Appendix: method procedure summaries

20 Analytical methods for food additives Table 2.1 cont’d b Method Matrix Sample preparation Method conditions Detection Reference Capillary zone Non-alcoholic Samples used as is or diluted A background solution 216 nm 5 electrophoresis beverages and with water consisting of 15 mM borate CZE fruit flavoured buffer at pH 10.5, hydrodynamic syrups injection and a 20 kV separation voltage Spectro- Beverages, Samples diluted in 5 mL acetate Analysed by spectrophotometry 427 nm 8 photometric gelatine, syrups buffer and diluted to 25 mL with using a Beckman DU-70 instrument water Solid-phase Colourings Sample solution mixed with 1 M The mixture was shaken for 15 min Absorbance 9 spectro- caramel, HCl, ethanol sufficient for a 10 then the gel beads were filtered off, measured at photometry confectionery conc., water and Sephadex DEAE packed into a 1 mm cell and 525 nm and A-25 gel absorbance measured 800 nm Rapid clean-up Various foods Liquid samples as is. Solid Colour separated on reverse phase TLC or 10 method for samples dissolved in water and C18 Sep-Pak cartridge and eluted spectrophotometric spectro- filtered through sintered glass with aqueous isopropanol solutions photometric and filter TLC methods Spectro- Soft drinks Ion-pair formation with octadecyl- Extraction of the ion-pair into 550 nm 11 photometric trimethylammonium bromide at n -butanol pH 5.6 E122: Azorubine 21 Table 2.2 Summary of statistical parameters for azorubine in foods Method Matrix Extent of validation Statistical parameters Reference Rapid clean-up Various foods AOAC Official Method Ref. JAOAC 1988, 71, 458. 10 method for 988.13 spectro- photometric and TLC methods IP-RP-HPLC Lemonade, Full collaborative trial see Table 2.3 2 cake crumb, skimmed milk RP-HPLC Bitter Performance of method Linear range of calibration 2–10 mgL, 3 established with standards Recoveries 93.6–106.3 CV 4.7 n=9 and validated with Bitter sample n=9 see Table 2.4 real samples IP HPLC Commercial Performance of method Calibration graph linear from 2–10 mgL SD 0.039 mgL 4 products established with standards RSD 2.32 Detection limit 7.6 ng Recovery 99.54 n=5 n=9 and validated with Real samples: Bitter: 34.3±0.1 mgL commercial food products Syrup: 146.2±0.3 mgkg 22 Analytical methods for food additives Table 2.2 cont’d Method Matrix Extent of validation Statistical parameters Reference CZE cf Non-alcoholic Performance of method Calibration graph linear up to 4–200 mgL 5 HPLC 3 beverages and established and applied Detection limit 0.60 mgL flavoured to real samples Recoveries were 92.3–111.3 for 4–60 mgL dyes from synthetic mixtures syrups Real samples: Bitter: 37.5±0.2 mgL CZE, 35.0±0.2 mgL HPLC n=3 Strawberry syrup: 141.9±0.4 mgkg CZE, 137.9±0.3 mgkg HPLC n=3 Spectro- Soft drinks Performance of method Linear range 0–40 µgmL Recovery 98 n=6 11 photometric established and applied RSD 1.1 for 8 µgmL n=10 to real samples Strawberry flavoured drink: 3.90 µgmL n=3 {4} RSD 0.1 Results agree with manufacturers’ values {} SP spectro- Colourings, Performance of method Concentration range 12–650 µgL Detection limit 3.38 µgL 9 photometry caramel, established and applied RSD 1.3 for samples containing 250 µgL confectionery to 4 real samples n=3 Caramel: 107.99±0.3 mgL Spectro- Beverages, Performance of method Calibration graph linear up to 32 mgL 8 photometric gelatine, established and applied Replicate samples 8 mgL n=9 RSD 3.44 syrups to real samples Detection limit 0.72 mgL Recovery 95.3 n=10 HPLC Confectionery Method applied to Detection limit 12 µgL 7 confectionery HPLC Yogurt Method specific for Recovery 98 6 yogurt Table 2.3 Performance characteristics for azorubine in collaborative trial samples 2 Sample Lemonade Cake crumb Skimmed milk Analyte Azorubine Azorubine Azorubine No. of laboratories 10 9 9 Units mgkg mgkg mgkg Mean value 24.5 35.1 51.5 72.8 84.4 81.1 S r 1.64 3.68 14.81 RSD r 5.5 5.92 17.89 r 4.59 10.31 41.46 S R 2.05 7.69 20.32 RSD R 6.87 12.37 24.56 R 5.73 21.53 56.91 Ho R 10.72 1.44 2.98 Key Mean The observed mean. The mean obtained from the collaborative trial data. r Repeatability within laboratory variation. The value below which the absolute difference between two single test results obtained with the same method on identical test material under the same conditions may be expected to lie with 95 probability. S r The standard deviation of the repeatability. RSD r The relative standard deviation of the repeatability S r × 100Mean. R Reproducibility between-lab variation. The value below which the absolute difference between two single test results obtained with the same method on the identical test material under different conditions may be expected to lie with 95 probability. S R The standard deviation of the reproducibility. RSD R The relative standard deviation of the reproducibility S R × 100mean. Ho R The HORRAT value for the reproducibility is the observed RSD R value divided by the RSD R value calculated from the Horwitz equation. Table 2.4 Performance characteristics for azorubine in bitter samples 3 Sample Bitter kas Bitter kalty Analyte Azorubine Azorubine Quantification method Direct Standard Direct Standard measurement addition measurement addition Number of determinations 2 2 2 2 Units mgL mgL Mean value 33.3±0.1 32.8±0.2 18.5±0.1 17.5±0.3 Statistical parameters for assay Number of determinations 9 Calculated by Peak height Peak area Units mgL SD 0.041 0.040 RSD ±2.40 ±2.44 Detection limit 4.1 1.9 3 E141: Copper complexes of chlorophylls and chlorophyllins

3.1 Introduction

The major food groups contributing to dietary intake of copper complexes of chlorophylls and chlorophyllins are sugar confectionery, desserts, sauces and condiments, cheese and soups and soft drinks. The ADI for copper complexes of chlorophylls and chlorophyllins is 15 mgkg body weightday. Sodium copper chlorophyllin Cu-Chl-Na is not a single substance but a mixture mainly consisting of copper chlorin e 6 and copper chlorin e 4 . Copper chlorin e 6 is less stable and in some cases disappears as a result of pH and heat treatment during the manufacturing process of foods, whereas copper chlorin e 4 is relatively stable under these conditions and can be used as an indicator substance for the analysis of Cu-Chl-Na. 1

3.2 Methods of analysis

The only references that could be found for copper complexes of chlorophylls and chlorophyllins were in Japanese

1, 2

and both are HPLC methods. A summary of them is given in Table 3.1, together with the matrices for which the method is applicable. Statistical parameters for these methods, if available, are summarised in Table 3.2.

3.3 Recommendations

There are no recent methods published for copper complexes of chlorophylls and chlorophyllins in foods; therefore these need to be developed and validated by collaborative trial.

3.4 References

1 ‘Investigation to find an indicator substance for the analysis of sodium copper chlorophyllin in foods’, Yasuda K, Tadano K, Ushiyama H, Ogawa H, Kawai Y, Nishima T. Journal of the Food Hygienic Society of Japan 1995 366, 710–716. [Japanese] 2 ‘Determination of sodium copper chlorophyllin in foods’, Amakawa E, Ogiwara T, Takeuchi M, Ohnishi K, Kano I. Annual Report of Tokyo Metropolitan Research Laboratory of Public Health . 1993 44, 131–137. [Japanese] 26 Analytical methods for food additives Table 3.1 Summary of methods for Cu complexes of chlorophylls and chlorophyllins in foods Method Matrix Sample preparationextraction Method conditions Detection Reference HPLC Boiled bracken, agar-agar, Sample homogenised after pH adjustment Inertsil ODS-2 column with Photodiode array 1 chewing gum to 3–4 with 0.1 M HCl and extracted with MeOH–H 2 O 97:3 mobile at 405 nm ethyl ether, concentrated to dryness. phase containing 1 acetic Residue dissolved in MeOH acid HPLC Chewing gum, candies, Sample was suspended in citrate buffer Chemcosorb 5-ODS-UH Photodiode array at 2 processed seaweeds, pH 2.6, homogenised after adding ethyl column with MeOH–H 2 O– 625 nm processed edible wild plants, acetate–acetone 5:1. Extracted with 1 acetic acid 100:2:0.5 mobile chocolate aq ammonia solution. Ethanol added to phase aqueous layer Table 3.2 Summary of statistical parameters for Cu complexes of chlorophylls and chlorophyllins in foods Method Matrix Extent of validation Statistical parameters Reference HPLC Chewing gum, candies, processed Requires further validation Determination limit 5 ngg 2 seaweeds, processed edible wild Recoveries in spiked food samples plants, chocolate 90.7–102.5 Sodium copper chlorophyllin detected at levels of 4.3–85.3 ngg in 2 types of chewing gum and 2 types of candy produced in the UK 4 E150c: Caramel class III

4.1 Introduction