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 [email protected] 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 0 C. 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 λ 1 and λ 2 . 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 C H and A H , 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 Absorbance BHAadded, μg/mL 0 0,2 0,4 0,6 0,8 1 1,2 -30 -20 -10 0 10 20 30 Absorbance BHAadded, 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 Absorbance 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 Absorbance 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

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Page 1: SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF … poster sunu.pdf · simultaneous spectrophotometric determination of phenolic antioxidant (bha and bht) in pharmaceutical preparations

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

[email protected]

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