14. Repeat steps 10 to 13 in a model system containing known amounts of analyte and
standard to determine the relative recovery factors of analyte versus standard through-
out the isolation procedure.
The ratio of the analyte to the standard added, divided by the ratio of the analyte to the
standard measured, is the recovery factor. Multiplying the resulting measurements by the
appropriate recovery factor will improve accuracy by reducing some of the matrix effects.
ALTERNATE
PROTOCOL
QUANTIFICATION OF AROMA COMPOUNDS BY ISOTOPE DILUTION
ASSAY (IDA)
Aroma compounds can be quantified with high accuracy by using their corresponding
isotope labeled analogs as internal standards, e.g., the concentration of dimethylsulfide
in a product will be determined with d6-dimethylsulfide. Due to the almost identical
chemical as well as physical properties all possible losses of the analyte during the
isolation, extraction, and concentration steps are fully compensated for. Since many
standards are not available solvent-free in a high enough purity, the procedure involves
(1) the determination of the concentration of labeled standards, (2) homogenization of
isotopically labeled standard throughout the sample, and (3) GC-MS analysis in different
MS modes depending on the nature of the compound (fragmentation pattern) and
sensitivity needed. Although chemical ionization in positive and negative mode may have
certain advantages in terms of selectivity and sensitivity, electron impact ionization for
MS is sufficient for many cases.
Materials
Isotope labeled standards: a few aroma volatiles are commercially available from
CDN Isotopes and Cambridge Isotope Laboratories (CIL); custom synthesis is
available from Aspen Research Laboratories and Aldrich; the standards may
also be synthesized in-house
Purified aroma compounds to be quantified
Ester of high GC purity (e.g., methyloctanoate)
Organic solvents (highest purity)
Food sample to be analyzed
Extraction apparatus or headspace adsorption device
Gas chromatograph with MS detector (EI mode and optional CI mode;
Hewlett-Packard)
Gas chromatograph with FID (Hewlett-Packard)
NOTE: Store standards highly diluted (ppm) in organic solvents, preferably CH
2
Cl
2
or
methanol (for thiols) and in a freezer. Compounds like vinylguaiacol or furfurylthiol are
stable under these conditions.
NOTE: Solution of labeled standards stored in the freezer should be brought up to room
temperature before opening the vials and using them. If standard solutions are in a very
low-boiling solvent, e.g., pentane, use them refrigerated to facilitate pipetting.
Determine purity and concentration of labeled standard for non-commercially avail-
able standards by GC-FID
1. Inject 0.5
µl of a solution containing the labeled standard (A*) at a concentration of
0.005% and determine the purity of the solution.
Possible impurities in the standard solutions should be identified by GC-MS. Impurities
can be tolerated if they do not interfere with the quantitation.
Current Protocols in Food Analytical Chemistry
G1.3.4
Identification and
Quantification of
Aroma
Compounds