Identification of In Vitro Metabolites of Synthetic Phenolic Antioxidants BHT, BHA, and TBHQ by LC-HRMS/MS

Butylated hydroxytoluene (BHT) and its analogs, butylated hydroxyanisole (BHA) and tert-butyl-hydroquinone (TBHQ), are widely used synthetic preservatives to inhibit lipid oxidation in the food, cosmetic and pharmaceutical industries. Despite their widespread use, little is known about their human exposure and related biotransformation products. The metabolism of these compounds was investigated using in vitro incubations with human and rat liver fractions. Liquid chromatography coupled to high-resolution tandem mass spectrometry was employed to detect and characterize stable and reactive species formed via oxidative metabolism, as well as phase II conjugates. Several oxidative metabolites have been detected, as well as glutathione, glucuronide, and sulfate conjugates, many of which were not previously reported. A combination of accurate mass measurements, MS/MS fragmentation behavior, and isotope-labeling studies were used to elucidate metabolite structures.


Introduction
Synthetic phenolic antioxidants were developed in the late 1940s [1]. They have been used in food, pharmaceuticals, cosmetic, and petrochemical industries to increase shelf life and to improve the quality, freshness, taste, and texture of consumer products [2]. They are widely used to trap free radicals and delay lipid oxidation in various products [1]. Despite their widespread use, very little is known about human exposure or environmental emissions, which has led to public concern about their health effects and environmental contamination [3]. Butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butyl-hydroquinone (TBHQ) are among the most used synthetic phenolic antioxidants [4].
BHT, the most frequently used synthetic phenolic antioxidant, is added to food, pharmaceuticals, and cosmetics, as well as an additive in rubber, plastics, mineral oil, and printing inks [5,6]. Reports on BHT toxicity and side effects have been somewhat contradictory. Some studies have shown positive effects of BHT, such as enhancing the intracellular levels of glutathione and related enzymes in rat [7], protecting against cancer due to its antioxidant activity [8], and having tumor reducing effects [6]. On the other hand, it has been shown to cause renal and hepatic damage in rats, increase liver weight, decrease the activities of several hepatic enzymes and exhibit toxic effects in lung tissue [6]. BHT toxicity has been mainly attributed to its metabolism. For instance, Nagai et al. reported that BHT-quinone, one of the major metabolites of BHT, cleaves DNA strands [9]. Moreover, Kupfer et al. [10] demonstrated lung toxicity and tumor production caused by hydroxylated metabolites of BHT.

Metabolism of BHT
For the purpose of studying the various metabolic routes of BHT, an LC-MS/MS method was optimized. A biphenyl solid-core column using 5 mM ammonium acetate and acetonitrile as mobile phases A and B, respectively, yielded significant signal increase over using formic acid as an additive, as well as ameliorating peak shapes for many of the metabolites found in this study. BHT eluted with a retention time of 14.3 min with our optimized gradient ( Table 1). The high-resolution MS/MS spectrum of deprotonated BHT (m/z 219.1760) with a collision energy of 30 V, presented in Figure 1, exhibits only one major fragment ion at m/z 203.1429 (C 14 H 19 O − ), via the loss of CH 4 . Otherwise, this molecule is quite resistant to fragmentation, and when collision energy was increased to form other structurally characteristic fragments, most of the signal was lost and no clear product ions were observed. Both human and rat microsomes yielded similar metabolic profiles for all tested compounds and incubations conditions. Throughout the manuscript, representative chromatograms from the human incubations are shown. All metabolites were confirmed with accurate mass measurements within 5 ppm of the theoretical exact masses. When BHT was incubated under oxidative conditions, several metabolites were detected, as shown in Figure 2. Two hydroxylated BHT metabolites (BHT + O) were detected, eluting at 13.4 and 14.1 min ( Table 1). The comparison of their HRMS/MS spectra showed a common water loss at m/z 217.159 confirming that, in both cases, the oxygen is not added to the aromatic ring. To investigate whether the oxygen is added to the para-methyl or on a t-butyl group, isotope-labeled BHTs were also incubated (  Table 2), proving that the oxidation occurs on a t-butyl group. Di-hydroxylated BHT was also detected at 11.9 min ( Figure 2) with m/z 251.1660 (C 15 H 23 O 3 − , 2.9 ppm), and its MS/MS spectrum showed two water losses (Table 1). Both BHT-d 3 and BHT-d 20 analogs lost one deuterium atom, therefore one oxygen is added on the methyl group and the second one is on the t-butyl, in accordance with the two BHT + O isomers mentioned above. BHT-aldehyde via the oxidation of the p-methyl group was also detected at 14.0 min (Table 1) [6], confirmed by the fact that BHT-d 3 lost all three labels during this metabolic transformation, while BHT-d 20 did not lose any. * fragment ions of >20% intensity relative to base peak are listed here in bold, base peak in each spectrum is underlined.  Another two oxidative metabolites were detected at 9.8 and 13.5 min, both corresponding to C 15 H 21 O 3 − (m/z 249.150). The isomer eluting at 9.8 min showed a characteristic fragment at m/z 205.1592 corresponding to the loss of CO 2 (Table 1), and lost the three labels from BHT-d 3 (Table 2), proving it to be BHT-acid, where the methyl group is oxidized to a carboxylic acid. To further confirm this, the commercial standard of BHT−acid was purchased and showed the same retention time ( Figure S1) and MS/MS fragmentation behavior. The second isomer (Table 1), at 13.5 min, presented a water loss consistent with an oxidation on a methyl group, as well as the loss of CH 2 O. The analogous metabolite from BHT-d 20 had lost one deuterium (m/z 268.2689), while BHT-d 3 lost its three labels (m/z 249.1504). Taken together, these results confirm this isomer as hydroxylated BHT−aldehyde (BHT−aldehyde + O). DBP (2,6-di-tert-butylphenol) is a known metabolite of BHT. It is also used as a synthetic phenolic antioxidant in plastics and food packaging [30]. DBP was detected in oxidative incubations at 14.2 min with m/z 205.1600 (Table 1 and Figure 2). This metabolite has been confirmed by the synthetic DBP standard, which was also incubated under oxidative conditions ( Figure S1). Further oxidation of DBP can form BHT-hydroquinone (BHQ), which was also detected in BHT incubations at 8.3 min (Table 1 and Figure 2). The loss of all three deuterium labels from BHT-d 3 and none from BHT-d 20 confirm the para-hydroquinone structure ( Table 2) of BHQ. Another two oxidative metabolites were detected at 9.8 and 13.5 min, both corresponding to C15H21O3 − (m/z 249.150). The isomer eluting at 9.8 min showed a characteristic fragment at m/z 205.1592 corresponding to the loss of CO2 (Table 1), and lost the three labels from BHT-d3 (Table 2), proving it to be BHT-acid, where the methyl group is oxidized to a carboxylic acid. To further confirm this, the commercial standard of BHT−acid was purchased and showed the same retention time ( Figure S1) and MS/MS fragmentation behavior. The second isomer (Table 1), at 13.5 min, presented a water loss consistent with an oxidation on a methyl group, as well as the loss of CH2O. The analogous metabolite from BHT-d20 had lost one deuterium (m/z 268.2689), while BHT-d3 lost its three labels (m/z 249.1504). Taken together, these results confirm this isomer as hydroxylated BHT−aldehyde (BHT−aldehyde + O). Numerous metabolites of BHT have been described and metabolic pathways proposed [6,31]. A major metabolic pathway is initiated by the oxidation of the p-methyl group, leading to the formation BHT-aldehyde and BHT-acid by stepwise oxidation. The acidic form is decarboxylated to DBP, from which BHQ is likely formed. Another metabolic pathway is initiated by the oxidation of the t-butyl group. The double oxidation forms BHT + 2O, being further oxidized into hydroxylated BHT-aldehyde (BHT-aldehyde + O) ( Figure 3). Thompson et al. [32] studied the oxidative metabolism of BHT by hepatic and pulmonary rodent microsomes and identified several of these metabolites by LC-UV, GC-MS and using radiolabeled BHT ( 14 C-BHT). The oxidative pathways of BHT have also been studied in vivo in different species, including the oxidation of the p-methyl group as a major metabolic route in rat, rabbit, and monkey, while the oxidation of t-butyl groups has been described as the predominant pathway in human and mouse [33]. Zhang et al. [29] reported that BHT and its metabolites, including BHT-acid, BHQ, and BHT-aldehyde were present in metabolism-related organs (e.g., liver and kidney) in mouse. synthetic DBP standard, which was also incubated under oxidative conditions ( Figure S1). Further oxidation of DBP can form BHT-hydroquinone (BHQ), which was also detected in BHT incubations at 8.3 min (Table 1 and Figure 2). The loss of all three deuterium labels from BHT-d3 and none from BHT-d20 confirm the para-hydroquinone structure ( Table 2) of BHQ. Numerous metabolites of BHT have been described and metabolic pathways proposed [6,31]. A major metabolic pathway is initiated by the oxidation of the p-methyl group, leading to the formation BHT-aldehyde and BHT-acid by stepwise oxidation. The acidic form is decarboxylated to DBP, from which BHQ is likely formed. Another metabolic pathway is initiated by the oxidation of the t-butyl group. The double oxidation forms BHT + 2O, being further oxidized into hydroxylated BHTaldehyde (BHT-aldehyde + O) (Figure 3). Thompson et al. [32] studied the oxidative metabolism of BHT by hepatic and pulmonary rodent microsomes and identified several of these metabolites by LC-UV, GC-MS and using radiolabeled BHT ( 14 C-BHT). The oxidative pathways of BHT have also been studied in vivo in different species, including the oxidation of the p-methyl group as a major metabolic route in rat, rabbit, and monkey, while the oxidation of t-butyl groups has been described as the predominant pathway in human and mouse [33]. Zhang et al. [29] reported that BHT and its metabolites, including BHT-acid, BHQ, and BHT-aldehyde were present in metabolism-related organs (e.g., liver and kidney) in mouse. When GSH was added under oxidative conditions, three adducts were detected (Figure 2), corresponding to BHT-2H + GSH at 8.5 min, BHT + O-2H + GSH at 7.6 min, and DBP-2H + GSH at When GSH was added under oxidative conditions, three adducts were detected (Figure 2), corresponding to BHT-2H + GSH at 8.5 min, BHT + O-2H + GSH at 7.6 min, and DBP-2H + GSH at 8.2 min. HRMS/MS spectra for these adducts were dominated by characteristic peaks from the GSH moiety (m/z 306.077, 272.089, 254.078, 160.007, 143.046, and 128.035) ( Table 1). The most prominent fragment ion for BHT-2H + GSH and BHT + O-2H + GSH was m/z 306.07 (Table 1), corresponding to deprotonated GSH. These two metabolites are proposed to result from the formation of the quinone methide intermediate followed by the addition of GSH on the methylene carbon. This hypothesis is supported by results from incubations with isotope-labeled BHT analogs. For BHT-2H + GSH, BHT-d 3 lost one deuterium, and none were lost from BHT-d 20. Tajima et al. [34] had also identified this metabolite by 13 C-NMR in rat bile. For BHT + O−2H + GSH, both BHT-d 3 and BHT-d 20 lost one deuterium, confirming the same mechanism as above with the t-butyl being hydroxylated as well. This GSH adduct was also described by Madsen et al. where they compared electrochemical and enzymatic formation of several reactive metabolites [35].
The deprotonated ion of DBP-2H + GSH did not form m/z 306 upon CID and instead had a unique fragment ion at m/z 237.1316, assigned as deprotonated DBP with the sulfur of GSH still attached. The HR-MS spectra of isotope-labeled analogs showed the loss of the three labels from BHT-d 3, while none were lost from BHT-d 20 ( Table 2). This supports the structure where the SG group replaces the methyl group. The formation of this metabolite is explained by a radical pathway, initiated by the decarboxylation of BHT-acid to form DBP · , followed by GSH trapping. Glutathione is able to scavenge radicals by its electron-donating ability, enabling it to neutralize such reactive species. This GSH adduct had not been described in previous studies.
The only sulfate metabolite detected could be assigned as BHQ + SO 3 , eluting at 9.0 min ( Figure 2). Its HRMS/MS spectra presented characteristic peaks of the sulfate conjugates at m/z 80.9645, m/z 79.9576 and m/z 221.1539 corresponding to the HSO 3 − ion, the sulfonate radical ion (SO 3 − ), and the neutral loss of the SO 3 radical. This metabolite had not been reported previously.

Metabolism of BHA and TBHQ
BHA is an analog of BHT and has been used in different industries alone or in combination with BHT and other antioxidants. TBHQ is a metabolite of BHA through O-demethylation and a powerful synthetic phenolic antioxidant. BHA and TBHQ were also incubated under the same conditions studied for BHT. Table 3  Two hydroxylated BHA metabolites (BHA + O) were detected ( Figure S2) at 11.4 and 11.8 min (Table 3). No significant differences were seen in their MS/MS spectra. These hydroxylated BHA isomers were attributed to the fact that BHA is a mixture of two isomers, 2-BHA (10%) and 3-BHA (90%). Armstrong et al. [36] were the first to identify 3-BHA + O by incubating pure 3-BHA (99.5%) with RLM under oxidative conditions, using 1 H-NMR. Hydroxylated TBHQ (TBHQ + O) were also detected ( Figure S3 [36] also described a di-BHA metabolite [36]. BHA dimer was also found to form in rat intestine and by incubating BHA with rat intestine peroxidase, as well as horseradish peroxidase [37]. No dimer was detected for TBHQ under our conditions, which may be explained by its preference to form the quinone reactive metabolite. Identical GSH adducts were detected for BHA and TBHQ, namely two isomers of TBHQ-2H + GSH (at 5.1 and 5.4 min), TBHQ-4H + GSH (at 6.4 min), and a di-glutathione adduct, TBHQ-4H + 2GSH (at 1.7 min) ( Figures S2 and S3). The HRMS/MS spectra from these GSH adducts were dominated by characteristic fragment ions of deprotonated GSH (m/z 272, 254, 210, 179, 166, 143, and 128), with the exception of m/z 197.066 (C 10 H 13 O 2 S − ) for the two isomers, where the sulfur atom of GSH is still bound to deprotonated TBHQ (Table 3). These GSH adducts were also detected in rat bile and urine following TBHQ administration [38]. Peters et al. [38] reported three GSH conjugates of TBHQ in vivo, including 5-(GS)-TBHQ, 6-(GS)-TBHQ, and 3,6-(GS) 2 -TBHQ by LC-MS and 1 H-NMR. They also suggested that these conjugates could represent nephrotoxic metabolites, and may be responsible for the tumor-promoting effects of TBHQ and BHA [38]. Two novel BHA glutathione adducts have been detected here, namely BHA-2H + GSH (at 5.8 min) and BHA + O-2H + GSH (at 5.0 min) ( Figure S2). The first is suggested to form via an epoxide followed by GSH addition and loss of a water molecule, while the second is likely formed via the ortho-quinone reactive metabolite.
Four BHA glucuronide conjugates were detected, namely TBHQ + gluc, BHA + gluc, and two isomers of BHA + O + gluc, at 3.4, 5.6, 5.7, and 6.6 min, respectively (Table 3). By comparing the MS/MS spectra of the two BHA + O + gluc isomers, the peak at 5.7 min showed a water loss at m/z 353.1259 (C 17 H 20 O 7 − , 2.1 ppm) proving that the oxygen is added to the tert-butyl group, and not in the ring as for the isomer detected at 6.6 min. These two novel metabolites have not been previously characterized. TBHQ also formed the same glucuronide conjugate as BHA (TBHQ + gluc) at 3.4 min (m/z 341.1240) with fragment ions mostly from the glucuronide moiety. Several sulfate conjugates were detected for BHA, including two isomers corresponding to BHA + SO 3 at 7.2 and 8.3 min. Their MS/MS spectra yielded very similar fragments suggesting that one is 3-BHA + SO 3 and the other 2-BHA + SO 3 ( Table 3) . One peak corresponding to BHA + O + SO 3 was detected at 8.4 min. A common sulfate conjugate between BHA and TBHQ (TBHQ + SO 3 ) was also detected at 5.7 min. These same sulfate conjugates were previously detected in human urine using GC-MS [39]. The proposed biotransformation products of BHA and TBHQ are summarized in Figure 4.  The major metabolic pathway for BHA is reported to be via the conjugation of the free hydroxyl group with both glucuronic acid and sulfate [39]. Conning et al. [33] reported that glucuronide conjugation predominates in rat, rabbit, and human, whereas sulfation is the major phase II reaction in dog. The major TBHQ metabolites found in rat bile and urine were TBHQ-glucuronide and TBHQsulfate [38].

Metabolism of DBP and BHT Acid
DBP and BHT acid standards were used to confirm these two metabolites of BHT, as mentioned above. The oxidative metabolism for these two compounds was also studied to help support the complex metabolic pathway proposed for BHT.
Under oxidative conditions, DBP formed previously uncharacterized oxidative metabolites, including a major metabolite hydroxylated of the t-butyl group (DBP + O), as well as two dihydroxylated forms and a carboxylic acid metabolite (DBP + 2O−2H). Figure S1 shows the extracted ion chromatograms for DBP and BHT-acid oxidative metabolites, as well as a proposed scheme for their formation, supported by high-resolution MS/MS spectra for each metabolite.
The BHT-acid standard confirmed this BHT metabolite and helped distinguish it from the novel The major metabolic pathway for BHA is reported to be via the conjugation of the free hydroxyl group with both glucuronic acid and sulfate [39]. Conning et al. [33] reported that glucuronide conjugation predominates in rat, rabbit, and human, whereas sulfation is the major phase II reaction in dog. The major TBHQ metabolites found in rat bile and urine were TBHQ-glucuronide and TBHQ-sulfate [38].

Metabolism of DBP and BHT Acid
DBP and BHT acid standards were used to confirm these two metabolites of BHT, as mentioned above. The oxidative metabolism for these two compounds was also studied to help support the complex metabolic pathway proposed for BHT.
Under oxidative conditions, DBP formed previously uncharacterized oxidative metabolites, including a major metabolite hydroxylated of the t-butyl group (DBP + O), as well as two di-hydroxylated forms and a carboxylic acid metabolite (DBP + 2O−2H). Figure S1 shows the extracted ion chromatograms for DBP and BHT-acid oxidative metabolites, as well as a proposed scheme for their formation, supported by high-resolution MS/MS spectra for each metabolite.
The BHT-acid standard confirmed this BHT metabolite and helped distinguish it from the novel (BHT-aldehyde) + O. BHT-acid incubations showed that the formation of DBP and BHQ was non-enzymatic, and allowed a novel hydroxylated (BHT-acid + O) metabolite to be characterized ( Figure S1).
By compiling the results from in vitro biotransformations of BHT, BHA, TBHQ, DBP, and BHT-acid, comprehensive schemes of the many metabolic transformations for these small synthetic antioxidants have been proposed. Using an untargeted high-resolution tandem mass spectrometry approach to decipher the metabolism of analogous compounds, while incorporating isotope labeling, proved to be a powerful method to elucidate structures of all detected metabolites.

Oxidative Metabolites and GSH Adducts
BHT and analogs were incubated at 20 µM with human and rat liver microsomes (1 mg/mL protein) containing 5 mM GSH and a NADPH-regenerating system (5 mM MgCl 2 , 0.5 mM NADP + , 10 mM glucose-6-phosphate and 2 units/mL glucose-6-phosphate dehydrogenase) at 37 • C for 1 h in 100 mM phosphate buffer, pH 7.4. Control samples were prepared without NADPH regenerating system and/or without GSH. Adding an equal volume of cold acetonitrile quenched the reaction. Incubation mixtures were centrifuged for 8 min at 14,000 rpm, at 4 • C. The supernatants were diluted (1:1) in water prior to LC-MS/MS analysis.

Phase II Metabolism-Glucuronidation and Sulfation
To study the glucuronide conjugates, all compounds (20 µM) were incubated with HLM and RLM (1 mg/mL protein), NADPH-regenerating system (as above), and 5 mM UDPGA at 37 • C for 1h. Each compound (20 µM) was also incubated with human and rat S9 fractions (2 mg/mL) in phosphate buffer, containing 1 mM of PAPS with the NADPH-regenerating system, to study the formation of sulfates conjugates. All samples were incubated at 37 • C for 1h, quenched, and centrifuged as above. Supernatants were diluted as above and subjected to LC-MS/MS analysis.

LC-HRMS/MS Analysis and Data Processing
LC-MS/MS analyses were performed using a Shimadzu Nexera HPLC coupled to a Sciex 5600 TripleTOF ® (quadrupole-time-of-flight) system (Concord, ON, Canada), in negative electrospray mode.
Chromatographic separation was performed using a Phenomenex Kinetex biphenyl (100 × 2.1 mm, 2.6 µm) column, using mobile phases of 5 mM ammonium acetate in water and 100% ACN, at 0.25 mL/min and a column temperature of 40 • C. The injection volume was 25 µL. The HPLC gradient was as follows: 5% B held for 0.5 min, linearly increased to 30% at 8 min, up to 50% at 12 min, and 90% at 13 min, held for an additional 2 min.
Ion source parameters were as follows: ionization voltage at 5000 V, curtain gas of 35 psi, drying and nebulizer gases each at 50 psi, source temperature of 450 • C, and declustering potential of 60 V. TOF-MS spectra were acquired (with 250 ms accumulation time), followed by MS/MS in information-dependent acquisition (IDA) mode on the 5 most intense ions using dynamic background subtraction (175 ms each). Nitrogen was used as collision gas and collision energy was 30 ± 10 V. Metabolites that did not have high-quality MS/MS in IDA mode, targeted MS/MS mode was used in a second injection.
MetabolitePilot 2.0 (Sciex) software was employed to screen samples for potential metabolites using a set of known biotransformations, including oxidative reactions, GSH, glucuronide, and sulfate conjugates. PeakView 2.2 and MasterView 1.1 (Sciex) were also used for processing LC-MS/MS data to confirm and expand the list of features based on mass accuracy, isotope pattern, and MS/MS analysis.

Conclusions
The metabolism of BHT and several analogs were investigated in vitro using human and rat liver microsomes and S9 fractions. Many oxidative metabolites, GSH adducts, glucuronide, and sulfate conjugates were detected with excellent mass accuracy, some of which had not been previously reported. Structures of biotransformation products were elucidated by HRMS/MS data and supported using isotope-labeled analogs. These results have enabled many biotransformation products to be determined, which are potentially involved in the toxicity of these compounds. Knowledge of all these possible metabolites would be useful in assessing environmental exposure to these compounds.