Specific Method for Spectrophotometric Determination of Gossypol
Simultaneous reactions of aniline on the aldehyde groups and ammonium molybdate on the ortho-diphenolic groups of gossypol were studied in tartaric acid buffer solution. The results indicated in the range 300-520 nm that the spectrum of dianilino-dimolybdate of gossypol complex obtained presents two peaks of absorption at 340 and 450 nm whereas the dianilino-gossypol and the dimolybdate of gossypol present one peak at 444 and 420 nm, respectively. The pH effect on the intensity of the absorption indicated that the optimum pH was 6.5. Gossypol is the only compound in the cottonseed extract having two peaks of absorption in the range 300-520 nm. The calibration curve was linear over the range 0.634-4.953 ppm (r2 = 0. 9949) with the detection limit of 0.474 ppm. The stability of the complex was 90 min. The method was sensitive, specific and accurate for the determination of total gossypol in glanded cottonseed, crude, neutral and refined glanded cottonseed oil.
In many African tropical regions, glanded cottonseed oil is the main
source of food lipids. The quality of this refined glanded cottonseed
oil is very uncertain because crude glanded cottonseed oil contains gossypol
(1) (Fig. 1), a toxic constituent for men and animals
species (Yuan and Shi, 2000; Yildirim-Aksoy et al., 2004). The
presence of gossypol in cottonseed is also a major economic problem because
it is a factor reducing the value of glanded cottonseed oil on the market
place due to dark colour (Osman et al., 1976; El-Nockrashy et
al., 1976; Wan, 1996). Gossypol is a pigment not only found in cotton
plant material, but also in many Malvaceae species used in traditional
medicine (Angela et al., 2005). Its derivatives exhibit many benefits
biological properties (Blackstaffe et al., 1997; Bushunow et
al., 1999; Dao et al., 2003; Abe et al., 2004). In both
the nutritional and pharmacological point of view, the determination of
gossypol is important. The quantitative HPLC developed is the sensitive
and specific method for the analysis of gossypol but is an expensive method
(Hron et al., 1999; Gamboa et al., 2001; Benbouza et
al., 2002; Cai et al., 2004) in African cottonseed oil industries.
The spectrophotometric method (AOCS, 1997) for determining gossypol involved
reaction of gossypol with aniline to form a yellow dianilino-gossypol
(2) (Fig. 1). The AOCS method is relatively inexpensive
and a fast method for determining gossypol but lacks sensitivity and specificity
(Mahoney et al., 1985). The selective reaction of ammonium molybdate
with o-diphenolic group was used for determining gossypol as gossypol
dimolybdate (3) (Fig. 1) in glanded cottonseed (Tchatchueng
et al., 1992). In the present study an improved spectrophotometric
method was developed for quantitative and specific analysis of gossypol.
The developed method is based on the simultaneous reaction of ammonium
molybdate and aniline on the gossypol to form a yellow complex called
dianilino-dimolybdate of gossypol (4) (Fig. 1).
MATERIALS AND METHODS
A spectronic 20D+ of Milton Roy and Co (USA) was used for the measurement
of absorbance. For the pH measurements, PH-meter consort p207 of bioblock
equipped with calomel glass electrode was used. Eppendorf micropipette
of 100-1000 μL was used for appropriate volume of a sample solution
and reagent additions. A A200s Sartorius analytic balance, accurate to
0,0001 g was used for weighing all the compounds required. Officially
calibrated Pyrex glassware was used throughout this study and a prolabo
water bath accurate to 2 °C for heating the solution.
Materials and Reagents
Glandless cottonseeds, crude, neutral and refined glanded cottonseed
oils used were kindly supplied by the Cotton Development Society Sodecoton
of Garoua and Maroua in Cameroon. All chemicals from Sigma France S.A:
Aniline A9880, Ammonium molybdate A7302, Caffeic acid C0625, DMSO D8779,
Gossypol G4382, Kaempferol K0125, Myricetin M6760, Quercetin Q0125, Tartaric
acid T8277, Glacial acetic acid A0808, Zinc dust 19834-0010 (Acros), acetonitrile,
ethyl alcohol and isopropyl alcohol were of analytical reagent-grade and
were used as received. Doubly distilled water was used throughout.
A 30.2 ppm ethyl alcohol Stock solution of gossypol acetic (24.92 ppm
pure gossypol) was prepared and stored in borosilicate glass vessel in
refrigerator at 4 °C. Every day working solutions were obtained by
appropriate dilution of stock solution. Reagent 1 was an 800 ppm solution
of ammonium molybdate in a mixture of isopropyl alcohol and dimethylsulfoxyde
(1:1). The binary solvent was a good solvent for ammonium molybdate, aniline
and gossypol. Reagent 2 was 1% freshly redistilled aniline under zinc
dust in a mixture of isopropyl alcohol and dimethylsulfoxyde (1:1). This
reagent can be stored in a brown bottle in a refrigerator and used for
three or four days. The acid buffer media of a given pH was prepared by
mixing various volumes of 0.2 mol L-1 of tartaric acid in DMSO/isopropyl
alcohol (1:1) and 0.2 mol L-1 sodium hydroxide solutions to
the required pH value (2-8) under a pH-meter control.
To construct the calibration curve and measure the absorbance at the
maximum wavelength, place aliquots of gossypol acetic standard solution
in 10 mL calibrated flasks in order to obtain a final concentration range
of 0.298-4.985 ppm (pure gossypol). Add 2 mL of buffer solution of a given
pH, 1 mL of reagent 1 and 1 mL of reagent 2. Incubate the resulting solution
for 15 min in a water- bath at 70 °C accurate to 2 °C. After cooling,
dilute to volume with appropriate buffer solution. Obtain he spectrum
of the complex by recording the absorbance from 320 to 520 nm. Measure
the absorbance of the complex at 450 nm against a blank. For the analysis
of the sample, the procedure described above was followed except that
the standard solution of gossypol was replaced by the sample solution
of gossypol firstly extracted according to the method developed by Marquié
and Bourrély (1991).
Application and Comparison with the Reference AOCS Method
For the analysis of the samples and the comparison purpose, the proposed
method and standard AOCS method (1997) were simultaneously tested for
glanded cottonseeds, crude, neutral and refined glanded cottonseed oils.
The contents of gossypol in the samples were obtained with a calibration
RESULTS AND DISCUSSION
Basis of the Proposed Method
The shame of the reactions that are on the basis of the proposed method
is presented in Fig. 1. Gossypol (1) reacts with aromatic
amines such as aniline to form dianilino-gossypol (2). This reaction has
been the basis of the colorimetric analysis of gossypol (Smith, 1958).
Tchatchueng et al. (1992) using the reaction of molybdenum ions
on o-diphenolic group demonstrated that gossypol could be determined by
means of the molybdate gossypol complex (3). Combining the reactions of
aniline and molybdenum ions on gossypol at pH 6.5, dianilino-dimolybdate
of gossypol (4) was obtained (Fig. 1).
Figure 2 shows two possible structures of o-diphenolic
compounds. One o-diphenolic function can react with one molybdenum ion
giving a compound of structure (6) or two o-diphenolic functions can react
with one molybdenum ion giving a compound of structure (7) depending on
the pH and the o-diphenolic compound structure. The structure of the gossypol
molybdate complex was established by the mole-ratio method using spectrophotometric
analysis (Fig. 3). The experiments were performed at
pH 6.5. The result obtained was 2.1:1. This result confirms the proposed
structure of dimolybdate of gossypol (3).
To evaluate the influence of other cottonseed phenolic compounds such as
flavonoids, ortho-diphenolic acids on the determination of gossypol, known amounts
of the desired compound were added in the standard solution of gossypol (2 ppm)
under the optimal conditions of the proposed procedure.
||Structure of gossypol complexes, (1) gossypol, (2) dianilinogossypol,
(3) dimolybdate of gossypol, (4) dianilino-dimolybdate de gossypol
|| Metallic ortho-diphenolic complexes
|| Determination of mole-ratio of molybdenum and gossypol
The tolerance limits of other phenolic compounds were taken as the quantity
that caused an error not more than ± 5% in the absorbance (Table
1). These tolerance limits expressed as the ratio of interferants and gossypol
was: Myricetin (0.3), Quercetin (0.5), Kaempferol (2), Caffeic acid (2). According
to Stipanovic et al. (1977, 1988), the sum of the content of all these
interferants could not practically found in the cottonseed extract. These compounds
could not interfere with the method in the studied experimental conditions.
Fingerprint of Gossypol and Specificity of the Method
The spectrum of dianilino-gossypol (2) has one peak at 444 nm and
dimolybdate of gossypol (3) has also one peak at 420 nm. The spectrum
of dianilino-dimolybdate of gossypol (4) has two maxima at 450 and 340
nm (Fig. 4).
In the cottonseed extract, other terpenoid aldehydes identified besides
gossypol are divided into two groups: the first group that contained methoxylated
gossypol and some of its derivatives: 6-methoxygossypol (9), 6,6-dimethoxygossypol
(10), methoxyhemigossypol (11), methoxyhemigossypolone (12), heliocides
of B series (13) cannot undergo simultaneous reaction with aniline and
molybdenum ions. The second group of compounds such as hemigossypol (14),
hemigossypolone (15) and heliocides of H series (16) (Fig.
5) in spite of getting simultaneous reaction of aniline and molybdenum
ions like gossypol have the molar absorption coefficient very lower (ε
= 7000-8300) than the molar absorption coefficient of aniline derivative
of gossypol (ε = 41450).
|| Spectra of dianilino-dimolybdate of gossypol
||Other terpenoid aldehydes in cottonseed: (9) 6-methoxygossypol,
(10) 6,6dimethoxygossypol, (11) 6-methoxyhemigossypol, (12) 6-methoxy
gossypolone, (13) helioside B1 and B2, (14) hemigossypol,
(15) hemigossypolone, (16) helioside H1 and H2
In addition to this according to Stipanovic et al. (1977), the content
of other terpenoid aldehydes besides gossypol in the cottonseed was less than
10%. This result associated with the interference study show that gossypol is
the only terpenoid aldehyde in the cottonseed products presenting in the studied
experimental conditions a spectrum having two maxima at 450 and 340 nm in the
range 300-520 nm.
|| Effect of pH on the absorption of gossypol complex, Concentration
of gossypol (2 mg L-1)
|| Recovery and reproducibility of the determination of 0.298
to 4.985 mg L-1 of gossypol
The peak at 340 nm is the fingerprint of gossypol. The proposed method is specific
for the determination of gossypol in cottonseed and cottonseed products.
Effect of the pH on the Absorption of Dianilino-Dimolybdate of Gossypol
The effect of pH on the intensity of the absorption of the dianilino-dimolybdate
of gossypol was studied over the pH range 2-8. As shown in Fig.
6, the pH of the medium had a great effect on the absorbance of the
dianilino-dimolybdate of gossypol. The experimental results obtained indicate
that the maximum absorbance was at pH 6.5 that was used in subsequent
Precision and Accuracy
The accuracy and precision of the proposed method were evaluated by
performing replicate analyses (n = 5) at each of six different concentrations
of gossypol in the range of the calibration graph (0.634-4.953 ppm). The
relative standard deviation and the recovery of gossypol were calculated.
The standard errors for the five replicate determinations were less than
3% showing good reproducibility of the method. The recovery percentage
was found to be in the range 95.7-99.4% giving certitude that the proposed
method is accurate (Table 2).
Calibration Graph, Detection and Determination Limits
The calibration graph obtained using the proposed method under the optimized
conditions was constructed (Fig. 7). There is a linear relationship
between the absorbance and the gossypol concentration from 0.634 to 4.985 ppm.
The detection limit was obtained by analysing series standard solutions of gossypol
(0.298-4.985 ppm) under optimized conditions. The specificity of the method
is the appearance of two peaks of absorption.
|| Calibration graph with optimized conditions using proposed
|| Gossypol content in cottonseed and cottonseed oil
Then the detection limit was defined as concentration of gossypol ya
(0.474 ppm) under which only one peak at 450 nm was observed (Fig.
4). The determination limit was then ya+10Sa (Miller and Miller,
2000) and equal at 0.634 ppm.Then the detection limit was defined as concentration
of gossypol ya (0.474 ppm) under which only one peak at 450 nm was
observed (Fig. 4). The determination limit was then ya+10Sa
(Miller and Miller, 2000) and equal at 0.634 ppm.
Stability of Dianilino-Dimolybdate of Gossypol
The stability of the dianilino-dimolybdate of gossypol was determined
by analyzing a standard concentration of the compound (2 ppm) immediately
after preparation (reference value) and after storage times. Stability
was defined as being less than 2% loss of the initial absorbance in the
stated time. The absorbance of the dianilino-dimolybdate of gossypol (4)
remains constant for 1 h and decreases by 2% after 3 h and 5% after 5
h. The stability of the dianilino dimolybdate of gossypol was then 3 h.
The system gives ample time to measure the absorbance of large number
of samples during cottonseed industrial processes. The relatively short
reaction time of 30 min, the simplicity of the method are remarkable advantages
over the spectrophotometric method reported by others Fisher et al.
(1987), which requires more than 60 min and 10 steps to complete the reaction.
Application of the Method
The applicability of the proposed method for the quantification of
gossypol in a glanded cottonseed, crude, neutral and refined glanded cottonseed
oils was examined by analysing these products obtained from Cameroon cottonseed
industries. The results of the proposed method were statistically compared
with reference AOCS method (1997) and summarized in Table
3. The contents of the total gossypol obtained from the Maroua cottonseed
samples were slightly lower than those obtained from the Garoua cottonseed
samples. The AOCS methods produced higher estimation of total gossypol
in all cottonseed samples with lower reproducibility compared to the proposed
The specificity of the proposed method was characterised by two peaks
of absorption at 340 and 450 nm whereas standard AOCS methods and the
method developed earlier (Tchatchueng et al., 1992) give spectra
of the complex of gossypol showing a peak at 444 and 420 nm, respectively.
The fingerprint of the gossypol is the appearance of the peak at 340 nm.
The simplicity of the proposed method and the results obtained in the
application to real samples demonstrate that this new method can be conveniently
used for the determination of free or total gossypol during glanded cottonseed
oil process. The method cannot be used to determine gossypol in which
ortho-hydroxyl and aldehyde groups are not in the free form without pre-treatment
such as hydrolysis with sulphuric acid as reported by Carruth (1918).
The perspectives of the present study are the systematic study of bifunctional
molecule to search a probable specific spectrophotometric signature of
the molecule and practically, for the general project, we intend to selectively
extract using selective ion exchange resin the toxic gossypol from crude
glanded cottonseed oil before refining process.
This research was supported in part by the University of Ngaoundéré
through its budget line 566263 ENSAI and in part by financial support
N° 2001-132-2002 Cometes (French cooperation AUF).
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