensive sampling of potentially contaminated areas• Able to determine which samples to be analysed by LCMS (if required)

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Future work• Improve LOQ and LOD for MALDI and determine for NALDI • Improve MALDI-TOF MS acquisition

– Lower acquisition time per spot

• Evaluate alternative enrichment strategies for PFAS– Porous monolithic material for enrichment

• Investigate other matrix-free applications:– Porous monolithic surfaces – Laser desorption/ionisation (LDI)

• Poly(butyl methacrylate-co-ethylenedimethacrylate) monolith• Poly(styrene-co-divinyl benzene) monolith

University of Adelaide 20

Acknowledgements

Prof. Peter HoffmannDr. Mark CondinaDr. Dario ArruaDr. Lyron Winderbaum Silvana Napoli

Contact information: brooke.dilmetz@adelaide.edu.au

Yin Ying HoParul MittalChristopher Cursaro Matthew BriggsMitchell AcklandChao ZhangNoor Alia LokmanCarly Gregor

Dr. Richard Stewart