SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF … poster sunu.pdf · simultaneous...
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SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF
PHENOLIC ANTIOXIDANT (BHA AND BHT) IN PHARMACEUTICAL
PREPARATIONS AND CHEWING GUMS BY USING H-POINT STANDARD
ADDITION METHOD
Z.Kalaycıoğlu, Ö. Dülger, M. Üstün Özgür
Yıldız Technical University, Faculty of Science and Art, Department of Chemistry, 34210 İstanbul, TURKEY
ABSTRACT
Antioxidants are used to inhibit lipid oxidation and preservatives are used to inhibit bacterial and fungal growth [1]. To ensure food safety and safeguard the health of the general public, the use of synthetic phenolic antioxidants in food is strictly
controlled in many countries, whether singly, which is the usual case, or in combinations, the corresponding permitted levels range from 100 to 200 mg kg-1 [2]. In this study, a simple, rapid and sensitive spectrophotometric method was described for the
simultaneous determination of binary mixtures of BHA and BHT in pharmaceutical preparations and chewing gums, without prior separation steps using the H-Point standard addition method (HPSAM). In this method, the concentration of one
antioxidant was calculated from overlapping spectra at two appropriate wavelengths, where the other antioxidant was selected as interferent present the equal absorbance relationship. Absorbances at two pairs of wavelengths, 265 and 288 nm (when
BHA acts as analyte) or 288 and 293 nm (when BHT acts as analyte) were monitored, while adding standard solutions of BHA or BHT, respectively. In the literature, many UV absorption methods are presented (3, 4). Also, chromatography in all
forms, such as liquid (6), gas (7) . The proposed method was validated successfully applied to the determination of synthetic antioxidants (BHA and BHT) in synthetic binary mixtures with different concentration ratios and commercial products
(pharmaceutical capsules and chewing gums). The results were compared with the derivative spectrophotometric method which has been previously reported, and very similar values were found between the methods.
KEYWORDS: H-point standard addition method, synthetic antioxidants, BHA, BHT, pharmaceutical preparations, chewing gums, spectrophotometry.
EXPERIMENTAL
Apparatus
Spectrophotometric measurements were carried out with a Schimadzu Spectrophotometer 1800 using 1.00 cm
quartz cells. All spectra were recorded over the range 200-350 nm with 2 nm slit width, and 1200 nm/min
scanning speed.
Samples and Solutions
Chewing-Gum Samples: First, Vivident, Falım
Pharmaceutical Preparations: Metrin, Devit
Stock solutions of BHA (200 μg/mL) and BHT (500 μg/mL) were prepared in methanol. These solutions were
stored in the dark at 4 0C. BHA and BHT stock solutions were diluted to obtain standard solutions for the
preparation of calibration graphs in the concentration range of 4-20 μg/mL and 20-100 μg/mL, respectively.
(E-320), Butylated hydroxyanisole (E-321), Butylatedhydroxytoluene
Scheme 1. Chemical structure, number and names of the antioxidants used.
Preparation of Samples
A portion of 12 g chewing-gum samples was chopped and placed in an 100 mL erlenmeyer flask and 80 mL
methanol was added. The mixture was shaken in an ultrasonic water bath for 30 minutes, then the solution was
filtered. Appropriate volume of filtrates was transferred into 10 mL volumetric flask and diluted with methanol. 10
mL of Devit sample was placed in 100 mL erlenmeyer flask and 50 mL methanol was added. The mixture was
shaken magnetically for 30 minutes and the methanol phase was taken. Appropriate volume of sample was
transferred into 10 mL volumetric flasks and diluted with methanol. 15 g of Metrin sample was placed in 50 mL
erlenmeyer flask and 30 mL methanol was added. The mixture was shaken magnetically for 30 minutes and the
solution was centrifuged. The upper phase was taken. Appropriate volume of sample was transferred into 10 mL
volumetric flasks and diluted with methanol. The absorption spectra of the samples were given in Figure 1.
Figure 1. Absorption spectra of (a) 50 µg/mL BHT, Figure 2. Absorption spectra of (a) 50 µg/mL BHT and
(b) 20 µg/mL BHA, (c) mixture, (d) Metrin, (e) Devit (b) 20 µg/mL BHA. The black lines are selected
wavelengths (f) Vivident, (g) First, (h) Falım for applying HPSAM
RESULTS & DISCUSSIONS
The absorption spectra of BHA and BHT under the experimental conditions are shown in Figure 1. As can be seen in
Figure 2, BHT cannot be determined by direct absorbance measurement in the presence of BHA. However BHA can be
approximately determined by direct absorbance measurement. Therefore determination of BHA and BHT in the presence
of each other is impossible by classical spectrophotometry. We proposed H-point standard addition method.
Wavelength selection
In the proposed system, the determination of the concentration of the analytes requires the selection of two wavelengths
λ1 and λ2.
To select the appropriate wavelenght pair for using HPSAM the following principles should be applied. At these selected
wavelenghts the analyte signals must be linear with concentrations and the interferent signal must be equal, remaining
unchanged by changing the analyte concentration. As it is observed from Figure 2, the wavelenght pairs were 265-288
and 288-293 nm for determination of BHA and BHT respectively. . Two straight lines for concentration and absorption
were plotted and the values for the absorption and concentration at the intersection of these two lines (H-point), termed
CH and AH, respectively, were obtained by extrapolation (Fig. 3 and Fig. 4). Standard solutions of BHA and BHT were
initially tested to validate the applicability of the chosen wavelenghts. Figure 5, 6 are H-point standard addition
calibration lines constructed at two selected wavelengths (265-288) for determination of BHA in the presence of BHT.
Figure 7, 8 are H-point standard addition calibration lines constructed at two selected wavelenghts (288-293) for
determination of BHT in the presence of BHA. The experiments on binary samples of BHA and BHT antioxidants were
performed to check the reproducibility of the proposed method (Table 1). A recovery of 95-106.25% was obtained for the
BHA and BHT in the samples. The data observed with the current methods, excellent mean, % recovery values, close to
100% and their low standard deviations (RSD% ˂0,21) which represents high accuracy of the proposed analytical
methods.
Figure 3. H-point standard addition plot for the
simultaneous determination of BHA and BHT with
a constant concentration of BHA (12 μg/mL) and
BHT (80 μg/mL) and different BHA concentrations
(4-20 μg/mL).
0
0,2
0,4
0,6
0,8
1
1,2
-15 -10 -5 0 5 10 15 20 25
Ab
sorb
an
ce
BHAadded, μg/mL
0
0,2
0,4
0,6
0,8
1
1,2
-30 -20 -10 0 10 20 30A
bso
rban
ceBHAadded, μg/mL
H-point
Figure 5. H-point standard addition plot for fixed BHA
concentration (12 μg/mL) and different concentrations
of BHT (20-100 μg/mL) at wavelenghts 265 and 288
nm.
Figure 4. H-point standard addition plot for the
simultaneous determination of BHA and BHT with
a constant concentration of BHT (40 μg/mL) and
BHA (12 μg/mL) and different BHT concentrations
(20-100 μg/mL).
Figure 6. H-point standard addition plot for different
BHA concentrations (4-20 μg/mL) and fixed BHT
concentration (60 μg/mL) at wavelenghts 265 and 288
nm.
0
0,2
0,4
0,6
0,8
1
1,2
-60 -40 -20 0 20 40 60 80 100 120
Ab
sorb
an
ce
BHTadded, μg/mL
Figure 7. H-point standard addition plot for fixed
BHT concentration (40 μg/mL) and different
concentrations of BHA (4-12 μg/mL ) at wavelenghts
288 and 293 nm.
0
0,2
0,4
0,6
0,8
1
1,2
-150 -100 -50 0 50 100 150
Ab
sorb
an
ce
BHTadded, μg/mL
Figure 8. H-point standard addition plot for different
BHT concentrations (20-100 μg/mL) and fixed BHA
concentration (12 μg/mL) at wavelenghts 288 and
293 nm.
Application of Methods to the Samples
In order to apply the H-point standard addition method to the samples, appropriate volumes of filtrates were transferred
into 10 mL volumetric flasks and 12 μg/mL BHA and 20 μg/mL BHT were added. The different concentrations of BHA
(4-20 μg/mL) and the different concentrations of BHT (20-100 μg/mL) were added to these solutions and the volumes
were completed with methanol in 10 mL volumetric flask for the determination of BHA and BHT, respectively. The
absorption spectra of the solutions were recorded. The results were compared with the derivative spectrophotometric
method which has been previously reported, and very similar values were found between the methods. RSD values found
were well with the acceptable range indicating that the proposed method was excellent repeatibility. The results are shown
in Table 2.
Table 2. Determination of BHA and BHT content in chewing gums and pharmaceutical preparations by the proposed
method (HPSAM) and comparison method (derivative spectrophotometric method)
CONCLUSIONS
The suggested method shows that application of HPSAM can be well adopted for resolving binary mixtures of BHA and
BHT. The proposed method is comparable to the derivative spectrophotometric for determination of BHA and BHT in the
chewing gum samples and pharmaceutical preparations studied.
REFERENCES[1] Biparva, P., Ehsani, M., Hadjmohammadi M. R., 2012. Journal of Food Composition and Analysis 27, 87-94
[2] Madhavi DL, Deshpande SS, Salunkhe DK. 1996. Food antioxidants: technological, toxicological and health perspectives. New York: Marcel Dekker
[3] M.E. Komaitis, M. Kapel, J. Am. Oil Chem Soc. 62 (1985) 1371.[4] C.S.P. Sastry, S.G. Rao, B.S. Sastry, J. Food Sci. Technol. 29 (1992) 101. [5] Zhenfeng, Y. et al. (2002), A study on rapid determination method for BHA, BHT, PG and TBHQ in oil and containing oil products, Food and Fermentation Industries[6] Ding, M. et al. (2012) Food Chemistry, 131 (3), 1051-1055
Table 1. Determination of BHA and BHT in Binary Mixtures