2 Appendix: method procedure summaries

E310–12: Gallates 147 HPLC Foods Extracted with acetonitrile–2- CrestPac C18S Gradient elution system of UV at 280 nm 6 propanol–ethanol 2:1:1. Extract in 4.6 × 150 mm 5 acetic acid and freezer for 1 h then filtered. Filtrate MeOH–acetonitrile 50:50. concentrated under vacuum. 10 µL Flow rate 1 mLmin injected LC Foods and MeOH added to sample and sonicated Kromasil C18 Water–MeOH 10:90, Electrochemical 7 drugs and re-extracted. Extracts filtered on a 300 mm × 1 mm, 0.01 M LiClO 4 , pH 5.5 oxidation SepPak silica cartridge connected to 5 µm at 50 µLmin potential +0.8 V syringe. 0.2 µL injected versus Ag–AgCl LC Edible oils Microemulsion of oil prepared by Spherisorb ODS-2 0.1 M SDS, 2.5 UV at 284 nm 8 mixing 5 oil with 95 water–SDS– 125 × 4 mm, 5 µm n -propanol and 10 mM n -pentanol 37.5:12.5:50. 20 µL and guard column phosphate at pH 3. Flow injected 35 × 4.6 mm, 10 µm rate 1 mLmin LC Dairy products Samples prepared in mobile phase at Spherisorb ODS-2 0.090 M SDS, 6.6 UV at 284 nm 9 and dietetic following concentrations: liquid milk 125 × 4 mm, 5 µm n -propanol and 10 mM supplements 20 ; powdered milk and cream 1 ; and guard column phosphate at pH 3. Flow dietetic supplement 0.5 . Solutions 35 × 4.6 mm, 10 µm rate 1 mLmin filtered through 0.45 µm filter. 20 µL injected HPLC-ECD Foods Samples prepared in mobile phase. Lichrocart RP18 Isocratic CH 3 CN–THF– 3 electrodes, 10 Solutions filtered through 0.45 µm 250 × 4 mm, 5 µm H 2 O 60:25:15. Flow rate reference filter. 20 µL injected 0.9 mLmin electrode at 0.90 V, sensitivity is 50 nA 148 Analytical methods for food additives Table 11.1 cont’d b Method Matrix Sample preparation Method conditions Reference Micellar electrokinetic Cola beverages and Butyl paraben was used as an Additives were separated using a 20 mM berate buffer 11 chromatography jams internal marker with 35 mM sodium cholate, 15 mM sodium dodecyl MECC sulphate and 10 methanol added at pH 9.3 MECC vs HPLC Antioxidants 4 antioxidants were separated completely with excellent 12 resolution and efficiency within 6 min and picomole amounts of antioxidants were detectable using UV absorption. RP–HPLC separation not as efficient and requires larger sample amounts and longer separation time Stopped flow mixing Foods Sample dissolved in petroleum 2 solutions prepared to fill syringes in stopped flow module. 13 and a T-format ether and extracted 3 × with One based on lanthanide chelate with terbium in presence of luminescence 72 EtOH, filtered and Triton X-100 and tri-n-octylphosphine oxide for PG and the spectrometer diluted to volume. 0.7 mL used other based on a reaction between the oxidised form of Nile Blue and BHA. In each run 150 µL of each solution is mixed in the mixing chamber. The excitation wavelength used is 310 nm. Emission 545 nm for PG and 665 nm for BHA Stopped flow mixing Foods Sample dissolved in petroleum Based on kinetic behaviour of PG and BHA when reacted 14 and diode-array ether and extracted 3 × with with 3-methylbenzothiazolin-2-one hydrazone in the 72 EtOH, filtered and diluted presence of cerium IV and on the joint use of the stopped- to volume. 0.7 mL used flow mixing technique and a diode-array detector, which allows kinetic data to be obtained at 2 wavelengths simultaneously E310–12: Gallates 149 Voltammetric Foods For vegetable oil samples: 1.0 mL of 1.0 M perchloric acid and 0.1 mL MeOH added 15 equal volume of MeOH added, to aliquot and transferred to electrochemical cell and shaken, centrifuged. MeOH diluted to 10 mL with water. Stirred for 60 s after 2 s pause, extracts made to volume, a linear potential scan taken from 1–1300 mV at the glass 1.0 mL aliquot used. For solid carbon electrode vs Ag–AgCl with scan rate of 75 mVs. samples: ground to powder, The obtained linear sweep voltammograms LSV showed shaken with petroleum ether well-defined oxidation waves with a peak potential of and extracted as for oil samples 599 mV UV, TLC–UV, Hydrogenated 100 mgg BHA, BHT and PG UV methods not appropriate for products containing 2 or 16 colorimetry, TLC– vegetable fat added in fat more antioxidants andor interfering substances. TLC–UV colorimetry and TLC–colorimetry are more adequate; the colorimetric method can also be used, depending on the interfering substances and the colorimetric specificity. Colorimetric method is more economical and rapid than methods using TLC Colorimetric Food Dissolve in petroleum ether Dilute to 20 mL with ammonium acetate solution. Add 17 and extract with aqueous exactly 4 mL H 2 O and pipette 1 mL ferrous tartrate reagent ammonium acetate solution. into flask. Mix well and measure A at 540 nm against Filter ammonium acetate reagent blank. Calculate amount of PG from standard curve extract and transfer aliquot to conical flask PG = propyl gallate OG = octyl gallate DG = dodecyl gallate 150 Analytical methods for food additives Table 11.2 Summary of statistical parameters for gallates in foods Method Matrix Extent of validation Statistical parameters Reference LC Oils, fats and AOAC Official Method 983.15 Ref: Journal of AOAC International 1,2 butter oil Full collaborative trial 1993 76, 765 Details given in Table 11.3 HPLC Bakery products Precision of method established and Linear calibration curve in range 2–100 µgmL. Recovery 5 applied to 15 commercial samples calculated for the IS was 94.6 n=10. Cake spiked with 16 µgg. CV was 5.3 for PG, 5.7 for OG and 5.9 DG. Of 15 samples analysed none contained gallates Stopped flow Foods Precision of method established and Calibration graphs linear over range 0.09–3.5 µgmL. The 13 mixing and a applied to 10 commercial samples relative standard deviation was 2.3 . LOD was 0.03 µgmL T-format for PG. luminescence The method was applied to the determination of PG and spectrometer BHA in several commercial foods with recoveries ranging between 94.1 and 102.1 for PG Stopped flow Foods Precision of method established and Calibration graphs linear over range 1.6–27.5 µgmL. The 14 mixing and applied to 8 commercial samples relative standard deviations for both systems are close to diode-array 2 . LOD = 0.5 µgmL for PG. The method was applied to the determination of PG and BHA in several commercial foods with recoveries ranging between 97.2 and 103.5 Voltammetric Foods Precision of method established and Linear calibration graph obtained for PG in range 15 applied to 8 commercial samples 1.0–15.0 mgL. LOD 0.54 mgL. Recovery ranged from 85–118 for PG in spiked food samples E310–12: Gallates 151 LC Dairy products Precision of method established Linear calibration curve. LOD 0.05 ng, repeatability 0.7 9 and dietetic n=6 for microemulsion containing 5 µgg PG. Recovery supplements ranged from 96 to 103 n=3 for 5 foods spiked at 2 µgg PG i.e. powdered milk, cream, milk and 2 dietetic supplements. Linear calibration curve. LOD 0.2 ng, repeatability 0.5 n=6 for microemulsion containing 5 µgg OG. Recovery ranged from 99 to 103 n=3 for 5 foods spiked at 2 µgg OG i.e. powdered milk, cream, milk and 2 dietetic supplements RP-HPLC Pharmaceutical Precision of method established PG: Linear calibration curve in range 0.00006–5.1 µgmL. 4 formulations LOD = 0.14 pg Mean recovery of 99 from olive oil spiked at 3 levels 29.7, 212.2 and 424.4 mgkg PG n=5, range 95.3–100.9 . OG: Linear calibration curve in range 0.028–14.1 µgmL. LOD = 0.31 pg. Mean recovery of 99 from olive oil spiked at 3 levels 56.5, 197.6 and 395.3 mgkg OG n=5, range 92.5–105.0 . DG: Linear calibration curve in range 0.034–20.3 µgmL. LOD = 0.33 pg. Mean recovery of 99 from olive oil spiked at 3 levels 67.7, 203.1 and 473.8 mgkg DG n=5, range 95.7–105.7 HPLC Foods Precision of method established Recovery ranged from 84.9 to 96.5 n=3 for 5 foods 6 spiked at 100 µgg PG i.e. corn oil, butter oil, butter, niboshi, frozen shrimp. Recovery ranged from 87.4 to 96.2 n=3 for 5 foods spiked at 100 µgg OG. Recovery ranged from 87.8 to 91.9 n=3 for 5 foods spiked at 100 µgg DG LC Edible oils Precision of method established Linear calibration curve. LOD 2.5 ng, repeatability 2.5 8 n=5 for microemulsion containing 10 µgg PG. Linear calibration curve. LOD 5.9 ng, repeatability 1.9 n=5 for microemulsion containing 10 µgg OG LC Foods and drugs Precision of method established Linear calibration curve. LOD = 0.9 ppb. RSD n=5 7 repeatability 3 for PG Table 11.3 Performance characteristics for gallates in oils, lard and butter oil

1,2

Sample Oils Lard Analyte Propyl gallate Propyl gallate Propyl gallate Propyl gallate No. of laboratories 7 7 7 7 Units mgg mgg mgg mgg Spike value 193.7 96.7 19.4 96.9 Mean value 184 93.8 17.6 90.1 S r 16.0 4.50 2.01 3.18 RSD r 8.66 4.80 11.5 3.53 S R 16.0 4.50 2.52 3.18 RSD R 8.66 4.80 14.3 3.53 Recovery 95.2 96.9 90.9 93.0 Sample Lard Butter oil Analyte Propyl gallate Propyl gallate Propyl gallate Propyl gallate No. of laboratories 7 7 7 7 Units mgg mgg mgg mgg Spike value 38.7 92.1 46.0 9.20 Mean value 34.6 89.3 46.9 9.53 S r 1.55 4.76 3.86 0.450 RSD r 4.48 5.33 8.23 4.72 S R 1.55 6.08 4.54 0.875 RSD R 4.48 6.81 9.67 9.17 Recovery 89.4 97.0 102 104 Sample Butter oil Analyte Octyl gallate Octyl gallate Octyl gallate No. of laboratories 7 7 7 Units mgg mgg mgg Spike value 89.2 43.7 8.76 Mean value 86.3 42.0 8.19 S r 3.80 2.89 1.69 RSD r 4.40 6.87 20.6 S R 4.37 2.89 1.69 RSD R 5.06 6.87 20.6 Recovery 96.8 96.2 93.5 Sample Butter oil Analyte Dodecyl gallate Dodecyl gallate Dodecyl gallate No. of laboratories 7 7 7 Units mgg mgg mgg Spike value 101.1 50.6 10.1 Mean value 96.7 48.8 9.76 S r 4.02 2.98 0.468 RSD r 4.16 6.12 4.80 S R 7.94 3.05 0.742 RSD R 8.21 6.24 7.61 Recovery 95.7 96.5 96.4 12 E320: BHA

12.1 Introduction

The major food groups contributing to dietary intake of BHA are cakes, cookies and pies, other fine bakeryware and emulsified sauces with the maximum permitted level of 400 mgkg being allowed in dietary supplements and chewing gum. The ADI for BHA is 0.5 mgkg body weightday.

12.2 Methods of analysis

There are numerous methods published for the determination of BHA in food- stuffs. The majority of these are applicable to foods and are GC, 1–4 HPLC, 5–14 micellar electrokinetic chromatography MECC, 15,16 spectrophotometric, 17–20 voltammetric 21 and TLC 22 methods. A summary of these methods is given in Table 12.1, together with the matrices to which they are applicable. If statistical parameters for these methods were available they have been summarised in Table 12.2. Two of these methods 1,5 have been adopted as AOAC official methods. The liquid chromatographic method for the analysis of BHA in oils, fats and butter oil was collaboratively trialled. 5,6 The method consists of phenolic antioxidants being extracted into acetonitrile. The extract is concentrated and diluted with 2-propanol. Antioxidants are separated by liquid chromatography and measured by UV detection at 280 nm. The procedure for this method is given in the Appendix and the performance characteristics are given in Table 12.3.

12.3 Recommendations

There are many methods available for the analysis of BHA in fatty foods and the decision as to what method should be used depends on the matrix to be analysed. The majority of these methods are for liquids i.e. oils and further method develop- ment may be required to adapt these methods to be applicable for all matrices.

12.4 References

1 ‘AOAC Official Method 968.17. Butylated hydroxyanisole and butylated hydroxytoluene in cereals, gas chromatographic method. IUPAC-AOAC Method’, AOAC Official Method of Analysis 2000 47.2.03 p 5. 2 ‘Gas chromatographic flow method for the preconcentration and simultaneous determi- nation of antioxidant and preservative additives in fatty foods’, González M, Gallego M, Valcárcel M. Journal of Chromatography A 1999 848, 529–536. 3 ‘Simultaneous analysis of preservatives in foods by gas chromatographymass spectrometry with automated sample preparation instrument’, Ochiai N, Yamagami T, Daishima S. Bunseki Kagaku 1996 456, 545–550. 4 ‘Gas chromatographic determination of synthetic antioxidants in edible fats and oils – a simple methylation method’, Choong Y M, Lin H J. Journal of Food and Drug Analysis 2001 91, 20–26. 5 ‘AOAC Official Method 983.15. Phenolic antioxidants in oils, fats and butter oil liquid chromatographic method, IUPAC-AOAC Method’, AOAC Official Method of Analysis 2000 47.2.02. 6 ‘Liquid-chromatographic method for the determination of 9 phenolic antioxidants in butter oil – collaborative study’, Page B D. Journal of AOAC International 1993 764, 765–779. 7 ‘Quantitative determination of butylated hydroxyanisole, butylated hydroxytoluene, and ter-butyl hydroquinone in oils, foods and biological fluids by high-performance liquid chromatography with fluorometric detection’, Yankah V V, Ushio H, Ohshima T, Koizumi C. Lipids 1998 3311, 1139–1145. 8 ‘Effect of pH on the retention behavior of some preservatives-antioxidants in reverse- phase high-performance liquid-chromatography’, Ivanovic D, Medenica M, Nivaudguernet E, Guernet M. Chromatographia 1995 4011–12, 652–656. 9 ‘Separation and determination of phenolic antioxidants by HPLC with surfactantn- propanol mobile phases’, Aparicio A, San Andres M P, Vera S. HRC-Journal of High Resolution Chromatography 2000 234, 324–328. 10 ‘Liquid chromatographic determination of phenolic antioxidants in bakery product’, Rafecas M, Guardiola F, Illera M, Codony R, Boatella J. Journal of Chromatography A 1998 8222, 305–309. 11 ‘Determination of 9 phenolic antioxidants in foods by high-performance liquid- chromatography’, Yamada M, Miyata M, Kato Y, Nakamura M, Nishijima M, Shibata

T, Ito Y. Journal of the Hygienic Society of Japan 1993 346, 535–541.

12 ‘Microbore liquid-chromatography with electrochemical detection for the control of phenolic antioxidants in drugs and foods’, Boussenadji R, Porthault M, Berthod A. Journal of Pharmaceutical and Biomedical Analysis 1993 111, 71–78. 13 ‘Direct injection of edible oils as microemulsions in a micellar mobile phase applied to the liquid chromatographic determination of synthetic antioxidants’, Noguers-Orti J F, Villanueva-Camanas R M, Ramis-Ramos G. Analytica Chimica Acta. 1999 3872, 127–134. 14 ‘Determination of synthetic antioxidants in dairy products and dietetic supplements by micellar liquid chromatography with direct sample injection’, Noguera-Orti J F, Villanueva-Camanas R M, Ramis-Ramos G. Chromatographia 2000 511–2, 53–60. 15 ‘Simultaneous determination of antioxidants, preservatives and sweeteners permitted as additives in food by micellar electrokinetic chromatography’, Boyce M C. Journal of Chromatography A 1999 847, 369–375. 16 ‘Comparison between capillary electrophoresis and high-performance liquid- chromatography separation of food grade antioxidants’, Hall C A, Zhu A, Zeece M G. Journal of Agricultural and Food Chemistry 1994 424, 919–921. 17 ‘Simultanous stopped-flow determination of butylated hydroxyanisole and propyl gallate using a T-format luminescence spectrometer’, Aguilar-Caballos M P, Gomez- Hens A, Perez-Bendito D. Journal of Agricultural and Food Chemistry 2000 482, 312–317. 18 ‘Simultaneous kinetic determination of butylated hydroxyanisole and propyl gallate by coupling stopped-flow mixing technique and diode-array detection’, Aguilar-Caballos M P, Gomez-Hens A, Perez-Bendito D. Analytica Chimica Acta 1997 3541–3, 173– 179. 19 ‘Spectrophotometric determination of butylated hydroxy anisole BHA in oils’, Sastry C S P, RAO S G, Sastry B S. Journal of Food Science and Technology-Mysore. 1992

292, 101–102.

20 ‘Determination of butylated hydroxyanisole and butylated hydroxytoluene in lard by derivative spectrophotometry’, Cabanillas A G, Diaz T G, Salinas F. Analusis. 1991

198, 262–265.

21 ‘Voltammetric determination of butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate and tert-butylhydroquinone by use of chemometric approaches’, Ni Y, Wang L, Kokot S. Analytica Chimica Acta 2000 412, 185–193. 22 ‘Development and comparison of analytical methods for BHA, BHT and PG determi- nation in foods’, Minim Y P R, Cecchi H M. Ciencia e Tecnologia de Alimento. 1995 152, 150–154. [Spanish]

12.5 Appendix: method procedure summaries

Analysis of oils, fats and butter oil 5,6 AOAC official method 983.15, phenolic antioxidants in oils, fats and butter oil, liquid chromatographic method, IUPAC–AOAC method Principle Antioxidants are extracted into acetonitrile. Extract is concentrated and diluted with 2-propanol. Antioxidants are separated by liquid chromatograph and measured by ultraviolet detection at 280 nm. Determination a Extraction – Accurately weigh to nearest 0.01 g 50 mL beaker containing c. 5.5 g liquid or butter oil or c. 3.0 g lard or shortening liquefied in bulk using 60 °C water bath or oven, and swirled or shaken to ensure homogeneity. Decant as much test portion as possible into 125 mL separatory funnel containing 20 mL 22.5 mL for lard or shortening saturated hexane. Reweigh beaker to determine test portion weight. Swirl to mix test portion with hexane, and extract with three 50 mL portions of saturated acetonitrile. If emulsions form, hold separatory funnel under hot tap water 5–10 s. Collect extracts in 250 mL separatory funnel and let combined extracts drain slowly into 250 or 500 mL round-bottom flask to aid removal of hexane-oil droplets. Note: at this point, 150 mL acetonitrile extract may be stored overnight, refrigerated. Evaporate to 3–4 mL, using flash evaporator with ≤40 °C water bath, within 10 min. Note: 1 Prolonged evaporation time may cause TBHQ losses. To decrease evaporation time, use efficient vacuum source and water-ice con- denser cooling. 2 Use 500 mL flask to reduce ‘bumping’ losses. Take care to ensure quantitative transfer of extract after evaporation. Using disposable pipette, transfer acetonitrile-oil droplet mixture to 10 mL glass-stoppered graduated cylinder. Rinse flask with small portions non-saturated acetonitrile. As rinse pools in flask bottom, pipette rinse to cylinder until 5 mL is collected. Rinse pipette through top and continue to rinse flask with small portions 2- propanol, transferring rinses to cylinder until 10 mL is collected. Mix cylinder contents. Note: Delay in analysing extracted test portion may cause TBHQ loss. b Chromatography – Using sample loop injection valve, inject 10 µL sample extract and elute with solvent gradient program for test extracts. Before and after every 3–4 test injections, or more frequently if differences between standard peak heights are found to be 5 , inject 10 µL antioxidant working standard solution 10 µLmL and elute with solvent gradient program for standards. For analyte peaks off scale or 3x standard, quantitatively dilute test extract with 2-propanol-acetonitrile 1 + 1 and reinject. Identify peaks by comparison with retention times of standard. For reagent blank determination, take 25 mL saturated hexane and follow extraction a, from ‘. . . extract with three 50 mL portions of saturated acetonitrile’. Inject 10 µL reagent blank extract and elute with solvent gradient program for samples. The reagent blank should have no peaks interfering with antioxidant determination. Use electronically determined peak height, or measure peak height to 0.1 mm, using blank gradient chromatogram as guide to follow baseline. Determine antioxidant peak heights and average antioxidant standard peak heights from duplicate injections before and after test injection, corrected for gradient blank. Calculation Calculate concentration of antioxidant as follows: Antioxidant, µgg = R x R s × C s W x × D [12.1] where: R x and R s are peak heights from test portion and standard, respectively C s is concentration standard, µgmL W x is test portion weight, gmL, in undiluted 10 mL test extract D is dilution factor, if injected solution is diluted For further information see AOAC official method 983.15.