Food Control 13 (2002) 117–123

www.elsevier.com/locate/foodcont

Determination of benzoic and sorbic acids in Brazilian food

S.A.V. Tfouni, M.C.F. Toledo *

Department of Food Science, Faculty of Food Engineering, State University of Campinas, UNICAMP,
Caixa Postal 6121, CEP 13081-970 Campinas-SP, Brazil
Received 18 July 2001; received in revised form 29 August 2001; accepted 30 August 2001

Abstract
Different brands of soft drink, fruit juice, margarine, yoghurt and cheese available on the Brazilian market were analysed for
benzoic and sorbic acids by high performance liquid chromatography (HPLC) with a photodiode array detector. The levels of
benzoic and sorbic acids were, respectively, in the range of not detected to 804 mg/l and not detected to 1371 mg/kg, respectively.
Only one sample presented a preservative level above that permitted by the legislation in force in Brazil. According to the results it
can be predicted that the real utilisation of benzoates and sorbates is significantly lower than the maximum authorised levels. Ó 2002
Elsevier Science Ltd. All rights reserved.

Keywords: Benzoic acid; Sorbic acid; HPLC

1. Introduction syth, 1989; Safford, Basketter, Allenby, & Goodwin,

Food preservation has always been of great impor-
tance. Nowadays this preservation is often made with
the use of chemical preservatives, among which benzoic
and sorbic acids, and their respective sodium, potassium
and calcium salts, are widely used. They are generally
used to inhibit yeast and mold growth, being also ef-
fective against a wide range of bacteria. These com-
pounds are most active in foods of low pH value and
essentially ineffective in foods at neutral pH values (Jay,
1996; Kimble, 1977; L€ uck, 1977; Sofos, 1995).
Despite the widespread use of these antimicrobial
agents, adverse effects such as metabolic acidosis, con-
vulsions and hyperpnoea were observed in experimental
animals and humans given very high doses of benzoic
acid. Some weak clastogenic activity was noted in in
vitro assays (WHO, 1997). The development of allergic
reactions to benzoates in humans, such as urticaria,
non-immunological contact urticaria and asthma, has
been reported in some studies (Hannuksela & Haahtela,
1987; Juhlin, 1981; Juhlin, Michaelsson, & Zeterstrom,
1972; Lahti, V€ a€an€anen, Kokkonen, & Hannuksela,
1987; Michaelsson & Juhlin, 1973; Rademaker & For-
*
Corresponding author. Tel.: +55-19-378-82170; fax: +55-19-328-
91513.
E-mail address: macecil@fea.unicamp.br (M.C.F. Toledo).
1990). Other studies showed that sorbic acid has low
toxicity, explained by the fact that it is rapidly metab-
olised by pathways similar to those of other fatty acids.
In humans a few cases of idiosyncratic intolerance to
sorbic acid have been reported (non-immunological
contact urticaria and pseudo-allergy) (Deuel, Calbert,
Anisfeld, McKeehan, & Blunden, 1954; Hannuksela &
Haahtela, 1987; Juhlin, 1981; Safford et al., 1990;
Walker, 1990).
The use of food additives in different countries is
limited by specific regulations. Brazil, as many coun-
tries, follows the recommendation of the Joint FAO/
WHO Expert Committee on Food Additives (JECFA)
on the safe use of food additives. According to JECFA,
the safety in use of an additive can be expressed in terms
of its acceptable daily intake (ADI), which represents
the amount of the substances that can be daily con-
sumed, even for a lifetime, without health hazards. The
ADI is expressed in mg of the additive/kg body weight
(WHO, 1987). Group ADIs of 0–5 and 0–25 mg/kg of
body weight have been established by JECFA for ben-
zoic acid and benzoates salts, benzyl acetate, benzyl al-
cohol and benzaldehyde, and for sorbic acid and
sorbates salts, respectively (FAO/WHO, 1999).
To assure the security of consumers the intake of
certain food additives, among which benzoic acid is in-
cluded, has been evaluated by JECFA. Nine member
states (Australia, China, Finland, France, Japan, New

0956-7135/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 5 6 - 7 1 3 5 ( 0 1 ) 0 0 0 8 4 - 6
118 S.A.V. Tfouni, M.C.F. Toledo / Food Control 13 (2002) 117–123
Zealand, Spain, the UK and the USA) provided JECFA
information on benzoates intake for assessment during
its 51st meeting. This Committee noted that mean intake
estimates based on maximum limits specified in national
standards were below the ADI for benzoates. However,
when the intake estimates were based on the maximum
limits and the range of uses specified in the draft General
Standard for Food Additives (GSFA), the ADI was
exceeded. The Committee observed that benzoates are
not likely to be used in all foods for which their use is
permitted, concluding that information on these aspects
could be used to revise both the intake assessment and
the maximum limits specified in the GSFA. The Com-
mittee also concluded that further information on levels
of benzoates in food at the time of consumption is
needed (WHO, 2000).
There are various methods for the analysis of ben-
zoates and sorbates in foodstuffs, such as thin layer
chromatography, UV spectroscopy, high performance
liquid chromatography (HPLC) and gas chromato-
graphy. Nowadays, HPLC is the most common ana-
lytical procedure for the detection and quantification of
these preservatives in foods and beverages (Bui &
Cooper, 1987; Burini & Damiani, 1991; Castellari, En-
sini, Arfelli, Spinabelli, & Amati, 1997; Chen & Fu,
1995; Lee, Rouseff, & Fisher, 1986; Leuenberger,
Gauch, & Baumgartner, 1979; Mannino & Cosio, 1996;
Monta~ no, Sanchez, & Rejano, 1995; Serrano, L opez, &
Revilla, 1991).
The objective of the present study was to measure the
concentration of benzoic and sorbic acids in certain food
categories in which their use is approved in Brazil. This
information combined with food consumption data will
be used to further estimate the average intake of these
preservatives in Brazil.
2. Materials and methods
2.1. Standards and chemicals
HPLC grade acetonitrile was purchased from Mal-
linckrodt. Other reagents (analytical grade) were pur-
chased from Merck. Commercial standards (Sigma
Chemical) of benzoic and sorbic acids were used. Stan-
dards were diluted in water. Water was obtained from a
Millipore Milli-Q water purification system. For the
filtration of the samples prior to injection, a Millex HV
0.45 lm filter (Millipore) was used.
2.2. Sample preparation
Different brands and batches of soft drinks (cola,
‘‘guarana’’ and orange types as well as their respective
diet versions), fruit juices (pineapple, mango, passion
fruit, grape, cashew, guava, ‘‘caj
a’’, West Indian cherry,
muscatel grape), margarine, strawberry yoghurts, petit
suisse, curd–cheese and processed cheese were purchased
from supermarkets in the city of Campinas, SP, Brazil,
in the period 1999–2000. Three batches of each brand
were homogenised and analysed in duplicate. Extraction
was necessary only for certain food categories, as de-
scribed later. Before injection into the liquid chro-
matograph, all samples were filtered through a 0.45 lm
filter.
2.2.1. Soft drink
Two hundred ml samples of carbonated soft drinks
were degassed in an ultrasonic bath for 5 min and di-
luted with water (Argoudelis, 1984; Veerabhadrarao,
Narayan, Kapur, & Sastry, 1987; Williams, 1986).
2.2.2. Fruit juice
Samples were centrifuged (2000 rpm) for 10 min and
a 1 ml aliquot of the supernatant was diluted with water
(Bennet & Petrus, 1977; Bui & Cooper, 1987).
2.2.3. Margarine
A 5 g sample was dissolved in 50 ml of diethyl ether
and extracted twice with 10 ml of 0.1 M sodium hy-
droxide solution in a separating funnel. The basic
aqueous extracts were acidified with 1 ml of 2.5 M sul-
phuric acid in a volumetric flask and diluted to volume
with water (Leuenberger et al., 1979).
2.2.4. Yoghurt and petit suisse
The extraction procedure was based on the method
described by Bui and Cooper (1987). A 5 g sample was
placed into a test tube with screw thread and 15 ml of
2% H3 PO4 (ortho-phosphoric acid) added. The solution
was then shaken in a tube rotator for 1 min, 30 ml of
acetonitrile were added and stirred again for 1 min. The
mixture was filtered through a Whatman no. 1 filter
paper and diluted with water.
2.2.5. Cheese
Forty ml of ethanol were added to 10 g sample of
cheese, and then stirred on a mechanical shaker for 30
min. The mixture was filtered through a Whatman no. 1
filter paper and diluted with water (Cecchi, 1988).
2.3. HPLC
The analysis was carried out using a HPLC apparatus
equipped with a Waters 600 Controller pump, a Waters
in-line degasser, a Waters 717 plus autosampler, a C18
column (Nova-Pak, 30 cm  3:9 mm i.d., 4 lm particle
size) and a Waters 996 photodiode array detector. The
mobile phase used consisted of 81% water, 17% aceto-
nitrile and 2% 0.005 M ammonium acetate buffered at
pH 4.2 with glacial acetic acid, at a flow rate of
1:0 ml min 1 . The injection volume was 20 ll. The
 S.A.V. Tfouni, M.C.F. Toledo / Food Control 13 (2002) 117–123
chromatogram was recorded for 15 min. The detection
of benzoic and sorbic acids was carried out at the
wavelengths of maximum absorption of the compounds,
228 and 260 nm, respectively. The peaks of benzoic and
sorbic acids in the samples were identified by their UV
spectrum (200–400 nm) and by comparing the retention
time with that of the standard.
2.4. Quantification
The external standard plot method was used. Du-
plicate injections of 20 ll benzoic acid and sorbic acid
standard solutions were used to construct linear re-
gression lines (peak ratio versus acid concentrations).
The concentration range of the standard curves was
0:2–10:0 lg ml 1 . The detection limit (expressed as
concentration) corresponded to the analyte concentra-
tion giving a signal equal to the blank signal plus three
standard deviations of the blank (Miller & Miller,
1993).
2.5. Recovery study
In order to verify the accuracy and precision of the
analytical procedure, recovery studies were carried out
by spiking selected samples of each matrix with stan-
dards. Benzoic and sorbic acid standard aqueous solu-
tions (for soft drinks, fruit juices, margarine and
yoghurt) or ethanol solutions (for cheese) were added at
three different concentrations. Samples of soft drinks
were spiked previously to dilution with water and those
of fruit juices before the centrifugation step. For mar-
garine, this procedure was conducted before dissolving
with diethyl ether, and for yoghurt and cheese, before
the addition of 2% H3 PO4 and ethanol. The ranges of
concentration added were: 0.2–0.8 mg benzoic acid/ml
soft drink, 0.05–0.2 mg sorbic acid/ml soft drink, 0.2–0.8
mg benzoic and sorbic acids/g margarine, 0.5–1.0 mg
benzoic and sorbic acids/ml fruit juice, 0.2–0.5 mg sorbic
acid/g yoghurt, 0.1–0.4 mg sorbic acid/g cheese. The
spiked samples as well as the unspiked controls were
analysed in duplicate.
Recoveries were calculated from the differences in
total amount of either benzoic or sorbic acid between
the spiked and unspiked samples.
3. Results and discussion
The analytical method used for the separation of
benzoic and sorbic acids in all samples was based on
Mannino and Cosio (1996). The C18 column used by the
authors was 10 cm in length and the mobile phase was
supplied at a flow rate of 0:5 ml min 1 . Since the C18
column used in this present work was 30 cm in length,
more acetonitrile (17%) than the quantity suggested
119
(10%) was used, and the flow rate was increased to
1:0 ml min 1 , in order to avoid a long run time. These
analytical conditions resulted in an effective separation
of the preservatives for all products analysed in a run
time of 15 min. The average retention time was 8 min for
benzoic acid and 10.7 min for sorbic acid. The identifi-
cation and purity of the peaks were confirmed by com-
paring their spectra (200–400 nm) with those of aqueous
solutions of the standards.
The extraction of the preservatives was based on
procedures described in the literature. Recovery for
benzoic acid and sorbic acid varied from 94.0% to
102.2% and from 86.1% to 101.0%, respectively (Table
1). In yoghurt and cheese recovery studies were only
carried out for sorbic acid, because benzoic acid was
not detected in these products. The detection limits for
benzoic and sorbic acid were, respectively, 0.06 and
0:05 lg ml 1 in soft drinks; 0.2 and 0:2 lg ml 1 in
fruit juice; 0.06 and 0:05 lg ml 1 in margarine; 0.1
and 0:2 lg ml 1 in yoghurt; 0.1 and 0:07 lg ml 1 in
cheese.
Mean concentrations of benzoic and sorbic acids are
given in Tables 2 and 3. The calculated CVs refer to
duplicates analysis of the homogenised samples (3 bat-
ches). Figs. 1–6 display histograms of benzoic and sorbic
acids levels in all analysed samples. Of all the products,
only one sample of processed cheese had sorbic acid in a
quantity above the maximum level allowed by the Bra-
zilian legislation (Brasil, 1999). Sorbic acid was found in
only one brand of a typical Brazilian soft drink
(‘‘guarana’’) and two brands of fruit juice (passion fruit
and West Indian cherry). Two out of three brands of
traditional cola did not contain any of the preservatives.
In general, traditional colas have a high sucrose con-
centration and very low pH values, which together in-
hibit microbial growth without the need of added
preservative. The use of either benzoic or sorbic acid was
noted in different brands of margarine, whereas only one
sample presented both preservatives. The analysis of
yoghurts and cheese confirmed the absence of benzoic
acid.
According to the results of this study, although both
preservatives are added to different foodstuffs in a wide
range of concentrations, none of the samples presented
Table 1
Recovery of benzoic and sorbic acids from different products
Product Mean recoverya (%) SD
Benzoic acid Sorbic acid
Cola soft drink 102.2  2.7 99.3  5.2
Pineapple juice 98.7  1.9 101.0  2.5
Margarine 94.0  1.9 94.6  2.8
Yoghurt – 86.1  2.8
Cheese – 89.1  4.0
a
Average of three different concentrations.
120 S.A.V. Tfouni, M.C.F. Toledo / Food Control 13 (2002) 117–123

Table 2
Benzoic acid levels in different food categories
Food category n Maximum permitted level Benzoic acid level (mg/kg or litre) % CV (range)
(mg/kg or litre)a
Mean Range
Soft drink 15 500 225 nd–338 0–6.8
Margarine 6 1000 276 nd–721 0–4.4
Fruit Juices 18 1000 495 nd–804 0–1.8
n: number of analysed samples; nd: not detected. Mean values were calculated assuming nd as zero.
a
Source: Brasil (1999).

Table 3
Sorbic acid levels in different food categories
Food category n Maximum permitted Sorbic acid level (mg/kg or litre) % CV (range)
level (mg/kg or litre)a
Mean Range
Soft drink 15 100 1.0 nd–47.1 0–2
Margarine 6 1000 355 nd–803 0–2.0
Fruit juices 18 1000 51 nd–450 0–0.68
Yoghurt 8 600 148 126–213 0.22–2.3
Petit suisse 3 1000 276 155–367 0.12–4.8
Cheese 6 1000 629 376–1371 0.51–5.9
n: number of analysed samples; nd: not detected. Mean values were calculated assuming nd as zero.
a
Source: Brasil (1999).

Fig. 1. Benzoic and sorbic acids levels in soft drink samples.

levels below the range of antimicrobial activity. The
minimum concentrations of benzoic and sorbic acids
required to inhibit microbial growth may vary due to
species, strains, pH of the substrate and other factors.
Most yeasts and molds are inhibited by 0.001–0.1%
sorbic acid, and by 0.002–0.07% benzoic acid (L€ uck,
1977).
The preservatives were detected in all food products
whose labels mentioned these additives, and were not
detected in the products that were not labelled as con-
taining them. A preference for using benzoic acid in
beverages was detected, despite the fact that both pre-
servatives are permitted for use in these products. All
samples (except one) contained benzoic and sorbic acids
 S.A.V. Tfouni, M.C.F. Toledo / Food Control 13 (2002) 117–123 121

Fig. 2. Benzoic and sorbic acids levels in margarine samples.

Fig. 3. Benzoic and sorbic acids levels in fruit juice samples.

at levels below the maximum permitted by the Brazilian
food legislation.
4. Conclusion
Although the evidence provided is limited, it seems
logic to predict that the real utilisation of benzoates
and sorbates is significantly lower than the maximum
authorised levels. This information is particularly rel-
evant in the case of benzoates, for which intakes ex-
ceeding the ADI have been reported for high
consumers, based on maximum limits specified in na-
tional standards and on model diets (WHO, 2000).
Therefore, in order to reduce an overestimation, it is
recommended that whenever possible the intake esti-
mates of additives be refined by a more precise ap-
proach using analytical data.
122 S.A.V. Tfouni, M.C.F. Toledo / Food Control 13 (2002) 117–123
Fig. 4. Sorbic acid levels in yogurt samples.
Fig. 5. Sorbic acid levels in petit suisse samples.
Fig. 6. Sorbic acid levels in cheese samples.
Acknowledgements
Financial support from FAPESP (Proc. 198/13818-0)
and scholarship from CAPES are gratefully acknowl-
edged.
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