Práticas recomendadas para quantificação de compostos voláteis em flavorizantes

Prévia do material em texto

Recommended practices
Received: 15 December 2015, Accepted: 16 December 2015 Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/ffj.3311
IOFI recommended practice for the use of
predicted relative-response factors for the
rapid quantification of volatile flavouring
compounds by GC-FID
T. Cachet,* H. Brevard, A. Chaintreau, J. Demyttenaere, L. French,
K. Gassenmeier, D. Joulain, T. Koenig, H. Leijs, P. Liddle, G. Loesing,
M. Marchant, Ph. Merle, K. Saito, C. Schippa, F. Sekiya and T. Smith
Abstract: This recommended practice enables the quantifica
chromatography with flame-ionization detection, without ha
tion of volatile compounds in flavourings to be made by gas
ving authentic compounds available, and also in many cases
it can avoid time-consuming calibration procedures. The relative-response factors (RRF) can be predicted from the molecular
formula of the compound, and this approach can be applied to compounds containing the atoms C, H, O, N, S, F, Cl, Br, I,
and Si, providing that the molecular formula and number of benzene rings in the analytes are known. The purity of chemically-
defined flavouring substances or chromatographic standards can also be estimated using these predicted RRF, and this proce-
dure can also be used to quantify (poly)hydroxylated compounds, after their derivatization into trimethylsilyl ethers or esters.
Copyright © 2016 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: flavourings; quantitative analysis; GC-FID; predicted relative response factors
* Correspondence to: Dr. T. Cachet, IOFI, 6 Avenue des Arts, 1210 Brussels,
Belgium. E-mail: tcachet@iofiorg.org
IOFI (International Organization of the Flavor Industry), Working Group on
Methods of Analysis
Introduction
The Working Group on Methods of Analysis (WGMA) of
the International Organization of the Flavor Industry (IOFI)
has previously published a recommended practice for the
quantitative determination of specific volatile substances in
flavourings and other complex mixtures such as essential oils,
using gas chromatography with flame-ionization detection
(GC-FID).[1]
In the scientific literature on flavours, fragrances, and essential
oils, the raw percentages of peak areas are often used as such, or
in association with that of an internal standard (ISTD), assuming
that all response factors are equal to unity - a practical approach
but one that has been shown to lead to poor accuracy.[2]
On the other hand, quantifying by rigorous methods (internal
standardization, internal normalization) is accurate but time-
consuming, because it requires the establishment of calibration
curves, or the experimental determination of response factors
relative to a given ISTD).[1] Even this does not solve the challenge
of compounds that are not available in the pure state to be used
as standards, or those that are not stable enough to be stored
before use for the determination of their relative-response fac-
tors (RRF).
The present technique enables the quantification of volatile
compounds in flavourings to be made by GC-FID, without having
authentic compounds available, and also in many cases it can
avoid time-consuming calibration procedures. The relative-
response factors can be predicted from the molecular formula of
Flavour Fragr. J. 2016 Copyright © 2016 John
the compound, and this approach can be applied to compounds
containing the atoms C, H, O, N, S, F, Cl, Br, I, and Si, providing that
the molecular formula and number of benzene rings in the
analytes are known. This procedure also makes it possible to quan-
tify (poly)hydroxylated compounds, after their derivatization into
trimethylsilyl ethers or esters.
This technique, described in a number of publications,[2–5]
with applications reported in the flavour, fragrance, and other
domains,[6–12] can also be used to estimate the purity of
chemically-defined flavouring substances or chromatographic
standards using these predicted RRF.
Principle
The flavouring or individual compound (as such or after derivatiza-
tion) is injected together with an internal standard, and their
responses are corrected using their predicted RRF. The value of
the latter is calculated from themolecular formula and the number
of benzene rings in the analytes.
Wiley & Sons, Ltd.
T. Cachet et al.
Chemicals
Solvent
Ethanol, toluene, acetonitrile, ethyl acetate, and methyl pivalate, all of
analytical grade, are typical solvents for this determination. Low-boiling
solvents (methanol, dichloromethane, pentane, diethyl ether, etc.) yield
less accurate results and are not recommended.[3]
Internal standard
Methyl octanoate (MO), with a purity of 99 % is used as the internal
standard (ISTD). Other internal standardsmay be used, if their RRF com-
pared to methyl octanoate is accurately determined (see below).
Apparatus
This technique is applicable with any gas chromatograph
equipped with an FID.
Injector
Use a split/splitless standard injector equipped with a tubular liner.
The latter can be empty or may contain a plug of silanized quartz
wool. Experience has shown that cup-splitter liners or PTV injectors
are not always suitable for this approach, in particular for high
molecular-weight compounds.
The injector temperature should be 250 °C (this temperature
influences the RRF of high-boiling compounds[2]). Inject the samples
(1μl) using an autosampler, a 10μl syringe, and a typical split ratio of
1/50 to 1/100. Modifying this ratio may alter the RRF of high-boiling
compounds.[2]
Detector
The FID temperature should be 250 °C, fueled with 40ml/min
hydrogen, 450ml/min air, and a make-up of 30ml/min nitrogen.
The detector temperature influences the RRF values of high-
boiling compounds.[2]
Column
Typically a bonded apolar or semi-polar column can be used. Polar
phases such as polyethylene glycol are also suitable, but the RRF of
high-boiling compounds may be modified.[2]
Quantification procedure
Sample concentration
Both the ISTD and the flavouring or individual compound to be
quantified are diluted in the solvent at a concentration of below
10%. Lower concentrations do not alter the RRF values, but higher
ones do.
Oven program
Typical conditions for such an analysis with a non-polar column
are an initial oven temperature of 50 °C for 5min, then ramp at
3 °C/min up to 120 °C, ramp at 5 °C/min up to 250 °C, and finally
ramp at 15 °C up to 300 °C for 20min. Other programs are possible,
below the limit of 300 °C (or less depending on the maximum
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working temperature of the column, but a lower temperature
may alter RRF values).
Measurement
Inject the diluted sample and determine the areas corresponding
to the analyte and the ISTD (Ai and AMO, respectively).
Prediction of the RRF
The predicted RRF of an analyte RRFi
Pred is calculated using its mo-
lecular formula:
RRFPredi ¼ 103 MWi=MWISTDð Þð�61:3þ 88:8nC þ 18:7nH
�41:3nO þ 6:4nN þ 64:0nS � 20:2nF � 23:5nCl
þ51:6nBr � 1:75nI þ 39:9nSi þ 127nBenzÞ�1
where nC, nH, nO, etc. are the number of carbon, hydrogen, oxygen,
etc. atoms in the compound, nbenz is the number of benzene rings,
and MWi and MWISTD are the molecular masses of the analyte and
the internal standard, respectively.
The calculation can be automated with a spreadsheet, an exam-
ple of which is given in the supplementary material for this publi-
cation. In the case of most compounds occurring in flavourings,
this can of course be simplified to:
RRFPredt ¼ 103 MWi=MWISTDð Þð�61:3þ 88:8nC þ 18:7nH � 41:3nO
þ6:4nN þ 64:0nS þ 127nBenzÞ�1
The mean accuracy of these predicted RRF is± 6%, as deter-
mined using a database of 490 compounds.[3]
Quantification
Using the mass of ISTD added to the sample (mMO), the peak area
of the analyte and the ISTD, respectively (Ai and AMO), and the
predicted RRF, determine the mass of the analyte in the sample:
mi ¼ RRFPredi mMO
Ai
AMO
Use of other internal standards
If anotherISTD, X, is used, the analyte amount can be determined
as follows:
RRFPredi=X ¼
RRFPredi=MO
RRFMeasX=MO
Where:
RRFPredi=X = the predicted RRF of the substance i relative to X
RRFPredi=MO = the predicted RRF of the substance i relative to MO
RRFMeasX=MO = the measured RRF of the ISTD X relative to MO
The mean RRF of the alternative internal standard X relative to
MO (RRFMeasi=MO) has been determined experimentally by 11 laborato-
ries for four compounds, with the results shown in Table 1. Note
that such a conversion implies a decrease of accuracy.
Flavour Fragr. J. 2016Wiley & Sons, Ltd.
Table 1. Mean RRF of alternative internal standards
Predicted RRF Mean experimental RRF RSD Bias vs. predicted
2-nonanol 0.851 0.861 1.5% 3.0%
tetralin 0.709 0.702 1.5% 3.5%
1,4-dibromobenzene 1.919 1.924 1.2% -0.5%
undecanol 0.825 0.826 2.9% -0.4%
Table 2. Quantification of a model mixture by 10 laboratories
(all constituents in the range of 100-1000mg/kg).
Compound RSD Bias
isoamyl acetate 5.8% 2.0%
1,8-cineole 3.7% 8.9%
linalol 3.0% 4.2%
4-methylacetophenone 3.2% 4.9%
anisaldehyde 5.1% -4.5%
citronnelol 4.5% 1.8%
eugenol 6.2% 0.0%
coumarin 8.6% 1.9%
ethyl decanoate 4.7% 2.6%
beta-caryophyllene 5.2% 8.2%
methyl isoeugenol 6.9% -3.9%
pentadecane 4.6% 5.6%
Hedione®=Methyl dihydrojasmonate
(2 isomers)
9.9% -4.4%
IOFI recommended practice - use of predicted relative-response factors
Purity estimation
Quick procedure
The purity Pi of a chemically-defined flavouring substance or a
chromatographic standard can be estimated, without having the
pure reference compound available, and without knowing the
identity of impurities.
Apparent RRF determination
Determine the apparent RRF of i by weighing a massmi of the sub-
stance of unknown purity (mi ¼ m′i þmx, wherem′i, andmx are the
unknown masses of the pure substance and any impurities,
respectively):
RRFappi ¼
miAMO
mMOAi
Purity estimate
Calculate the purity as the ratio of the predicted to the apparent
RRF value:
Pi ¼ RRF
Pred
i
RRFappi
where RRFPredi is calculated as above:
Table 3. Quantification of a lime essential oil by 10 laborato-
ries; concentrations are expressed as the mean percentage in
the oil
Compound Mean RSD Mean RSD
alpha-pinene 1.48% 5.0%
camphene 0.71% 8.8% 0.70% a 7.2% a
beta-pinene 2.70% 4.2%
myrcene 1.56% 3.6%
1,4-cineole 1.90% 3.7%
alpha-terpinene 2.37% 3.5%
limonene 49.52% 4.0%
gamma-terpinene 12.32% 3.4%
terpinolene 9.87% 2.9%
alpha-fenchol 0.76% 5.3%
terpinen-1-ol 1.06% 4.0%
b b
Full procedure
This procedure requires that all impurities are detectable in the
course of the GC analysis (i.e. absence of any non-volatiles) and
that all of them are identified.
Quantification of all constituents
The amounts of the target compound and all its impurities are
determined according to the quantification procedure above.
Purity
The purity is then estimated as the ratio of the target-compound
amount to the sum of all constituent amounts:
Pi ¼ mi∑mi
(E)-beta-terpineol 0.86% 12.9% 0.80% 7.8%
terpinen-4-ol 0.85% 10.9% 0.88% b 2.8% b
alpha-terpineol 9.57% 2.7%
gamma-terpineol 1.30% 4.0%
decanal 0.14% 20.9%
neral 0.28% 9.8%
geranial 0.39% 8.8%
caryophyllene 0.50% 6.1%
alpha-bergamotene 0.51% 6.2%
awithout one outlier
bwithout three outliers exhibiting a co-elution
Trimethylsilyl derivatives
Just before the derivatization procedure, prepare two solutions at
0.5g/kg (exactly weighed) in moisture-free pyridine, one with the
ISTD (methyl octanoate), the second one with the compound to
be derivatized.
In another vial, mix 0.6mL of the latter plus 0.4mL of pyridine
(both exactly weighed), and 200μL of BSTFA/1% TMCS. Seal the
vial, and maintain it at 50 °C for 1 h. Cool down at room tempera-
ture and add 0.6mL (exactly weighed) of the ISTD solution. After
mixing, inject 1μL of this solution in the GC-FID instrument.
Flavour Fragr. J. 2016 Copyright © 2016 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/ffj
T. Cachet et al.
Reproducibility
Model mixture
Table 2 shows the results for the quantification of a model
mixture by 10 laboratories (all constituents were in the range
of 100-1000mg/kg).
Lime essential oil
Table 3 shows the results for the quantification of a lime essential
oil by 10 laboratories.
Down to a concentration of 1% in the oil (≈1 ng in the
detector), the precision, expressed as the RSD, is better than
5%, and better than 10% down to a concentration of 0.5%.
The precision decrease as a function of the concentration is
mainly due to chromatographic reasons (co-elutions, active col-
umn sites, integration accuracy, etc.).
The use of experimentally-determined RRF clearly remains the
most accurate means whenever pure authentic standards are
available. However, when this is not the case, or when a complex
multi-component mixture makes the experimental RRF measure-
ment too time-consuming, the predicted RRF are a valid alterna-
tive, with a mean accuracy of 6.0%.[3]
Acknowledgements
We gratefully acknowledge the contribution of Esméralda Cicchetti,
of Cosmo Ingredients International, for her participation in the inter-
laboratory reproducibility tests of this recommended practice.
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Supporting information
Additional supporting information may be found in the online
version of this article at the publisher’s web site.
Glossary of terms
Symbol Meaning
Ai area of the compound i to be quantified
AMO area of methyl octanoate
ISTD internal standard
mi mass of the compound i to be quantified
MO methyl octanoate; mMO mass of methyl octanoate
Pi purity of compound i
RRF relative-response factor
RRFAppi apparent RRF of compound i (assuming it is 100% pure)
RRFMeasX=MO measured response factor of internal standard X relative
to methyl octanoate
RRFi
Pred predicted relative response factor of compound i
RRFPredi=X predicted response factor of compound i relative to the
internal standard X
RRFPredi=MO predicted response factor of compound i relative to
methyl octanoate
RSD relative standard deviation
Flavour Fragr. J. 2016Wiley & Sons, Ltd.
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