Gas Chromatographic Determination of Butylated Hydroxyanisole, Butylated Hydroxytoluene and Tertiarybutyl Hydroquinone in Soybean Oil

Oils containing different levels of the most common phenolic antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiarybutyl hydroquinone (TBHQ) from 0–200 ppm were analyzed by a new gas chromatographic method. The antioxidants in oils were isolated by nitrogen gas purging, collected on Tenax GC coated with polymetaphenoxylene, and then separated and quantitated by gas chromatography. This method requires neither extraction of antioxidants from oil nor derivatization of antioxidants. The correlation coefficients (r) between gas chromatographic peak heights or peak area and their concentrations in oils were 0.99 for BHA, BHT, and TBHQ. This simple gas chromatographic method can determine as little as 10 ppm pf BHA, BHT, or TBHQ in oils in an hour.     

Determination of synthetic phenolic antioxidants in food items using reversed-phase HPLC

A HPLC with gradient elution method for the determination of the synthetic phenolic antioxidants (SPAs) propyl gallate (PG), tertiary butyl hydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) in food items is described. A C18 column served as the stationary phase; the gradient elution was formed by acetonitrile and water:acetic acid (1%). The UV detector was set at 280 nm. Under the recommended conditions, separation of the four SPAs was achieved in less than 8 min. Analytical characteristics of the HPLC method such as limit of detection, linear range, and reproducibility were evaluated. Extraction parameters were optimized for the recoveries of the SPAs in different types of food items (cooking oil, margarine and butter, and cheese). Before the HPLC separation, the SPAs were extracted with methanol/acetonitrile (1:1, v/v) and were subjected to vortex/ultrasonic treatment. The extracts were next kept in a freezer (∼2 h) to precipitate co-extracted components. Recoveries of the SPAs when spiked to cooking oil, margarine, butter and cheese at 50 and 200 mg l⁻¹ were in the ranges 93.3–108.3% for PG, 85.3–108.3% for TBHQ, 96.7–101.2% for BHA and 73.9–94.6% for BHT. The method was applied to the determination of SPAs in 38 food items (16 cooking oils, ten margarine, six butter and six cheese samples). The levels of SPAs in positive samples are all below the legal limits of Malaysia.     

Cloud-point extraction and reversed-phase high-performance liquid chromatography for the determination of synthetic phenolic antioxidants in edible oils

A cloud-point extraction (CPE) method using Triton X-114 (TX-114) nonionic surfactant was developed for the extraction and preconcentration of propyl gallate (PG), tertiary butyl hydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) from edible oils. The optimum conditions of CPE were 2.5% (v/v) TX-114, 0.5% (w/v) NaCl and 40 min equilibration time at 50 °C. The surfactant-rich phase was then analyzed by reversed-phase high-performance liquid chromatography with ultraviolet detection at 280 nm, using a gradient mobile phase consisting of methanol and 1.5% (v/v) acetic acid. Under the studied conditions, 4 synthetic phenolic antioxidants (SPAs) were successfully separated within 24 min. The limits of detection (LOD) were 1.9 ng mL(-1) for PG, 11 ng mL(-1) for TBHQ, 2.3 ng mL(-1) for BHA, and 5.9 ng mL(-1) for BHT. Recoveries of the SPAs spiked into edible oil were in the range 81% to 88%. The CPE method was shown to be potentially useful for the preconcentration of the target analytes, with a preconcentration factor of 14. Moreover, the method is simple, has high sensitivity, consumes much less solvent than traditional methods, and is environment-friendly. Practical Application: The method established in this article uses less organic solvent to extract SPAs from edible oils; it is simple, highly sensitive and results in no pollution to the environment.     

Determination and confirmation of five phenolic antioxidants in foods by LC/MS and GC/MS

Identification and determination of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) by means of LC/MS and GC/MS were examined. These five phenolic antioxidants were detected as their pseudo-molecular ions [M-H]- by LC/MS using a Shim-pack FC-ODS column with drying gas. Moreover, BHA, BHT and TBHQ were detected based on their mass fragment ions by GC/MS. Decomposition of TBHQ, NDGA and PG during analysis could be prevented by the addition of L-ascorbic acid (AsA) to the extraction solvent. All five antioxidants were extracted from nikuman, olive oils, peanut butter, pasta sauce and chewing gum with a mixture of acetonitrile-2-propanol-ethanol (2:1:1) containing 0.1% AsA (AsA mixture), which had been cooled in a freezer and filtered. One part filtrate and 5 parts water were mixed and placed on a Mega-Bond Elut C18 cartridge, except in the case of chewing gum. Lipids in foods were removed on a C18 cartridge by washing with 5 mL of 5% acetic acid, and antioxidants were eluted with 5 mL of AsA mixture. The antioxidants spiked into nikuman, olive oil, peanut butter, pasta sauce and chewing gum were successfully identified and their concentrations determined by LC/MS, and GC/MS with good recoveries.     

ENHANCEMENT OF THE SHELF-LIFE OF FLAVORED BUTTER OILS BY SYNTHETIC ANTIOXIDANTS

The shelf-life of flavored butter oils was enhanced by adding permitted antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) at 0.02% level. Such samples competed well with conveitional ghee. Tertbuthydroquinone (TBHQ) (permitted antioxidant for vegetable fats) showed the greatest antioxidant potency and could be recommended even at a very low level (0.005%) for imparting a longer shelf-life to flavored butter oils.     

Migration analysis,antioxidant, and mechanical characterization of polypropylene-based active food packaging films loaded with BHA,BHT, and TBHQ

Polypropylene (PP) based active composite films were prepared by adding butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), and tertiary butylated hydroquinone (TBHQ) antioxidants using the extrusion molding process. All concentrations of BHT, 2% to 3% BHA, and 3% TBHQ significantly increased the tensile strength (TS) of the composite films compared with control films. Increasing antioxidant concentration decreased TS values for BHT films, whereas an opposite trend was observed for BHA and TBHQ films. BHA at < 2%, BHT at > 2%, and TBHQ at all added concentrations significantly reduced elongation at break (E_b) of the composite films compared to control films. Water vapor permeability (WVP) of 1% BHT film was not significantly different from control. However, other antioxidants especially at increased concentrations significantly increased WVP values. TBHQ films with 300% to 662% increase had the highest WVP and BHT films with 5% to 81% increase had the lowest WVP among composite films. All three antioxidants had a negative effect on the transparency of the films; however the effect of BHA at higher concentrations was greater. The antioxidants did not change the color attributes of the films. Films containing all antioxidants showed 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, which increased with increase in their concentration, especially for those containing 3 wt.% BHT and TBHQ. Overall, incorporating BHA and BHT into a PP matrix improved mechanical, barrier, antioxidant properties, and film appearance and consequently were proposed for the development of antioxidant active PP films. TBHQ film is not recommended for food packaging because of its weak mechanical properties (lower E_b and TS values, higher WVP, and greater migration).     

SYNERGISTIC ACTIVITY OF CAPELIN PROTEIN HYDROLYSATES WITH SYNTHETIC ANTIOXIDANTS IN A MODEL SYSTEM

Capelin protein hydrolysates (CPHs) were examined in a β-carotene-linoleate model system together with synthetic antioxidants, namely butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butyl hydroquinone (TBHQ). Addition of CPHs was at 0.25 and 1 mg per 5 mL in the above emulsion. BHA and BHT were added at levels of 2.5 and 5 μg and TBHQ at 5 and 10 μg. Absorbance of model emulsion system at 470 nm was recorded every 30 min for 2 h. CHPs and synthetic antioxidants inhibited oxidation of linoleic acid effectively. In the experiment with BHA, the influence of CPHs was significant (p<0.01) for the samples incubated for 60, 90, and 120 min; and in experiments with BHT and TBHQ during the entire incubation period. The influence of added BHA and BHT on inhibition of bleaching of β-carotene in the emulsion system was significant (p <0.01) during the incubation period and after 60, 90 and 120 min when TBHQ was used. A synergistic effect was observed only for CPHs and TBHQ when incubation time was 60, 90, and 120 min.     

Estimates of the theoretical maximum daily intake of phenolic antioxidants BHA, BHT and TBHQ in Brazil

The theoretical maximum daily intakes (TMDI) of the phenolic antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertbutyl hydroquinone (TBHQ) in Brazil were estimated using food consumption data derived from a household economic survey and a packaged goods market survey. The estimates were based on maximum levels of use of the food additives specified in national food standards. The calculated intakes of the three additives for the mean consumer were below the ADIs. Estimates of TMDI for BHA, BHT and TBHQ ranged from 0.09 to 0.15, 0.05 to 0.10 and 0.07 to 0.12mg/kg of body weight, respectively. To check if the additives are actually used at their maximum authorized levels, analytical determinations of these compounds in selected food categories were carried out using HPLC with UV detection. BHT and TBHQ concentrations in foodstuffs considered to be representive sources of these antioxidants in the diet were below the respective maximum permitted levels. BHA was not detected in any of the analysed samples. Based on the maximal approach and on the analytical data, it is unlikely that the current ADI of BHA (0.5mg/kg body weight), BHT (0.3mg/kg body weight) and TBHQ (0.7mg/kg body weight) will be exceeded in practice by the average Brazilian consumer.     

Quantitative determination of the phenolic antioxidants using voltammetric techniques

Synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ), show sensitivity to voltammetric waves. The waves of these antioxidants, however, are seriously overlapped and it is difficult to determine them simultaneously. The influence of different parameters (working electrode, supporting electrolyte, pH, voltammetric technique) was evaluated in a quantitative simultaneous determination of three antioxidants in alcoholic mixtures and real sample foods. Glassy carbon (GC) and platinum (Pt) working electrodes were investigated as mediators of oxireduction reactions. Two supporting electrolytes were investigated: Britton-Robinson 0.1 mol l⁻¹ buffer (pH 2.0) and HCl 0.1 mol l⁻¹ (pH 2.0) both with 2 g l⁻¹ (p/v) of methanol. In this paper, voltammetric conditions for the analysis of up to three-component mixtures of antioxidant present at levels: 2.0-100.0 mg l⁻¹ for BHA, 4.0-100.0 mg l⁻¹ for TBHQ and 2.0-20.0 mg l⁻¹ for BHT at GC in HCl 0.1 mol l⁻¹ and 8.0-120.0, 10.0-130.0 and 4.0-30.0 mg l⁻¹ for BHA, TBHQ and BHT, respectively, at Pt in the same supporting electrolyte. The results show that for real food samples, the parameters investigated were satisfactory for quantitative determination using square wave voltammetry (SWV) without chemometric approaches and without suffering overlapping problems.     

Estimated daily intakes of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) antioxidants in Korea.

The study was conducted to establish the estimated daily intake (EDI) of antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) in Korea. The EDIs were obtained from two sources. One of the estimations was based on the analytical determination of BHA, BHT and TBHQ in 12 food categories (ten food categories for TBHQ) and on individual dietary intake data obtained from the National Health and Nutrition Survey in 1998 (n=11 525, age > 1 year). The other EDIs of BHA, BHT and TBHQ were based on the maximum permitted levels specified in national food standards in Korea and on individual dietary intake data obtained from the National Health and Nutrition Survey in 1998 (n=11 525, age > 1 year). To establish the EDIs based on the analytical determination and on individual dietary intake data, 133 food samples in 12 food categories were selected from the foods considered to be representative sources of BHA, BHT and TBHQ in the Korean diet. Selected samples were analysed by GC with FID. BHA was not detected in any of the samples analysed. BHT and TBHQ were detected in the samples, but the levels were significantly lower than their maximum limits. The EDIs1 of BHT, and TBHQ for average consumers were 0.0156(-3), and 0.0012(-3) mg kg(-1) body weight bw day(-1) and as a proportion of the ADI were 0.0052 and 0.0002%, respectively. For 95th percentile consumers, the EDIs of BHT and TBHQ were 0.0080 and 0.0006 mg kg(-1) bw day(-1), and as a proportion of the ADI were 2.67 and 0.09%, respectively. EDIs for BHA, BHT and TBHQ based on the maximum permitted levels and on individual dietary intake data were 0.04, 0.04 and 0.04 mg kg(-1) bw day(-1), respectively. The EDIs of BHA, BHT and TBHQ for average consumers ranged from 6.00 to 14.42% of the ADI of each antioxidant. According to these results, the EDIs of BHA, BHT and TBHQ in Korea were significantly lower than ADI of these antioxidants established by the JECFA.     

Simultaneous voltammetric determination of phenolic antioxidants in food using a boron-doped diamond electrode

A method for the simultaneous determination of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) in food was developed using square-wave voltammetry (SWV). The determination of these phenolic antioxidants was carried out using a cathodically pre-treated boron-doped diamond electrode (BDD) and an aqueous-ethanolic (30% ethanol, v/v) 10 mmol L⁻¹ KNO₃ solution (pH_cond. 1.5) as supporting electrolyte. In the SWV measurements using the BDD electrode, the oxidation peak potentials of BHA and BHT present in binary mixtures were separated by about 0.3 V. The attained detection limits for the simultaneous determination of BHA and BHT (0.14 and 0.25 μmol L⁻¹, respectively) are lower than the ones by voltammetric techniques reported in the literature. The proposed method was successfully applied in the simultaneous determination of BHA and BHT in food products, with results similar to those obtained using a high-performance liquid chromatography method, at a 95% confidence level.     

Qualitative and Quantitative Effects of Antioxidants on the Flavor Stability of Oil

Qualitative and quantitative effects of the common antioxidants, BHA, BHT, PG and TBHQ on the rate of dissolved free oxygen disappearance in soybean oil during storage were studied. The order of effectiveness was BHA, BHT, PG and TBHQ with TBHQ as the most effective. Statistical analyses of the results showed that the effects of BHA and BHT were not significantly different from each other at the 5% level, but BHA or BHT was different from PG or TBHQ, and PG was different from TBHQ. The antioxidant effectiveness of levels of 0, 50, 100, 150 and 200 ppm BHA, BHT, PG or TBHQ was significantly different from one another at the 5% level. The higher the amounts of antioxidants added, the slower the rate of dissolved oxygen disappearance in the oil.     

电分析法同时快速测定植物油中TBHQ、BHA和BHT的研究

研究抗氧化剂TBHQ、BHA和BHT在玻碳电极上的伏安性质,三者的循环伏安图表明:3个峰形良好的氧化峰分别对应:TBHQ的氧化峰电位为590 mV,BHA的氧化峰电位为710 mV,BHT的氧化峰电位为1 050 mV,只有一个还原峰,电位为-90 mV。通过实验优化了电分析法测定植物油样抗氧化剂的分析技术参数。借助最小偏二乘法(PLS),利用电化学分析法预测了人工模拟试样中的3种抗氧化剂的含量,预测结果及加标回收率结果较好。利用所开发的方法测定了实际油样中的抗氧化剂的含量,其回收率在94%~104.1%,结果表明该法同时测定植物油中2种及以上的抗氧化剂是可行的,而且无需提取、分离及清洗等预处理程序,应用潜力巨大。     

气相色谱--质谱法测定XO酱中BHA、BHT和TBHQ

采用气相色谱—质谱(全扫描方式)测定XO酱中丁基羟基茴香醚(BHA)、二丁基羟基甲苯(BHT)和叔丁基对苯二酚(TBHQ)。样品用甲醇振荡萃取，以DB—5MS为分析柱。3种组分的回收率在85％～99％之间，相对标准偏差小于8．4％；样品中BHA、BHT和TBHQ的检测限分别为0．05、0．05和0．10(mg／kg)。该法简单、快速、准确，可用于XO酱等基质复杂的富油食品中BHA、BHT和TBHQ的检测和确证。     

Determination of phenolic antioxidants by micellar electrokinetic capillary chromatography with electrochemical detection

A new and efficient method for the determination of synthetic phenolic antioxidants (SPAs) has been developed by using micellar electrokinetic capillary chromatography (MECC) with electrochemical detection. Under the optimum conditions, all analytes were successfully separated within 13 min at the separation voltage of 18 kV in a 20 mmol/L borate running buffer (pH 7.4) containing 25 mmol/L sodium dodecyl sulfate. The excellent linearity was obtained in the concentration range from 5.0 × 10⁻⁴ to 2.0 × 10⁻⁶ mol/L and the detection limits (S/N = 3) of propyl gallate (PG), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) range from 2.9 × 10⁻⁷ to 2.7 × 10⁻⁶ mol/L. This method has been proved to be effective and successfully applied for the determination of SPA in food products, providing a promising and convenient entry to monitor the superscale use of phenolic antioxidants.     

油脂及其制品中BHA、BHT、PG和TBHQ快速测定方法的研究

研究了采用高效液相色谱仪同时测定油脂及食品中BHA、BHT、PG、TBHQ的检测方法。样品经甲醇提取、过滤后即可直接测定。检测波长 ,2 80nm ;流动相 ,V(甲醇 )∶V(质量分数 1%乙酸 ) =40∶60 ;梯度 ,40 %～ 10 0 %甲醇 /7 5min ,10 0 %甲醇保持 4 5min。 4种组分的回收率在 85 8%～ 10 1 5 %之间 ,相对标准偏差 (RSD) <5 5 4%。该法简单、快速、准确 ,可用于油脂及其制品中BHA、BHT、PG和TBHQ的日常检测。     

加速氧化测试辣椒茎叶提取物对大豆油氧化稳定性的影响

食用油氧化稳定性是产品的一个重要指标,影响产品的安全性和货架期。采用加速氧化测定法,通过测定过氧化值(PV)、β-茴香胺值(p-AV)、共轭二烯烃(CD)和共轭三烯烃(CT)等指标研究辣椒茎叶提取物(PLSE)对大豆油氧化稳定性的影响。结果显示,在加速氧化测定条件下空白对照组大豆油4个指标逐渐增加,抗氧化剂BHT、BHA、TBHQ和提取物PLSE对大豆油氧化具有不同程度的抑制作用;10、100、200mg/kg剂量组PLSE对大豆油4个氧化指标的抑制作用均小于TBHQ(200 mg/kg),10、100、200 mg/kg剂量组PLSE对大豆油氧化指标PV和p-AV的抑制作用强于BHT(200 mg/kg)和BHA(200 mg/kg);而仅200 mg/kg剂量组PLSE对大豆油氧化指标CD和CT的抑制作用强于BHT和BHA。     

Antioxidant and pro-oxidant activity of green tea extracts in marine oils

The activity of green tea extracts (GTE) on the oxidation of refined, bleached and deodorized (RBD) seal blubber oil (SBO) and menhaden oil (MHO) was examined under Schaal oven conditions at 65 °C. Progression of oxidation was monitored using weight gain, peroxide value (PV) and 2-thiobarbituric acid-reactive substances (TBARS) data. GTE exhibited a pro-oxidant effect in both oils examined, perhaps due to the catalytic effect of their chlorophyll constituents. Therefore, in follow-up experiments, a column chromatographic technique was employed to remove chlorophyll from GTE. The resultant dechlorophyllized green tea extract (DGTE) was applied to both SBO and MHO at 100, 200, 500 and 1000 ppm levels. The antioxidant activity of DGTE was compared with the effects of the commonly-used antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) at 200 ppm and α-tocopherol at 500 ppm. DGTE at ≥200 ppm exhibited excellent antioxidant activity in both oils and its efficacy was higher than that of BHA, BHT and α-tocopherol, but less than that of TBHQ.     

THERMAL STABILITY OF SOME COMMERCIAL NATURAL AND SYNTHETIC ANTIOXIDANTS AND THEIR MIXTURES

The thermal stability of some commercially available antioxidants alone or in binary or ternary mixtures was investigated in order to find the optimal combination of antioxidants for food processing. The synthetic antioxidants used were butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and tertiary-butyhydroquinone (TBHQ). Other antioxidants such as ascorbyl palmitate (AP), mixed tocopherols (TOCO) and monoacylglycerol citrate (MGC) were also employed. The effectiveness of these antioxidants and their combinations was assessed using the Rancimat test by measuring the induction period for the oxidation of sunflower oil after heating at 180C for 1 h and comparing it to the oxidation kinetics of the oil without added antioxidants. All antioxidants and their binary or ternary mixtures showed different degrees of thermal instability. TBHQ individually, among all the examined antioxidants, showed the highest thermal stability. On the other hand, AP as an antioxidant in sunflower oil exhibited low stability during heating. The thermal stability of AP could be enhanced by the addtion of BHA or BHT in binary mixtures at a ratio of 1:3 (w/w). In addition, the ternary mixture of AP, TOCO and MGC (65:25:10) in sunflower oil also showed a higher stability to thermal inactivation. However, the ternary mixture containing 0.13% AP, 0.05% TBHQ and 0.02% MGC provided the optimal protection during thermal treatment. Although a combination of 0.13% BHT, 0.05% AP and 0.02% MG was very effective synergistically at room temperature, it showed a higher susceptibility to thermal inactivation. It was     

气相色谱法测定饼干中的抗氧化剂BHA、BHT与TBHQ

建立了气相色谱法同时测定饼干中的3种抗氧化剂丁基羟基茴香醚（BHA）、二丁基羟基甲苯（B肿）和特丁基对苯二酚（TBHQ）含量的方法。样品采用无水乙醇提取,经净化、离心后直接进样测定。试验得出方法最低检出限BHA和BHT为1．0mg／kg,TBHQ为2．0mg／kg,3种抗氧化剂加标回收率在86．70％-103．02％之间,相对偏差均小于3％,线性范围在2μg／mL-200μg／mL之间,相关系数均大于0．999。该方法简便、快速、稳定、可靠,适用于饼干中BHA、BHT和TBHQ含量的同时快速检测。