• Christinawaty Indonesia National Agency of Drug and Food Control, Jl. Percetakan Negara No.23, Jakarta, Indonesia
  • Sophi Damayanti Pharmacochemistry Research Group School of Pharmacy ITB Bandung Institute of Technology


Antioxidants are food additives in oil and fat products usually to decrease the process of rancidity by preventing the oxidation such as in margarine. BHA, BHT, propyl gallate, and TBHQ are commonly used antioxidants. Combinations of antioxidants are generally used in the food product. Therefore, analytical method which is capable to measure the antioxidants simultaneously, is required. This study was aimed to obtain analytical method for determination of BHA, BHT, propyl gallate, and TBHQ using HPLC in margarine. The analysis was using Waters column Xbridge C18 (4.6 x 150 mm, 5μm) and 280 nm ultraviolet detector. The mobile phase was a mixture of 1% acetic acid in acetonitrile and 1% acetic acid (3:7). The flow rate was set at 1.7 ml/min. The calibration curve of BHA, BHT, propyl gallate and TBHQ were linear with correlation coefficient r = 0.9999, 0.9996, 0.9998 and 0.9998, respectively. Limit of detection and limit of quantitation of BHA, BHT, propyl gallate and TBHQ were 2.6217 and 7.9445 µg/ml; 4.1837 and 12.6777 µg/ml; 2.494 and 7.559 µ/ml; 2.1272 and 6.4461 µg/ml, respectively. Inter day precision of the method was expressed as RSD with result; BHA, BHT, propyl gallate, and TBHQ were 0.1553; 0.4576; 0.0237; 4.8106 %; respectively whereas recovery using standard addition method were 105.38; 107.87; 86.50; 83.64 % respectively. Based on the obtained results, it can be concluded that this method has been successfully developed and validated for the determination of antioxidants BHA, BHT, propyl gallate, and TBHQ in margarine.


1. Madhavi DL, Deshpande SS, Salunkhe DK, editors. Food Antioxidants, New York, Marcell Dekker Inc: 1996
2. SN Mahindru. Food Safety: A Techno Legal Analysis. New Delhi, MDH Ltd: 2000
3. Potter NN and Hotchkiss H. Food Science, 5th ed, New York, Chapman & Hall: 1995.
4. Race S. Antioxidants The Truth about BHA, BHT, TBHQ and Other Antioxidants Used as Food Additives. London, Tigmor Books: 2009.
5. Carocho M and Isabel CFRF. A Review on Antioxidants, Prooxidants and Related controversy: Natural and Synthetic Compounds, Screening and Analysis Methodologies and Future Perspectives, Food and Chemical Toxicology 2013; 51, 15-25.
6. Hasenhuettl GL, Hartel RW. Food Emulsifier and Their Applications, New York, Chapman & Hall. International Thompson Publishing: 1997.
7. Perrin C and Meyer L. Quantification of Synthetic Phenolic Antioxidants in Dry Foods by Reversed-phase HPLC with Photodiode Array Detection. Food Chemistry 2002; 77, 93-100.
8. Ding M and Zou J. Rapid Micropreparation Procedure for The Gas Chromatographic–Mass Spectrometric Determination of BHT, BHA And TBHQ In Edible Oils. Journal of Food Chemistry 2012; 131, 1051-1055.
9. Saad B, Sing YY, Asri MN, Hashim N, Salhin AMA, Idris MS, Fariza SS, Md Talib K, Ahmad K. Determination Of Synthetic Phenolic Antioxidants In Food Items Using Reversed-Phase HPLC. Journal of Food Chemistry 2007; 105, 389-394.
10. Sin, Della WM, Wong YC, Mak CY, Sze ST, Yao WY. Determination Of Five Phenolic Antioxidants In Edible Oils Method Validation And Estimation Od Measurement Uncertainty. Journal of Food Composition and Analysis 2006; 19, 784-791.
11. ICH Harmonized Tripartit Guideline. Text On Validation of Analytical Procedure; 1994.
12. Ahuja SM and Dong W. Handbook of Pharmaceutical Analysis by HPLC Separation Science and Technology. London & New York, Elsevier Academic Press: 2005.
13. Ermer J and Miller JHM.Editor. Performance Parameters, Calculations and Tests, in Method Validation in Pharmaceutical Analysis: A Guide to Best Practice (), Weinheim, Wiley-VchVerlag GmbH & Co.:2005.
14. Kromidas S. HPLC Made to Measure A Practical Handbook For Optimization, Weinheim, Wiley-Vch Verlag GmbH & Co: 2006.
15. Kazakevich Y, LoBrutto R. HPLC for Pharmaceutical Scientists. New Jersey, John Wiley and Sons, Inc. Publication: 2007..
16. Adamovics JA. Chromatographic Analysis of Pharmaceuticals, 2nd ed. New Jersey, Marcel Dekker Inc: 1997.
17. Ibrahim S. Berbagai Pendekatan Pengujian Kelinieran Kurva Baku pada Metode Analisis Instrumental. Acta Pharm. Ind 2005; 30, 1-5.
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How to Cite
Christinawaty, and S. Damayanti. “SIMULTANEOUS DETERMINATION METHOD OF BHA, BHT, PROPYL GALLATE, AND TBHQ IN MARGARINE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 8, no. 4, July 2015, pp. 209-11,
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