Analytical Means to Understand Furan Formation

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1 Analytica 2008, Munich, April 1-4, 2008 Analytical Means to Understand Furan Formation Imre Blank Nestlé Product Technology entre, rbe, Switzerland 12/04/2008

Survey on furan in heat-treated food (FDA, May 2004) EFSA/IAR/FDA assessment: Furan is carcinogenic to rats and mice

(IAR): Possibly carcinogenic to humans (2B) anned vegetables Juices Sauce Bolognese Baby food ~ 10 µg/kg ~ 15 µg/kg

2 Survey on furan in heat-treated food (FDA, May 2004) EFSA/IAR/FDA assessment: Furan is carcinogenic to rats and mice Furan-induced carcinogenicity is probably due to a genotoxic mechanism International Agency for Research on ancer (IAR): Possibly carcinogenic to humans (2B) anned vegetables Juices Sauce Bolognese Baby food ~ 10 µg/kg ~ 15 µg/kg ~ 40 µg/kg ~ 40 µg/kg ~ 70 µg/kg ~ 125 µg/kg Up to ~200 µg/kg found in food (canned, jarred) (FDA, June 7, 2004; Further data needed for reliable risk assessment on toxicity & exposure FDA has issued a dietary message advising consumers to continue to eat a balanced diet PT rbe - Innovating with 12/04/2008 2

3 utline of the presentation 1. Quantification of furan : literature survey Using d 4 -furan as internal standard (Isotope Dilution Assay) ccurrence 2. Labelling studies : extract from our studies Using specifically labelled precursors (Monitoring of isotopomers) Formation mechanism PT rbe - Innovating with 12/04/2008 3

4 Furan as a chemical 4 H 4 Aromatic heterocyclic organic compound Relative molecular mass of g/mol olourless liquid at room temperature Relatively stable chemical Very volatile (boiling point of 31.4 ) PT rbe - Innovating with 12/04/2008 4

5 1 Analytical methods based on Isotope Dilution Assay Method LD (µg/kg) LQ (µg/kg) Linearity (µg/kg) Precision V (%) Recovery (%) Application Reference 1. Static headspace G/ITMS, split, PorarisQ, 80 n.d n.d. 6 (n=6) 8 (n=6) n.d. Baby food offee Reinhard et al G/MS(SIM), splitless, P-PoraBND Q 0.1 n.d (R 2 >0.999) ( µg/kg) offee brew, rice soup Becalski et al., SPME G/MS, split, P-PoraBND Q (R 2 =0.9953) ( µg/kg) offee brew Ho, 2005 G/MS(SIM), splitless, HP-PLT Q (R ) 7-16 (coffee) (2-10 µg/kg) offee brew range juice Goldmann et al., 2005 G/MS, split, HP-PLT Q (R 2 =0.9915) Apple juice Peanut butter Nyman et al., 2006 G/MS(SIM), splitless, HP-InnoWAX (R 2 =0.998) Baby food Bianchi et al., 2006 G/IT-MS, splitless, BPX-volatiles (R 2 >0.999) 6-10 n.d. offee, baby food, etc. Altaki et al., 2007 G/MS(SIM), split, ZebronWAX (R 2 >0.999) 5-8 (models, n=6) (2-10 µg/kg) Model systems, pap, juice Limacher et al., 2007 PT rbe - Innovating with Blank (2008) 12/04/2008 5

6 1 Quantification of furan SPME-G-MS method: rapid and robust - Addition of deuterated furan to the sample, - Sodium chloride-assisted extraction into the headspace (SPME), - ryofocussing and fibre desorption, - G-MS analysis. Furan is quantified by the use of an external calibration curve, achieving a - Decision limit (α) of 17 pg - Detection capability (β) of 43 pg as absolute furan concentration PT rbe - Innovating with 12/04/2008 6

7 1 hoice of the SPME fibre The AR/PDMS fiber shows decisive advantages in terms of sensitivity (based on m/z 72) in the order AR/PDMS > DVB/AR/PDMS > PDMS/DVB > polyacrylate, W/DVB AR/PDMS : arboxen / polydimethylsiloxane DVB/AR/PDMS : Divinylbenzene / carboxen / polydimethylsiloxane PDMS/DVB W/DVB : Polydimethylsiloxane / divinylbenzene : arbowax / divinylbenzene PT rbe - Innovating with 12/04/2008 7

8 1 Quantitative data and accuracy Broad application, e.g. fruits juices, baby foods in jars, canned foods, pet food, coffee and coffee substitutes. Typical amounts of furan found in selected foodstuffs range from about 1 µg/kg (fruit juice) to 110 µg/kg (baby food containing cooked vegetables). In-house validation data show good precision and accuracy of the method: - typical repeatability of 5-16% in different food matrices, and - trueness determined in orange juice (87%) and coffee (93%) PT rbe - Innovating with 12/04/2008 8

9 1 Effect of matrix complexity: Petfood MS (SIM) chromatogram of a dry pet food sample showing interference in the m/z 39 trace and a poor baseline for m/z 42. Reliable quantitation is possible using the ion traces m/z 68 and m/z 72. PT rbe 12/04/2008 Goldmann - Innovating et al. (2005) with 9

10 1 Effect of analytical parameters onditions: 75µm AR/PDMS fibre extraction time: 60 min desorption time: 5 min PT rbe - Innovating with 12/04/

11 1 Example: Honey SPME G/MS chromatograms of (A) total-ion current (TI), (B) reconstructed ion current m/z 68 for furan, () reconstructed ion current of m/z 72 for furan-d 4, PT rbe - Innovating with Altaki et al. (2007) 12/04/

12 1 Effect of volatility: Baby food Decrease in furan concentration in a hot water heated canned baby food (on standing) with stirring - 85% PT rbe - Innovating with 12/04/

13 1 Effect of volatility: Baby food Loss of inherent furan in a non-heated and heated baby food (open jar, no stirring) over time (5.5 h) - 50% - 85% PT rbe - Innovating with Goldmann et al. (2005) 12/04/2008

14 1 Effect of thermal generation during sample preparation hange of furan peak response during equilibration of green coffee, tomato juice and orange juice at temperatures between for a fixed time of 30 min PT rbe 12/04/2008 Senyuva - Innovating & Gökmen with (2005) 14

15 1 Take-home message 1. Quantification of furan Using d 4 -furan as internal standard (Isotope Dilution Assay) Adaptation of the method to the food sample is important omplications might be due to volatility, complexity of the food sample, incorporation of d 4 -furan (homogeneity), physical properties of furan and d 4 -furan, thermal formation during sample preparation ccurrence PT rbe - Innovating with 12/04/

16 2 Furan can be generated from Ascorbic acid (Vitamin ) PUFAs (Poly unsaturated fatty acids) Sugars Sugars and amino acids (Maillard reaction) PT rbe - Innovating with (Perez Locas & Yaylayan,J. Agric. Food hem. 2004, 52, 6830) 12/04/

17 2 Furan formation in dry roasting model systems Furan [µmol/mol] (10 min at 200 ) Ascorbic acid Sugar Maillard precursors PUFA Ascorbic acid Ascorbic acid + Erythrose Ascorbic acid + Phenylalanine Ascorbic acid + Glucose Dehydroascorbic acid Glucose Fructose Arabinose Erythrose Glucose + Alanine + Threonine Fructose + Alanine + Serine Glucose + Phenylalanine Fructose + Phenylalanine Linoleic acid Linolenic acid Linolenic acid + Ascorbic acid Linolenic acid + Glucose Phospatidylcholine PT rbe - Innovating with Limacher et al. (unpublished) 12/04/

18 2 Formation from ascorbic acid Model system M (68) M+1 (69) M+2 (70) M+3 (71) M+4 L-[1- H ]-ASA H Dry 100 % 0 % 0 % 0 % 0 % L-[2- ]-ASA H aq. ph % 0 % 0 % 0 % 0 % L-[6- ]-ASA H (72) aq. ph 7 0 % 100 % 0 % 0 % 0 % L-ASA + D-[U- 6 ]-Glc (1:1) Dry 73 % 0 % 0 % 0 % 27 % L-ASA + D-[U- 6 ]-Glc (1:1) aq. ph 7 84 % 0 % 0 % 0 % 16 % L-ASA + D-[U- 6]-Glc (1:1) aq. ph 4 99 % 0 % 0 % 0 % 1 % -1 and -2 not incorporated into Furan (composed of -3 to -6) No recombination of ascorbic acid fragments Glucose contributes to the total Furan amount (dry, aq. ph 7) PT rbe - Innovating with Limacher et al. (2007) 12/04/

19 2 Labelling studies: Ascorbic acid Furan Model system Ratio 2 / 2 L-[1- ]-Ascorbic acid 1 : 1.79 L-[2- ]-Ascorbic acid 1 : 0.25 L-[6- ]-Ascorbic acid 1 : 0.16 HH (M) HH (M+1) (m/z = 46) (m/z = 47) L-[1- ]-ASA 80 % 20 % L-[2- ]-ASA 68 % 32 % L-[6- ]-ASA 84 % 16 % H H H H H H H H H H H H H - H 2 H 2 H H H H H H H H H H H H Furan formation in three steps: 1. Decarboxylation 2. Dicarbonyl cleavage 3. yclization/dehydration H PT rbe - Innovating with - H 2 12/04/ H 100 % 100 % 100 %

20 2 Binary mixtures result in reduced furan amounts (ph 4, 121, 25 min) Furan (rel. concentration) 57 µmol/mol 100% 48% 33% 40% 30% Ascorbic acid Dehydroascorbic acid Ascorbic acid + Erythrose Ascorbic acid + Glucose Ascorbic acid + Phenylalanine Because of competing reactions Binary Furan systems Ascorbic acid generates less furan in binary systems, even in the presence of other furan precursors (e.g. Glucose - 60 % Furan) PT rbe - Innovating with Limacher et al. (2007) 12/04/

21 2 Ascorbic acid is unlikely to be a precursor of furan in vegetables Spiking experiments with [6- ]-labelled ASA (50 mg/100 g, 123, 22 min) Pumpkin purée arrot juice range juice ph after spiking Deviation in furan level [%] % + 38 % - 16 % [1- ]-Furan content after spiking [%] 0 % 4 % 3 % Probably pro-oxidative effect of ASA because lebelled ASA is (almost) not incorporated Decrease of the furan content most probably due to competing pathways Hypothesis: Polyunsaturated fatty acids (PUFAs) PT rbe - Innovating with Limacher et al. (2007) 12/04/

22 onclusion: ASA seems to influence furan formation but is most likely not a direct precursor of furan Mitigation concepts PUFA-rich products should not be fortified with ascorbic acid prior to heat-treatment because of - probably pro-oxidative effect - decreasing ph (favouring furan formation from PUFA) PT rbe - Innovating with 12/04/

23 2 Furan increases almost linearly with the linoleic acid amount Spiking of linoleic acid (PUFA) to pumpkin vegetables (ph = 6.3, 22 min, 123 ) 250 Furan [µg/kg] % Furan % Furan Addition of 4200 mg/100 g linoleic acid Addition of 2300 mg/100 g linoleic acid Addition of 00 mg/100 g linoleic acid No addition of linoleic acid (natural content: 500 mg/100 g) % % 0 PT rbe - Innovating with Limacher et al. (unpublished) 12/04/

24 2 PUFA as key precursor validated by spiking of 18 -linolenic acid Spiking of linoleic acid (PUFA) to pumpkin vegetables (ph = 6.3, 22 min, 123 ) Furan [µg/kg] factor 5.6 more furan + 1 g/100 g 18 -linolenic acid M (68) M+1 (69) Furan M+2 (70) M+3 (71) M+4 (72) 15 % 0 % 0 % 0 % 85 % H 15 % factor of % PT rbe - Innovating with Limacher et al. (unpublished) 12/04/

2 Furan formation from PUFAs: Three levels of precursors 1 R 12 11 10 9 PUFA H Polyunsaturated fatty acids (including their acylglycerols) 2 R autoxidative degradation 12 12 10 10 R 11 9 11 9

25 2 Furan formation from PUFAs: Three levels of precursors 1 R PUFA H Polyunsaturated fatty acids (including their acylglycerols) 2 R autoxidative degradation R alkenal 2,4-alkadienal Efficient reduction of furan in model systems by BHT ( 99%) Unsaturated aldehydes autoxidative degradation H maleinaldehyde 4-hydroxy-2-butenal disproportionation Low molecular carbonyls cyclization dehydration furan 12 PT rbe - Innovating with Perez Locas & Yaylayan (2004); Limacher et al. (2006) 12/04/

26 2 Furan formation from 2,4- alkadienal R ,4-alkadienal 2 1 R= methyl or ethyl System ph4 (121 /25min) Precursor 2,4-nonadienal 2,4-decadienal Furan distribution (%) D0 D1 D H 4 B R A 2 3 d2-furan 4-hydroperoxide A B 4 R R 8 ph7 (121 /25min) dry (200 /5min) H 2,4-nonadienal 2,4-decadienal 2,4-nonadienal 2,4-decadienal 8 6 R % major R H 5 4 R H d1-furan minor furan % PT rbe - Innovating with Novotny et al. (unpublished) 12/04/ %

27 onclusion: PUFAs are major precursors of furan in food Mitigation concepts - Addition of antioxidants (e.g. BHT, tocopherols, ) optimization of concentration/composition of antioxidants is necessary for each product - De-aeration prior to heat-treatment (removal of 2 ) - Addition of PUFA after the heat-treatment (e.g. by aseptic filling) PT rbe - Innovating with 12/04/

28 2 Take-home message 2. Labelling studies Using specifically labelled precursors (Monitoring of isotopomers) Formation mechanism First level of understanding in model systems using labelled precursors formation mechanism Verification of hypotheses in food systems indispensable spiking experiments Furan can be generated by many precursors PUFAs, carotenes, Vit., sugars, amino acids, etc. mitigation studies by careful optimisation of recipe and process parameters PT rbe - Innovating with 12/04/

29 References: Recent reviews Yaylayan, V.A. (2006) Precursors, formation and determination of furan in food, J. ons. Protect. Food Safety, 1, 5-9. rews,. and astle, L. (2007) A review of the occurrence, formation and analysis of furan in heat-processed foods, Trends in Food Science & Technology, 18, Blank, I. (2008) Furan in processed foods. In Bioactive ompounds in Foods - Natural Toxicants and Process ontaminants; Gilbert, J., Senyuva, H., Eds.; Blackwell Publishing: xford; pp and Anita LIMAHER ndrej NVTNY Tomas DAVIDEK Josef KERLER PT rbe - Innovating with 12/04/

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