Spectrometri c studi es on st abi l i t y of tenuazoni c aci d (TEA) sol ut i on

in organi c sol vents

Combi na M*, Dalcero AM, and Torres A
Departamento de Microbiologfa e Inmunologfa. Facultad de Ciencias
Exactas, Ffsico-Qufmicas y Naturales. Universidad Nacional de Rfo
Cuarto. Ruta 8 km 601 (5800) Rfo Cuarto, C6rdoba, Argentinia
*Address for correspondence
Microb. Mariana Combina
Estaci6n Experimental Agropecuaria Mendoza
Instituto Nacional de Tecnologfa Agropecuaria (INTA)
San Martin 3857 CC3
(5507) Luj&n de Cuyo, Mendoza. Argentina
E-mail: cimza @ inta.gov.ar
Abst ract
The st abi l i t y of tenuazoni c aci d sol ut i on at di fferent temperatures
and storage ti mes was studi ed usi ng methanol , methanol -water (8:2
v/v), benzene and benzene- acet oni t r i l e ( 9 8 : 2 v / v ) a s sol vent s.
Sol ut i ons were anal ysed by a spect r omet r i c met hod TeA U.V.-
spect r um was r ecor ded. Resul t s i ndi cat ed t hat t he opt i mum
temperature f or l ong-ti me storage peri od of tenuazoni c aci d sol ut i on
in any sol vent assayed i s -20~ Benzene and benzene-acetoni tri l e
(98:2 v/v) coul d be advi sed to make tenuazoni c aci d sol ut i on whi ch
wi l l be stored less than 2 mont hs at 4~ Methanol and methanol -
water (8:2 v/v) are not recommended because a l ow st abi l i t y of TeA
sol ut i on i n thi s sol vents.
I nt roduct i on
Natural occurrence ofAlternaria alternata and its mycotoxins in several commodities
and commercial products has been reported. Tenuazonic acid (TEA) is the most studied
Alternaria toxic metabolite because of its biological activity and high field from fungal
cultures. High level of this toxin has been detected on sunflower seeds at harvest time
and storage in Argentina (1, 2, 3). It has been also found in tomato and tomato paste,
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Mycotoxi n Research. Vol. 14 (1998)
sorghum, olives, mandarin, orange and others food products (4, 5, 6, 7).
This toxin exhibits a wide range of effects on different organisms. It shows antitumor
activity against human adenoearcinoma- 1 and antiviral and antibacterial activities against
several microorganisms. Some adverse side effect has been observed in many test animals,
including guinea pigs, mice, rabbits, dogs and rhesus monkeys when the toxin was
administered to them. They induced salivation, emesis, anorexia, erythema,
gastrointestinal hemorrhages, convulsions, increase in packed cell volume, and many
other effects (8, 9, 10, 11).
The principal mode of action of TeA appeared to be the inhibition of protein synthesis
by suppressing the release of newly formed proteins from the ribosome (12). Further
investigations on protein synthesis inhibition showed that TeA acted at peptide bond
formation step where the 60S unit, site of the peptidyltransferase centre, appeared to be
involved (13).
TeAis not stable as its acidic form. Upon storage it converts slowly into a mixture of
tenuazonic acid and D-allo-tenuazonic acid, commonly called isotenuazonic acid, so it
is normally stored as its copper salt (14). This fact becomes troublesome if the toxin has
to be periodically used as standard solution in different assays.
Chloroform has been traditionally considered a good solvent to store mycotoxin
standards. Many authors have reported that chloroform is a solvent with an intermediate
polarity where the toxins can reach a good solubility. Nevertheless, the fact that
chloroform is very volatile prompted us to investigate other solvents. Some authors
have suggested methanol and benzene for some other toxins since they show less loss
due to evaporation (15, 16).
The objective of this work was to establish the best conditions to storage a solution
of TeA for extended periods without decomposition, in order to keep a stable and
homogeneous product that could be used as reference in different mycotoxin studies.
Materials and Methods
T e n u a z o n i c a c i d s t a n d a r d
Tenuazonic acid-Copper salt (3-Acetyl-5-sec-butyl-4-hydroxy-3-pyrrolin-2-one) was
provided by Sigma Chemical Co., St. Luis, M.O., U.S.A.
The standard was prepared as required by reconversion of the Cu (II) salt to tenuazonic
acid as reported by Scott and Kanhere (17). The concentration of standard was performed
using a spectrometric method, recording U.V. spectrum of TeA solutions from 200 to
350 nm (~,) by measuring absorbance at wavelength of maximum absorption (280 nm).
TeA concentration was calculated using the following equation:
lag TeA/ml = A. MW.1000/e.
A= absorbance at 280 nm
MW= molecular weight (TeA MW= 197.1051)
efmolar absorptivity coefficient in methanol (TeAc280 ffi 14500) (18).
Stock solution containing 20 mg TeA/ml was prepared in methanol.
Solvents
The analytical grade compounds assayed were methanol, benzene and the mixtures
methanol-water (8:2 v/v) and benzene-acetonitrile (98:2 v/v).
55
Stability studies
Aliquots (1 ml) of TeA stock solution were dispensed in Teflon cap-tubes and
concentrated to dryness. Five millilitres of each solvent were added to eyery tubes,
closed with Teflon caps and Parafilm | and stored at 25~ 4~ and -20~ for 3, 8, 23
and 28 weeks in darkroom.
Each time, 3 tubes corresponding to the different temperatures were removed,
evaporated to dryness and resuspended in 5 ml of methanol. Tenuazonic acid
determinations were done by spectrometry, considering the characteristic spectrum and
absorbance at 280 nm of TeA solution (Figure 1). The U.V. spectrum was only considered
when it not showed any change at all over the wavelength considered.
Apl icaci6n: BARRIDO
Nomb~-e an&lisis: HE25_I
A~'ch ivo datos: TA
Lone onda initial: 28g.g
Lone onda Final: 358. 8
0. 110
A| S
0, 002
I i I J !
l
200. 0 L0 ( NI l )
!
SS0,0
F i g u r e 1 - Ab s o r b a n c e spect r um of TeA sol ut i on i n me t h a n o l
Results and Discussion
The results of Tea stability in solution showed that optimum storage temperature
was -20~ at which toxin concentration remained constant during the 28 weeks of
storage, independently of the solvent considered (Table 1). This was not surprising since
all chemical reactions are slowed considerably at low temperatures. After 3 weeks storage,
total degradation of TeA was observed at 25~ and 4~ in methanol and methanol-
water (8:2 v/v) solutions. A considerable decrease of TeA levels was observed in benzene
solutions, particularly at the highest temperature, with a decrease of 30% after 3 weeks
storage. Whereas at 4~ the toxin levels remained constant up to 8 weeks storage.
The stability of TeA in benzene-acetonitrile (98:2 v/v) solutions was acceptable at
4~ during the 8 weeks, after this time, a slow breakdown of the toxin began (Table 1).
At 25~ the partial TeA degradation occurred faster than at 4~ but the total amount of
breakdown product at 28-week storage did not exceed 60%. It was only in this solvent
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Mycotoxin Research. Vol. 14 (1998)
system that characteristical spectrum of TeA remained constant over the storage time, in
spite of the reduction of its levels.
In mycotoxin research stock toxin solutions are used for different experimental
protocols. While toxins generally are sold lyophilised from manufacturers, they are
usually reconstituted with methanol or ethanol for storage at 4~ or at lower temperature.
There is little information on stability of mycotoxins in organic solvents. Most of them
were performed for aflatoxins. Chloroform, benzene and methanol were recommended
for a good solubility and storage of aflatoxin solutions (19, 20). Among other mycotoxins,
We and Chu (21) studied the instability of some trichotecenes in methanol. They found
that trichotecenes tested were transformed to several products after a prolonged exposure
to methanol. In relation to T-2 mycotoxin, it has been found that the stability of this
toxin in aqueous media decreased with increased temperature (22). Fumonisin B, and
B: stability was studied in acetonitrile-water and methanol, the first solvent system was
recommended as solvent for reference solutions of fumonisin (23).
Considering the limits of the methodology employed, we could recommend to use
only the solvent and temperature combination which level toxin remained constant.
Table I - Concent r at i on of tenuazoni c aci d (pg/ml) soluti ons i n different solvent systems
Solvent T*C Storage period (weeks)
Systems 0 3 8 23 28
Methanol 25 3.834-0.06* ND ND ND ND
4 3.934-0.06" ND ND ND ND
-20 3.80-I-0.1 * 3.73+0.06 3.80 3.77 3.80
Methanol:
Water (8:2)
25 4.03 ND ND ND ND
4 3.934-0.05* ND ND ND ND
-20 3.97 4.03 3.90~0.1 3.97 4.0
Benzene
25 4.03 2.90 ND ND ND
4 4.13 3.97 3.90 ND ND
-20 3.77 3.77 3.87+0.06 3.87 3.77
Benzene: 25 4.03 3.03 2.57 1.47~-0.15 1.304-0.2
Aeetonitrile 4 3.80-~).1" 3.771-0.06 3.66 2.87 2.73
(98:2) -20 3.87 3.934-0.06 4.03+0.06 3.83 3.804-0.2
ND: not detected. Change oftenuazonic acid spectrum
Data represent means 1SD (n=3)
*Values measurements at room temperature.
57
Temperature as -20~ could be recommended for long-time storage of TeA solution in
any of the solvent systems tested. Also benzene and benzene-acetonitrile (98:2 v/v)
could be advised to make TeAsolutions which will stored relatively short-time (2 months)
at 4~ Methanol and methanol-water (8:2 v/v) are not recommended for storage of
TeA solutions.
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Manuscript received May 14, 1998; accepted Sept. 10, 1998.
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