Method Development and Validation of Hormone Residues Determination in Meat by LC-MS/MS

Abstract

       Hormones, both natural and synthetic, play an important role in controlling growth, sex, physical characteristics and stimulating or inhibiting an immune system as well as controlling formation and decomposition of metabolisms of the body. Incorrect usage of hormones for purposes of growth promoting agents and increasing muscles in food-producing animals may result in having hormone residues in edible tissues. The residues can possibly induce breast cancer and uterine cancer, therefore, those compounds are prohibited to use in the European Union (EU) to protect consumers from the exposure to carcinogenic residues in animal products. In this study, a method for determination of 4 hormone substances, namely, Diethylstilbesterol (DES), zeranol, 17 α-estradiol and 17 β-estradiol by LC-MS/MS techniques was developed and validated. The compounds were extracted from meat samples with methanol and fat was further removed by liquid-liquid extraction technique with hexane and then cleaned up by solid phase extraction with Oasis HLB. As a result of method validation, limit of detection (LOD) was 0.1 μg/kg for all substances. In addition, our study demonstrated that limits of quantitation (LOQ) for DES and zeranol were 2.0 μg/kg, while those for 17 α-estradiol, 17 β-estradiol were 6.0 μg/kg. A linearity of working range of DES and zeranol was between 2.0-10.0 μg/kg, while that of 17 α-estradiol and 17 β-estradiol was between 6.0-15.0 μg/kg with coefficient of determination, R², at more than 0.96. An accuracy of the method expressed as mean of % recovery was in a range of 73-105% and its precision as shown by % Relative Standard Deviation (RSD) was 5.4-26.3%. The study concluded that characteristics of this method were suitable for application testing laboratory as required by the notification of The Ministry of Public Health Regulation No. 303 (B.E. 2550) on Establishment of Maximum Residue Levels of Veterinary Medical Products in Foodstuffs of Animal Origin. Moreover,
a survey of hormone residues, conducted from October 2014 to September 2015, in 50 samples of chicken and beef using the developed method indicated that no four hormone residues were detected in any samples.