12. Method Validation
Validation was carried out in terms of specificity, linearity,
precision, limit of detection (LOD) and quantification
(LOQ), accuracy and robustness. Finally, the applicability
of the method to the determination of targeted flavorings
in a number of commercial samples was demonstrated.
The method performance was established by spiking
experiments with blank matrices (solid – oat flakes;
semi-solid – pure tomato sauce; and liquid – water with
ethanol) with a mixture of targeted compounds.
12.1 Specificity
Using Selected Reaction Monitoring (SRM) the specificity
is confirmed based on the presence of the transition ions
(quantifier and qualifier) at the correct retention times
corresponding to those of the respective flavoring standards.
The measured peak area ratios of qualifier/quantifier ion
have to be in close accordance with the ion ratios of the
standards as indicated in Table 3.
12.2 Linearity and Calibration Curve
The linearity of calibration curves is assessed over the range
from 0.01–2.0 mg/kg (for six flavorings) and 0.1–2.0 mg/kg
for coumarin. In all cases, the correlation coefficients of
linear functions has to be > 0.99. The calibration curves
are created from seven matrix-matched calibration
standards which are injected in each batch in duplicate.
12.3 Precision
The relative standard deviation (% RSD) was determined by
injecting six replicates of spiked samples of three different
matrices at two different levels. For the liquid matrix,
aqueous ethanol (40%) was used as the blank matrix
(with the addition of various amounts of saccharose to
simulate liqueurs and energy drinks), for semi-solid matrices
pure tomato sauce was used, and for the solid matrix oat
flakes. The samples were spiked at 0.1 and 1 mg/kg levels
and six replicate analyses were analyzed. For six flavorings
(
β
-asarone, estragole, menthofuran, methyl eugenol,
pulegone and thujone) the first level of addition was
0.1 mg/kg, and for coumarin the addition was at 1 mg/kg.
The second level of addition for six flavorings was 1 mg/kg,
again with coumarin being at a higher level of 10 mg/kg.
The results that establish method precision are shown in
Table 4, indicating RSDs from 2 to 21%. All precisions are
acceptable for a regulatory method, with liquid and semi-
solid foods offering a better performance than solid foods.
12.4 Limits of Detection (LOD) and Quantification (LOQ)
Limits of detection and quantification were estimated
following the IUPAC approach which consisted of analyzing
the blank sample to establish noise levels and then estimating
LODs and LOQs for signal/noise, 3 and 10 respectively.
The values for three matrices (solid, semi-solid and liquid)
are shown in Table 5 and, in all cases, these values far exceed
requirements to test for compliance to regulatory limits in
which 0.5 mg/kg is the lowest level which is controlled.
12.5 Accuracy
Accuracy was evaluated by comparing found values with
spikes by standard addition. The optimization method
was used to analyze three types of matrix. For the liquid
matrix, spiking was into 40% aqueous ethanol, for a
semi-solid matrix pure tomato sauce was used, and for a
solid matrix oat flakes were used. The samples were spiked
at levels of 0.1 and 1 mg/kg in six replicates. For six
flavorings (
β
-asarone, estragole, menthofuran, methyl eugenol,
pulegone and thujone) at 0.1 mg/kg and for coumarin at
1 mg/kg for level 1, and six flavorings at 1 mg/kg and
10 mg/kg for coumarin for level 2. The results in Table 6
show good accuracy, except in the case of solid matrices
for which overestimations are indicated.
13. Conclusion
This single laboratory validated method is capable of
determining levels of any one of seven biologically active
flavoring substances which have use restrictions in composite
foodstuffs. The method can cover all food types based on
a generic approach of selecting the category of either a
liquid, semi-solid or solid matrix, and then following the
optimized conditions for that category. The method has a
sensitivity which far exceeds regulatory requirements and
the use of MS/MS for detection guarantees a high level of
confidence in correct identification based on ion ratios.
We recommend this method for use for enforcement of
limits of biologically active flavorings in foods.
14. References
1. For details of this research please see: Bousova K., Mittendorf K., Paez V.,
Senyuva H., A Solid-Phase Micro-Extraction GC-MS/MS Method for
Rapid Quantitative Analysis of Food and Beverages for the Presence of
Legally Restricted Biologically Active Flavorings.
J. AOAC.
Accepted for
publication, 2011.
2. Regulation (EC) No 1334/2008 of 16 December 2008 on flavorings and
certain food ingredients with flavoring properties for use in and on foods
and amending Council Regulation (EEC) No 1601/91, Regulations (EC)
No 2232/96 and (EC) No 110/2008 and Directive 2000/13/EC.
Official Journal of the European Union
. (2008)
L 354
/34-50.
3. Code of Federal Regulations Title 21CFR189.180 [Revised as of April 1, 2010]
4. Kataoka, H., Lord, H. L., & Pawliszyn, J. (2000).
J. Chromatogr.,
A,
880
, 35-62.
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