Mechanism Of Thermal Loss Of Tertiary Butyl Hydroquinone(TBHQ) In Oil And Multispectroscopic Studies On The Interaction Of TBHQ With Bovine Serum Albumin(BSA)

The effect of acid value(AV) of soybean oil(SBO) and different types of free fatty acid(FFA) on tert-butylhydroquinone(TBHQ) and butylated hydroxytoluene(BHT) losses during heating and the related mechanisms of that were systematically studied. The method for simultaneous and accurate analysis of TBHQ and 2-tert-butyl-1,4-benzoquinone(TQ) in edible oils by normal-phase(NP) high performance liquid chromatography(HPLC) was developed. The interaction of TBHQ, BHT or TQ with bovine serum albumin(BSA) by spectroscopic methods was studies.The effect of AV of SBO and different types of FFA on TBHQ and BHT losses during heating was evaluated by heating experiment in the oven. The results showed as follows: as heating temperature increased and heating time was prolonged, losses of both TBHQ and BHT were significantly increased. The higher the AV of SBO at 120 and 180 ℃, the greater the TBHQ loss. The type of FFA also had an observable effect on TBHQ losses at both temperatures, whereas AV and FFA type had no effect on BHT losses. Employing HPLC, liquid chromatography/mass spectra(LC/MS), thin layer chromatography(TLC) and nuclear magnetic resonance(NMR), it was determined that esterification of TBHQ with FFA(C8:0, C18:0, C18:1) occurred at 120 and 180 and esterification reaction occurred at the 4℃-position of TBHQ. The related mechanisms of that was speculated.Due to relatively simple pretreatment, shorter analysis time, high accuracy, small precision and good repeatability, reverse phase(RP) HPLC can be applied to qualitative and quantitative analysis of TBHQ in oils & fats. However, when RP-HPLC was used to detect TQ, part of TQ was reduced to TBHQ by reduction status of hydrogen([H]) in mobile phase, indicating that RP-HPLC cannot detect TQ exactly. Therefore, an analytical method for the simultaneously exact detection of TBHQ and TQ in edible oils was developed. The TBHQ and TQ were extracted by methanol, and then separated and detected by normal-phase HPLC(NP-HPLC). The ranges of TBHQ and TQ were both 0.10~500.00 μg/mL, and correlation coefficients of TBHQ and TQ were both above 0.9999. The limit of detection(LOD) and limit of quantitation(LOQ) was respectively 0.30 and 0.91 μg/mL for TBHQ, 0.10 and 0.30 μg/mL for TQ. The recoveries of TBHQ and TQ were respectively 98.92-102.34% and 96.28-100.58% for SBO, 96.11-99.42% and 98.83-99.24% for lard, with the relative standard deviations(RSD) below 0.38% and 1.71% for SBO, 2.06% and 3.58% for lard. The recoveries of TBHQ/TQ at tested concentrations of 150/50, 100/100 and 50/150(mg/kg)/(mg/kg) were respectively 97.83-98.72% for SBO and 96.93-99.30% for lard, with the relative standard deviations below 0.96% for SBO and 2.25% for lard. Because of relatively simple pretreatment, high sensitivity, and good repeatability, the method was suitable for simultaneously exact analysis of TBHQ, TQ and TBHQ plus TQ in edible oils during storage. In addition, it can be applied to predict the originally added level of TBHQ in edible oils.At pH=7.40 condition, it was found by UV spectroscopy that the interaction of TBHQ, BHT or TQ with BSA occurred and new polymers were formed. Besides, it was found by spectrofluorimetry that the quenching mechanism of BSA by TBHQ, BHT or TQ was a static procedure. The binding of TBHQ, BHT or TQ with BSA was very firm. The binding site of TBHQ, BHT or TQ with BSA was 1. The negative value of ΔH, positive value of ΔS and the negative value of ΔG indicated that hydrogen bonding and hydrophobic interactions played major roles in the binding and the process was spontaneous.