SELECTED AROMATIC AMINES BY GAS CHROMATOGRAPHY MASS SPECTROMETRY: CHALLENGES OF MAINSTREAM CIGARETTE SMOKE

Alexandra Martin

October 3, 2013

What are PAAs and why are they important?

Smoke Collection 101

Brief history of PAA analysis

New challenges - FDA HPHC list SPE clean-up

GC-MS parameters – EI vs. NCI

Validation results LOD/LOQ

Accuracy

Precision

OUTLINE

WHAT ARE PAAS AND WHY ARE THEY IMPORTANT?

BACKGROUND Tobacco smoke contains more than 4,000

chemical compounds.

Characterization of this matrix has been the focus of scientists for many years.

One of the goals of this work has been to identify the compounds primarily responsible for serious health effects.

IARC Monographs on the Evaluation of Carcinogenic Risks to Humans - Classification

PAA CLASSIFICATIONS AND REPORTING REQUIREMENTS

Compound IARC Class

Health Canada

Brazil ANVISA US FDA

ortho-toluidine 1 X

2,6-dimethylaniline 2B X

ortho-anisidine 2B X

1-aminonaphthalene 3 X X X

2-aminonaphthalene 1 X X X

3-aminobiphenyl na X X

4-aminobiphenyl 1 X X X

THE ORIGINAL FOUR PAAS

1-aminonaphthalene 2-aminonaphthalene

3-aminobiphenyl 4-aminobiphenyl

NH2

NH2

NH2

NH2

ADDITIONS FOR FDA HPHC LIST

2,6-dimethylaniline o-anisidine

NH2

CH3

NH2

OCH3

NH2

CH3H3C

o-toluidine

SMOKE COLLECTION

CERULEAN 20-PORT SMOKING MACHINE

TOTAL PARTICULATE MATTER (TPM) FROM 5 KY 3R4F CIGARETTES

BRIEF HISTORY OF PAA ANALYSIS

AMERICAN HEALTH FOUNDATION METHOD “On the Analysis of Aromatic Amines in

Tobacco Smoke”. C. Patrianakos, K.D. Brunnemann and D. Hoffmann, American Health Foundation, Valhalla New York 10595.

Presented at the 31st TCRC, Greensboro, NC, October 1977.

C. Patrianakos and D. Hoffmann, “Chemical Studies on Tobacco Smoke LXIV. On the Analysis of Aromatic Amines in Cigarette Smoke”, J. Anal. Toxicol., 3, 150-154 (1979).

AHF METHOD (1977)

Smoke Collection - 210 cigs

Impingers w/ 5% HCl[Add ISTDs]

Liquid:Liquid Extraction w/ Ether

Dry and Concentrate

Derivatize (PFPA)

AHF METHOD (1977) CONTINUED…

Florisil – 10 g

Fraction (75 mL), Evaporate

Florisil – 30 g

Fraction 1 (500 mL) and Fraction 2 (400 mL)

GC-MS or GC-ECD Analysis

Wash, Dry, Evaporate

Evaporate each to 2 mL

HEALTH CANADA METHOD Health Canada Official Test Method T-102, 1999

Based upon Pieraccini et al 1992

Modifications CFP for trapping, not impinger Smaller volume solvent used Fewer evaporation steps Much better LOQs

G.Pieraccini, F. Luceri and G. Moneti, “New Gas-chromatographic/Mass Spectrometric Method for the Quantitative Analysis of Primary Aromatic Amines in Main- and Side-stream Cigarette Smoke. I”, Rapid Communications in Mass Spectrometry, 6, 406-409 (1992).

HC T-102 METHOD (1999)Smoke Collection - 10 cigs

Extraction of CFP w/ 5% HCl

[Add ISTDs]

Liquid:Liquid Extraction w/ Hexane

Derivatize (TMA / PFPA)

GC-MS Analysis

Florisil SPE (3 g)

NEW CHALLENGES: FDA HPHC LIST

GOALS OF NEW METHOD One method to determine all 7 PAAs.

No evaporation steps.

Evaluate SPE clean-up to reduce solvent use and improve reproducibility and efficiency of method.

Retain the advantages of NCI (lower detection limits, improved selectivity).

NEW SAMPLE PREPARATION SCHEME(ARISTA METHOD)

Smoke Collection (CFP)

Extraction of CFP w/ 5% HCl

[Add ISTDs]

SPE Clean-Up and Solvent Exchange (DCM)

Derivatize (PFPA)

GC-MS Analysis (NCI)

Selectivity

Can selectively retain/release compounds of interest.

Requires small volumes of solvent

New method uses 8-10 mL vs. 60-70 mL with liquid:liquid.

Efficiency

Can process many more samples in a batch with manifolds of up to 24 ports.

Effectively isolates and concentrates analytes of interest!

SPE ADVANTAGES

WHY IS CLEAN-UP IMPORTANT? Response

Interferences can swamp the system and reduce sensitivity.

Sample ThroughputDirty samples can destroy the chromatography and suppress the detector response and can increase down time due to system maintenance.

QuantitationWith GC/MS by NCI PAAs have simple spectra - quantitation is based on one significant daughter ion.

Clean-up and separation are critical!

KY 3R4F EXTRACTS(NO SPE CLEAN-UP)

KY 3R4F EXTRACTS(CATION EXCHANGE ONLY)

KY 3R4F EXTRACTS(CATION EXCHANGE PLUS SILICA)

WHICH WOULD YOU INJECT?

CHROMATOGRAMS OF 3R4F EXTRACTS(ISO SMOKING REGIME)

Cation plus Si SPE

Cation SPE only

CHROMATOGRAMS OF 3R4F EXTRACTS(ISO SMOKING REGIME)

Cation plus Si SPE

Cation SPE only

CHROMATOGRAMS OF STANDARD SOLUTIONS(WHY CLEAN SAMPLES ARE IMPORTANT!)

1

CHROMATOGRAMS OF STANDARD SOLUTIONS(WHY CLEAN SAMPLES ARE IMPORTANT!)

1

2

CHROMATOGRAMS OF STANDARD SOLUTIONS(WHY CLEAN SAMPLES ARE IMPORTANT!)

1

2

3

WHY DERIVATIZATION?

NH2

O

OF3CF2C CF2CF3

O

N

H

CF2CF3

OO

OHF3CF2C

4-aminobiphenyl

+

+

DETECTION - EI Electron Ionization

M + e- → M+ + 2e-

M+ produces a characteristic fragmentation pattern or a “mass spectrum” specific to each compound.

DETECTION – NCI Chemical Ionization (methane)

CH4 + e- → CH4+ + 2e-

The electrons have much lower energies. Most compounds cannot form stable negative ions but react with the CH4

+ (PCI).

If a compound can capture and hold an extra electron and retain a negative charge, it can be observed in NCI mode.

MF + e- → MF- → [M-H]- + HF

EI VS. NCI SPECTRA FOR O-TOLUIDINE

EI VS. NCI SPECTRA FOR 4-AMINOBIPHENYL

CHROMATOGRAM OF LOW STANDARD (EI VS. NCI)

EI

CHROMATOGRAM OF LOW STANDARD (EI VS. NCI)

EI

NCI

CHROMATOGRAMS OF A CIGAR EXTRACT(EI VS. NCI)

1-aminonaphthalene

2-aminonaphthalene

3-aminobiphenyl4-aminobiphenyl

o-toluidine

o-anisidine

2,6-DMA

o-toluidine

2,6-DMA

o-anisidine

1-aminonaphthalene

2-aminonaphthalene

3-aminobiphenyl

4-aminobiphenyl

EI

NCI

METHOD VALIDATION

VALIDATION PARAMETERS

o-toluidine

2,6-DMA

o-anisidine

3-Day Precision (n=20) 3.7% 6.0% 5.2%

Accuracy (n=49) 99.6% 95.4% 95.1%

LOD (ng/mL) 0.06 0.02 0.005

LOQ (ng/mL) 0.20 0.06 0.018

Linearity (ng/mL) 2.7 - 277 0.17 - 17 0.16 - 16

VALIDATION PARAMETERS

1-amn 2-amn 3-amb 4-amb

3-Day Precision (n=20) 4.3% 5.2% 5.5% 5.0%

Accuracy (n=49) 95.8% 100.8% 94.3% 91.5%

LOD (ng/mL) 0.03 0.01 0.005 0.003

LOQ (ng/mL) 0.09 0.04 0.015 0.010

Linearity (ng/mL) 0.65 - 65 0.34 - 34 0.16 - 16 0.093 - 9.3

METHOD COMPARISON(WE’VE COME A LONG WAY, BABY)

AHF (1977)

HC T-102

(1999)

Arista (2012)

No. of Cigarettes 210 10 5

Volume Organic Solvent (mL) > 3000 ~ 300 6 – 8

No. of SPE Steps 2 1 2

No. of Evaporation Steps 4 2 None

Final Volume (mL) 2 1 5

~ 4-AMB LOQ (ng/mL) 50 0.5 0.01

LCMSMS

J. Schubert, O. Kappenstein, A. Luch and T. G. Schulz, “Analysis of primary aromatic amines in mainstream waterpipe smoke using liquid chromatography-electrospray ionization tandem mass spectrometry”, J. Chrom. A, 1218, 5628-5637 (2011).

SUMMARY Achieving lower and lower detection limits

in complicated matrix requires both improving clean-up (SPE) and selectivity of the instrument (NCI).

The method presented here demonstrates excellent detection limits, selectivity, accuracy and precision.

It is robust, efficient and fit-for-purpose, accommodating the regulatory requirements of Health Canada, ANVISA and the US FDA.

THANK YOU!