Z Lebensm Unters Forsch (1996) 202:205-210
Gitte Sorensen 9 Soren Storgaard Jorgensen
A critical examination of some experimental variables in the
2.thiobarbituric acid (TBA) test for lipid oxidation in meat products
Received: 27 March 1995/Revised version: 9 June 1995
Abstract By comparison of various procedures for the
determination of TBARS (2-thiobarbituric-acid-reactive
substances) in non-cured meat samples, it was found that
a direct extraction procedure with trichloroacetic acid
gave significantly higher reaction yields than a rapid
Kjeltec steam distillation procedure (50 ml distillate in
2.5 min). When optimizing the distillation procedure
with regard to distillation principle (heating by thermal
conductivity or steam injection) and rate, the reaction
yield was more affected by changes in distillation prin-
ciple than by changes in distillation rate. Simple distilla-
tion (conduction heating) yielded significantly higher
TBA values than steam distillation at the same distilla-
tion rate. The reproducibility of the distillation proced-
ure was found to be about 5-10 times poorer than of the
extraction procedure and, because of the high reaction
yields and robustness of the extraction procedure, the
use of the extraction procedure is recommended with
food matrices such as non-cured meat samples with less
than 14-18% fat and no artificial colorants.
Key words 2-Thiobarbituric-acid-reactive
substances' Malondialdehyde 9Non-cured meat"
TBA-test 9TBARS
Introduction
The 2-thiobarbituric acid (TBA) test is probably
the most extensively used chemical method for the
G. Sorensen
The Royal Veterinary and Agricultural University,
KVL Center for Food Research, Thorvaldsensvej 40,
DK-1871 Frederiksberg C, Denmark
S. Storgaard Jorgensen (12N)
The Royal Veterinary and Agricultural University,
Chemistry Department, Thorvaldsensvej 40,
DK-1871 Frederiksberg C, Denmark
9 Springer-Verlag 1996
semi-quantitative estimation of lipid oxidation in
foods. The method is based on the spectrophotometric
measurement of a red chromophore formed by the
reaction of TBA with secondary products from
lipid oxidation of unsaturated fatty acids, with
malondialdehyde (MDA) being used as a calibration
standard.
The TBA test has been described with various differ-
ences by many workers, the most common procedure
used being that described by Tarladgis et al. [1], which
involves distillation of an acidified sample in order to
separate the TBA-reactive substances (TBARS) from
the food matrix. A more simple procedure is the
so-called extraction method in which the acidified
sample is filtered to give a clear aqueous solution [2].
There has been great interest in optimizing the TBA
method, particularly the more generally applicable dis-
tillation procedure, since the extraction procedure may
be inadequate in cases of coloured samples and samples
high in fat ( > 10%) [-3]. In the original work of Tar-
ladgis et al. [1], the distillation principle is based on
simple distillation, in which case the sample mixture is
heated by conductivity and not by steam. In more
recent work almost all distillation procedures have
been based on steam distillation where it is possible to
obtain very high distillation rates with the automated
distillation apparatus (Kjeldahl stills) commonly found
in modern laboratories. Thus Hoyland and Taylor 1-4]
have described a rapid steam distillation procedure in
which 50 ml distillate is collected in about 4 min as
opposed to 10 min by simple distillation [1]. In our
laboratory, distillation rates of up to 20 ml/min (50 ml
collected in 2.5 min) are a common procedure using the
Tecator Kjeltec distillation apparatus.
The results obtained by various TBA procedures are
known to be both method and operator dependent
[5, 6]. In the extraction procedure, only TBARS that
are instantaneously released from the food matrix into
an acid aqueous phase at room temperature are deter-
mined. In a distillation procedure, those TBARS that
206
are released during heat treatment at 100 ~ and distil-
led with water vapour are determined. Earlier research
[2, 7-10] has concluded that the distillation procedure
yields greater TBA values than does the extraction
procedure. This indicates that more TBARS are re-
leased at 100 ~ than at room temperature, but it is also
generally accepted that when using the distillation
procedure the total amount of TBARS in the sample
mixture is not transferred to the collected distillate.
Calculated TBA values are therefore corrected by
means of an experimentally determined percentage
recovery, in order to get a more reliable estimate of the
total amount of TBARS and to be able to use TBA
values on a comparative basis. Obviously this requires
a stable and reproducible distillation procedure. In
contrast, the extraction procedure will normally yield
about 100% recovery. In our laboratory, low and
poorly reproducible recoveries of added malondi-
aldehyde (MDA) were experienced when using the very
rapid Kjeltec distillation procedure.
The purpose of this work was to compare various
procedures for the determination of TBARS in meat
samples with respect to reaction yields, reproducibility
and ease of operation. The following variables were
examined: (1) extraction versus distillation; (2) reaction
temperature; (3) the influence on reaction of light and
of a cold pretreatment [11].
An attempt has been made to optimize the
distillation procedure with regard to distillation
principle (conduction and steam heating) and rate.
Materials and methods
Sample preparation
Meat. Commercially sold fresh ground beef with 14-18% fat was
used. To produce meat samples with two different levels of lipid
oxidation, one half of the sample was immediately vacuum-packed
in plastic bags and stored at - 18 ~ The other half was exposed to
air and light for 2 days to accelerate lipid oxidation and then
vacuum-packed in plastic bags and stored at - 18 ~ On the day of
analysis the meat samples were thawed in lukewarm water and
homogenized before analysis.
Standards. A stock solution of 0.002 M TEP (1,1,3,3-tetraethoxy-
propane, Merck 805797) in distilled water was prepared. Aliquots
of the TEP shock solution were used to make standard samples
with concentrations corresponding to 2 x 10 - 6 M and 10 x 1 0 - 6 M
TEP.
Experimental design
Optimization. In order to optimize the TBA method, a fractional
factorial experiment was designed with 4 factors at 2 levels each.
Treatment factors and levels are shown in Table 1,
Comparison of distillation principles and distillation rates. Steam
distillation (the mixture was heated by steam injection) with
distillation rates of 2.1 and 4.2 ml/min (volumes of 50 ml were
Table 1 Factors and levels in the optimization design experiment
Factor Description Level 1 Level 2
A TBA procedure Extraction Distillation"
B Pretreatment of TBA
reaction 5 ~ for 1 h b None
C Reaction temperature 70 ~ 100 ~
D Sensitivity of TBA
reaction to light Light No light
a Steam distillation on Kjeltec System 1002 Distilling Unit at a rate
of 20 ml/min
b According to [11]
collected in each distillation) were compared to simple distillation
(the mixture was heated by thermal conductivity) with distillation
rates of 3.3, 5, 10 and 20 ml/min.
All treatment parameters were tested on both meat and standard
samples with two levels of TBARS.
Extraction procedure
The extraction procedure was modified according to previous publi-
cations [2, 12]. Of the sample 10 g was homogenized with 30 ml of
a 7.5% trichloroacetic acid (TCA) solution containing
0.1% propylgallate (PG) and 0.1% ethylenediaminetetraacetic
acid, disodium salt (EDTA) for 30 s in an Ultra Turrax blender
(9500rpm) (Janke and Kunkel, Germany) and filtered through
a Whatman filter no. 42.
Distillation procedure
The distillation procedure was modified according to previous
publications [1, 13]. Of the sample, 10 g was homogenized with
50 ml distilled water containing 0.1% P G and 0.1% EDTA for 1 rain
in an Ultra Turrax blender (9500 rpm.). The mixture was transferred
quantitatively into a distillation tube by washing with an additional
47.5 ml distilled water containing 0.1% PG and 0.1% EDTA. The
mixture was acidified with 2.5 ml of a HC1 solution (concentrated
HCI: distilled water, 1 : 2) and five drops of Antifoam A were added.
Steam distillation was carried out using an automated distillation
apparatus (Kjeltec System 1002 Distilling Unit, Tecator, Sweden)
with a distillation rate of 20 ml/min, or using a laboratory designed
glass apparatus with distillation rates of 3.3, 5 and 10 ml/min. Simple
distillation was carried out with common glass equipment with
distillation rates of 2.1 and 4.2 ml/min. Volumes of 50 ml were
collected in each distillation.
Quantification of TBARS
Equal 5 ml volumes of filtrate or distillate and 0.02 M TBA solution
(Merck 8180, in distilled water) were mixed in glass stoppered tubes
and incubated in a water bath at 70 ~ or 100 ~ for 40 rain before
cooling to room temperature under running cold tap water. All
samples were analysed in duplicate. In accordance with the optim-
ization design experiment (Table 1), some samples were subjected to
a cold pretreatment at 5 ~ for 1 h before incubation in the water
bath. Sensitivity of the reaction to light was tested either with
a metal lid placed over the water bath (no light) or with two 60 W
light bulbs placed directly (5 cm) above the samples in the water bath
and covered with a reflecting lid to give an even distribution of light.
A calibration curve for each of the extraction and the distillation
procedures was constructed from a dilution series of the 0.002 M

TEP stock solution to a range from 2 x 10 6 M to 10 x 10 . 6 M
TEP. The dry matter content of the beef samples was measured
on an automatic moisture analyser (Sartorius MA30, Sartorius,
G6ttingen, Germany). All absorbances were converted to concentra-
tions of MDA by reference to the calibration curves and the corres-
ponding TBA values for standards were expressed as micromoles
MDA and for meat as micromoles MDA equivalents per kilogram
sample (TBARS) according to:
TBARS (Extraction)
i ( 1 0 0 % + % drymatter
CMDA" VTCA "Jr- 100%
Ms
CMDA " Vdis
TBARS (Distillation) (2)
Ms
where CMDAis the concentration of MDA (gM) as read from the
calibration curve; Ms is the mass of the sample (g); VTCA is the
volume of TCA (ml); and Vdisis the volume of the distillate (ml).
Spectrophotometric measurements
A HP 8452 A diodearray photometer (Hewlett Packard, Germany)
and 1-cm quartz cuvettes were used for all measurements. Absorb-
ances were recorded in the wavelength range 300 nm to 650 nm
against a reagent blank which had been subjected to the same
analytical procedure as described above for the samples. TBA values
were calculated from the absorbance measured at 532 nm, but the
entire spectrum was checked for the possible formation of other
components.
Statistical analysis
Statistical analysis was performed with SAS version 6.0. (SAS Insti-
tute, N.C., USA). Treatment effects were tested with standard analy-
sis of variance (General Linear Models), and Tukey's (HSD) Studen-
tized test was used to test for significant differences between mean
TBA values at the 5% level.
Recommended procedure (extraction, non-cured meat samples)
Homogenize 10 g of sample with 30 ml of a 7.5% TCA solution
containing 0.1% PG and 0.1% EDTA for 30 s in an Ultra Turrax
blender (9500 rpm) and filter through a Whatman filter no. 42. Mix
equal 5 ml volumes of filtrate and 0.02 M TBA solution in glass
stoppered tubes and incubate in a water bath at 100 ~ for 40 rain
before cooling to room temperature under running cold tap water.
Measure the absorbance at 532 nm against a reagent blank which
has been subjected to the same analytical procedure as described
above for the sample. Prepare a calibration curve from a dilution
series of 0.002M TEP to cover a range from l x 10-6M to
10x 10 6 M TEP, where the TEP aliquots are diluted with a
7.5% TCA solution containing 0.1% PG and 0.1% EDTA. The
calibration curve should be linear in this range. Calculate TBARS
from the equation given above.
Results and discussion
I n o r d e r t o q u a n t i f y t h e T B A R S in t e r m s o f T B A
values, t h e m e a s u r e d a b s o r b a n c e s h a v e b e e n c o n v e r t e d
to e q u i v a l e n t a m o u n t s o f M D A a n d t h e n c o r r e c t e d for
207
Table 2 Estimated significance levels of treatment parameters from
the analysis of variance of the results from the optimization design
experiment
Factor Description Meat Standard
A TBA procedure *** ***
(extraction/distillation)
B Cold pretreatment n.s. n.s.
C Reaction temperature * *
D Sensitivity to light n.s. n.s.
AxC n.s. **
A x Concentration *** n.s.
Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not
significant
~" 1 0 - - -
6
4
2 2
0--
Extraction Distillation
70oC 10OOC 70~C 100~
Fig. 1 Results from the optimization design experiment for both
standards and meat samples expressed as mean 2-thiobarbituric acid
(TBA) values sorted according to TBA procedure and reaction
temperature. Standards: 9 2x 10 -6 M MDA; [] 10x 10 -6 M
MDA. Meat samples: 9 low oxidative level; [] high oxidative level
s a m p l e size a n d f i l t r a t e / d i s t i l l a t e v o l u m e . T h e T B A
v a l u e s a r e n o t c o r r e c t e d for p e r c e n t a g e r e c o v e r y o f t h e
T B A m e t h o d , as is u s u a l l y d o n e , b e c a u s e of t h e p o o r
reproducibility of the steam distillation procedure. The
reproducibility of the distillation procedure, expressed
as t h e r e l a t i v e s t a n d a r d d e v i a t i o n , is a b o u t 5 - 1 0 t i m e s
p o o r e r t h a n t h a t o f t h e e x t r a c t i o n p r o c e d u r e a n d fur-
ther results indicate (data not shown) that when con-
structing a calibration curve with samples of TEP that
h a v e b e e n distilled, t h e w i t h i n - s a m p l e r e p r o d u c i b i l i t y is
e q u a l l y p o o r a n d t h e r e g r e s s i o n coefficient o f t h e c a l i b -
r a t i o n c u r v e is v e r y low, at a b o u t 0.88. W h e n c o n s t r u c t -
i n g a l i n e a r c a l i b r a t i o n c u r v e in t h e s a m e f a s h i o n for t h e
e x t r a c t i o n p r o c e d u r e , t h e r e g r e s s i o n coefficient is 0.99.
I n T a b l e 2 a r e l i s t e d s i g n i f i c a n c e levels of t h e t r e a t -
ment parameters from the analysis of variance which
were performed on the estimated TBA values from the
optimization design experiment. It was found that the
p a r a m e t e r s p r o c e d u r e a n d t e m p e r a t u r e h a v e a signifi-
c a n t effect o n t h e m e a s u r e d T B A v a l u e s for b o t h s t a n -
dards and meat samples. Figure 1 shows the results
f r o m t h e o p t i m i z a t i o n o f T B A m e t h o d s , a n d it is seen
208
Table 3 Reproducibility of percentage recovery of malondialdehyde
(MDA) from standard samples for 3 different TBA procedures
TBA procedure Percentage recovery of MDA (%)
Concentration of M D A in standard
sample
2 ~tM 10 pM
Extraction 102.7 _+ 5.6 (5.5) 98.9 __ 1.0 (0.9)
Kjeltec Steam distillation 22.8 __+8.5 (37.3) 18.2 _+ 2.5 (13.7)
(20 ml/min)
Simple distillation 76.1 • 8.5 (11.2) 72.5 i 0.8 (1.1)
(4.2 ml/min)
Results are expressed as the mean percentage recovery _+ SD of
4 determinations. In parentheses are shown the coefficient of vari-
ation (%)
that the extraction procedure yields much higher TBA
values than the rapid Kjeltec steam distillation proced-
ure. For standards, steam distillation yields about 20%
of extraction values while for meat the yield is about
42%. Although these findings are in contrast with those
in several publications [-2,7 10], the conflicting results
are not surprising considering the missing correction
for percentage recovery. It was found that the extrac-
tion procedure yields about 100% recovery while the
recovery is about 20% for the rapid steam distillation
procedure (Table 3), but with a large variation between
determinations and some inconsistency for low and
high concentrations of TEP. (Percentage recovery of
M D A is calculated as the concentration of M D A in
standard samples which have been extracted or distil-
led compared to the concentration of appropriate cal-
ibration samples.) Reports relating to the recovery of
a steam distillation method [-4] support these results, as
the recoveries have a tendency to vary with the concen-
tration of TEP.
The reaction temperature was found to be significant
at the 5% level, but the analysis of variance indicates
a statistical interaction between procedure and temper-
ature (Table 2). This is illustrated in Fig. 2 for both
meat and standard samples. The interaction effect is
found only to be significant for standard samples, but
Fig. 2 suggests that this could hold for meat samples as
well since the interaction plot for meat samples is very
similar to that of standard samples. The statistical
interaction between procedure and temperature indi-
cates that while the TBA values found when using the
distillation procedure at a reaction temperature of
70 ~ are lower than those found at a temperature of
100 ~ there seems to be no difference between TBA
values found with the two temperatures when using the
extraction procedure. This could be caused by a lower
pH in the filtrates after extraction due to the addition of
TCA. This finding suggests that the distillation proced-
ure is more sensitive to temperature changes during
analysis than the extraction procedure and supports
1.6
t
2.4
J 2.2 e
0.8:
0.4
1.8
S 0 \ 1.6 .~
%
-0.4 1.4
Extraction Distillation
T B A procedure
Fig. 2 Interaction effect between TBA procedure and reaction
temperature shown for both standards and meat samples. Stan-
d a r d s : - D - 70 ~ - I 1 - 100 ~ Meat samples: - - * - - 70 ~ - - A - -
100 ~
a similar conclusion to that drawn by others [4] who
stress the importance of using a uniform temperature in
the water bath when using the distillation procedure.
The optimization experiment showed no effect of
letting the filtrate or distillate react with TBA at 5 ~
for 1 h before incubation in water at 70 ~ or 100 ~
just as no difference could be detected, neither at
532 nm nor in any other part of the spectrum (300 nm
to 650 nm) between samples reacted in the dark or
under intense illumination.
Figure 3a, b presents the results from the comparison
between simple distillation and steam distillation with
different distillation rates for either procedure. For
both standard and meat samples the TBA values from
simple distillation are significantly higher than those
obtained by steam distillation where the automated
Kjeltec procedure yields the significantly lowest TBA
values. With the distillation equipment available there
seems to be no effect of lowering the distillation rate for
simple distillation from 4.2 to 2.1 ml/min, just as there
is no significant difference between TBA values ob-
tained with distillation rates of 3.3, 5 and 10 ml/min.
This could lead to the conclusion that it is the distilla-
tion principle, rather than the distillation rate, which
has the largest influence on the estimated TBA values.
Our findings coincide with those of Tarladgis et al. [-1]
who recommended the collection of the distillate from
simple distillation with the highest distillation rate pos-
sible. From relevant literature [-1,4, 14, 15] it is noticed
that a higher distillation rate is usually obtained by
using steam distillation instead of simple distillation,
but, at the same time, this will result in lower reaction
yields.
In Fig. 4 mean TBA values found with the two differ-
ent distillation principles with distillation rates of
20 ml/min and 4.2 ml/min and the extraction proced-
ure are compared. The automated steam distillation
procedure yields, as expected, the significantly lowest
TBA values for both meat and standard samples. For
 209

a) Standard further lipid oxidation during distillation despite the

a
use of ant 9 [10, 161.
This work corroborates the general experience that
the TBA test is highly influenced by procedural para-
.I meters, especially whether the TBARS are separated
from the sample matrix by distillation or by extraction.
The reaction yields from extraction are found to be
significantly higher than reaction yields found with
either distillation principle and it is seen that the reac-
5 10 15 20 tion yield from distillation is more affected by changes
Distillation rate in 9
in distillation principle than by changes in distillation
60 b) M e a t rate. It is notable that the Kjeltec steam distillation
procedure yields by far the lowest TBA values. This
could indicate that a u t o m a t e d Kjeldahl stills are not
4o
optimal for distillation of TBARS.
3?
<
The TBA test is often used as a measure of lipid
ca oxidation on a comparative basis, where a correction
20
u for percentage recovery of M D A is thought to justify
-------L the comparison of TBA values found with different
distillation procedures. Experimentally we found it dif-
5 10 15 20
Distillation rate in ml/min
ficult to establish a distillation procedure which was
stable and provided reliable and reproducible yields of
TBARS. This is particularly i m p o r t a n t for the compari-
Fig. 3a,b Comparison of different distillation principles and distilla-
tion rates. Mean TBA values labelled with the s a m e letter are not son of TBA values when the distillation procedure
significantly different (5% level), a Standards, simple distillation: * yields low amounts of TBARS and consequently a low
2 x 10.6 M MDA; 9 10 x 10-6 M MDA. Standards, steam distilla- percentage recovery as is typically seen at high distilla-
tion: [] 2 x 10.6 M MDA; 9 10 x 10-6 M MDA. b Meat samples, tion rates. As noted by Karastogiannidou and Ryley
simple distillation: * low oxidative levels; 9 high oxidative level.
Meat samples, steam distillation: [] low oxidative level; 9 high [15] there seems to be some inherent advantage in the
oxidative level reliability of a high reaction yield which, at the same
distillation rate, is shown to be obtained more easily
with simple distillation than with steam distillation.
C o n t r a r y to this, the extraction procedure has
10-
proven to be simple and very easy to operate. It was
30 possible to obtain clear filtrates from meat samples
with as much as 14-18% fat. Recoveries of M D A were
z" found to be a b o u t 100% and highly reproducible.
9
6- As mentioned earlier, the calculated TBA values in
z0
I this work have not been corrected for recovery of M D A
0~ and it is argued that due to the influence of procedural
4~ C~
-<
variables, the quantitative results from the TBA test are
~0
best evaluated without correction for recovery. As in all
empirical methods, it is essential to emphasize the im-
portance of a detailed description of all procedural
o-
Steam distill. Simple distill. Extraction details in order to evaluate the found TBA results
Kieltec 20 ml/min 4,2 ml/rnin
correctly. O u r results show that this should include the
Fig. 4 Comparison of two distillation procedures (rapid Kjeltec distillation principle as well.
steam distillation and simple distillation) and the extraction proced- T o our knowledge no single version of the TBA test
ure for both standards and meat samples. Mean TBA values labelled is suitable for application to all sample matrices, but
with the s a m e letter are not significantly different (5% level). Stan- considering the robustness of the extraction procedure
dards; 9 2x 10-6M MDA; [] 10x 10 6M MDA. Meat samples:
9 low oxidative level; [] high oxidative level c o m p a r e d to the distillation procedure, it is recom-
mended to use the extraction procedure with food
matrices such as non-cured meat products with less
than 1 4 - 1 8 % fat and no artificial colorants.
standard samples simple distillation gives significantly
lower TBA values than the extraction procedure, while,
for meat samples, no significant differences exist be- Acknowledgements We wish to thank Ib Skovgaard for helpful
advice with the experimental design and statistical treatment. This
tween the two procedures. In a food matrix this could work was financially supported by the Danish Ministry of Educa-
be due to insufficient extraction of the TBARS or tion and Research as a part of the FOTEK programme.
210
References
1. Tarladgis BG, Watts BM, Younathan MT, Dugan LR (1960)
J Am Oil Chem Soc 37:44-48
2. Vyncke W (1975) Fette Seifen Anstrichm 77:239-240
3. Hoyland DV, Taylor AJ (1991) Food Chem 40:271 291
4. Hoyland DV, Taylor AJ, (1989) Int J Food Sci Technol 24:
153-161
5. Gray JI, Monahan FJ (1992) Trends Food Sci Technol 3:
315-319
6. Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Had-
ley M (1993) Free Radical Biol Med 15:353-363
7. Witte VC, Krause GF, Bailey ME (1970) J Food Sci 35:582-585
8. Siu GM, Draper HH (1978) J Food Sci 43:1147-1149
9. Salih AM, Smith DM, Price JF, Dawson LE (1987) Poult Sci 66:
1483-1488
10. Shahidi F, Hong C (1991) J Food Biochem 15:97-105
11. Kosngi H, Kojima T, Kikugawa K (1989) Lipids 24:873-881
12. Vyncke W (1970) Fette Seifen Anstrichm 72:1084 1087
13. Tarladgis BG, Pearson AM, Dugan LR (1964) J Sci Food Agric
35:1248-1254
14. Ke PJ, Cervantes E, Robles-Martinez C (1984) J Sci Food Agric
35:1248-1254
15. Karastogiannidou C, Ryley J (1994) Int J Food Sci Technol 29:
19-22
16. Rhee KS (1978) J Food Sci 43:1776-1781