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Optimization of 2,4- dinitrophenylhydrazine (DNPH) derivatization conditions for the determination of carbonyl compounds in e-vapor products Lena Jeong , John Miller, Niti Shah Altria Client Services I Lena Jeong | Postdoctoral Fellow l

  1. Optimization of 2,4- dinitrophenylhydrazine (DNPH) derivatization conditions for the determination of carbonyl compounds in e-vapor products Lena Jeong , John Miller, Niti Shah Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 1

  2. Background ▪ FDA requires reporting of carbonyls in e-vapor products*: ▪ No CRM available for measuring carbonyls in e-vapor products - CRM 74: mainstream cigarette smoke - CRM 86: tobacco and tobacco products ▪ Carbonyl compounds react with 2,4-dinitrophenylhydrazine (DNPH) the presence of an acidic catalyst to form the respective hydrazones *FDA Premarket Tobacco Product Applications for Electronic Nicotine Delivery Systems Final Guidance for Industry. 2019. CRM: CORESTA recommended method Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 2

  3. Current Methods DNPH conc. Acid Diluent CRM 74 11.65 mM 2.05 M phosphoric acid 50/50 ACN/H 2 O Altria 17.5 mM 1.82 M perchloric acid ACN (published) Challenges with current methods: • High background for formaldehyde in current DNPH • Low and unstable recovery for acrolein J.W. Flora et al., Method for the Determination of Carbonyl Compounds in E-Cigarette Aerosols, Journal of Chromatographic Science , 55 (2017), 1421-148 Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 3

  4. 1. Formaldehyde Contamination in DNPH DNPH ~30% H 2 O High background signals for Formaldehdye • Problematic for low level quantitation • Often requires recrystallization • Lot-to-lot variation in background levels 0.12 µg/puff • Limited availability (issue for high volume testing) Alternate DNPH type desired Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 4

  5. 1. Formaldehyde Contamination in DNPH DNPH-HCl DNPH ~30% H 2 O 0.12 µg/puff < 0.01 µg/puff Switching to HCl salt form dramatically reduced background carbonyl levels Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 5

  6. 2. Low and Unstable Acrolein Recovery CRM 74 Altria published 100 Desired recovery above 80% 80 Acrolein recovery (%) 60 Unstable acrolein-DNPH complex 40 20 Matrix: E-Liq pH 9.36 n = 3 0 0 10 20 30 40 50 60 70 80 90 100 Time (min) J.W. Flora et al., Method for the Determination of Carbonyl Compounds in E-Cigarette Aerosols, Journal of Chromatographic Science , 55 (2017), 1421-148 Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 6

  7. Investigation into Low Acrolein Recovery Polyderivatization of Acrolein* Polyderivatization increases under strong acidic conditions *S. Uchiyama, Y. Inaba, N. Kunugita, J. Chromatogr. A, 1217 (2010) 4383-4388. doi: 10.1016/j.chroma.2010.04.056 Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 7

  8. Method Optimization ▪ Need to optimize method to: - Reduce formaldehyde background using new DNPH-HCl - Obtain higher and more stable acrolein recovery ▪ Evaluate preparation of DNPH solution: - Acid type/concentration - DNPH concentration - Solvent ratio Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 8

  9. Derivatization Optimization Evaluation of pH and DNPH concentration ▪ Concentration of DNPH (9 mM; 4.5 mM; 1.8 mM) in ACN solution prepared with 1.5 % (v/v) of - 1.82 M perchloric acid pH 0.04 - 0.1 M sodium citrate buffer pH 3 - 0.1 M sodium citrate buffer pH 6 Literature reports that acidity of derivatization solution has a significant impact on reaction rate and stability Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 9

  10. Effect of pH 9 mM TCI DNPH; pH 0.04 9 mM DNPH HCl; pH 0.04 9 mM TCI DNPH; pH3 9 mM TCI DNPH; pH6 9 mM DNPH HCl; pH 6 9 mM DNPH HCl; pH 3 100 Acrolein recovery (%) 80 60 40 20 n = 3 Matrix: Water 0 0 10 20 30 40 50 60 70 80 90 100 Time (min) Low pH results in decomposition of acrolein-DNPH complex Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 10

  11. Effect of DNPH Concentration 9 mM TCI DNPH; pH3 4.5 mM TCI DNPH; pH3 1.8 mM TCI DNPH; pH3 9 mM DNPH HCl; pH 3 4.5 mM DNPH HCl; pH 3 1.8 mM DNPH HCl; pH 3 100 Acrolein recovery (%) 80 60 40 20 n = 3 Matrix: Water 0 0 20 40 60 80 100 Time (min) DNPH concentration is directly related to the derivatization rate Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 11

  12. Derivatization Optimization Effect of Water Content DNPH conc. Acid Diluent CRM 74 11.65 mM 2.05 M phosphoric acid 50/50 ACN/H 2 O Altria 17.5 mM 1.82 M perchloric acid ACN (published) ▪ How is derivatization rate affected in presence of added water? Varying diluent ratios tested: - 0/100 H 2 O/ACN - 25/75 H 2 O/ACN - 50/50 H 2 O/ACN Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 12

  13. Water Content Comparison 9 mM TCI DNPH; pH6; ACN 9 mM DNPH HCl; pH 6; ACN 9 mM TCI DNPH; pH6; 25/75 H2O/ACN 9 mM DNPH HCl; pH 6; 25/75 H2O/ACN 9 mM TCI DNPH; pH6; ACN 9 mM TCI DNPH; pH6; 50/50 H2O/ACN 9 mM DNPH HCl; pH 6; 50/50 H2O/ACN 100 100 80 80 Acrolein recovery (%) Acrolein recovery (%) 60 60 40 40 20 20 n = 3 Matrix: Water 0 0 0 0 10 10 20 20 30 30 40 40 50 50 60 60 70 70 80 80 90 90 100 100 Time (min) Time (min) Addition of protic solvent slows derivatization reaction Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 13

  14. Optimized Method vs. CRM 74 CRM 74 9 mM TCI DNPH; pH 3; ACN 9 mM DNPH HCl; pH 3; ACN CRM 74 100 100 80 80 Acrolein recovery (%) Acrolein recovery (%) 60 60 40 40 20 20 Derivatization too slow. pH shifting with sample addition. n = 3 0 0 0 0 10 10 20 20 30 30 40 40 50 50 60 60 70 70 80 80 90 90 100 100 Time (min) Time (min) Sample matrix: 50/50 PG/GLY with 2.5% nicotine ( pH 9.36 ) Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 14

  15. Comparison of Methods: Acrolein Recovery Optimized Method Optimized Method Optimized Method CRM 74 Altria published Altria published 100 100 100 Needed buffer capacity achieved! 80 80 80 Acrolein recovery (%) Acrolein recovery (%) Acrolein recovery (%) 60 60 60 DNPH conc. Acid (type; %; Diluent (vendor) conc.) 40 40 40 CORESTA 11.65 mM 2.9% of 2.05 M 50/50 (No. 74) phosphoric acid ACN/H2O Altria 17.5 mM 1.5% of 1.82 M ACN (published) perchloric acid 20 20 20 Optimized 9 mM (HCl) 5% of 100 mM ACN method citrate buffer pH 3 n = 3 0 0 0 0 0 0 10 10 10 20 20 20 30 30 30 40 40 40 50 50 50 60 60 60 70 70 70 80 80 80 90 90 90 100 100 100 Time (min) Time (min) Time (min) Basic e-liquid: 50/50 PG/GLY with 2.5% nicotine (pH 9.36) Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 15

  16. New Method Performance with Varying Sample pH Water Basic e-liq Acidic e-liq 100 Acrolein recovery (%) 80 60 40 20 n = 3 0 0 20 40 60 80 100 Time (min) Basic e-liquid: 50/50 PG/GLY with 2.5% nicotine (pH 9.36) Acidic e-liquid: 50/50 PG/GLY with benzoic acid (pH 3.72) Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 16

  17. Comparison of Methods: Percent Recovery Derivatization time: 10 min n = 3 Formaldehyde Acetaldehyde Acrolein Crotonaldehyde CRM 74 100% ± 2.8% 78.7% ± 0.91% 74.5% ± 1.9% 100% ± 2.4% Altria published 87.2% ± 1.3% 77.7% ± 2.6% 72.5% ± 2.3% 103 ± 2.0% Optimized method 102% ± 0.84% 80.7% ± 2.9% 85.6% ± 2.4% 103% ± 3.3% Sample matrix: 50/50 PG/GLY with 2.5% nicotine (pH 9.36) Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 17

  18. Summary - Learnings ▪ Switching to DNPH HCl form dramatically reduced background levels of formaldehyde ▪ Highly acidic DNPH solution results in polyderivatization of acrolein-DNPH (formation of AD1) ▪ Use of buffer to control the pH improves and stablizes acrolein recovery for over 90 min derivatization time ▪ Addition of protic solvent (H 2 O) as diluent slows down the derivatization reaction Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 18

  19. Conclusions ▪ DNPH-HCl form reduces background levels of formaldehyde and improves quantitation of carbonyls in e-vapor aerosol ▪ The DNPH derivatization method was optimized to give acceptable recovery levels for all aldehydes including acrolein ▪ New conditions allow for better stability of acrolein to extend aerosol collections Altria Client Services I Lena Jeong | Postdoctoral Fellow l Tobacco Science Research Conference, 2019 I 19

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