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