Off-odors Quantification & Quality Control
Application to Pet Food Ingredients
Results obtained at Alpha MOS Laboratory (Toulouse, France) with pet food samples supplied by a
manufacturer.
Objective
Palatability enhancers are frequently used in pet
food products. The quality of these ingredients
greatly impacts the final product and thus the
success among pets.
The quality control consists of detecting and
quantifying the compounds that mainly
contribute to the flavor, as well as unwanted off-
odors.
To achieve that, gas chromatography is one of
the most commonly employed method. This
Application Note
application note investigates the possibility to
use an electronic nose as a fast, sensitive and
reliable technique to quantify off-odors in pet
food ingredients, while comparing global sensory
profiles.
Equipment
HERACLES Flash Gas Chromatography
Electronic Nose
The HERACLES Electronic Nose (Alpha MOS,
France Fig. 1) is based on the technology of
ultra fast chromatography. It features two metal
columns of different polarities (non polar RXT-5
and slightly polar RXT-1705, length = 10m,
diameter = 180m, Restek) mounted in parallel
and coupled to 2 Flame Ionization Detectors
(FID). Therefore, 2 chromatograms are obtained
simultaneously, allowing a sharper identification
of the chemical compounds. It allows headspace
or liquid injection modes.
The integrated solid adsorbent trap thermo-
regulated by Peltier cooler (0-260C) achieves
an efficient pre-concentration of light volatiles
and shows a great sensitivity (in the pg range).
With fast column heating rates (up to 600C/min),
results are delivered within seconds and the
analysis cycle time is around 5 to 8 minutes.
ANH Fig. 1: Ultra Fast GC based HERACLES Electronic Nose
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The electronic nose is coupled to an
autosampler (HS 100, CTC Analytics) to
automate sampling and injection.
The instrument is operated through Alpha Soft
software. In addition to classical chromatography
functionalities, it provides chemometrics data
processing tools such as sample fingerprint
analysis and comparison, qualitative and
quantitative models, quality control cards.
AroChembase: Kovats Index library for
chemical pre-screening & sensory
characterization
The HERACLES e-nose was additionally
equipped with AroChembase module (Alpha
MOS, France) that can be used within Alpha Soft
E-Nose software. It consists of a library of
chemical compounds with name, formula, CAS
number, molecular weight, Kovats retention
Index, sensory attributes and related
bibliography.
It allows to pre-screen the chemical compounds
and give sensory features by directly clicking on
the chromatograms peaks generated by
HERACLES e-nose.
Samples & analytical conditions
Sixteen samples (S1 to s16) of pet food products
were analyzed in triplicate each (table 2).
An alkane solution (n-hexane to n-hexadecane)
was analyzed in order to convert retention time
in Kovats indices and thus to identify the volatile
compounds using AroChemBase.
To quantify the 4 off-odor compounds involved
(hexanal, p-cresol, isovaleric acid, indole), a
calibration was achieved by standard addition of
standard solutions to a reference pet food
sample (S1). The following pure compounds
were used: hexanal (Aldrich, ref 11.560-6), p-
cresol (Acros, ref. 405740050), isovaleric acid
(Acros, ref. 156690100) and indole (Acros, ref.
122150100).
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 Main Analytical Conditions Off-odor compounds quantification
For each of the 4 chemical compounds, a
Parameter Value calibration curve (fig. 4) was set up by standard
Sample 1g in 20mL vial addition method on S1 sample.
Sample incubation 110C
Injected volume 5000 l
Hexanal
Trap temperature 50C
Column temperature 40C (5s) to 270c
(21s) by 200 C/min
Acquisition duration 95 s
Detector temperature 260C
Time between 2 analyses 8 min

Table 2: HERACLES e-nose parameters for pet food odor

analysis (headspace injection)
p-cresol

Results: off-odors quantification

Chromatograms
The chromatograms obtained on Heracles e-
nose showed a good resolution of hexanal,
isovaleric acid, p-cresol and indole (Fig. 3).

Indole

T ime (s)

Isovaleric acid

T ime (s)

Fig. 3: Chromatograms of a pet food product on the 2

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columns of HERACLES

Figure 4: Calibration models for hexanal, p-cresol, indole,

isovaleric acid

All compounds showed a high linearity of

quantification on the range 0 to 100 g/g, with
correlation coefficients between 0.996 and 0.999.
Therefore, these calibration curves can be used
to quantify the amount of these 4 compounds in
the pet food ingredients studied (table 5).

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 Column RTX-5 : 790, 908, 1064, 1281
Column RTX-1701 : 895, 1034, 1294, 1550
Sample Hexanal Indole Isovaleric p-cresol Increasing concentration of hexanal

S1 20 2,7 24 3,5 S11

Increasing concentrations of S10
S2 7 7,5 55 2,6 the 3 other off-odors
S8

S3 9 9,2 95 2,3 Lower concentrations

Higher of hexanal
S4 14 0,0 38 2,1 concentration
of hexanal S3
S12
S5 16 2,8 52 1,3
S16
S6 5 3,3 37 1,8 S2
S9
Medium concentrations of hexanal
S7 10 4,8 21 2,1 S13 S7
S15 S5
S6
S8 34 5,7 182 8,5 S14 S1
S4

S9 17 3,3 51 3,5
S10 30 5,0 162 3,6 Figure 7: Global odor map of palatability enhancers
S11 33 6,7 182 4,6 (Principal Components Analysis) using selected peaks of off-
odors
S12 16 4,0 93 4,6
S13 70 2,5 102 3,3 The discrimination on PC1 axis is mainly linked
S14 77 2,7 135 2,1 with hexanal concentration: on the left part are
S15 48 2,8 104 2,0 located the samples with the highest content of
S16 137 2,9 208 2,7 hexanal, and on the right side samples with the
lowest content of hexanal.
The second axis PC2 is mainly linked with the
Table 5: Concentration of 4 off-odor molecules in palatability amount of other off-odors.
enhancers (in g/g or ppm m/m)

Quality Control Card

Repeatability checking
Based on HERACLES e-nose measurements,
The repeatability was determined on 10 the major peaks of pet food palatability
analyses of sample S1 (Table 6). Being less enhancers were selected and sample S1 taken
than 5%, the relative standard deviation (RSD) is as the reference quality to set up a Statistical
considered as acceptable. Quality Control model (fig. 8).

Isovaleric Column RTX-5 : 790, 908, 1064, 1281

Column RTX-1701 : 895, 1034, 1294, 1550
Chemical Hexanal Indole acid p-cresol
S16
Nbr of analyses 10 10 10 10
Mean
concentration
(ppm) 19,9 2,74 23,5 3,53 S14
S13
Standard
deviation (ppm) 0,9 0,05 1,1 0,07
Relative standard S10
S8
deviation (%) 4,4 1,6 4,6 1,9 S3 S15
S11
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S5 S12
S7 S9
S1
Table 6: Calculation of repeatability values for palatability S4
S2
enhancer quantification S6

Figure 8: Statistical Quality Control (SQC) chart of

Results: odor quality control palatability enhancers on Heracles e-nose with sample S1 as
the reference quality
Overall odor profiling
Upon selecting the major chemical compounds
on HERACLES e-nose chromatograms, a This Quality Control Card shows that samples
general odor map was established (fig. 7). S2, S4, S5, S6, S7 and S9 appear in the green
band of acceptable quality determined from S1
grade. All other samples are out of specifications.

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 Volatile compounds identification &
sensory characterization

The nature of volatile compounds detected in

the headspace of palatability enhancers was
investigated using Kovats retention indices
(Table 9) and AroChemBase library. Most of
the identified volatile compounds correspond to
aldehydes, ketones and alcohols. Sensory
attributes can be found out thanks to this
analysis.

KI on KI on Sensory
MXT-5 MXT-1701 Possible matches attributes
661 747 methyl butanal
700 791 pentanal almond like
763 877 pentanol green
801 894 hexanal green
890 991 2-heptanone cheezy
902 997 heptanal green
964 1077 1-heptanol fresh
994 1037 psi-cumene herbaceous
1004 1096 octanal rancid
1040 1160 2-acetyl pyridine popcorn
1040 1140 2-octenal green leaf
1102 1199 nonanal green

Table 9: Possible volatile compounds identified in the

headspace of palatability enhancers by Kovats indices

Conclusion
This application note showed that HERACLES
e-nose can be used to rapidly quantify off-
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odors in pet food ingredients from a chemical

and a sensory point of view. HERACLES
provides a sensitivity and reproducibility as
good as with SPME-Gas Chromatography.

This makes this system suitable for both

sensory characterization in R&D departments
and high throughput quality control at the
production floor.

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