2. Determine the GC-FID response of a known amount of the unlabeled aroma com-
pound (C
(A)
) and an ester (C
(E)
) of high GC purity (e.g., methyloctanoate) that does
not coelute with any impurities coming from the labeled standard solution. Calculate
the response factor as follows:
f
resp
= C
(A)
/C
(E)
× F
(E)
/F
(A)
where C indicates concentration and F is the peak area.
3. Take a 200-
µl aliquot of labeled standard solution (A*) and 200 µl of ester (E) in
approximately the same concentration and determine the exact concentration of the
labeled standard by peak area comparison correcting for the previously determined
response factor according to the equation:
C
(A*)
= F
(A*)
/F
(E)
× C
(E)
× f
resp
where C
(A*)
is the concentration of labeled standard; F
(A*)
is the peak area of labeled
standard; F
(E)
is the peak area of the ester; C
(E)
is the concentration of the ester; and
f
resp
is the response factor [F
resp
= C
(A)
/C
(E)
× F
(E)
/F
(A)
].
The concentrations of these standard solutions should be checked regularly depending on
the expected chemical stability.
Add standards
The sample should be liquid or a slurry of a finely ground solid sample.
4. Add an aliquot (10 to 500
µl) of the organic solution containing the labeled standard,
keeping the ratio of analyte (A) in the food sample and its labeled analog (A*) in the
range of 0.2- to 5-fold.
The volume of standard added should be kept to a minimum if headspace sampling or SPME
sampling is performed. For extraction work-up procedures, the amount of solvent added
via the standard may not be critical at all.
5. Stir the solution vigorously for 30 min after adding the standard and before proceed-
ing with the work-up.
6. Transfer the sample to an extraction apparatus or headspace adsorption device for the
isolation of volatiles.
Quantify by GC-MS
7. Inject 0.5
µl of a solution containing the labeled standard (A*) at a concentration of
0.005% by GC-MS (EI mode).
8. Study the fragmentation pattern of the labeled compound and compare it with the
compound to be quantified.
9. Choose selective mass traces of labeled and unlabeled compound for subsequent
quantification using single ion monitoring (SIM mode).
The mass traces of the unlabeled and labeled counterparts should be specific and prefer-
ably of a high intensity. For compounds showing a strong molecular ion in MS-EI the
respective molecular mass ions will be used.
In the absence of a strong molecular ion, and if the EI-spectra of labeled standard and
analyte are too, other similar ionization modes need to be considered (CI positive/negative;
different reactant gases).
10. Establish a calibration curve, based on selected mass traces, by injecting a series of
solutions containing the same amount of labeled standard and varying amounts of
the analyte over a concentration range of 0.2- to 5-fold.
To obtain the calibration curve, the amount ratio of unlabeled compound/labeled com-
pound (x-axis) is plotted against the ratio of peak area of the mass trace of the unlabeled
Current Protocols in Food Analytical Chemistry
G1.3.5
Smell Chemicals