Article pubs.acs.org/JAFC
Simultaneous Analysis of Tertiary Butylhydroquinone and 2-tert-Butyl-1,4-benzoquinone in Edible Oils by Normal-Phase High-Performance Liquid Chromatography Jun Li, Yanlan Bi,* Wei Liu, and Shangde Sun Lipid Technology and Engineering, School of Food Science and Engineering, Henan University of Technology, Lianhua Road, Zhengzhou, Henan 450001, People’s Republic of China S Supporting Information *
ABSTRACT: During the process of antioxidation of tertiary butylhydroquinone (TBHQ) in oil and fat systems, 2-tert-butyl-1,4benzoquinone (TQ) can be formed. The toxicity of TQ was much more than that of TBHQ. In the work, a normal-phase highperformance liquid chromatography (NP-HPLC) method for the accurate and simultaneous detection of TBHQ and TQ in edible oils was investigated. A C18 column was used to separate TBHQ and TQ, and the gradient elution solutions consisted of n-hexane containing 5% ethyl acetate and n-hexane containing 5% isopropanol. The ultraviolet (UV) detector was set at dual wavelength mode (280 nm for TBHQ and 310 nm for TQ). The column temperature was 30 °C. Before the NP-HPLC analysis, TBHQ and TQ were first extracted by methanol, subjected to vortex treatment, and then filtered through a 0.45 μm membrane filter. Results showed that linear ranges of TBHQ and TQ were both within 0.10−500.00 μg/mL (R2 > 0.9999). The limit of detection (LOD) and limit of quantification (LOQ) of TBHQ and TQ were below 0.30 and 0.91 μg/mL and below 0.10 and 0.30 μg/mL, respectively. The recoveries of TBHQ and TQ were 98.92−102.34 and 96.28−100.58% for soybean oil and 96.11− 99.42 and 98.83−99.24% for lard, respectively. These results showed that NP-HPLC can be successfully used to analyze simultaneously TBHQ and TQ in the oils and fats. KEYWORDS: TBHQ, TQ, NP-HPLC, oxidation, reduction
■
INTRODUCTION Tertiary butylhydroquinone (TBHQ) is a kind of synthetic antioxidant, which has been widely used in edible oils and oleaginous foods.1,2 However, the maximum legal level established by the U.S. Food and Drug Administration for synthetic antioxidants in oils or foods was 0.02% of the oil content of the food.3 Moreover, some studies have indicated that the safety of TBHQ had been queried as a result of its potential risk.4,5 In addition, during the storage of edible oils and oleaginous foods with TBHQ at room temperature, TBHQ can be easily oxidized to 2-tert-butyl-1,4-benzoquinone (TQ) in the presence of metal ions (such as Fe3+ and Cu2+), oxygen, hydroperoxides, etc.6 In the frying system, TBHQ can also be oxidized to TQ by oxygen, hydroperoxides, etc. (Figure 1).7 However, the level of TQ in the frying system was less than that of room temperature because of rapid volatilization of TBHQ.8−10 Besides, some relevant studies also indicated that TQ has high toxicity, and its toxicity was much more than that of TBHQ.11−15 Braeuning et al.11 had reported that TBHQ and TQ were both toxic and the level of TQ making murine 3T3 cells dead was only one tenth of that of TBHQ. Morimoto et al.12 found that 0.0001% TQ can cause DNA damage, and the DNA damaging activity of TQ was 100 times higher than that of TBHQ. Schilderman et al.13 reported that TQ had the capability of inducing oxidative DNA damage. Matsunaga et al.14 found that TQ was more cytotoxic toward endothelial cells than TBHQ. Kahl et al.15 also found that toxicity of TQ was more than that of TBHQ. Therefore, considering the safety of edible oils with TBHQ, it was very necessary that a method for © 2015 American Chemical Society
accurate qualitative and quantitative detections of TQ should be developed. However, there were only a few studies on the qualitative analysis of TQ separated from the oxidation products of TBHQ in heated air and heated vegetable oils.1,16−19 No relevant studies on the quantitative detection method of TQ in edible oils was found. At present, RP-HPLC equipped with different detection systems was the most common analytical method for qualitative and quantitative detections of TBHQ in edible oils and oleaginous foods.20−22 During the analysis process, TQ can be reduced to TBHQ by water and acid from mobile phases of reverse-phase high-performance liquid chromatography (RPHPLC), which resulted in the content of TQ detected lower than that of actual TQ and the content of TBHQ detected higher than that of actual TBHQ. Therefore, it was crucial to detect accurately contents of TBHQ and TQ to prevent the redox reaction between TBHQ and TQ. In the work, a normal-phase (NP)-HPLC method was developed to analyze accurately and quantitatively TBHQ and TQ in edible oils. The method can be used to evaluate the original added load of TBHQ in edible oils. Moreover, TBHQ and TQ were analyzed in three kinds of commercial edible vegetable oils, including soybean oil (SBO), edible blended oil, and sunflower seed oil. Received: Revised: Accepted: Published: 8584
June 17, 2015 September 6, 2015 September 12, 2015 September 12, 2015 DOI: 10.1021/acs.jafc.5b03002 J. Agric. Food Chem. 2015, 63, 8584−8591
Article
Journal of Agricultural and Food Chemistry
Figure 1. Two pathways of TBHQ converting to TQ.
■
MATERIALS AND METHODS
Chemicals. Standards of TBHQ (purity > 99.0%) and TQ (purity > 98.0%) were purchased from Sigma-Aldrich (St. Louis, MO). Fresh refined SBO (acid value of 0.13 mg of KOH/g and peroxide value of 1.22 mmol/kg) with no synthetic antioxidants was obtained from Henan Sunshine Oils and Fats Co., Ltd. (Zhengzhou, China). Fresh refined lard (acid value of 0.10 mg of KOH/g and peroxide value of 1.03 mmol/kg) with no antioxidants was purchased from Tianjin Tianyuan Oils & Fats Co., Ltd. (Tianjin, China). Liquid chromatography (LC) solvents (methanol, n-hexane, isopropanol, ethyl acetate, and acetonitrile) were of HPLC grade, obtained from VBS Biologic, Inc. (New York), and used after filtration by a 0.45 μm organic membrane. Glacial acetic acid (purity > 99.8%, HPLC grade) was purchased from Kerch Chemical Reagent Co., Ltd. (Tianjin, China). All other solvents were analytical-grade and used without further purification. Apparatus. The RP-HPLC spectra were measured with a 2695 separation module and an ultraviolet (UV)/visible-2489 detector (Waters, Milford, MA). The NP-HPLC spectra were measured with a 2695 separation module and an UV/visible-2487 detector (Waters, Milford, MA). The vortex mixer (Staufen, German), SCQ-250B ultrasonic apparatus (Shanghai, China), and 800 low-speed centrifuge (Jintan, China) were also used in the experiments. Preparation and Storage of TBHQ or TQ Standard Solutions. To prepare a standard stock solution, 0.10 g (accurately weighted to 0.1 mg, similarly hereinafter) of TBHQ or TQ was individually weighted into a 100 mL amber volumetric flask and dissolved with methanol. The stock solutions were stored in a refrigerator (4 °C) and away from light for a maximum of 1 month. Before use, standard working solutions of variable concentrations (0.10, 1.00, 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, 200.00, and 500.00 μg/mL) were prepared daily by diluting appropriate volumes of the stock solution in methanol. Preparation of SBO or Lard Containing 2000 mg/kg of TBHQ or TQ. Lard was preheated at 40 °C until completely melted. A total of 0.12 g of TBHQ or TQ was added to 59.88 g of refined SBO or lard and then shaken until homogeneous. Preparation of SBO or Lard Containing 50, 100, and 200 mg/kg of TBHQ or TQ. Lard was preheated at 40 °C until completely melted. A total of 1.50, 3.00, and 6.00 g of SBO or lard containing 2000 mg/kg of TBHQ or TQ were diluted to 60.00 g with control SBO or lard and then shaken until homogeneous. Preparation of SBO or Lard Containing 150 mg/kg of TBHQ Plus 50 mg/kg of TQ, 100 mg/kg of TBHQ Plus 100 mg/kg of TQ, and 50 mg/kg of TBHQ Plus 150 mg/kg of TQ. Lard was preheated at 40 °C until completely melted. A total of 4.50, 3.00, or 1.50 g of SBO or lard containing 2000 mg/kg of TBHQ and 1.50, 3.00, or 4.50 g of SBO or lard containing 2000 mg/kg of TQ were diluted to 60.00 g with SBO or lard without TBHQ and TQ and then shaken until homogeneous.
Figure 2. UV absorption spectra of TBHQ and TQ. Extraction Procedure. The extraction procedure was in accordance with previous methods.16,21,23 A total of 0.50 g of each of the oil samples was weighed into a 10 mL test tube, and 4 mL of methanol was added. The mixture was agitated for 3 min using a vortex mixer and then centrifuged at 3000 rpm for 2 min at room temperature. The supernatant was quantitatively transferred to a 10 mL flask. The extraction procedure was repeated twice, adding 3 mL of methanol each time. Finally, sufficient methanol was added to achieve a 10 mL solution. The solution was filtered through a 0.45 μm membrane before HPLC analysis. Analysis of TBHQ and TQ. The conditions of RP-HPLC analysis were as follows:16,24,25 the injection volume for TBHQ or TQ samples was 20 μL; the column was Symmetry C18 (4.6 × 250 mm, 5 μm, Waters); the solvent system was methanol−H2O containing 0.5% AcOH (65:35, v/v); the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 35 °C. Methanol and H2O containing 0.5% AcOH were both degassed for 15 min by an ultrasonic apparatus. The conditions of NP-HPLC analysis were as follows: the injection volume for TBHQ or TQ samples was 20 μL; the column was Sunfire Prep Silica (4.6 × 250 mm, 5 μm, Waters); the solvent system was n-hexane containing 5% ethyl acetate (solvent A) and n-hexane containing 5% isopropanol (solvent B); the gradient elution was isocratic mode of 8% solvent B for 10 min, linear gradient from 8 to 100% solvent B for 5 min, and then isocratic mode of 100% solvent B for 12 min; the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 30 °C. Solvents A and B were both degassed for 40 min by an ultrasonic apparatus. Identification of compounds was 8585
DOI: 10.1021/acs.jafc.5b03002 J. Agric. Food Chem. 2015, 63, 8584−8591
Article
Journal of Agricultural and Food Chemistry
Figure 3. Superimposed RP-HPLC chromatograms of variable concentrations of (A) TQ standard solutions, (B) methanol extracting solutions of SBO adding 50, 100, and 200 mg/kg of TQ, and (C) methanol extracting solutions of lard adding 50, 100, and 200 mg/kg of TQ. Analysis conditions: the injection volume for TQ samples was 20 μL; the column was Symmetry C18 (4.6 × 250 mm, 5 μm, Waters); the solvent system was methanol−H2O containing 0.5% AcOH (65:35, v/v); the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 35 °C. Methanol and H2O containing 0.5% AcOH were both degassed for 15 min by an ultrasonic apparatus. achieved by comparing their retention times to those of standards. Data were collected and processed using Empower 2.0 software (Waters Corporation). Statistical Analyses. All experiments were performed at least in duplicate. All data were presented as the mean ± standard deviation (SD).
absorption wavelengths of TBHQ were 206, 227, and 292 nm. The characteristic absorption wavelengths of TQ were 248 and 310 nm. According to the American Oil Chemists’ Society (AOCS) method,24 280 nm was selected as the appropriate detection wavelength for TBHQ. As a result of 248 nm less than the UV cutoff wavelength of ethyl acetate (254 nm)26 and weak absorption of TQ at 280 nm (Figure 2), 310 nm was selected as an appropriate detection wavelength for TQ. Finally,
■
RESULTS AND DISCUSSION Wavelength Screening. The UV absorption spectra of TBHQ and TQ were shown in Figure 2. The characteristic 8586
DOI: 10.1021/acs.jafc.5b03002 J. Agric. Food Chem. 2015, 63, 8584−8591
Article
Journal of Agricultural and Food Chemistry dual wavelengths (280 and 310 nm) were selected to detect TBHQ and TQ. Determination of TBHQ by RP-HPLC. Specificity, Linearity, Limit of Detection (LOD), and Limit of Quantification (LOQ) of TBHQ. Specificity of the RP method for TBHQ detection was verified by comparing the chromatograms of TBHQ standard solution, oil samples, and lard samples before and after spiking with TBHQ. In oil and lard blank samples, no signal of TBHQ was detected. The calibration curve was constructed by plotting the measured peak area versus the concentration of TBHQ. Excellent linearity was obtained within the concentration range of 0.10−500.00 μg/mL for TBHQ (R2 > 0.9999). The instrumental LOD was estimated and defined as the average response (n = 3) of the lower concentration level of the calibration curve for TBHQ plus 3 times the standard deviation.27 The instrumental LOQ was taken as 10/3.3 times LOD.28 The results showed that LOD and LOQ of TBHQ were below 0.06 and 0.18 μg/mL, respectively, which were both lower than that reported by Saad et al.20 Intra- and Interday Precision and Accuracy of TBHQ. The precision of the RP-HPLC system was demonstrated by intraand interday variation studies.29 In the intraday studies, six repeated injections of TBHQ standard solutions (5.00, 10.00, and 20.00 μg/mL) were made in 1 day and its relative standard deviation (RSD) and bias were calculated. In the interday variation studies, three repeated injections of TBHQ standard solutions (5.00, 10.00, and 20.00 μg/mL) were performed on 3 consecutive days and its RSD and bias were also calculated. Results showed that intra- and interday precisions of TBHQ with the RP-HPLC system were acceptable, with its RSD lower than 0.58% and bias lower than 1.52% (Table S1 of the Supporting Information). Recoveries and Precision of TBHQ in SBO or Lard. Recoveries of TBHQ were determined using SBO or lard spiking with TBHQ. The recovery experiments were carried out at three levels of 50, 100, and 200 mg/kg (n = 6), and the percentage recoveries were calculated and presented in Table S2 of the Supporting Information. Recoveries of TBHQ in SBO and lard were in the ranges of 96.45−99.02 and 94.23−99.56%, respectively, with the RSD less than 0.64 and 0.45%. Determination of TQ by RP-HPLC. Specificity of TQ. Verification of specificity of the RP-HPLC method for TQ detection was similar to that of TBHQ. In oil or lard blank samples, no signal of TQ was detected. However, in TQ standard solution (Figure 3A) and oil samples (Figure 3B) and lard samples (Figure 3C) spiking with TQ, there were all signals of TBHQ and the peaks were symmetric, which indicated that, during the injection, TQ was reduced to TBHQ by reduced hydrogen ([H]). Reduction ratios of TQ were calculated (Figure S1 of the Supporting Information), and specific results were presented in Tables 1 and 2. The [H] was from the mobile phase (water and acid solution) of the RP-HPLC system (Figure S2 of the Supporting Information). Determination of TBHQ and TQ by NP-HPLC. Specificity, Linearity, LOD, and LOQ of TBHQ and TQ. Verification of specificity of the NP-HPLC method for TBHQ and TQ detections was also similar to that of the RP-HPLC method for TBHQ. In oil or lard blank samples, no signal of TBHQ and TQ was detected. In TBHQ standard solution, no signal of TQ was detected (Figure 4A). In TQ standard solution, no signal of TBHQ was also detected (Figure 4B).
Table 1. Reduction Ratios and Precision of Variable Concentrations of TQ Standard Solutions with the RPHPLC Systema concentration of TQ (μg/mL) 1.00 2.00 5.00 10.00 20.00 50.00 100.00 200.00 500.00
reduction yield of TQ (%)b
RSD (%)
± ± ± ± ± ± ± ± ±
3.77 1.44 4.37 5.23 7.73 10.99 9.77 4.41 11.64
9.55 9.70 8.70 8.03 7.24 5.55 4.30 3.17 1.89
0.36 0.14 0.38 0.42 0.56 0.61 0.42 0.14 0.22
a Analysis conditions: the injection volume for TQ samples was 20 μL; the column was Symmetry C18 (4.6 × 250 mm, 5 μm, Waters); the solvent system was methanol−H2O containing 0.5% AcOH (65:35, v/v); the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 35 °C. Methanol and H2O containing 0.5% AcOH were both degassed for 15 min by an ultrasonic apparatus. bMean ± SD.
Table 2. Reduction Ratios and Precision of the Spiked TQ from SBO and Lard with the RP-HPLC Systema sample SBO
lard
concentration of TQ (mg/kg) 50 100 200 50 100 200
reduction yield of TQ (%)b
RSD (%)
± ± ± ± ± ±
5.89 7.15 10.13 0.62 2.30 2.74
7.98 8.26 5.94 5.71 6.14 5.15
0.47 0.59 0.60 0.04 0.14 0.14
Analysis conditions: the injection volume for TQ samples was 20 μL; the column was Symmetry C18 (4.6 × 250 mm, 5 μm, Waters); the solvent system was methanol−H2O containing 0.5% AcOH (65:35, v/v); the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 35 °C. Methanol and H2O containing 0.5% AcOH were both degassed for 15 min by an ultrasonic apparatus. bMean ± SD. a
These results indicated that the redox reaction of TBHQ and TQ did not happen during the NP-HPLC analysis. Constructions of calibration curves of TBHQ and TQ with the NP-HPLC system were similar to that of TBHQ with the RP-HPLC system. Excellent linearity was obtained within the concentration range of 0.10−500.00 μg/mL for TBHQ and TQ (R2 > 0.9999). The estimations and definitions of instrumental LOD and LOQ with the NP-HPLC system were similar to that of the RPHPLC system. Results showed that LOD and LOQ of TBHQ were below 0.30 and 0.91 μg/mL, respectively, which were both lower than those of Saad et al.20 The LOD and LOQ of TQ were below 0.10 and 0.30 μg/mL, respectively. Intra- and Interday Precision and Accuracy of TBHQ and TQ. The demonstration of the precision of the NP-HPLC system was similar to that of the RP-HPLC system. Corresponding RSD and bias were calculated and presented in Table S3 of the Supporting Information. Results showed that intra- and interday precisions of TBHQ and TQ with the NP-HPLC system were acceptable, with the RSD lower than 1.76% and the bias lower than 5.55%. Selection of the Extraction Solvent. In general, phenolic antioxidants in edible oils can often be extracted using some polar solvents, such as methanol, acetonitrile, etc.30 Nonpolar 8587
DOI: 10.1021/acs.jafc.5b03002 J. Agric. Food Chem. 2015, 63, 8584−8591
Article
Journal of Agricultural and Food Chemistry
Figure 4. NP-HPLC chromatograms of (A) TBHQ and (B) TQ standard solutions. Analysis conditions: the injection volume for TBHQ or TQ samples was 20 μL; the column was Sunfire Prep Silica (4.6 × 250 mm, 5 μm, Waters); the solvent system was n-hexane containing 5% ethyl acetate (solvent A) and n-hexane containing 5% isopropanol (solvent B); the gradient elution was isocratic mode of 8% solvent B for 10 min, linear gradient from 8 to 100% solvent B for 5 min, and then isocratic mode of 100% solvent B for 12 min; the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 30 °C. Solvents A and B were both degassed for 40 min by an ultrasonic apparatus.
Table 3. Recoveries and Precision of the Spiked TBHQ or TQ from SBO and Lard Determined by NP-HPLCa TBHQ (n = 6) sample
added (mg/kg)
SBO
50 100 200 50 100 200
lard
TQ (n = 6)
recovery (%)b 98.92 100.69 102.34 96.17 99.42 96.11
± ± ± ± ± ±
RSD (%)
added (mg/kg)
0.38 0.38 0.36 1.28 0.57 2.06
50 100 200 50 100 200
0.38 0.39 0.37 1.23 0.56 1.98
recovery (%)b 100.58 96.68 96.28 98.83 99.24 99.00
± ± ± ± ± ±
RSD (%)
1.73 0.29 0.55 3.54 1.28 0.39
1.71 0.30 0.58 3.58 1.29 0.39
Analysis conditions: the injection volume for TBHQ or TQ samples was 20 μL; the column was Sunfire Prep Silica (4.6 × 250 mm, 5 μm, Waters); the solvent system was n-hexane containing 5% ethyl acetate (solvent A) and n-hexane containing 5% isopropanol (solvent B); the gradient elution was isocratic mode of 8% solvent B for 10 min, linear gradient from 8 to 100% solvent B for 5 min, and then isocratic mode of 100% solvent B for 12 min; the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 30 °C. Solvents A and B were both degassed for 40 min by an ultrasonic apparatus. bMean ± SD. a
Table 4. Recoveries and Precision of the Spiked TBHQ and TQ from SBO and Lard Determined by NP-HPLCa TBHQ/TQ sample
added ((mg/kg)/(mg/kg))
SBO
150/50 100/100 50/150 150/50 100/100 50/150
lard
recovery of TBHQ (%)b
RSD (%)
± ± ± ± ± ±
1.00 0.91 0.87 0.59 2.33 0.67
95.12 96.73 97.41 98.42 96.73 94.14
0.95 0.88 0.85 0.58 2.26 0.63
recovery of TQ (%)b 101.57 97.86 97.57 101.61 98.85 97.87
± ± ± ± ± ±
0.17 0.18 0.17 0.80 2.19 0.92
RSD (%) 0.17 0.18 0.17 0.79 2.21 0.94
recovery of TBHQ plus TQ (%)b
RSD (%)
± ± ± ± ± ±
0.96 0.22 0.51 0.43 2.25 0.86
98.72 97.96 97.83 99.30 97.81 96.93
0.95 0.22 0.50 0.43 2.20 0.83
Analysis conditions: the injection volume for TBHQ and TQ samples was 20 μL; the column was Sunfire Prep Silica (4.6 × 250 mm, 5 μm, Waters); the solvent system was n-hexane containing 5% ethyl acetate (solvent A) and n-hexane containing 5% isopropanol (solvent B); the gradient elution was isocratic mode of 8% solvent B for 10 min, linear gradient from 8 to 100% solvent B for 5 min, and then isocratic mode of 100% solvent B for 12 min; the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 30 °C. Solvents A and B were both degassed for 40 min by an ultrasonic apparatus. bMean ± SD. a
solvents, such as n-hexane, can often be used to remove the oil matrix coextracted. However, because of the polarity of TQ of
less than that of TBHQ, TQ can be easily dissolved in nonpolar solvents, such as n-hexane. To obtain a specific distribution 8588
DOI: 10.1021/acs.jafc.5b03002 J. Agric. Food Chem. 2015, 63, 8584−8591
Article
Journal of Agricultural and Food Chemistry
Table 5. Contents of TBHQ and TQ in SBO or Lard Containing 200 mg/kg of TBHQ during Storage at Room Temperature Determined by NP-HPLCa SBO storage time (week) 0 2 4 6 8
C2 (mg/kg)b 197.35 183.50 182.98 180.79 178.59
± ± ± ± ±
C3 (mg/kg)b
0.56 0.93 0.14 0.37 1.21
0.00 11.17 14.17 16.61 16.15
± ± ± ± ±
0.00 0.51 0.42 0.68 0.81
lard C1 (mg/kg)b 197.35 194.67 197.15 197.40 194.74
± ± ± ± ±
C0 (mg/kg)
0.56 1.44 0.56 1.05 2.02
200 200 200 200 200
C2 (mg/kg)b 198.50 154.37 146.99 108.22 87.25
± ± ± ± ±
0.19 1.36 0.31 2.01 0.94
C3 (mg/kg)b 0.00 41.79 51.07 77.25 95.32
± ± ± ± ±
C1 (mg/kg)b
0.00 0.83 0.71 0.89 0.64
198.50 196.16 198.06 185.47 182.57
± ± ± ± ±
C0 (mg/kg)
0.19 2.19 1.02 2.90 1.58
200 200 200 200 200
Analysis conditions: the injection volume for TBHQ and TQ samples was 20 μL; the column was Sunfire Prep Silica (4.6 × 250 mm, 5 μm, Waters); the solvent system was n-hexane containing 5% ethyl acetate (solvent A) and n-hexane containing 5% isopropanol (solvent B); the gradient elution was isocratic mode of 8% solvent B for 10 min, linear gradient from 8 to 100% solvent B for 5 min, and then isocratic mode of 100% solvent B for 12 min; the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 30 °C. Solvents A and B were both degassed for 40 min by an ultrasonic apparatus. C0, original added load of TBHQ in SBO or lard; C1, content of TBHQ plus TQ detected in SBO or lard; C2, content of TBHQ detected in SBO or lard; and C3, content of TQ detected SBO or lard. bMean ± SD. a
Table 6. Contents of TBHQ and TQ in Some Commercial Vegetable Oils Determined by NP-HPLCa original commercial oils sample type SBO
edible blended oil sunflower seed oil
TBHQ (mg/kg)b
TQ (mg/kg)
± ± ± ± ± ±
−c − − − − −
64.46 26.19 73.44 19.36 90.18 57.85
0.12 0.30 0.55 1.70 0.67 2.47
commercial oils stored for 2 months without seal
TBHQ plus TQ (mg/kg)b 64.46 26.19 73.44 19.36 90.18 57.85
± ± ± ± ± ±
0.12 0.30 0.55 1.70 0.67 2.47
TBHQ (mg/kg)b 56.28 23.18 64.75 16.07 76.62 46.34
± ± ± ± ± ±
0.41 0.09 0.29 0.15 1.32 0.85
TQ (mg/kg)b 7.02 3.53 9.96 3.82 12.18 10.09
± ± ± ± ± ±
0.39 0.52 0.11 0.22 0.48 0.42
TBHQ plus TQ (mg/kg)b
conversion yield of TBHQ (%)
± ± ± ± ± ±
10.89 13.48 13.62 19.73 13.51 17.44
63.30 26.72 74.72 19.89 88.79 56.43
0.44 0.61 0.18 0.37 1.80 0.43
Analysis conditions: the injection volume for TBHQ and TQ samples was 20 μL; the column was Sunfire Prep Silica (4.6 × 250 mm, 5 μm, Waters); the solvent system was n-hexane containing 5% ethyl acetate (solvent A) and n-hexane containing 5% isopropanol (solvent B); the gradient elution was isocratic mode of 8% solvent B for 10 min, linear gradient from 8 to 100% solvent B for 5 min, and then isocratic mode of 100% solvent B for 12 min; the flow rate was 0.8 mL/min; the dual detection wavelengths were 280 nm for TBHQ and 310 nm for TQ; and the column temperature was 30 °C. Solvents A and B were both degassed for 40 min by an ultrasonic apparatus. bMean ± SD. c“−” represents no TBHQ or TQ found. a
ratio of TQ in n-hexane and methanol (or acetonitrile), 5 mL of n-hexane was added to 5 mL of 500.00 μg/mL TQ methanol solution (or acetonitrile solution). The mixtures were mixed for 2 min using a vortex mixer and then centrifuged at 3000 rpm for 2 min at room temperature. After filteration using 0.45 μm organic membrane, the n-hexane fraction and methanol fraction (or acetonitrile fraction) were analyzed. Results showed that the distribution ratios of TQ in n-hexane and methanol were 44.91 and 54.62%, respectively; the distribution ratios of TQ in n-hexane and acetonitrile were 33.84 and 66.16%, respectively. Therefore, in the work, if nonpolar solvents were used to remove the oil matrix coextracted, approximately half or onethird of TQ was removed from methanol or acetonitrile extracting solution and dissolved in nonpolar solvents. In addition, mobile phases of NP-HPLC were low polarity or nonpolar solvents,31,32 which can easily dissolve the oil matrix coextracted. The oil matrix coextracted could be eluted from stationary phase by mobile phases, which has little effect on the lifetime of the silica gel column and the baseline (Figure S3 of the Supporting Information). Therefore, the process of removing the oil matrix coextracted could be left out. In addition, considering the higher toxicity and cost of acetonitrile than that of methanol,33 methanol was used to extract TBHQ and TQ from the edible oils. Recoveries and Precision of TBHQ or TQ in Edible Oil Samples. The determination of recoveries of TBHQ or TQ with the NP-HPLC system was similar to that of TBHQ with
the RP-HPLC system. The recoveries were calculated and presented in Table 3. Recoveries of TBHQ in SBO and lard were in the ranges of 98.92−102.34 and 96.11−99.42%, with the RSD less than 0.38 and 2.06%, respectively. Recoveries of TQ in SBO and lard were in the ranges of 96.28−100.58 and 98.83−99.24%, with the RSD less than 1.71 and 3.58%, respectively. Recoveries and Precision of TBHQ and TQ in Edible Oil Samples. Recoveries of TBHQ and TQ were determined using SBO and lard spiking with TBHQ and TQ. The recovery experiments were carried out at three levels of 150/50, 100/ 100, and 50/150 (mg/kg)/(mg/kg) (n = 6), and the recoveries of TBHQ and TQ were calculated and presented in Table 4. Recoveries of TBHQ and TQ in SBO and lard were in the ranges of 97.83−98.72 and 96.93−99.30%, with the RSD less than 0.96 and 2.25%, respectively, which indicated that the NP-HPLC method can be used to evaluate the original added load of TBHQ in edible oils (Figure S4 of the Supporting Information). Analysis of Contents of TBHQ and TQ in SBO and Lard during Storage at Room Temperature. On the basis of the NP-HPLC method, the contents of TBHQ and TQ in SBO and lard containing 200 mg/kg of TBHQ during storage at room temperature were determined. Results showed that, with the increase of the storage time, the contents of TBHQ in SBO and lard decreased gradually, while the contents of TQ in SBO and lard increased (Table 5). For example, the original added 8589
DOI: 10.1021/acs.jafc.5b03002 J. Agric. Food Chem. 2015, 63, 8584−8591
Journal of Agricultural and Food Chemistry load of TBHQ in SBO was 197.35% without TQ. After SBO samples were stored for 1 and 2 months, the contents of TBHQ were 182.98 and 178.59 mg/kg, respectively, and the contents of TQ were 14.17 and 16.15 mg/kg, respectively. The original added load of TBHQ in lard was 198.50% without TQ as well. After lard samples were stored for 1 and 2 months, the contents of TBHQ were 146.99 and 87.25 mg/kg, respectively, and the contents of TQ were 51.07 and 95.32 mg/kg, respectively. Meanwhile, contents of TBHQ converting to TQ in SBO were less than those in lard, which might be attributed to the presence of tocopherols (approximately 1000 mg/kg34) in SBO. Partial oxidation of tocopherols can consume partial oxygen dissolved in SBO, which can slow the conversion of TBHQ to TQ. Analysis of TBHQ and TQ in Some Commercial Vegetable Oils. Some commercial oil samples were analyzed using the NPHPLC method. Results showed that TBHQ existed in all original oil samples but TQ was not detected, which might be no oxygen presented in the commercial bottled oils before opening. However, when the package of the original samples was unclosed and stored at 25 °C for 2 months, TQ can be detected (Table 6). Conversion ratios of TBHQ to TQ were in the ranges of 10−20% in all commercial vegetable oils tested. These results indicated that, after the continuous exposure of oils in air, TBHQ can be converted to TQ in the presence of oxygen and free radicals of oils. In addition, none of the positive samples violated the legal limit (200 mg/kg3). In conclusion, in comparison to the RP-HPLC system, the NP-HPLC method overcame the reduction of TQ, can simultaneously and accurately analyze the contents of TBHQ and TQ in edible oils, and was also successfully applied to evaluate the original added load of TBHQ in edible oils.
■
■
ABBREVIATIONS USED
■
REFERENCES
TBHQ, tertiary butylhydroquinone; TQ, 2-tert-butyl-1,4benzoquinone; SBO, soybean oil; HPLC, high-performance liquid chromatography; RP, reverse phase; NP, normal phase; [H], reduced hydrogen; LOD, limit of detection; LOQ, limit of quantification; SD, standard deviation; RSD, relative standard deviation
(1) Hamama, A. A.; Nawar, W. W. Thermal decomposition of some phenolic antioxidants. J. Agric. Food Chem. 1991, 39, 1063−1069. (2) Ramanathan, L.; Das, N. P. Studies on the control of lipid oxidation in ground fish by some polyphenolic natural products. J. Agric. Food Chem. 1992, 40, 17−21. (3) U.S. Food and Drug Administration (FDA). Code of Federal Regulations 21, Food and Drugs; Office of the Federal Register (OFR): Washington, D.C., 1980. (4) Macari, E. R.; Schaeffer, E. K.; West, R. J.; Lowrey, C. H. Simvastatin and t-butylhydroquinone suppress KLF1 and BCL11A gene expression and additively increase fetal hemoglobin in primary human erythroid cells. Blood 2013, 121, 830−839. (5) Eskandani, M.; Hamishehkar, H.; Ezzati Nazhad Dolatabadi, J. Cytotoxicity and DNA damage properties of tert-butylhydroquinone (TBHQ) food additive. Food Chem. 2014, 153, 315−320. (6) Choe, E.; Min, D. B. Mechanisms and factors for edible oil oxidation. Compr. Rev. Food Sci. Food Saf. 2006, 5, 169−186. (7) Kim, C. M.; Pratt, D. E. Degradation products of 2-tertbutylhydroquinone at frying temperature. J. Food Sci. 1990, 55, 847− 847. (8) Buck, D. F. Antioxidants in soya oil. J. Am. Oil Chem. Soc. 1981, 58, 275−278. (9) Asap, T.; Augustin, M. A. Effect of TBHQ on quality characteristics of RBD olein during frying. J. Am. Oil Chem. Soc. 1986, 63, 1169−1172. (10) Marmesat, S.; Morales, A.; Velasco, J.; Dobarganes, M. C. Action and fate of natural and synthetic antioxidants during frying. Grasas Aceites 2010, 61, 333−340. (11) Braeuning, A.; Vetter, S.; Orsetti, S.; Schwarz, M. Paradoxical cytotoxicity of tert-butylhydroquinone in vitro: what kills the untreated cells? Arch. Toxicol. 2012, 86, 1481−1487. (12) Morimoto, K.; Tsuji, K.; Iio, T.; Miyata, N.; Uchida, A.; Osawa, R.; Kitsutaka, H.; Takahashi, A. DNA damage in forestomach epithelium from male F344 rats following oral administration of tert-butylquinone, one of the forestomach metabolites of 3-BHA. Carcinogenesis 1991, 12, 703−708. (13) Schilderman, P.; Rhijnsburger, E.; Zwingmann, I.; Kleinjans, J. C. S. Induction of oxidative DNA damage and enhancement of cell proliferation in human lymphocytes in vitro by butylated hydroxyanisole. Carcinogenesis 1995, 16, 507−512. (14) Matsunaga, T.; Endo, S.; Takemura, M.; Soda, M.; Yamamura, K.; Tajima, K.; Miura, T.; Terada, T.; El-Kabbani, O.; Hara, A. Reduction of cytotoxic p-quinone metabolites of tert-butylhydroquinone by human aldo-keto reductase (AKR) 1B10. Drug Metab. Pharmacokinet. 2012, 27, 553−558. (15) Kahl, R.; Weinke, S.; Kappus, H. Production of reactive oxygen species due to metabolic activation of butylated hydroxyanisole. Toxicology 1989, 59, 179−194. (16) Li, J.; Bi, Y. L.; Liu, W.; Sun, S. D.; Liu, C. F.; Ma, S. M. Effect of acid value on TBHQ and BHT losses in heating oils: identification of the esterification products of TBHQ and free fatty acids. J. Am. Oil Chem. Soc. 2014, 91, 1763−1771. (17) Kurechi, T.; Aizawa, M.; Kunugi, A. Studies on the antioxidants XVIII: oxidation product of tertiary butyl hydroquinone (TBHQ) (I). J. Am. Oil Chem. Soc. 1983, 60, 1878−1882. (18) Lin, F. S.; Warner, C. R.; Fazio, T. Alteration of phenolic antioxidants in heated vegetable oil. J. Am. Oil Chem. Soc. 1981, 58, A789−A792.
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03002. Calculation of reduction ratios of TQ (Figure S1), reduction reaction of TQ by protonation (Figure S2), baseline of NP-HPLC before and after elution of the sample directly extracted with methanol (Figure S3), equation for calculating the original added load of TBHQ (Figure S4), values of the intra- and interday precision and accuracy assay of TBHQ determined by RP-HPLC (Table S1), recoveries and precision of the spiked TBHQ from SBO and lard determined by RP-HPLC (Table S2), and values of the intra- and interday precision and accuracy assay of TBHQ and TQ determined by NPHPLC (Table S3) (PDF)
■
Article
AUTHOR INFORMATION
Corresponding Author
*Telephone/Fax: +86-0371-67758022. E-mail: yanlanbi@ hotmail.com. Funding
This work was financially supported by the National Natural Science Foundation of China (31271883). Notes
The authors declare no competing financial interest. 8590
DOI: 10.1021/acs.jafc.5b03002 J. Agric. Food Chem. 2015, 63, 8584−8591
Article
Journal of Agricultural and Food Chemistry (19) Kurechi, T.; Kunugi, A. Studies on the antioxidants XIX: Photooxidation products of tertiary butyl hydroquinone (TBHQ) (II). J. Am. Oil Chem. Soc. 1983, 60, 1882−1887. (20) Saad, B.; Sing, Y. Y.; Nawi, M. A.; Hashim, N.; Mohamedali, A.; Saleh, M. I.; Sulaiman, S. F.; Talib, K. M.; Ahmad, K. Determination of synthetic phenolic antioxidants in food items using reversed-phase HPLC. Food Chem. 2007, 105, 389−394. (21) Perrin, C.; Meyer, L. Quantification of synthetic phenolic antioxidants in dry foods by reversed-phase HPLC with photodiode array detection. Food Chem. 2002, 77, 93−100. (22) Li, X. Q.; Ji, C.; Sun, Y. Y.; Yang, M. L.; Chu, X. G. Analysis of synthetic antioxidants and preservatives in edible vegetable oil by HPLC/TOF-MS. Food Chem. 2009, 113, 692−700. (23) Guo, L.; Xie, M. Y.; Yan, A. P.; Wan, Y. Q.; Wu, Y. M. Simultaneous determination of five synthetic antioxidants in edible vegetable oil by GC−MS. Anal. Bioanal. Chem. 2006, 386, 1881−1887. (24) American Oil Chemists’ Society (AOCS). AntioxidantsLipid chromatographic method. Official Methods and Recommended Practices of the American Oil Chemists’ Society, 5th ed.; AOCS Press: Champaign, IL, 1997; Official Method Ce 6-8. (25) AOAC International. Phenolic antioxidants in oils, fats and butter oil liquid chromatographic method. Official Methods of the Association of Official Analytical Chemists; AOAC Press: Rockville, MD, 1994; Official Method 983.15. (26) Yao, S.; Luo, J. G.; Huang, X. F.; Kong, L. Y. Application of preparative high-speed counter-current chromatography/preparative high-performance liquid chromatography mode in rapid separation of saponins. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2008, 864, 69−77. (27) Koesukwiwat, U.; Jayanta, S.; Leepipatpiboon, N. Validation of a liquid chromatography-mass spectrometry multi-residue method for the simultaneous determination of sulfonamides, tetracyclines and pyrimethamine in milk. J. Chromatogr. A 2007, 1140, 147−156. (28) Wang, J. Y.; Wu, H. L.; Sun, Y. M.; Gu, H. W.; Liu, Z.; Liu, Y. J.; Yu, R. Q. Simultaneous determination of phenolic antioxidants in edible vegetable oils by HPLC−FLD assisted with second-order calibration based on ATLD algorithm. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2014, 947−948, 32−40. (29) Chen, M.; Hu, X. J.; Tai, Z. G.; Qin, H.; Tang, H. N.; Liu, M. S.; Yang, Y. L. Determination of four synthetic phenolic antioxidants in edible oils by high-performance liquid chromatography with cloud point extraction using Tergitol TMN-6. Food Anal. Method. 2013, 6, 28−35. (30) Antolovich, M.; Prenzler, P.; Robards, K.; Ryan, D. Sample preparation in the determination of phenolic compounds in fruits. Analyst 2000, 125, 989−1009. (31) Ansari, G. A. S. High-performance liquid chromatographic separation of the isomers of butylated hydroxyanisole. J. Chromatogr. A 1983, 262, 393−396. (32) Panfili, G.; Fratianni, A.; Irano, M. Normal phase highperformance liquid chromatography method for the determination of tocopherols and tocotrienols in cereals. J. Agric. Food Chem. 2003, 51, 3940−3944. (33) Concha-Herrera, V.; Torres-Lapasió, J. R.; Vivó-Truyols, G.; García-Á lvarez-Coque, M. C. A comparative study of the performance of acetonitrile and methanol in the multi-linear gradient separation of proteic primary amino acids. Anal. Chim. Acta 2007, 582, 250−258. (34) Lim, H.; Woo, S.; Kim, H. S.; Jong, S.-K.; Lee, J. Comparison of extraction methods for determining tocopherols in soybeans. Eur. J. Lipid Sci. Technol. 2007, 109, 1124−1127.
8591
DOI: 10.1021/acs.jafc.5b03002 J. Agric. Food Chem. 2015, 63, 8584−8591
