Meat Science 98 (2014) 569573

Contents lists available at ScienceDirect

Meat Science
journal homepage: www.elsevier.com/locate/meatsci

TBARs distillation method: Revision to minimize the interference from

yellow pigments in meat products
P. Daz, M.B. Linares, M. Egea, S.M. Auqui, M.D. Garrido
Tecnologa de los Alimentos, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain

a r t i c l e

i n f o

Article history:
Received 22 April 2013
Received in revised form 9 April 2014
Accepted 14 June 2014
Available online 21 June 2014
Keywords:
TBA distillation method
Yellow pigment
Interference
Meat products
Sucrose

a b s t r a c t
The aim was to study the effect of the incubation method and TBA reagent (concentration/solvent) on yellow pigment interference in meat products. Distillates from red sausage, sucrose, malondialdehyde and a mixture of sucrosemalondialdehyde were reacted with four different TBA solutions at ve different temperature/time
relations. Two TBA solutions were prepared at 20 mM using 90% glacial acetic acid or 3.86% perchloric acid. In addition, an 80 mM TBA solution was prepared using distilled water adjusted to pH 4 and another using 0.8% TBA in
distilled water. The temperature/time relations were: (1) 35 min in a boiling water bath; (2) 70 C/30 min;
(3) 40 C/90 min; (4) room temperature (r.t.) (24 C) in dark conditions for 20 h; and (5) 60 min in a boiling
water bath. The results showed that aqueous or diluted acid solutions of TBA reagent and the application of
100 C for less than 1 h provided the best conditions to minimize the presence of yellow pigments and maximize
pink pigment formation in meat products.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
The 2-thiobarbituric acid (TBA) reaction as an index of lipid oxidation
was rst described by Kohn and Liversedge (1944) in their studies of the
oxidation products in animal tissues. Until then, the nature of the substance reacting with TBA and the cause of pink colour formation were unknown. Bernheim et al. (1948) suggested a structure composed of three
carbon atoms and an aldehyde or ketone group, and it was not until the
early 1950s (Patton & Kutz, 1951) that malondialdehyde (MDA) was
established as being responsible for the formation of a pink complex
with TBA that showed maximum absorbance at 532 nm. MDA, the principal product of lipid peroxidation, has since become the centre of attention when monitoring the oxidation of meat and meat products (Raharjo
& Sofos, 1993). However, the reaction between TBA and MDA is not specic and other carbonyl compounds resulting from lipid peroxidation
may react with TBA to form a pink complex, leading to an overestimation
of the results (Rey et al., 2005). Schmidt (1959) established a generic formula for these compounds: HO(CR_CH)xCHO, where x = 0, 1, and 2
and R may be any substituent; hence the term thiobarbituric acid reactive
substances (TBARs).
To the problem related with the lack of specicity, may be added
the existence of other molecules that may react with TBA to generate
yellow or orange complexes with an absorbance maximum at 440
460 nm. The overlapping of peaks also leads to overestimation of absorbance at 532 nm, which corresponds to the pink complex. Indeed,
several compounds present in meat products can react with TBA to
Corresponding author. Tel.: +34 868884708; fax: +34 868887167.
E-mail address: mgarrido@um.es (M.D. Garrido).

http://dx.doi.org/10.1016/j.meatsci.2014.06.012
0309-1740/ 2014 Elsevier Ltd. All rights reserved.

form yellow or orange complexes, such as water soluble proteins

and peptides (Schmedes & Holmes, 1989; Shamberger et al., 1977),
aldehydes (Kosugi et al., 1988), sugars (Wang et al., 2002), nitrites
and nitrates (Ulu, 2004), metal chelators (Gutteridge & Quinlam,
1983), polyphenolicantioxidants (Raharjo & Sofos, 1993), pigments
(Bird & Draper, 1984; Shamberger et al., 1977), amino acids, additives and fatty acids (Hoyland & Taylor, 1991).
Various alternatives exist to eliminate the interference produced by
these pigments and to increase specicity and detection limits; for example, the quantication of MDA by high-performance liquid chromatographic (HPLC) (Candan & Tuzmen, 2008), the addition of
sulphanilamide to eliminate the interference produced by nitrites in
cured meat products (Ulu, 2004) or ltration of the extract in solidphase cartridges (C18) before the TBA-MDA reaction takes place
(Raharjo et al., 1993). Also, aldehydes can react with free amino groups
of protein to give uorescent Schiff bases which can be measured by
spectroscopy (Gatellier et al., 2007). There are also mathematical
methods based on the differences in absorbance between pink and yellow pigments (Yu & Russell, 1962) and on the third-derivative signal at
532 nm (Botsogloum, Fletouris, Papageorgiu, Vassilopoulos, Mantis &
Trakatellis, 1994).
However, the most widely used methods to quantify the TBARs
index are distillation (Tarladgis et al., 1960) and aqueous acid extraction
(Botsoglou et al., 1994). In the case of interference caused by the nonvolatile compounds present in meat products e.g. colourants such as paprika, distillation is recommended even though it is less accurate and
slower (Ganhao et al., 2011; Raharjo & Sofos, 1993; Raharjo & Sofos,
1993; Yu & Russell, 1962). Although these methods are widely cited in
the bibliography, the solvent and temperature/time relations applied

570

P. Daz et al. / Meat Science 98 (2014) 569573

differ between authors. Although the specic reactivity of TBA with

MDA is not affected by pH or the type of acid used (Janero, 1990), the
reactivity with the peroxidized lipids present in meat is produced at a
pH of between 3 and 4 and is affected by the type of acid present during
incubation (Asakawa & Matsushita, 1979). The temperature is another
factor which affects TBA reactivity. At room temperature, MDA may
react with TBA to produce a pink pigment (Tarladgis et al., 1962), but
for maximal colour development from peroxidized lipids, exposure to
high temperatures (80120 C) for time periods ranging from 10 min
to over 1 h is necessary (Bernheim et al., 1948; McKnight & Hunter,
1965; Ottolenghi, 1959; Sinnhuber et al., 1958). It is also known
that the reaction is faster as the TBA concentration increases (Wang
et al., 2002).
The reactivity of TBA with substances that produce yellow pigments
is similarly affected by these factors. However, the matrix of meat products is complex and it is difcult to identify any particular compound as
being solely responsible for the interference. Sucrose, which may exceed 4% of the content of meat products, is one such compound, leading
Wang et al. (2002) to study the effect of TBA concentration and the temperature/time relation on the interference caused by sucrose in cooked
ground beef meat. They used the aqueous acid extraction method and
standard solutions of sucrose and MDA to reproduce the development
of yellow and pink pigments. However, the solvent used in preparing
the TBA reagent may also affect the reaction, and the effect may further
differ depending on whether distillation or the aqueous acid extraction
method is used. All these variables must be taken into account when
choosing the most suitable incubation treatment to be used in cases
where substances may exist that will interfere with the determination.
Tarladgis et al. (1960) and Botsoglou et al. (1994) proposed higher incubation temperatures and shorter times than Wang et al. (2002), while
Ganhao et al. (2011) used room temperature for periods of up to 20 h.
TBA reagent (concentration/solvent) and incubation method are parameters which must be analysed together if we are to know the conditions that favour the formation of pink pigments and minimize the
formation of yellow pigments in meat products.
The objective of the present study therefore was to establish the optimal conditions (incubation temperature/time and TBA reagent) that
minimize the interference produced by sucrose and maximize the formation of pink pigment during TBARs determination using the distillation method in a meat product containing paprika.
2. Material and methods
2.1. Fresh red sausage
Fresh red sausage is a typical Spanish meat product, which is elaborated with pork lean/fat and a mixture of spices, mainly paprika. The
samples used in this study were manufactured simulating commercial
processing conditions. A relation 60/40% of pork lean/fat was minced
(5 mm) using a P3298 cutter (Braher International, San Sebastian,
Spain). The spice mixture was composed of paprika, salt, garlic,
Pimpinellaanisum, black pepper, lactose and dextrose, which were dissolved in water before use. All ingredients plus minced meat were homogenized for 5 min using an RM-60 mixer (MaincaGranollers,
Spain).The fresh sausages meat was stuffed into lamb casings weighing
40 g. The samples were packaged in a transparent polystyrene tray BA85 (Sena, Sociedad de Envases Alimentarios, Aduna, Spain) without lm
and stored at room temperature for 15 days to ensure high levels of lipid
oxidation.
2.2. MDA working solution
The MDA working solution was prepared through acid hydrolysis of
1,1,3,3-tetraethoxypropane (TEP) following the procedure described by
Botsoglou et al. (1994). A quantity (73.2 mg) of TEP (Acros Organics,
New Jersey, USA) was weighed into a screw-capped test tube and

10 ml of 0.1 N HCl (Panreac, USA) was added. The test tube was immersed in a boiling water bath for 5 min and quickly cooled with
water bath. The hydrolyzed TEP solution was transferred into a 100 ml
volumetric ask and diluted to volume with water to obtain the MDA
stock solution (239 l/ml). The MDA working solution (2.39 l/ml)
was obtained by pipetting a 1 ml aliquot into another 100 ml volumetric
ask and diluting to volume with distilled water.
2.3. Samples and distillation method
A 10 g sample of meat product and 50 ml of distilled water were homogenized (Heidolph Silent Crusher, J.P. Selecta) and added to a spherical ask. Also, three different samples were prepared by adding 2.5 ml
of sucrose (BDH AnalaR) solution (10%) (S), 5 ml of MDA working solution (M) or a mixture of both (S + M). Then, one drop of silicone antifoaming agent was added plus 2.5 ml HCl (4 N) (Panreac, USA) before
diluting to 100 ml with distilled water. The samples were distilled in
triplicate following the procedure described by Tarladgis et al. (1960).
The samples were then distilled and the rst 50 ml of distillate was collected. Then, 2 ml of distillate and 2 ml of TBA solution were added to a
test tube and incubated at specic temperature/time relations (Ganhao
et al., 2011). At the same time, a reagent blank was prepared for each
TBA solution and incubation treatment through the addition of 3 ml of
distilled water and 3 ml of TBA solution. After incubation, the test
tubes were cooled in a water bath for 10 min, and the absorption spectrum was scanned from 400 to 600 nm with an UV2 UNICAM UV/Vis
Spectrometer. The yellow colour of the sucroseTBA and pink colour
of the MDATBA were measured at 444 and 532 nm, respectively, and
the results were expressed as absorbance units (Wang et al., 2002).
2.4. TBA solution and temperature/time of incubation
Four different TBA solutions were tested at ve different temperature/
time relations. Two TBA solutions were prepared at 20 mM using 90% glacial acetic acid (J.T. Baker, Deventer, Netherlands) (Tarladgis et al., 1960)
or 3.86% perchloric acid (Panreac, USA) (Ganhao et al., 2011). In addition,
an 80 mM TBA solution was prepared using distilled water adjusted to
pH 4 (Wang et al., 2002) and another using 0.8% TBA in distilled water
(Botsoglou et al., 1994). The temperature/time relations were:
(1) 35 min in a boiling water bath (Tarladgis et al., 1960);
(2) 70 C/30 min (Botsoglou et al., 1994); (3) 40 C/90 min (Wang
et al., 2002); (4) room temperature (r.t.) (24 C) in the dark for 20 h
(Ganhao et al., 2011); and (5) 60 min in a boiling water bath
(Tarladgis et al., 1960).
3. Results and discussion
Table 1 shows the mean values and standard deviations of the absorbance recorded at 444 nm and 532 nm for the reaction between TBA
and the distillate from red sausage stored for 15 days. This wide range
of absorbances measured at 444 and 532 nm reects the need to unify
the criteria used for TBA reagent and the incubation treatment when
there are potential interferants, such as sucrose.
Table 2 shows the mean values and standard deviations of the absorbance measurements at 444 and 532 nm for the reaction of TBA and distillates from the standard dissolutions. The treatments that favoured the
TBAsucrose reaction, and therefore the least advisable, were those involving acid solutions (glacial acetic acid and perchloric acid) of TBA
(20 mM) and incubation temperatures lower than 100 C. Although
some of these treatments also show maximum absorbance at 532 nm,
especially when perchloric acid (3.86%) was used, they were discarded
because of the high values recorded at 444 nm. Wang et al. (2002) recommended treatment at 40 C/90 min since above 50 C sucrose may
react with TBA. These results show that the reaction not only depends
on temperature but also on the pH of the TBA solution. In all the treatments where the pH of TBA was below 4, the yellow colour developed

P. Daz et al. / Meat Science 98 (2014) 569573

571

Table 1
Mean values and standard deviations of the absorbance at 444 nm and 532 nm for the reaction between TBA and the distillate of red sausage stored for 15 days.
TBA

Absorbance

100 C/35 min

0.8% distilled water

A444
A532
A532 A444
A444/A532
A444
A532
A532 A444
A444/A532
A444
A532
A532 A444
A444/A532
A444
A532
A532 A444
A444/A532

0.183
0.351
0.168
0.521
0.399
0.457
0.058
0.874
0.148
0.181
0.033
0.816
0.180
0.593
0.413
0.304

20 mM acetic acid glacial 90%

80 mM distilled water pH = 4

20 mM perchloric acid 3.86%

40 C/90 min

0.021 b, y
0.014 c, x
0.007 b, y
0.039 b, w
0.004 a, z
0.032 b, x
0.028 c, y
0.051 a, z
0.020 b, xy
0.011 d, y
0.008 c, y
0.058 a, yz
0.003 b, z
0.008 a, x
0.004 a, x
0.001 c, w

0.335
0.128
0.206
2.609
0.899
0.061
0.837
14.776
0.096
0.082
0.014
1.178
0.968
0.351
0.617
2.756

70 C/30 min

0.023 c, x
0.011 b, z
0.012 b, w
0.039 b, y
0.006 b, y
0.009 c, z
0.003 d, z
2.117 a, x
0.008 d, yz
0.013 bc, z
0.006 a, zw
0.097 b, xy
0.009 a, x
0.008 a, z
0.017 c, w
0.087 b, x

0.265
0.265
0.005
1.003
1.077
0.154
0.923
6.990
0.185
0.142
0.043
1.311
0.648
0.530
0.118
1.223

100 C/60 min

0.012 c, x
0.020 b, y
0.008 a, z
0.030 b, z
0.020 a, x
0.012 c, y
0.008 d, w
0.411 a, y
0.009 d, x
0.016 c, y
0.006 b, w
0.079 b, x
0.005 b, y
0.012 a, xy
0.007 c, y
0.018 b, z

0.162
0.377
0.215
0.429
0.334
0.539
0.204
0.620
0.145
0.281
0.136
0.516
0.205
0.596
0.396
0.336

r.t./20 h

0.011 b, y
0.011 b, x
0.000 b, x
0.017 bc, w
0.030 a, z
0.020 a, x
0.011 b, x
0.034 a, z
0.019 b, xy
0.015 c, x
0.004 c, x
0.041 ab, z
0.006 b, z
0.009 a, x
0.016 a, x
0.016 c, w

0.292
0.083
0.209
3.525
1.146
0.132
1.014
8.720
0.074
0.069
0.005
1.073
1.019
0.474
0.545
2.155

0.011 c, x
0.004 b,z
0.006 b, w
0.052 b, x
0.006 a, x
0.013 b, yz
0.007 d, h
0.798 a, y
0.006 d, z
0.001 b, z
0.007 a, z
0.104 c, xy
0.022 b, x
0.037 a, y
0.015 c, z
0.121 bc, y

a, b, c: Effect (P b 0.05) of TBA solution for the same treatment.

x, y, z, w: Effect (P b 0.05) of treatment for the same TBA solution.

less as the temperature increased. However, when the aqueous TBA solution (80 mM) was adjusted to pH = 4, as used by Wang et al. (2002),
the absorbance at 444 nm for the sucrose distillate did not signicantly
(P N 0.05) differ between the different incubation treatments and was
always below 0.031(Table 2). However, in the sucrose/MDA mixture
there were statistically signicant (P b 0.05) differences between treatments, although the values were also low (below 0.038). This low reactivity between TBA and sucrose was also observed between TBA and
MDA, the lowest values (P b 0.05) of absorbance at 532 nm being observed for the distillate of MDA and the sucrose/MDA mixture. The use
of aqueous solutions of TBA (80 mM) adjusted to pH 4 produced low interference on the part of the yellow pigments, although the pink pigments were still not maximal if we compare it with other TBA
TREATMENT combinations.
To select the most suitable TBATREATMENT combination, we
established the following condition: that the ratio between the absorbances at 444 nm and 532 nm (A444/A532) should be close to zero to ensure that the combination favoured the formation of pink rather than
yellow pigments. Furthermore, as a complement to this condition, we

established that the difference between the absorbances at 532 nm

and 444 nm (A532 A444) should be the maximum with regard to the
other treatments and greater than zero to minimize interference from
the yellow pigment. The effect of the TBATREATMENT interaction on
these variables was determined by ANOVA (see Table 3). Choosing a
priori the TBATREATMENT combinations with a A444/A532 b 1 and the
maximum positive value for A532 A444, the rst treatments to be selected were those using a solution of 0.8% TBA (distilled water) and
20 mM TBA (3.86% perchloric acid) and the treatments at 100 C lasting
35 min and 60 min, which showed statistically signicant (P b 0.05) differences in the value of A532 A444 compared with the other treatments, especially in the meat product. Jointly, these treatments
fullled the criteria of A444/A532 close to zero (0.300.34 in the meat
product and 0.140.16 for the standards). Figs. 1 and 2 show the absorbance spectra for the distillates of red sausage and the sucrose/MDA
mixture, respectively, using the 20 mM TBA solution in 3.8% perchloric
acid for the different treatments assayed. As can be seen, the behaviour
was similar in both cases, the temperature of 100 C for 3560 min
being the most effective at minimizing the interference at 444 nm,

Table 2
Mean values and standard deviations of the absorbance at 444 nm and 532 nm for the reaction between TBA and the distillates of sucrose, MDA and mixture of both.
TBA
0.8% distilled water

20 mM acetic acid glacial 90%

80 mM distilled water pH = 4

20 mM perchloric acid 3.86%

Absorbance
S444
M532
(S + M)444
(S + M)532
(S + M)532
(S + M)444/(S
S444
M532
S + M444
S + M532
(S + M)532
(S + M)444/(S
S444
M532
S + M444
S + M532
(S + M)532
(S + M)444/(S
S444
M532
S + M444
S + M532
(S + M)532
(S + M)444/(S

100 C/35 min

(S + M)444
+ M)532

(S + M)444
+ M)532

(S + M)444
+ M)532

(S + M)444
+ M)532

0.016
0.161
0.022
0.209
0.187
0.111
0.062
0.159
0.044
0.201
0.156
0.225
0.030
0.095
0.038
0.102
0.064
0.368
0.062
0.204
0.035
0.260
0.224
0.136

a, b, c: Effect (P b 0.05) of TBA solution for the same treatment.

x, y, z, w: Effect (P b 0.05) of treatment for the same TBA solution.

40 C/90 min
b, y

0.011
0.014 b
0.013 yz
0.026 a
0.039 a
0.074 b
0.011 a, y
0.008 b, y
0.006 y
0.024 a, x
0.030 ab, xy
0.059 ab
0.004 ab
0.007 c, xy
0.008 x
0.009 b, xy
0.007 b, yz
0.050 y, a
0.001 a, w
0.006 a, y
0.006 z
0.014 a, x
0.007 a, x
0.017 w, ab

0.070
0.173
0.067
0.227
0.159
0.300
0.212
0.030
0.171
0.042
0.129
4.424
0.014
0.064
0.010
0.064
0.053
0.166
0.695
0.150
0.510
0.198
0.311
2.568

70 C/30 min
c, x

0.007
0.023 a
0.008 c, x
0.021 a
0.029 a
0.062
0.023 b, x
0.003 b, w
0.029 b, x
0.015 b, y
0.044 b, z
2.228
0.001 c
0.008 b, y
0.007 c, y
0.013 b, y
0.012 a, yz
0.022 z
0.007 a, x
0.004 a, z
0.028 a, x
0.003 a, y
0.025 c, w
0.097 x

0.043
0.175
0.044
0.224
0.180
0.199
0.214
0.046
0.177
0.063
0.114
2.975
0.031
0.054
0.028
0.057
0.029
0.491
0.381
0.195
0.239
0.261
0.021
0.918

100 C/60 min

c, xy

0.005
0.016 a
0.007 b, xyz
0.023 a
0.030 a
0.052 b
0.023 b, x
0.003 b, w
0.027 a, x
0.017 b, y
0.044 c, z
1.228 a
0.006 c
0.007 b, y
0.003 b, xy
0.004 b, y
0.001 b, z
0.013 ab, x
0.006 a, z
0.007 a, y
0.012 a, y
0.003 a, x
0.015 b, y
0.056 ab, z

0.029
0.164
0.013
0.173
0.160
0.079
0.031
0.192
0.016
0.211
0.194
0.078
0.021
0.085
0.011
0.099
0.088
0.116
0.057
0.182
0.039
0.250
0.211
0.158

r.t./20 h
xy

0.013
0.001 a
0.008 b, z
0.023 b
0.032 ab
0.059
0.013 y
0.003 a, x
0.007 b, y
0.003 ab, x
0.003 a, x
0.004
0.011
0.012 b, y
0.007 b, y
0.008 c, xy
0.007 b, xy
0.002 z
0.008 w
0.006 a, y
0.006 a, z
0.006 a, x
0.000 a, x
0.012 w

0.064
0.174
0.060
0.171
0.111
0.359
0.226
0.117
0.209
0.155
0.054
1.483
0.007
0.137
0.011
0.137
0.126
0.081
0.609
0.230
0.472
0.280
0.192
1.688

0.008 c, x
0.013 b
0.006 c, xy
0.025 ab
0.031 a
0.089
0.031 b, x
0.007 c, z
0.035 b, x
0.055 ab, xy
0.090 ab, yz
0.756
0.006 c
0.013 bc, x
0.001 c, y
0.013 b, x
0.015 a, x
0.018 z
0.008 a, y
0.003 a, x
0.003 a, x
0.006 a, x
0.009 b,z
0.047 y

572

P. Daz et al. / Meat Science 98 (2014) 569573

Table 3
ANOVA of variables A532 A444 and A444/A532 as a function of the combination TBA Treatment.
Fresh red sausage

Sucrose + MDA

A532 A444

A532 A444

A444/A532

A444/A532

TT

Mean

ANOVA

TT

Mean

ANOVA

TT

Mean

ANOVA

TT

Mean

ANOVA

41
44
14
24
11
34
21
31
13
35
32
33
43
12
15
45
42
22
23
25

0.41
0.40
0.21
0.20
0.17
0.14
0.06
0.03
0.00
0.00
0.01
0.04
0.12
0.21
0.21
0.54
0.62
0.84
0.92
1.01

A
A
B
BC
BC
C
D
DE
DEF
DEF
EF
F
G
H
H
I
J
K
L
M

22
25
23
15
42
12
45
33
43
32
35
13
21
31
24
11
34
14
44
41

14.78
8.72
6.99
3.52
2.76
2.61
2.15
1.31
1.22
1.18
1.07
1.00
0.87
0.82
0.62
0.52
0.52
0.43
0.34
0.30

A
B
B
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C

41
44
24
11
13
14
12
21
35
15
34
31
32
33
43
25
23
22
45
42

0.22
0.21
0.19
0.19
0.18
0.16
0.16
0.16
0.13
0.11
0.09
0.06
0.05
0.03
0.02
0.05
0.11
0.13
0.19
0.31

A
AB
AB
AB
AB
AB
AB
AB
ABC
ABC
ABC
ABCD
ABCD
ABCD
BCD
CDE
DEF
DEF
EF
F

22
23
42
45
25
43
33
31
15
12
21
13
32
44
41
34
11
35
14
24

4.42
2.97
2.57
1.69
1.48
0.92
0.49
0.37
0.36
0.30
0.22
0.20
0.17
0.16
0.14
0.12
0.11
0.08
0.08
0.08

A
AB
AB
AB
AB
AB
B
B
B
B
B
B
B
B
B
B
B
B
B
B

T T: TBA Treatment: XY.

X = TBA (1 = TBA 0.8% distilled water; 2 = TBA 20 mM glacial acetic acid 90%; 3 = TBA 80 mM distilled water pH = 4; and 4 = TBA 20 mM perchloric acid 3.86%.
Y = Treatment (1 = 100 C/35 min; 2 = 40 C/90 min; 3 = 70 C/30 min; 4 = 100 C/60 min; 5 = r.t./20 h).
ANOVA: Different indices reect statistically signicant differences (P b 0.05).

while absorbance at 532 nm was maximal. These ndings do not agree

with those of Ganhao et al. (2011), who concluded that the r.t./20 h
treatment with the same type of TBA was best. However the results of
this paper were based on the interference caused by coloured pigments
(avonoids) from red fruit, which have a similar pink colour to the TBA
MDA complex. The absorbance of these pigments not only showed a
maximum at 532 nm but also showed a mean absorbance of 0.300 between 420 and 460 nm, which was not further analysed by the authors.
However, in our case, the distillates obtained from red sausage and the
standard solutions were transparent and there were no coloured pigments. Taking into consideration the interference produced by the yellow pigments, the choice of incubation treatment differed from that
made by Ganhao et al. (2011). The treatments with acid solutions of
TBA and mild temperatures for long times favoured the formation of
TBA-sucrose, making them unadvisable.
The use of acids and a temperature of 100 C may produce changes
in the TBA molecule, involving the appearance of a yellow-orange

colour with maximum absorbance at 450 and 490 nm (Tarladgis et al.,

1962). Such changes may increase the absorbance of the TBAsucrose
complex in treatments involving these conditions. However, treatments
that involved glacial acetic acid (90%) or perchloric acid (3.86%) and a
temperature of 100 C led to lower absorbance values at 444 nm than
the other temperature/time combinations studied. The use of a blank
in the same conditions ruled out the possibility of an increase in absorbance as a result of changes in the TBA molecule (Tarladgis et al., 1962).
To avoid this problem, Botsoglou et al. (1994) used an aqueous solution
of TBA (0.8%), the only problem with this being its instability and the
possibility of precipitation with room temperature incubations. For
this reason, Wang et al. (2002) recommended adjusting the solution
to pH = 4, using in their case a concentration similar to that used by
Botsoglou et al. (1994). The aqueous solutions of 0.8% TBA gave acceptable results based on the established criteria, especially at 100 C. These
results were similar to those obtained with 20 mM TBA in 3.86%
perchloric acid for distillates of sucrose, MDA and a mixture of both. In

Fig. 1. Absorbance spectra for the distillate of red sausage using a solution of TBA (20 mM)
in perchloric acid (3.86%) and different incubation treatments.

Fig. 2. Absorbance spectra for the distillate of sucrose and MDA using a solution of TBA
(20 mM) in perchloric acid (3.86%) and different incubation treatments.

P. Daz et al. / Meat Science 98 (2014) 569573

the case of the distillate of red sausage, treatments involving the 0.8% TBA
in distilled water were less advised because of the statistically (P b 0.05)
lower values for the difference between A444and A532.

4. Conclusion
Aqueous solutions or very dilute acid solutions with a pH below 1.60
(pH of the 0.8% TBA solution in distilled water) are the most suitable for
reducing interference produced by yellow pigments during the quantication of TBARs in fresh meat products when temperatures of 100 C
and times of less than 1 h are used for incubation.

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