SELECTED AROMATIC AMINES BY GAS CHROMATOGRAPHY MASS SPECTROMETRY: CHALLENGES OF MAINSTREAM CIGARETTE SMOKE Alexandra Martin October 3, 2013 OUTLINE 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 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 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 US FDA X THE ORIGINAL FOUR PAAS 1-aminonaphthalene 2-aminonaphthalene 3-aminobiphenyl 4-aminobiphenyl ADDITIONS FOR FDA HPHC LIST o-toluidine 2,6-dimethylaniline o-anisidine 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… Wash, Dry, Evaporate Florisil – 10 g Fraction (75 mL), Evaporate Florisil – 30 g Fraction 1 (500 mL) and Fraction 2 (400 mL) Evaporate each to 2 mL GC-MS or GC-ECD Analysis 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) Florisil SPE (3 g) GC-MS Analysis 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) SPE ADVANTAGES 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! WHY IS CLEAN-UP IMPORTANT? Response Interferences can swamp the system and reduce sensitivity. Sample Throughput Dirty samples can destroy the chromatography and suppress the detector response and can increase down time due to system maintenance. Quantitation With 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? O NH2 + F3CF2C O O CF2CF3 4-aminobiphenyl O O CF2CF3 + N H F3CF2C OH 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+ + 2eThe 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 4AMINOBIPHENYL 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) o-toluidine EI 1-aminonaphthalene 2,6-DMA 2-aminonaphthalene 3aminobiphenyl 4-aminobiphenyl oanisidine o-toluidine NCI 2,6-DMA 1-aminonaphthalene oanisidine 2-aminonaphthalene 3aminobiphenyl 4-aminobiphenyl METHOD VALIDATION VALIDATION PARAMETERS otoluidine 2,6DMA oanisidine 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 2.7 - 277 0.17 - 17 0.16 - 16 Linearity (ng/mL) 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 T102 (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!