Development of a UHPLC-MS/MS-based data-mining method for rapid profiling and characterization of magnolol metabolites in rat urine and plasma

Beibei Ma; Tianyu Lou; Tingting Wang; Ruiji Li; Jinhui Liu; Shangyue Yu; Hailuan Pei; Shiqiu Tian; Yilin Li; Yudong Guo; Zijian Wang; Zhaozhou Lin; Zhibin Wang; Jing Wang; Yingying Gao

doi:10.1016/j.arabjc.2020.102954

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Original article

2020

:14;

202102

doi:

10.1016/j.arabjc.2020.102954

Development of a UHPLC-MS/MS-based data-mining method for rapid profiling and characterization of magnolol metabolites in rat urine and plasma

Beibei Ma^{^a}, Tianyu Lou^{^a}, Tingting Wang^{^a}, Ruiji Li^{^a}, Jinhui Liu^{^a}, Shangyue Yu^{^a}, Hailuan Pei^{^a}, Shiqiu Tian^{^a}, Yilin Li^{^a}, Yudong Guo^{^b}, Zijian Wang^{^c}, Zhaozhou Lin^{^d}, Zhibin Wang^{^d,⁎}, Jing Wang^{^a,⁎}, Yingying Gao^{^e}

a School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100029, China

b Beijing Institute for Drug Control, Beijing 102206, China

c Beijing Research Institution of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China

d Beijing Tongrentang Research Institute, Beijing 100071, China

e China National Accreditation Service for Conformity Assessment, Beijing 100062, China

⁎Corresponding authors. wangzhibin4804@126.com (Zhibin Wang), crystal_wj@163.com (Jing Wang),

Received: 2020-10-22, Accepted: 2020-12-13,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Magnolol, an essential bioactive natural lignans isolated from Magnolia Officinalis, has various pharmacological activities, such as antibacterial, anti-inflammatory, antioxidation and anti-tumor. In this study, a practical strategy based on ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was established to reveal the metabolic fate of magnolol in rat plasma and urine. First of all, in the data-dependent scanning (DDS) acquisition mode, the ESI-MSⁿ data sets of biological samples and reference standard were obtained by using a high-quality online data analysis method. Subsequently, off-line data-mining techniques including multiple mass defect filters (MMDFs) and high-resolution extracted ion chromatograms (HREICs) were applied to screen major-to-trace metabolites of magnolol at the full-scan ESI-MS¹ level. Finally, a total of 177 metabolites (prototype compound included) were preliminarily observed and characterized according to accurate mass measurement, the characteristic fragmentation patterns, chromato-graphic behaviors, and corresponding Clog P values. The results showed that magnolol experienced a variety of biotransformation reactions in rats, including hydroxylation, methoxylation, dehydrogenation, carboxylation, sulfonation, glucuronide conjugation, glucose conjugation, N-acetylcysteine (NAC) conjugation, glutathione conjugation and their composite reactions. In conclusion, this study not only greatly expanded the understanding of the therapeutic material basis and pharmacological mechanism of magnolol, but also provided new ideas for its toxicity evaluation, safety monitoring and drug delivery forms design.

Keywords

Magnolol

UHPLC-LTQ-Orbitrap MS

Data-mining techniques

Metabolites

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1 Introduction

Magnolol (shown in Fig. 1), as a typical biphenylene lignans, is mainly extracted and isolated from magnolia plants, such as Magnolia officinalis Rehd. et Wils. and Magnolia officinalis (Rehd. et Wils.) Cheng subsp. biloba (Rehd.et Wils.) Law(Kawahara et al., 2020; Li et al., 2020; Santos et al., 2020; Zhang et al., 2020 ). In recent years, there has been increasing interest and research on the health benefits and extensive pharmacological properties of magnolol, such as antibacterial, anti-inflammatory, anti-tumor, antiviral, cardiovascular regulation and neuroprotection(Fukuyama et al., 2020; Kim et al., 2020; Kiseleva et al., 2020; Lovecká et al., 2020; Tao et al., 2020; Yuan et al., 2020 ). Therefore, it is used for the treatment and alleviation of various intractable diseases, including liver cancer, breast cancer, ischemic brain injury, chronic bronchitis and Alzheimer disease(Chen et al., 2020; Elhabak et al., 2020; Guo et al., 2020; Upadhyay et al., 2020; Xian et al., 2020 ). Moreover, as previously reported, magnolol could not only attenuate cisplatin-induced muscle wasting by M2c macrophage activation (Lee et al., 2020), but also prevent acute alcoholic liver damage by activating PI3K/Nrf2/PPARγ and inhibiting the NLRP3 signaling pathway (Liu et al., 2019). However, to our best knowledge, little is known until now about the biotransformation of magnolol in vivo, which is essential to explain and predict a variety of events related to the efficacy and toxicity of magnolol.

In the past decade, ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) has been broadly used in multi-component analysis and drug metabolism research of traditional Chinese medicine due to its advantages of high resolution, high quality accuracy and wide dynamic range (Stojković et al., 2020; Vasić et al., 2019; Zengin et al., 2020 ). By combining the full scan-parent ion list-dynamic exclusion (FS-PIL-DE) data acquisition method with multi-channel off-line LC-MS data-mining technology, it is possible to identify and capture trace metabolites overwhelmed by complex background noise or matrix interference. Along with the advancement of science and the shining of wisdom, multifarious data processing methods have emerged, including mass defect filter (MDF), multiple mass defect filters (MMDFs), high-resolution extracted ion chromatograms (HREICs), diagnostic product ions (DPIs), neutral loss fragments (NLFs), and background subtraction (BS), which further improve the accuracy and efficiency of metabolite identification (de Benzi, 2020; Lee et al., 2020; Shang et al., 2020; Stavrianidi, 2020; Xiao et al., 2020 ).

Herein, based on the combination of UHPLC-LTQ-Orbitrap mass spectrometer with multiple data-mining techniques, an integrated and effective strategy was established for the comprehensive identification and characterization of major-to-trace metabolites in plasma and urine of Sprague-Dawley (SD) rats after oral administration of magnolol. At the same time, this developed method would be further adopted to elucidate different biotransformation pathways of magnolol in rats.

2 Experiment

2.1

2.1 Chemicals and reagents

The reference standard of magnolol was purchased from Chengdu Must Biotechnology Co. Ltd (Sichuan, China). Its structure was fully elucidated by comparing its spectral data (ESI-MS and ¹H, ¹³C NMR) with those published literature values. And its purity (≥98%, confirmed by HPLC-UV analysis) was suitable for UHPLC-HRMS analysis.

HPLC grade acetonitrile, methanol, and formic acid (FA) were all obtained from Thermo Fisher Scientific (Fair Lawn, NJ, USA). All other analytical grade chemicals were available from the workstation at the Beijing Chemical Works (Beijing, China). All deionized water used throughout the experiment was purified through the Milli-Q Gradient Å 10 system (Millipore, Billerica, MA, USA). Grace Pure^TM SPE C18-Low solid-phase extraction cartridges (200 mg/3 mL, 59 μm, 7 Å) were purchased from Grace Davison Discovery Science (Deerfield, IL, USA).

2.2

2.2 Animals

Eight male Sprague-Dawley (SD) rats weighing 220 g-240 g were purchased from Beijing Weitong Lihua Experimental Animals Company (Beijing, China). The animals were housed in a control room with standard temperature (24 ± 2℃) and humidity (70 ± 5%), and maintained in a 12-h light/12-h dark treatment. After a week of adaptation, the rats were randomly divided into two groups: Group A (n = 4), drug group for plasma and urine; Group B (n = 4), control group for blank plasma and urine. They were fasted for 12 h with free access to water prior to the experiment. The animal experimentation protocols were approved by the institutional Animal

Care and Use Committee at Beijing University of Chinese Medicine. The animal facilities and procedures were strictly in accordance with the Guide for the Care and Use of Laboratory Animals.

2.3

2.3 Drug administration and biological samples preparation

Magnolol was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) aqueous solution and given to Group A rats by gavage at a dose of 300 mg/kg body weight. Each rat in Group B was orally administered an equivalent amount of 0.5% CMC-Na aqueous solution. Blood samples (0.5 mL) were respectively collected from the suborbital venous plexus of rats at 0.5, 1.0, 1.5, 2.0, and 4.0 h after administration. After that, each sample was transferred to a heparinized micro-centrifuge tube and centrifuged at 3500 rpm (4℃) for 15 min to obtain plasma. Urine samples were collected over 0–24 h after oral administration. Finally, all biological samples from the same group were merged into a collective sample.

The solid-phase extraction (SPE) method, which could precipitate and concentrate proteins and solid residues, was applied to prepare all biological samples. Plasma and urine samples (1 mL) were separately added into the SPE cartridges, which were sequentially pretreated with methanol (5 mL) and deionized water (5 mL). Then, the SPE cartridges were washed with deionized water (5 mL) and methanol (3 mL) in turn. The methanol eluate was collected and evaporated to dryness under nitrogen at room temperature. The residue was re-dissolved in 100 μL of acetonitrile/water (10:90, v/v), and then centrifuged at 14000 rpm (4℃) for 15 min. The supernatant was used for further UHPLC-HRMS analysis.

2.4

2.4 Instrument and conditions

The LC analysis was carried out on the DIONEX Ultimate 3000 UHPLC system (Thermo Fisher Scientific, MA, USA), which was equipped with a binary pump, an automatic sampler and a column compartment. The chromatographic separation was performed at room temperature using a Waters ACQUITY BEH C18 column (2.1 × 100 mm i.d., 1.7 μm; Waters Corporation, Milford, MA, USA). Acetonitrile (solvent B) and 0.1% FA aqueous solution (solvent A) were used as the mobile phase. The flow rate was set to 0.30 mL/min and the linear gradient was as follows: 0–2 min, 5%-20% B; 2–27 min, 20%-85% B; 27–30 min, 85% B; 30–32 min, 85%-5% B; 32–35 min, 5% B. The injection volume was 2 μL.

High-resolution ESI-MS and MS/MS spectral analysis were performed on an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, MA, USA) that was connected to a UHPLC instrument via the ESI interface. Samples were analyzed in negative ion mode with the tuning method set as follows: sheath gas (nitrogen) flow rate of 40 arb, auxiliary gas (nitrogen) flow rate of 20 arb, capillary temperature of 350 °C, electrospray voltage of 4.0 kV, capillary voltage of 35 V, and tube lens voltage of 110 V. Centroided mass spectra was obtained within the mass range of m/z 50–800.

In the full-scan (FS) experiment, the HRMS data was recorded at mass resolving power of 30,000 full width at half maximum (FWHM, calculated for m/z 200). To minimize the total analysis time, the data-dependent MS/MS scanning was performed to trigger the fragmentation mass spectra of the target ions. The collision energy of the collision-induced dissociation (CID) was adjusted to 40% of the maximum value. The function of dynamic exclusion to prevent repetition was enabled, and the repeat count was set to 5 with the dynamic repeat time at 30 s, and the dynamic exclusion duration was 60 s. In addition, the parent ion list-dynamic exclusion (PIL-DE) dependent acquisition mode was also used as a complementary method to obtain the ESI-MSⁿ stages of the screened metabolite candidates. Data-dependent ESI-MS² analyses were triggered by the three most-abundant ions in the list of predicted metabolite precursors while ESI-MS³ analyses of the most-abundant product ions were followed.

2.5

2.5 Peak selections and data processing

The Thermo Xcalibur 2.1 workstation was applied to collect and process the HR-ESI-MS¹ and MSⁿ data. In order to obtain as many ESI-MS/MS fragment ions of magnolol metabolites as possible, the peaks detected with intensity over 10,000 were selected for further structural identification. The chemical formulas for all parent ions of the selected peaks were calculated from the accurate mass using a formula predictor by setting the parameters as follows: C [0–30], H [0–50], O [0–20], S [0–4], N [0–3] and ring double bond (RDB) equivalent value [0–15]. Other elements, such as P and Br, were not considered because they rarely appeared in the complex matrix.

3 Results and discussion

3.1

3.1 The establishment of analytical strategy

In this study, based on UHPLC-LTQ-Orbitrap MS analysis combined with data acquisition and various post-acquisition data processing techniques, a systematic and effective strategy (shown in Fig. 2) was proposed for the rapid screening and characterization of magnolol metabolites in rats. First of all, high-quality ESI-MS¹ analysis was performed and ESI-MSⁿ datasets for biological samples and reference standard were obtained in data-dependent scanning (DDS) acquisition mode. Secondly, for the subsequent post-acquisition data-mining processing, a combination of HREICs and MMDFs method was adopted for the comprehensive screening of magnolol metabolites. Among them, HREICs were utilized to identify known (as previously reported in literatures) and predicted metabolites (based on common biotransformation reactions), while MMDFs were employed to acquire specific HR-MS¹ information for unknown and unpredictable metabolites. Then, the ESI-MSⁿ datasets of all screened metabolite candidates were collected by the full scan-parent ion list-dynamic exclusion (FS-PIL-DE) data acquisition method. Afterwards, according to the chromatographic retention times, accurate mass measurements, characteristic mass fragmentation behaviors and the corresponding Clog P values, the structures of magnolol metabolites were tentatively expounded. Finally, the metabolic pathways of magnolol in rats were positively summarized based on the identified metabolites and the corresponding biotransformation reactions.

Summary diagram of the developed analytical strategy and methodology.

3.2

3.2 Fragmentation behaviors of the magnolol in negative ion mode

In order to better understand the characteristic fragmentation behaviors of magnolol, a comprehensive analysis of the reference standard solution was implemented using UHPLC-LTQ-Orbitrap MS to obtain the unique DPIs and NLFs of magnolol. In the negative ion mode, magnolol showed [M−H]^- ion at m/z 265.12357 (C₁₈H₁₇O₂, 4.77 ppm) in the ESI-MS¹ spectrum. In its ESI-MS² spectrum, several diagnostic product ions at m/z 247, m/z 237, m/z 224 and m/z 197 were generated due to the loss of H₂O (18 Da), CO (28 Da), C₃H₅ (41 Da) and C₃H₅ + C₂H₃ (68 Da), respectively. Besides, the NLFs of 2 Da (m/z 247 → m/z 245 → m/z 243) and 26 Da (m/z 247 → m/z 221) were both observed in the ESI-MS³ spectrum, which further illustrated the fragmentation patterns of m/z 247. If different kinds of biotransformation reactions occurred on the original drug, the new DPIs at m/z 247 + X, m/z 237 + X, and m/z 224 + X (X = molecular weight of substituent groups, such as 14 (CH₂), 30 (OCH₂), and 162 (Glc)) derived from the newly produced compounds would provide an important basis for the structural identification of the metabolites. The proposed fragmentation pathways of magnolol were illustrated in Fig. 3, and the ESI-MSⁿ spectra of magnolol in negative ion mode were displayed in Fig. 4.

The mass fragmentation behaviors of magnolol.

ESI-MSn spectra of magnolol in negative ion mode: (A) MS1 spectrum; (B) MS2 spectrum; (C) MS3 spectrum.

3.3

3.3 Implement of MMDFs data-mining methods

In order to reduce the potential interferences of endogenous substances, the MMDFs approach was chosen as a crucial complement to the HREICs method to entirely obtain the HR-MS¹ datasets of low-level predicted and unpredictable metabolites. For the MMDFs method, the most critical step was to set up metabolite templates, which commonly included drug filter, substructure filter, and conjugate filter (Zhang et al., 2015). Since magnolol is difficult to generate two small molecules through the cleavage reaction, three templates were founded in parallel to screen relevant compounds:1) parent drug template (m/z 266.13013) and its conjugation templates (m/z 346.08695 for sulfate conjugation, m/z 442.16222 for glucuronide conjugation); 2) hydroxylated magnolol template (m/z 282.12504) and its conjugation templates (m/z 362.08186 for sulfate conjugation, m/z 458.15713 for glucuronide conjugation);3) carboxylated magnolol template (m/z 296.10431) and its conjugation templates (m/z 376.06113 for sulfate conjugation, m/z 472.13640 for glucuronide conjugation). According to the above-mentioned metabolites screening templates, each MDF window was set to ± 50 mDa around the mass defects of the templates over a mass range of ± 50 Da around the filter template masses. Based on the similarity of mass defects of metabolites and their core substructures, the MMDFs method could mine major-to-trace metabolites to the greatest extent from complex background noise and endogenous components.

3.4

3.4 Identification of magnolol metabolites

A total of 177 metabolites were detected and characterized in rat urine and plasma using UHPLC-LTQ-Orbitrap MS technology in combination with the established strategy. Among them, 166 metabolites were found in the urine, while 26 metabolites were derived from plasma. The chromatographic and mass spectral data for all metabolites were presented in Table 1, and their corresponding HREICs were shown in Fig. 5.

Table 1 Summary of magnolol metabolites in rat urine and plasma.

Peak	t_R/min	Formula [M + H]⁺	Theoretical Mass m/z	Experimental Mass m/z	Error (ppm)	MS/MS fragment ions	Identification/Reactions	U	P
M0	17.34	C₁₈H₁₇O₂	265.12229	265.12357	4.77	MS²[2 6 5]:265(1 0 0),247(60),245(11),224(1) MS³[2 4 7]:206(1 0 0),245(38),203(15),247(14)	Magnolol	+	+
M1	2.92	C₁₈H₂₁O₆	333.13319	333.13571	7.34	MS²[3 3 3]:273(1 0 0),213(32),226(3)	Loss of 2C₂H₂, tetramethoxylation	+	−
M2	3.39	C₁₈H₂₁O₆	333.13319	333.13589	7.88	MS²[3 3 3]:273(1 0 0),213(22),315(4),281(3)	Loss of 2C₂H₂, tetramethoxylation	+	−
M3	3.49	C₂₁H₁₉O₉	415.10249	415.10593	8.60	MS²[4 1 5]:295(1 0 0),267(8),325(2),200(1) MS³[2 9 5]:267(1 0 0),253(1)	Loss of C₃H₄, oxidation, dehydrogenation, dihydroxylation, glucose conjugation	+	−
M4	3.67	C₁₈H₁₉O₈S	395.07949	395.08267	7.99	MS²[3 9 5]:315(1 0 0),339(6),377(2) MS³[3 1 5]:297(1 0 0),237(49),255(7),267(4)	Loss of C₂H₂, dimethoxylation, hydroxylation, sulfonation	+	−
M5	3.77	C₁₈H₁₉O₅	315.12269	315.12534	8.38	MS²[3 1 5]:297(1 0 0),237(63),255(6),267(2),272(1) MS³[2 9 7]:237(1 0 0),219(4),222(2)	Loss of C₂H₂, dimethoxylation, hydroxylation	+	−
M6	3.87	C₁₅H₉O₄	253.04971	253.05196	9.58	MS²[2 5 3]:253(1 0 0),224(55),209(40),225(34),197(20)	Loss of C₃H₄, oxidation, dehydrogenation, dihydroxylation	+	−
M7	3.87	C₁₈H₁₇O₅	313.10701	313.10971	8.50	MS²[3 1 3]:295(1 0 0),237(26),253(8),235(2) MS³[2 9 5]:237(1 0 0),265(64),235(56),223(42),267(12)	Loss of 2CH₂, dimethoxylation, hydroxylation	+	−
M8	3.87	C₂₁H₁₉O₉	415.10249	415.10587	8.46	MS²[4 1 5]:253(1 0 0),252(15),295(6),315(3),229(2) MS³[2 5 3]:224(1 0 0),225(30)	Loss of C₃H₄, oxidation, dehydrogenation, dihydroxylation, glucose conjugation	+	−
M9	3.87	C₂₁H₁₇O₁₀	429.08179	429.08514	8.20	MS²[4 2 9]:253(1 0 0),175(39),411(6),349(5)	Loss of C₃H₄, oxidation, dehydrogenation, dihydroxylation, glucuronide conjugation	+	−
M10	3.97	C₁₆H₁₁O₅	283.06019	283.06284	9.68	MS²[2 8 3]:268(1 0 0),255(2),220(1) MS³[2 6 8]:240(1 0 0),224(1)	Loss of C₂H₂, oxidation, carboxylation, hydroxylation	+	−
M11	4.08	C₁₆H₁₅O₅	287.09149	287.09375	8.19	MS²[2 8 7]:227(1 0 0),209(9),269(8),209(6),219(2),237(1) MS³[2 2 7]:209(1 0 0),181(22)	Loss of C₃H₄ + CH₂, dimethoxylation, hydroxylation	+	−
M12	4.28	C₁₈H₁₉O₅	315.12269	315.12555	9.04	MS²[3 1 5]:297(1 0 0),237(81),255(5),267(4) MS³[2 9 7]:237(1 0 0),219(3),270(2)	Loss of C₂H₂, dimethoxylation, hydroxylation	+	−
M13	4.28	C₂₁H₁₉O₉	415.10249	415.10596	8.68	MS²[4 1 5]:253(1 0 0),252(70),295(6),335(5),292(4)	Loss of C₃H₄, oxidation, dehydrogenation, dihydroxylation, glucose conjugation	+	−
M14	4.48	C₂₄H₂₅O₁₀	473.14422	473.14786	7.69	MS²[4 7 3]:297(1 0 0),436(5),427(4),269(3),275(2) MS³[2 9 7]:279(1 0 0),250(11),261(7)	Dihydroxylation, glucuronide conjugation	+	−
M15	4.57	C₁₅H₉O₅	269.04459	269.04712	9.92	MS²[2 6 9]:269(1 0 0),181(48),225(44),201(38),241(21)	Loss of C₃H₄, oxidation, carboxylation, hydroxylation	+	−
M16	4.57	C₁₈H₁₉O₅	315.12269	315.12524	8.06	MS²[3 1 5]:267(1 0 0),297(4),255(2),224(1) MS³[2 6 7]:249(1 0 0),247(29),239(28),221(10)	Loss of C₂H₂, dimethoxylation, hydroxylation	+	−
M17	4.57	C₁₈H₁₉O₈S	395.07949	395.08282	8.37	MS²[3 9 5]:315(1 0 0),267(12) MS³[3 1 5]:267(1 0 0),297(5)	Loss of C₂H₂, dimethoxylation, hydroxylation, sulfonation	+	−
M18	4.57	C₁₅H₁₁O₄	255.06532	255.06772	9.94	MS²[2 5 5]:225(1 0 0),214(33),211(25),240(10)	Loss of C₃H₄ + CH₂, dehydrogenation, methoxylation, hydroxylation	+	−
M19	4.68	C₁₈H₁₇O₅	313.10701	313.10983	8.88	MS²[3 1 3]:253(1 0 0),295(23),235(7),207(6) MS³[2 5 3]:235(1 0 0),207(84),225(10)	Loss of 2CH₂, dimethoxylation, hydroxylation	+	−
M20	4.88	C₁₅H₉O₄	253.04971	253.05193	9.46	MS²[2 5 3]:253(1 0 0),209(61),224(58),225(50),197(22)	Loss of C₃H₄, oxidation, dehydrogenation, dihydroxylation	+	−
M21	4.88	C₁₅H₉O₇S	333.00652	333.00919	8.53	MS²[3 3 3]:253(1 0 0),200(5),232(1) MS³[2 5 3]:211(1 0 0),209(36),225(29),197(11)	Loss of C₃H₄, oxidation, dehydrogenation, dihydroxylation, sulfonation	+	−
M22	5.00	C₁₆H₁₁O₅	283.06019	283.06271	9.22	MS²[2 8 3]:268(1 0 0),66(2),255(1) MS³[2 6 8]:240(1 0 0),239(4),224(1)	Loss of C₂H₂, oxidation, carboxylation, hydroxylation	+	−
M23	5.00	C₁₈H₁₇O₆	329.10189	329.10480	8.62	MS²[3 2 9]:137(1 0 0),311(31),219(23),269(6),226(5)	Tetrahydroxylation	+	−
M24	5.00	C₂₉H₃₃O₁₆	637.17634	637.18103	7.41	MS²[6 3 7]:461(1 0 0),619(1) MS³[4 6 1]:239(1 0 0),299(85),281(80),263(13)	Loss of C₂H₂, dimethoxylation, glucuronide conjugation, glucose conjugation	+	−
M25	5.10	C₂₄H₂₉O₉	461.18059	461.18439	8.20	MS²[4 6 1]:239(1 0 0),299(93),281(78)	Loss of C₂H₂, dimethoxylation, glucose conjugation	+	−
M26	5.20	C₁₅H₉O₅	269.04459	269.04700	9.48	MS²[2 6 9]:241(1 0 0),213(29),225(10),253(7) MS³[2 4 1]:213(1 0 0),197(8)	Loss of C₃H₄, oxidation, carboxylation, hydroxylation	+	−
M27	5.43	C₂₄H₂₇O₁₀	475.15989	475.16415	9.00	MS²[4 7 5]:299(1 0 0),175(3),239(1) MS³[2 9 9]:239(1 0 0),281(7),221(4)	Loss of C₂H₂, dimethoxylation, glucuronide conjugation	+	−
M28	5.54	C₁₅H₉O₅	269.04459	269.04712	9.92	MS²[2 6 9]:269(1 0 0),225(46),181(41),241(26),197(18)	Loss of C₃H₄, oxidation, carboxylation, hydroxylation	+	−
M29	5.54	C₂₄H₂₉O₉	461.18059	461.18381	6.94	MS²[4 6 1]:299(1 0 0),239(23),267(12)	Loss of C₂H₂, dimethoxylation, glucose conjugation	+	−
M30	5.63	C₁₈H₁₉O₅	315.12269	315.12534	8.38	MS²[3 1 5]:255(1 0 0),200(11),267(10),214(9)	Loss of C₂H₂, dimethoxylation, hydroxylation	+	−
M31	5.73	C₂₁H₂₂O₅NS	400.12132	400.12466	8.35	MS²[4 0 0]:279(1 0 0),341(26),200(14),333(10)	Hydroxylation, cysteine conjugation	+	−
M32	5.82	C₁₈H₁₉O₄	299.12771	299.13010	7.74	MS²[2 9 9]:239(1 0 0),281(3),221(2),133(1),263(1) MS³[2 3 9]:221(1 0 0),219(18),133(13),198(2),209(1)	Loss of C₂H₂, dimethoxylation	+	+
M33	5.82	C₁₈H₁₉O₇S	379.08459	379.08713	6.67	MS²[3 7 9]:299(1 0 0),222(2),281(1) MS³[2 9 9]:239(1 0 0),221(2),281(2),225(1)	Loss of C₂H₂, dimethoxylation, sulfonation	+	+
M34	5.95	C₁₈H₁₃O₂	261.09107	261.09323	8.52	MS²[2 6 1]:233(1 0 0),205(15),231(7),203(6)	Oxidation, dehydrogenation	+	−
M35	6.06	C₂₄H₂₅O₁₀	473.14422	473.14767	7.29	MS²[4 7 3]:297(1 0 0),201(7),239(6),235(5)	Dihydroxylation, glucuronide conjugation	+	−
M36	6.18	C₁₈H₁₉O₅	315.12269	315.12521	7.97	MS²[3 1 5]:267(1 0 0),297(6),255(5),237(1) MS³[2 6 7]:249(1 0 0),239(23),221(8),224(1)	Loss of C₂H₂, dimethoxylation, hydroxylation	+	−
M37	6.26	C₁₈H₁₉O₄	299.12771	299.13013	7.84	MS²[2 9 9]:239(1 0 0),281(3),221(2),133(1),263(1) MS³[2 3 9]:221(1 0 0),219(14),133(9),237(4)	Loss of C₂H₂, dimethoxylation	+	+
M38	6.26	C₁₈H₁₉O₇S	379.08459	379.08719	6.83	MS²[3 7 9]:299(1 0 0),297(1),242(1) MS³[2 9 9]:239(1 0 0),281(3),221(2),263(1)	Loss of C₂H₂, dimethoxylation, sulfonation	−	+
M39	6.29	C₁₄H₉O₂	209.05991	209.06119	7.10	MS²[2 0 9]:66(1 0 0),209(82),181(74),208(41),163(30)	Loss of C₃H₄ + CH₂, dehydrogenation	+	−
M40	6.29	C₁₄H₉O₃	225.05481	225.05592	5.77	MS²[2 2 5]:207(1 0 0),66(84),200(30),157(29),189(23)	Loss of C₃H₄ + CH₂, dehydrogenation, Hydroxylation	+	−
M41	6.29	C₁₅H₉O₄	253.04971	253.05106	6.03	MS²[2 5 3]:253(1 0 0),224(3),209(3),225(2),197(1)	Loss of C₃H₄, oxidation, dehydrogenation, dihydroxylation	+	−
M42	6.29	C₁₇H₁₅O₃	267.10151	267.10275	4.42	MS²[2 6 7]:249(1 0 0),239(38),133(13),221(13),247(4) MS³[2 4 9]:221(1 0 0),231(58),247(23),195(12)	Loss of CH₂, hydroxylation	+	−
M43	6.40	C₁₉H₂₁O₅	329.13829	329.14038	6.17	MS²[3 2 9]:299(1 0 0),297(6),237(65),269(41),239(11)	Loss of C₂H₂, trimethoxylation	+	−
M44	6.40	C₂₄H₂₇O₁₀	475.15989	475.16324	7.09	MS²[4 7 5]:299(1 0 0),175(10) MS³[2 9 9]:239(1 0 0),281(5),221(2)	Loss of C₂H₂, dimethoxylation, glucuronide conjugation	+	−
M45	6.40	C₁₅H₁₁O₄	255.06532	255.06677	6.21	MS²[2 5 5]:254(1 0 0),237(4),225(3),209(1) MS³[2 5 4]:224(1 0 0),192(17),210(14)	Loss of C₃H₄ + CH₂, dehydrogenation, methoxylation, hydroxylation	+	−
M46	6.40	C₁₉H₁₉O₆	343.08342	343.08679	6.57	MS²[3 4 3]:229(1 0 0),283(44),325(43),317(25),328(24)	Loss of CH₂, dimethoxylation, dihydroxylation	+	−
M47	6.52	C₁₆H₁₅O₃	255.10159	255.10394	9.29	MS²[2 5 5]:254(1 0 0),237(5),210(4),225(3),226(2) MS³[2 5 4]:224(1 0 0),192(17),210(14),225(13)	Loss of C₂H₂, hydroxylation	+	−
M48	6.62	C₁₅H₉O₅	269.04459	269.04208	−8.81	MS²[2 6 9]:240(1 0 0),241(17),225(4),197(1)	Loss of C₃H₄, oxidation, carboxylation, hydroxylation	+	−
M49	6.62	C₁₆H₁₁O₅	283.06019	283.06235	7.95	MS²[2 8 3]:268(1 0 0),255(1) MS³[2 6 8]:240(1 0 0),239(5),224(3)	Loss of C₂H₂, oxidation, carboxylation, hydroxylation	+	−
M50	6.62	C₁₅H₁₃O₄	257.08099	257.08304	8.58	MS²[2 5 7]:135(1 0 0),239(3),215(2),242(1) MS³[1 3 5]:91(1 0 0)	Loss of C₂H₂ + CH₂, dihydroxylation	+	−
M51	6.73	C₁₈H₁₇O₄	297.11211	297.11447	7.86	MS²[2 9 7]:237(1 0 0),267(86),225(75),279(19),261(11)	Loss of 2CH₂, dimethoxylation	+	−
M52	6.73	C₂₄H₂₃O₁₀	471.12859	471.13153	6.28	MS²[4 7 1]:295(1 0 0),382(3),221(4) MS³[2 9 5]:251(1 0 0),233(13),277(3)	Carboxylation, glucuronide conjugation	+	−
M53	6.73	C₁₈H₂₁O₄	301.14349	301.14542	6.59	MS²[3 0 1]:283(1 0 0),240(82),258(19),273(14)	Hydrogenation, dihydroxylation	+	−
M54	6.73	C₁₉H₂₁O₅	329.13829	329.14059	6.81	MS²[3 2 9]:269(1 0 0),311(16),267(2),299(1) MS³[2 6 9]:251(1 0 0),241(44),233(15)	Loss of C₂H₂, trimethoxylation	+	−
M55	6.73	C₁₈H₁₇O₇S	377.06892	377.07141	6.52	MS²[3 7 7]:297(1 0 0),263(2),341(1) MS³[2 9 7]:237(1 0 0),225(82),267(58),253(31)	Loss of 2CH₂, dimethoxylation, sulfonation	+	−
M56	6.84	C₁₆H₁₅O₅	287.09149	287.09399	9.02	MS²[2 8 7]:227(1 0 0),66(92),200(37),192(22)	Loss of C₃H₄ + CH₂, dimethoxylation, hydroxylation	+	−
M57	6.84	C₂₄H₂₇O₁₀	475.15989	475.16385	8.37	MS²[4 7 5]:299(1 0 0),457(13),281(7),175(4) MS³[2 9 9]:239(1 0 0),221(6),281(3)	Loss of C₂H₂, dimethoxylation, glucuronide conjugation	+	−
M58	6.84	C₂₄H₂₅O₁₀	473.14422	473.14822	8.45	MS²[4 7 3]:297(1 0 0),267(3),383(2),307(2) MS³[2 9 7]:267(1 0 0),279(51),237(19)	Loss of 2CH₂, dimethoxylation, glucuronide conjugation	+	−
M59	6.95	C₂₄H₂₉O₉	461.18059	461.18427	7.94	MS²[4 6 1]:299(1 0 0),239(87),281(50),263(11) MS³[2 9 9]:239(1 0 0)	Loss of C₂H₂, dimethoxylation, glucose conjugation	+	−
M60	7.05	C₁₇H₁₅O₂	251.10661	251.10907	9.61	MS²[2 5 1]:233(1 0 0),231(10),66(2),133(1) MS³[2 3 3]:231(1 0 0),206(45)	Loss of CH₂	+	−
M61	7.05	C₁₈H₁₇O₃	281.11721	281.11996	9.74	MS²[2 8 1]:263(1 0 0),245(43),133(7),233(4),261(2) MS³[2 6 3]:245(1 0 0),243(5),235(4),133(3)	Hydroxylation	+	−
M62	7.05	C₁₈H₁₇O₅	313.10701	313.10928	7.12	MS²[3 1 3]:298(1 0 0),285(2),269(1) MS³[2 9 8]:283(1 0 0),270(51),255(28)	Loss of 2CH₂, dimethoxylation, hydroxylation	+	−
M63	7.05	C₁₇H₁₃O₆	313.07059	313.07333	8.51	MS²[3 1 3]:298(1 0 0),285(3),191(2),269(1),253(1) MS³[2 9 8]:283(1 0 0),270(54),255(16),239(3),268(2)	Loss of C₂H₂, oxidation, carboxylation, hydroxylation, methoxylation	+	−
M64	7.17	C₂₁H₂₂O₅NS	400.12132	400.12454	8.05	MS²[4 0 0]:279(1 0 0),216(8),200(7),267(6)	Hydroxylation, cysteine conjugation	+	−
M65	7.38	C₁₇H₁₇O₃	269.11729	269.11871	5.53	MS²[2 6 9]:66(1 0 0),251(90),239(71),225(52),221(29)	Loss of C₂H₂, methoxylation	+	−
M66	7.38	C₂₄H₂₅O₁₀	473.14422	473.14774	7.43	MS²[4 7 3]:297(1 0 0),200(7),222(5),351(4)	Dihydroxylation, glucuronide conjugation	+	−
M67	7.49	C₁₇H₁₅O₂	251.10661	251.10878	8.46	MS²[2 5 1]:233(1 0 0),231(9),210(1),183(1) MS³[2 3 3]:231(1 0 0),206(7),189(3)	Loss of CH₂	+	−
M68	7.49	C₁₈H₁₇O₃	281.11721	281.11963	8.57	MS²[2 8 1]:263(1 0 0),245(41),133(7),233(4),261(2) MS³[2 6 3]:245(1 0 0),133(13),243(5),235(4)	Hydroxylation	+	−
M69	7.49	C₁₄H₁₁O₄	243.06539	243.06720	8.29	MS²[2 4 3]:66(1 0 0),175(79),214(66),109(65),225(43),181(28)	Loss of C₃H₄ + CH₂, dihydroxylation	+	−
M70	7.60	C₁₈H₁₅O₂	263.10661	263.10895	8.72	MS²[2 6 3]:245(1 0 0),133(28),235(4),243(2) MS³[2 4 5]:245(1 0 0),217(94),243(35),204(10)	Dehydrogenation	+	−
M71	7.60	C₁₅H₁₁O₅	271.06029	271.06250	8.85	MS²[2 7 1]:165(1 0 0),151(5),66(2),137(1),227(1) MS³[1 6 5]:137(1 0 0),121(28),109(8),93(7)	Carboxylation, loss of C₃H₄, hydroxylation	+	−
M72	7.60	C₁₈H₁₇O₄S	329.08422	329.08661	7.30	MS²[3 2 9]:249(1 0 0),208(16),222(2) MS³[2 4 9]:235(1 0 0),221(44),208(15)	Dehydroxylation, sulfonation	+	−
M73	7.72	C₁₆H₁₃O₂	237.09111	237.09323	9.38	MS²[2 3 7]:209(1 0 0),196(37),222(14),235(5),193(4),219(2) MS³[2 0 9]:168(1 0 0),194(87),207(57),181(27)	Loss of C₂H₂, dehydrogenation	+	−
M74	7.72	C₁₇H₁₃O₃	265.08581	265.08841	9.39	MS²[2 6 5]:237(1 0 0),209(13),196(10),247(6),221(2) MS³[2 3 7]:209(1 0 0),196(57),235(9),193(4)	Loss of CH₂, dehydrogenation, hydroxylation	+	−
M75	7.72	C₁₇H₁₅O₄	283.09641	283.09906	9.09	MS²[2 8 3]:265(1 0 0),225(15),237(7),239(2),221(1) MS³[2 6 5]:237(1 0 0),209(10),247(9),221(5)	Loss of CH₂, dihydroxylation	+	−
M76	7.72	C₁₈H₁₇O₅	313.10701	313.10971	8.50	MS²[3 1 3]:283(1 0 0),265(3),295(1) MS³[2 8 3]:265(1 0 0),225(64),237(4)	Loss of 2CH₂, dimethoxylation, hydroxylation	+	−
M77	7.72	C₁₈H₁₇O₇S	377.06892	377.07181	7.59	MS²[3 7 7]:297(1 0 0),315(51),271(14),359(10)	Dihydroxylation, sulfonation	+	−
M78	7.83	C₂₄H₂₅O₉	457.14931	457.15280	7.64	MS²[4 5 7]:281(1 0 0),439(11),175(3) MS³[2 8 1]:263(1 0 0),245(34),133(8),233(4)	Hydroxylation, glucuronide conjugation	+	−
M79	7.95	C₁₈H₁₇O₃	281.11721	281.11850	4.55	MS²[2 8 1]:263(1 0 0),245(22),133(7),253(2),235(1) MS³[2 6 3]:245(1 0 0),133(32),243(5),235(4)	Hydroxylation	+	+
M80	7.95	C₁₈H₁₇O₆S	361.07401	361.07550	4.06	MS²[3 6 1]:281(1 0 0),331(4),333(2) MS³[2 8 1]:263(1 0 0),245(17),133(5)	Hydroxylation, sulfonation	+	−
M81	7.95	C₂₄H₂₃O₁₀	471.12859	471.13135	5.89	MS²[4 7 1]:453(1 0 0),427(90),295(76),361(23)	Carboxylation, glucuronide conjugation	+	−
M82	7.95	C₁₇H₁₇O₃	269.11729	269.11807	3.16	MS²[2 6 9]:269(1 0 0),240(4),225(3),201(3),197(2),241(1)	Loss of C₂H₂, methoxylation	+	−
M83	7.95	C₂₄H₂₅O₁₀	473.14422	473.14642	4.64	MS²[4 7 3]:297(1 0 0),175(13),295(7),417(4)	Dihydroxylation, glucuronide conjugation	+	−
M84	8.06	C₁₄H₉O₃	225.05481	225.05600	6.13	MS²[2 2 5]:181(1 0 0),197(99),169(26),207(7)	Loss of C₃H₄ + CH₂, dehydrogenation, Hydroxylation	+	−
M85	8.06	C₂₄H₂₅O₉	457.14931	457.15140	4.58	MS²[4 5 7]:281(1 0 0),295(23),439(7),175(4) MS³[2 8 1]:263(1 0 0),245(23),133(14),235(3)	Hydroxylation, glucuronide conjugation	+	−
M86	8.06	C₁₅H₉O₅	269.04459	269.04614	6.28	MS²[2 6 9]:269(1 0 0),181(4),241(3),225(2),197(1)	Loss of C₃H₄, oxidation, carboxylation, hydroxylation	+	−
M87	8.06	C₁₅H₁₁O₅	271.06029	271.05988	−0.81	MS²[2 7 1]:270(1 0 0),151(12),182(6),226(5),202(4),242(2) MS³[2 7 0]:270(1 0 0),226(40),198(21),224(17)	Carboxylation, loss of C₃H₄, hydroxylation	+	−
M88	8.06	C₁₆H₁₃O₄	269.08092	269.07828	−9.50	MS²[2 6 9]:269(1 0 0),181(4),225(3),197(2)	Loss of 2CH₂, dihydroxylation	+	−
M89	8.17	C₁₈H₁₇O₆S	361.07401	361.07623	6.08	MS²[3 6 1]:281(1 0 0),305(10),333(3),331(1) MS³[2 8 1]:263(1 0 0),245(11),133(5),233(1)	Hydroxylation, sulfonation	+	−
M90	8.17	C₂₄H₂₃O₁₀	471.12859	471.13153	6.28	MS²[4 7 1]:295(1 0 0),277(44),453(18),361(11) MS³[2 9 5]:251(1 0 0),231(3),277(2),233(1)	Carboxylation, glucuronide conjugation	+	−
M91	8.17	C₂₀H₂₁O₅	341.14029	341.14039	5.69	MS²[3 4 1]:281(1 0 0),237(20),219(10),209(6),297(5)	Dimethoxylation, hydroxylation	+	−
M92	8.17	C₁₈H₁₃O₃	277.08582	277.08783	6.89	MS²[2 7 7]:233(1 0 0),259(20),249(11),219(5)	Dehydrogenation, hydroxylation	+	−
M93	8.17	C₁₈H₁₇O₇S	377.06892	377.06985	2.39	MS²[3 7 7]:297(1 0 0),315(18),271(11),359(8)	Dihydroxylation, sulfonation	+	−
M94	8.21	C₁₈H₁₉O₄	299.12771	299.13028	8.34	MS²[2 9 9]:239(1 0 0),281(3),221(3),133(1),263(1) MS³[2 3 9]:221(1 0 0),219(19),133(11),198(1)	Loss of C₂H₂, dimethoxylation	+	+
M95	8.21	C₁₆H₁₅O₂	239.10667	239.10878	8.88	MS²[2 3 9]:221(1 0 0),133(27),219(10),211(3),237(2)	Loss of C₂H₂	+	+
M96	8.28	C₁₆H₁₃O₂	237.09111	237.09271	7.19	MS²[2 3 7]:209(1 0 0),66(12),196(11),222(4),207(3) MS³[2 0 9]:167(1 0 0),207(74),181(72),168(35)	Loss of C₂H₂, dehydrogenation	+	−
M97	8.28	C₁₈H₁₇O₃	281.11721	281.11908	6.61	MS²[2 8 1]:263(1 0 0),245(13),133(4),237(1) MS³[2 6 3]:245(1 0 0),133(18),243(3),235(2)	Hydroxylation	+	−
M98	8.28	C₂₄H₂₅O₉	457.14931	457.15167	5.17	MS²[4 5 7]:281(1 0 0),439(68),295(55),175(6) MS³[2 8 1]:263(1 0 0),245(15),133(9)	Hydroxylation, glucuronide conjugation	+	−
M99	8.28	C₁₉H₂₁O₅	329.13829	329.13983	4.50	MS²[3 2 9]:314(1 0 0),254(9),269(2),299(1) MS³[3 1 4]:299(1 0 0),285(3),254(2)	Loss of C₂H₂, trimethoxylation	+	−
M100	8.40	C₁₄H₉O₃	225.05481	225.05637	7.77	MS²[2 2 5]:195(1 0 0),66(39),197(24),207(23),181(10)	Loss of C₃H₄ + CH₂, dehydrogenation, Hydroxylation	+	−
M101	8.40	C₁₈H₁₇O₆S	361.07401	361.07660	7.10	MS²[3 6 1]:281(1 0 0),305(8),343(7),331(1) MS³[2 8 1]:263(1 0 0),245(13),133(2)	Hydroxylation, sulfonation	+	−
M102	8.40	C₂₀H₂₁O₅	341.14029	341.14120	8.35	MS²[3 4 1]:281(1 0 0),219(15),237(14),253(12)	Dimethoxylation, hydroxylation	+	−
M103	8.40	C₁₅H₁₁O₅	271.06029	271.06219	7.71	MS²[2 7 1]:227(1 0 0),253(31),229(17),243(8)	Carboxylation, loss of C₃H₄, hydroxylation	+	−
M104	8.40	C₁₆H₁₃O₆	301.07079	301.07254	6.23	MS²[3 0 1]:283(1 0 0),274(34),251(17),255(12)	Loss of 2CH₂, tetrahydroxylation	+	−
M105	8.51	C₁₈H₁₃O₃	277.08582	277.08838	8.88	MS²[2 7 7]:233(1 0 0),187(57),223(21),259(9)	Dehydrogenation, hydroxylation	+	−
M106	8.51	C₂₄H₂₅O₁₀	473.14422	473.14780	7.56	MS²[4 7 3]:175(1 0 0),297(62),383(23),295(11)	Dihydroxylation, glucuronide conjugation	+	−
M107	8.61	C₁₈H₁₅O₃	279.10151	279.10410	9.06	MS²[2 7 9]:261(1 0 0),233(26),231(21),259(8),205(4) MS³[2 6 1]:233(1 0 0),231(57),207(28),205(14)	Dehydrogenation, hydroxylation	+	−
M108	8.61	C₁₉H₂₁O₅	329.13829	329.14050	6.53	MS²[3 2 9]:311(1 0 0),279(18),224(7),314(4) MS³[3 1 1]:251(1 0 0),225(77),253(18)	Loss of C₂H₂, trimethoxylation	+	−
M109	8.61	C₁₈H₁₃O₂	261.09107	261.09326	8.63	MS²[2 6 1]:66(1 0 0),233(46),231(39),209(26)	Oxidation, dehydrogenation	+	−
M110	8.61	C₁₉H₁₉O₅	327.12274	327.12515	7.49	MS²[3 2 7]:297(1 0 0),282(27),309(2) MS³[2 9 7]:282(1 0 0),225(11),279(4),237(1)	Loss of 2CH₂, trimethoxylation	+	−
M111	8.61	C₁₈H₁₇O₄S	329.08422	329.08221	−6.07	MS²[3 2 9]:315(1 0 0),249(39),301(18),302(16) MS³[3 1 5]:235(1 0 0),274(77),288(18),208(3)	Dehydroxylation, sulfonation	+	−
M112	8.72	C₁₈H₁₇O₅	313.10701	313.10944	7.63	MS²[3 1 3]:283(1 0 0),251(60),295(54),267(39),285(30)	Loss of 2CH₂, dimethoxylation, hydroxylation	+	−
M113	8.84	C₁₈H₁₇O₄	297.11211	297.11490	9.30	MS²[2 9 7]:253(1 0 0),282(17),251(7),237(2),279(1)	Loss of 2CH₂, dimethoxylation	+	−
M114	8.84	C₁₇H₁₇O₂	253.12232	253.12457	8.94	MS²[2 5 3]:225(1 0 0),224(42),235(4) MS³[2 2 5]:207(1 0 0),196(28),209(17)	Loss of CH₂, hydrogenation	+	−
M115	8.84	C₁₉H₁₉O₅	327.12274	327.12540	8.25	MS²[3 2 7]:297(1 0 0),282(26),171(3),229(2) MS³[2 9 7]:282(1 0 0),225(8)	Loss of 2CH₂, trimethoxylation	+	−
M116	8.96	C₁₆H₁₃O₂	237.09111	237.09314	9.00	MS²[2 3 7]:209(1 0 0),196(35),207(26),222(11),219(3) MS³[2 0 9]:193(1 0 0),168(83),181(44)	Loss of C₂H₂, dehydrogenation	+	−
M117	8.96	C₁₇H₁₃O₃	265.08581	265.08829	8.94	MS²[2 6 5]:237(1 0 0),207(14),247(12),196(5),209(4) MS³[2 3 7]:209(1 0 0),207(58),235(34),193(18),196(12)	Loss of CH₂, dehydrogenation, hydroxylation	+	−
M118	8.96	C₁₇H₁₅O₄	283.09641	283.09903	8.99	MS²[2 8 3]:239(1 0 0),265(61),221(51),237(31),225(14) MS³[2 3 9]:221(1 0 0),133(67),219(21),237(11)	Loss of CH₂, dihydroxylation	+	−
M119	9.08	C₁₇H₁₅O₃	267.10151	267.10379	8.31	MS²[2 6 7]:249(1 0 0),247(5),207(3),225(1),235(1) MS³[2 4 9]:207(1 0 0),247(20),191(16),221(13)	Loss of CH₂, hydroxylation	+	−
M120	9.08	C₁₆H₁₅O₃	255.10159	255.10365	8.15	MS²[2 5 5]:237(1 0 0),209(5),196(4),187(3)) MS³[2 3 7]:209(1 0 0),196(32),222(18),193(12)	Loss of C₂H₂, hydroxylation	+	−
M121	9.08	C₂₃H₂₃O₈	427.13877	427.14175	7.04	MS²[4 2 7]:295(1 0 0),277(8),391(2) MS³[2 9 5]:251(1 0 0),233(6),277(1)	Carboxylation, glucuronide conjugation, decarboxylation	+	−
M122	9.30	C₁₈H₁₇O₄	297.11211	297.11429	7.25	MS²[2 9 7]:237(1 0 0),225(75),267(41),279(25),261(11) MS³[2 3 7]:209(1 0 0),223(18),219(8)	Loss of 2CH₂, dimethoxylation	+	−
M123	9.42	C₁₆H₁₃O₄	269.08092	269.08319	8.75	MS²[2 6 9]:225(1 0 0),207(10),251(4),181(3) MS³[2 2 5]:207(1 0 0),205(30)	Loss of 2CH₂, dihydroxylation	+	−
M124	9.53	C₁₇H₁₅O₃	267.10151	267.10400	9.09	MS²[2 6 7]:249(1 0 0),239(36),133(15),221(13),247(4) MS³[2 4 9]:249(1 0 0),221(96),247(47)	Loss of CH₂, hydroxylation	+	−
M125	9.65	C₁₅H₁₃O₄	257.08099	257.08307	8.69	MS²[2 5 7]:239(1 0 0),109(71),163(39),242(30),213(28),224(9) MS³[2 3 9]:145(1 0 0),224(77),221(48),211(47),196(30)	Loss of C₂H₂ + CH₂, dihydroxylation	+	−
M126	9.65	C₁₆H₁₅O₅	287.09149	287.09363	7.77	MS²[2 8 7]:272(1 0 0),269(5),219(2) MS³[2 7 2]:152(1 0 0),123(8),257(5)	Loss of C₃H₄ + CH₂, dimethoxylation, hydroxylation	+	−
M127	9.76	C₁₈H₁₅O₂	263.10661	263.10895	8.72	MS²[2 6 3]:245(1 0 0),133(13),235(2),221(1) MS³[2 4 5]:245(1 0 0),243(48),218(37)	Dehydrogenation	+	−
M128	9.87	C₁₈H₁₅O₃	279.10151	279.10416	9.28	MS²[2 7 9]:261(1 0 0),233(25),231(21),259(7),205(4) MS³[2 6 1]:233(1 0 0),231(68),217(37),205(10)	Dehydrogenation, hydroxylation	+	−
M129	9.87	C₁₆H₁₅O₅	287.09149	287.09366	7.87	MS²[2 8 7]:269(1 0 0),272(61),219(28),258(13),227(3)	Loss of C₃H₄ + CH₂, dimethoxylation, hydroxylation	+	−
M130	9.87	C₂₃H₂₄O₆NS	442.13192	442.13541	7.97	MS²[4 4 2]:279(1 0 0),162(2),313(1) MS³[2 7 9]:261(1 0 0),233(27),231(23),259(10)	Hydroxylation, N-acetylcysteine conjugation	+	−
M131	9.98	C₁₇H₁₅O₃	267.10151	267.10312	5.80	MS²[2 6 7]:249(1 0 0),239(23),133(9),221(7),247(2)	Loss of CH₂, hydroxylation	+	−
M132	9.98	C₁₇H₁₇O₃	269.11729	269.11856	4.98	MS²[2 6 9]:239(1 0 0),207(26),251(17),221(15),225(8)	Loss of C₂H₂, methoxylation	+	−
M133	10.09	C₁₇H₁₅O₂	251.10661	251.10831	6.59	MS²[2 5 1]:233(1 0 0),231(33),221(5),247(1) MS³[2 3 3]:191(1 0 0),187(69),207(52),203(29)	Loss of CH₂	+	−
M134	10.20	C₁₉H₁₉O₅S	359.09482	359.09735	7.18	MS²[3 5 9]:279(1 0 0),344(39),264(31),261(12)	Methylation, sulfonation	+	−
M135	10.64	C₁₇H₁₅O₂	251.10661	251.10841	6.98	MS²[2 5 1]:233(1 0 0),231(9),210(1),223(1) MS³[2 3 3]:231(1 0 0),207(19),215(8),192(5)	Loss of CH₂	+	−
M136	10.64	C₁₈H₁₇O₃	281.11721	281.11923	7.15	MS²[2 8 1]:263(1 0 0),245(45),133(7),233(5),237(2) MS³[2 6 3]:245(1 0 0),133(8),243(6),235(3)	Hydroxylation	+	+
M137	10.64	C₁₈H₁₅O₄	295.09641	295.09866	7.37	MS²[2 9 5]:251(1 0 0),233(7),231(1),267(1) MS³[2 5 1]:233(1 0 0),231(9),210(1)	Carboxylation	+	−
M138	10.64	C₁₈H₁₇O₄	297.11211	297.11380	5.60	MS²[2 9 7]:297(1 0 0),279(47),224(14),282(11),267(1)	Loss of 2CH₂, dimethoxylation	+	−
M139	10.64	C₁₈H₁₃O₃	277.08582	277.08774	6.57	MS²[2 7 7]:233(1 0 0),205(60),259(32),217(17)	Dehydrogenation, hydroxylation	+	−
M140	10.89	C₁₆H₁₃O₂	237.09111	237.09305	8.62	MS²[2 3 7]:209(1 0 0),196(11),66(7),164(5),207(4) MS³[2 0 9]:207(1 0 0),193(49),181(37)	Loss of 2CH₂	+	−
M141	10.89	C₁₈H₁₇O₄	297.11211	297.11462	8.36	MS²[2 9 7]:225(1 0 0),237(90),279(38),253(35),235(27) MS³[2 2 5]:207(1 0 0),205(14)	Loss of 2CH₂, dimethoxylation	+	−
M142	10.89	C₂₀H₂₁O₅	341.14029	341.14050	6.30	MS²[3 4 1]:281(1 0 0),237(20),299(6),209(5),313(2) MS³[2 8 1]:237(1 0 0),261(9),239(6),253(4)	Dimethoxylation, hydroxylation	+	−
M143	10.89	C₁₇H₁₇O₂	253.12232	253.12433	8.00	MS²[2 5 3]:235(1 0 0),207(23),233(7),212(2) MS³[2 3 5]:207(1 0 0),233(14),205(10)	Loss of CH₂, hydrogenation	+	−
M144	11.00	C₁₉H₁₉O₅	327.12274	327.12509	7.31	MS²[3 2 7]:312(1 0 0),309(33),268(24),291(15) MS³[3 1 2]:297(1 0 0),282(59),271(7),241(4)	Loss of 2CH₂, trimethoxylation	+	−
M145	11.02	C₁₈H₁₅O₂	263.10661	263.10904	9.06	MS²[2 6 3]:245(1 0 0),133(23),235(5),243(3) MS³[2 4 5]:245(1 0 0),217(71),243(53),204(50)	Dehydrogenation	−	+
M146	11.02	C₁₈H₁₉O₃	283.13291	283.13510	7.87	MS²[2 8 3]:265(1 0 0),217(24),263(17),247(15),255(14) MS³[2 6 5]:247(1 0 0),245(44),220(15)	Hydration	+	+
M147	11.02	C₁₇H₁₅O	235.11177	235.11391	9.22	MS²[2 3 5]:194(1 0 0),66(74),157(69),220(33),200(22)	Loss of CH₂, dehydroxylation	−	+
M148	11.11	C₁₈H₁₇O₃	281.11721	281.11951	8.14	MS²[2 8 1]:263(1 0 0),245(23),133(8),235(1) MS³[2 6 3]:245(1 0 0),133(29),235(5),243(2)	Hydroxylation	+	+
M149	11.22	C₁₉H₁₉O₄	311.12772	311.13019	7.73	MS²[3 1 1]:293(1 0 0),211(21),275(19),183(17),296(13) MS³[2 9 3]:278(1 0 0),275(98),249(51),171(14)	Methoxylation, hydroxylation	+	−
M150	11.24	C₁₈H₁₅O₂	263.10661	263.10901	8.95	MS²[2 6 3]:245(1 0 0),133(8),243(5),235(2) MS³[2 4 5]:218(1 0 0),245(92),243(85),217(53)	Dehydrogenation	+	+
M151	11.34	C₁₆H₁₃O₃	253.08601	253.08818	8.93	MS²[2 5 3]:235(1 0 0),207(23),233(7),212(2),225(1) MS³[2 3 5]:207(1 0 0),233(14),208(13),194(5)	Loss of 2CH₂, hydroxylation	+	−
M152	11.34	C₁₈H₁₅O₇	343.08342	343.08380	7.49	MS²[3 4 3]:325(1 0 0),299(55),315(14),271(9)	Carboxylation, trihydroxylation	+	−
M153	11.57	C₁₇H₁₅O₃	267.10151	267.10403	9.21	MS²[2 6 7]:249(1 0 0),247(6),207(3),226(1) MS³[2 4 9]:207(1 0 0),206(47),221(3)	Loss of CH₂, hydroxylation	+	−
M154	12.02	C₁₈H₁₇O₃	281.11721	281.11981	9.21	MS²[2 8 1]:263(1 0 0),245(38),133(6),233(4),237(3) MS³[2 6 3]:245(1 0 0),133(16),243(4),219(2)	Hydroxylation	+	−
M155	12.08	C₂₄H₂₅O₈	441.15441	441.15805	8.29	MS²[4 4 1]:265(1 0 0),175(10),307(2),176(1) MS³[2 6 5]:247(1 0 0),245(24),243(1),224(1)	Glucuronide conjugation	+	+
M156	12.08	C₁₈H₁₇O₂	265.12229	265.12479	9.37	MS²[2 6 5]:247(1 0 0),265(24),245(19),224(1) MS³[2 4 7]:245(1 0 0),247(61),204(57),221(54)	Magnolol isomer	+	+
M157	12.13	C₁₇H₁₅O₂	251.10661	251.10884	8.70	MS²[2 5 1]:233(1 0 0),231(10),189(2),133(1)	Loss of CH₂	+	−
M158	12.13	C₁₅H₁₁O₄	255.06532	255.06746	8.92	MS²[2 5 5]:213(1 0 0),211(42),187(19),227(7),193(4)	Loss of C₃H₄, dehydrogenation, dihydroxylation	+	−
M159	12.31	C₁₈H₁₅O₃	279.10151	279.10406	8.92	MS²[2 7 9]:261(1 0 0),233(82),259(4),251(3) MS³[2 6 1]:233(1 0 0),259(6),219(5),218(4)	Dehydrogenation, hydroxylation	−	+
M160	12.36	C₁₈H₁₃O₂	261.09107	261.09320	8.40	MS²[2 6 1]:233(1 0 0),66(8),179(2) MS³[2 3 3]:231(1 0 0),207(51),205(48)	Oxidation, dehydrogenation	+	−
M161	12.47	C₁₆H₁₁O₅	283.06019	283.06259	8.80	MS²[2 8 3]:268(1 0 0),213(4),250(1) MS³[2 6 8]:267(1 0 0),239(42),240(36),224(31)	Loss of C₂H₂, oxidation, carboxylation, hydroxylation	+	−
M162	12.63	C₁₈H₁₇O₅S	345.07911	345.08191	8.08	MS²[3 4 5]:265(1 0 0),301(5),283(2),246(1) MS³[2 6 5]:247(1 0 0),245(30),224(3)	Sulfonation	−	+
M163	12.65	C₁₈H₁₇O₂	265.12229	265.12482	9.48	MS²[2 6 5]:247(1 0 0),245(19),217(2),224(1) MS³[2 4 7]:245(1 0 0),219(59)	Magnolol isomer	−	+
M164	12.91	C₁₅H₁₁O₅	271.06029	271.06247	8.74	MS²[2 7 1]:256(1 0 0),66(22),225(13),209(6)	Loss of C₂H₂ + CH₂, dehydrogenation, trihydroxylation	+	−
M165	13.59	C₁₈H₁₇O₃	281.11721	281.11996	9.74	MS²[2 8 1]:263(1 0 0),261(20),207(4),252(1) MS³[2 6 3]:206(1 0 0),207(90),233(86),231(69)	Hydroxylation	+	−
M166	13.60	C₂₈H₃₂O₁₁N₃S₂	650.14732	650.14221	−7.79	MS²[6 5 0]:570(1 0 0),305(45),345(29),265(7)	Sulfonation, glutathione conjugation	−	+
M167	14.39	C₁₉H₁₉O₄	311.12772	311.13040	8.40	MS²[3 1 1]:293(1 0 0),237(15),209(12),291(5) MS³[2 9 3]:249(1 0 0),275(38),247(16)	Methoxylation, hydroxylation	+	−
M168	14.50	C₁₉H₁₇O₄	309.11219	309.11499	9.23	MS²[3 0 9]:291(1 0 0),233(39),261(25),277(17),265(10) MS³[2 9 1]:263(1 0 0),231(80),273(44),233(30),247(20)	Carboxylation, methylation	+	−
M169	14.62	C₁₈H₁₇O₆	329.10189	329.10278	2.48	MS²[3 2 9]:311(1 0 0),201(32),293(18),217(7),281(6)	Tetrahydroxylation	+	−
M170	14.87	C₁₉H₁₇O₄	309.11219	309.11484	8.75	MS²[3 0 9]:291(1 0 0),247(23),193(11),265(7),209(6) MS³[2 9 1]:247(1 0 0),273(93),193(37),229(18),275(6)	Carboxylation, methylation	+	−
M171	17.34	C₁₅H₁₁O₂	223.07551	223.07654	5.31	MS²[2 2 3]:205(1 0 0),195(9),179(3),207(2)	Loss of C₃H₄, dehydrogenation	−	+
M172	17.34	C₁₈H₁₅O	247.11181	247.11307	5.38	MS²[2 4 7]:206(1 0 0),220(14),245(8)	Dehydration	+	+
M173	17.34	C₂₈H₃₀O₉N₃S	584.16969	584.16510	−7.92	MS²[5 8 4]:279(1 0 0),305(76),582(48),265(6)	Carboxylation, glutathione conjugation	+	+
M174	17.34	C₁₇H₁₅O	235.11177	235.11293	5.05	MS²[2 3 5]:66(1 0 0),200(28),157(25),202(21)	Loss of CH₂, dehydroxylation	−	+
M175	17.34	C₂₉H₃₂O₁₀NS	586.17419	586.17169	−4.19	MS²[5 8 6]:526(1 0 0),371(10),441(5),175(1)	Glucuronide conjugation, N-acetylcysteine conjugation	−	+
M176	27.75	C₂₂H₁₉O₁₀	443.09739	443.09583	−3.26	MS²[4 4 3]:267(1 0 0),175(33),249(21),223(13)	Carboxylation, Loss of C₂H₂, dehydrogenation, glucuronide conjugation	−	+

Note: t_R: retention time; U: urine; P: plasma; +: detected; −: undetected.

High resolution extracted ion chromatograms for the multiple daidzein metabolites in rat urine and plasma (A-C for plasma and D-P for urine): (A) m/z 223.07551, 235.11177, 239.10667, 345.07911, 443.09739, 586.17419, 650.14732; (B) m/z 247.11181, 263.10661, 279.10151, 283.13291, 299.12771, 379.08459; (C) m/z 265.12229, 441.15441, 584.16969; (D) m/z 209.05991, 225.05481, 243.06539, 253.12232, 287.09149, 309.11219, 313.07059, 333.13319, 395.07949; (E) m/z 255.10159, 269.11729, 311.12772, 315.12269, 329.10189, 341.14029, 361.07401; (F) m/z 255.06532, 301.07079, 343.08342, 359.09482, 415.10249, 427.13877, 461.18059, 471.12859; (G) m/z 261.09107, 269.08092, 271.06029, 329.08422, 400.12132, 429.08179, 637.17634; (H) m/z 327.12274, 329.08422, 329.13829, 377.06892; (I) m/z 473.14422; (J) m/z 237.09111, 251.10661, 253.08601, 257.08099, 283.09641, 333.00652; (K) m/z 263.10661, 265.08581, 313.10701, 475.15989; (L) m/z 267.10151, 283.06019, 295.09641, 442.13192, 457.14931; (M) m/z 277.08582, 279.10151; (N) m/z 281.11721; (O) m/z 253.04971, 297.11211; (P) m/z 269.04459.

3.4.1

3.4.1 Identification of metabolites M0, M156 and M163

In negative ion mode, the metabolites M0, M156 and M163 exhibited the same theoretical [M−H]^- ions at m/z 265.12229 (C₁₈H₁₇O₂, error ≤ 9.50 ppm), which were eluted at 17.34, 12.08 and 12.65 min, respectively. Based on the comparison of the chromatographic retention time and MS/MSⁿ spectral data with the magnolol reference standard, M0 could be accurately identified as magnolol (Guo et al., 2019; Wu et al., 2006; Zhu et al., 2020 ). Since the major product ions of M156 and M163 (m/z 247, 245 and 224) were consistent with M0, they could be inferred as positional isomers of magnolol.

3.4.2

3.4.2 Identification of metabolites M32, M37, M47, M65, M82, M94, M95, M120, M132 and their secondary metabolites

M95, eluted at 8.21 min, was 26 Da lower than magnolol, which was presumed to be the product of magnolol lost CH = CH. In its ESI-MS² spectrum, a series of NLFs similar to magnolol were observed, such as 2 Da (m/z 239 → m/z 237), 18 Da (m/z 239 → m/z 221) and 28 Da (m/z 239 → m/z 211), which further confirmed our inference. Metabolites M47 and M120 had the same [M−H]^- ions at m/z 255.10159 (C₁₆H₁₅O₃, error ≤ 9.50 ppm), which was 16 Da higher than M95. Therefore, they were considered to be hydroxylation products of M95 because of their similar fragmentation behaviors. In the same way, M65 M82 and M132 (C₁₇H₁₇O₃, 30 Da higher than M95) were identified as methoxylated products of M95, while M32, M37 and M94 (C₁₈H₁₉O₄, 60 Da higher than M95) were inferred as di-methoxylated products of M95.

Meanwhile, in the HREIC, M43, M54, M99 and M108 were extracted at m/z 329.13829 (C₁₉H₂₁O₅, error ≤ 7.00 ppm) with retention times ranging from 6.40 to 8.61 min. They were 90 Da higher than M95, and the continuous NLFs of 30 Da (m/z 329 → m/z 299 → m/z 269 → m/z 239) occurred in the ESI-MS² spectra, which further confirmed that they were tri-methoxylated products of M95. Metabolites M5, M12, M16, M30 and M36 produced the same [M−H]^- ions at m/z 315.12269 (C₁₈H₁₉O₅, error ≤ 9.50 ppm), which were 16 Da higher than that of M32. Furthermore, the identical ion fragments with NLFs indicated that they were hydroxylation products of M32. In the same vein, M33 and M38 (m/z 379.08459, C₁₈H₁₉O₇S), M25, M29 and M59 (m/z 461.18059, C₂₄H₂₉O₉), M27, M44 and M57 (m/z 475.15989, C₂₄H₂₇O₁₀) were 80 Da, 162 Da and 176 Da higher than M32, respectively. In their ESI-MS² spectra, the same DPIs at m/z 299 ([M−SO₃−H]^-, [M−C₆H₁₀O₅−H]^- and [M−C₆H₈O₆−H]^-) demonstrated that they were sulfonated, glucose conjugated, and glucuronide conjugated products of M32, respectively.

The metabolite M24, with a retention time of 5.00 min, yielded a [M−H]^- ion at m/z 637.17634 (C₂₉H₃₃O₁₆, 7.41 ppm), which was 162 Da higher than that of M27. In the ESI-MSⁿ spectrum, the fragment ions appeared including [M−GluA−H]^- at m/z 461 and [M−GluA−Glc−H]^- at m/z 299, both indicated that M24 was the glucose conjugated product of M27. M4 and M17 were eluted at 3.67 min and 4.57 min separately, and possessed the same [M−H]^- ion at m/z 395.07949 (C₁₈H₁₉O₈S, error ≤ 8.50 ppm), which were 80 Da higher than M5. Therefore, M4 and M17 were inferred as the sulfonated products of M5.

Metabolites M50 and M125 were eluted at 6.62 min and 9.65 min, respectively. Both of them gave rise to a deprotonated molecular ion with the molecular formula C₁₅H₁₃O₄ at m/z 257.08099. Their ESI-MS² base peak ions at m/z 239 and m/z 242 were individually formed due to neutral loss of H₂O and CH₃, suggesting that they might be products of di-hydroxylated magnolol lost C₂H₂ + CH₂.

3.4.3

3.4.3 Identification of metabolites M61, M68, M79, M97, M136, M148, M154, M155, M162, M165 and their secondary metabolites

M61, M68, M79, M97, M136, M148, M154, and M165 were detected at 7.05 min, 7.49 min, 7.95 min, 8.28 min, 10.64 min, 11.11 min, 12.02 min, and 13.59 min, respectively, and had the same [M−H]^- ion located at m/z 281.11721 (C₁₈H₁₇O₃, error ≤ 10.00 ppm). All of them were 16 Da higher than magnolol, indicating that they could be identified as hydroxylation products of magnolol. In their ESI-MSⁿ spectra, the emergence of multiple identical NLFs, such as 18 Da (m/z 281 → m/z 263), 2 Da (m/z 263 → m/z 261) and 28 Da (m/z 263 → m/z 235), further corroborated the above conclusion.

M155 was eluted at 12.08 min and produced its [M−H]^- ion at m/z 441.15441 (C₂₄H₂₅O₈, 8.29 ppm). It was 176 Da higher than magnolol, and [M−GluA−H]^- at m/z 265 was also observed, so M155 was presumed to be the glucuronidation product of magnolol. Along the same lines and evidence, M162 could be identified as the sulfonated product of magnolol.

The metabolites M31 and M64 were eluted at 5.73 min and 7.17 min, and generated the same deprotonated molecular ion at m/z 400.12132 (C₂₁H₂₂O₅NS, error ≤ 8.50 ppm). In their ESI-MS² spectra, the [M−C₃H₆NO₂S−H]^- at m/z 279 were both detected, indicating that they might be the N-acetylcysteine conjugation products of M61. In the same manner, M78, M85 and M98 (m/z 457.14931, C₂₄H₂₅O₉), M80, M89 and M101 (m/z 361.07401, C₁₈H₁₇O₆S), M130 (m/z 442.13192, C₂₃H₂₄O₆NS), M149 and M167 (m/z 311.12772, C₁₉H₁₉O₄), M23 and M169 (m/z 329.10189, C₁₈H₁₇O₆) could be identified as the glucuronidation, sulfonation, N-acetylcysteine conjugation, methoxylation, and tri-hydroxylation products of M61, respectively.

All metabolites M14, M35, M66, M83 and M106 yielded the same [M−H]^- ions at m/z 473.14422 (C₂₄H₂₅O₁₀, error ≤ 8.00 ppm) and were 16 Da higher than M78, which proved that they were hydroxylated products of M78. In their ESI-MS² spectra, there were abundant product ions at m/z 297 due to the neutral loss of 176 Da, which provided an adequate basis for our deduction. Similarly, M77 and M93 (m/z 377.06892, C₁₈H₁₇O₇S) could be presumed as the hydroxylated products of M80, while M91, M102 and M142 (m/z 341.14029, C₂₀H₂₁O₅) were characterized as the methoxylated products of M149.

In negative ion mode, M53 was eluted at 6.73 min and its elemental composition was proposed to be C₁₈H₂₁O₄ (6.59 ppm). In its MS/MS spectrum, a series of characteristic product ions were observed, such as m/z 283 ([M−H₂O−H]^-), m/z 240 ([M−H₂O−C₃H₇−H]^-), m/z 273 ([M−CO−H]^-), and m/z 258 ([M−CO−CH₃−H]^-), so that M53 was clearly identified as the hydrogenation and dihydroxylation product of magnolol.

3.4.4

3.4.4 Identification of metabolites M42, M46, M75, M118, M119, M124, M131, M153 and their secondary metabolites

The metabolites M42, M119, M124, M131 and M153 were 14 Da lower than that of M61 with retention times of 6.29 min, 9.08 min, 9.53 min, 9.98 min and 11.57 min, suggesting that they might be the products of M61 lost CH₂. The NLFs of 18 Da, 28 Da and 2 Da were consistent with the MS/MS spectrum of magnolol, which validated our above hypothesis. M75 and M118 owned the same [M−H]^- ion at m/z 283.09641 (C₁₇H₁₅O₄, error ≤ 9.50 ppm), and they were 16 Da higher than M42, which meant that they were hydroxylation products of M42. Similarly, M46 could be identified as the di-methoxylated product of M75.

M140 was eluted at 10.89 min and gave rise to its [M−H]^- ion at m/z 237.09111 (C₁₆H₁₃O₂, 8.62 ppm), which was 28 Da lower than magnolol. Based on the predicted molecular formula, it could be inferred that M140 was the product of magnolol lost 2CH₂. The metabolite M151 was 16 Da higher than M140, which illustrated that it was the hydroxylation product of M140. According to the established line of reasoning, M88 and M123 were the di-hydroxylated products of M140, while M51, M113, M122, M138 and M141 were the di-methoxylated products of M140 (as speculated by the continuous NLFs of 30 Da).

M55, which was eluted at 6.73 min, was 80 Da higher than M51 in negative ion mode, suggesting that it might be the sulfonation product of M51. In the MS² spectra, the neutral loss of 80 Da (m/z 377 → m/z 297) and the fragmentation behaviors of m/z 297 both provided ample support for our inference. With this method for continuous practice, M58 (176 Da higher than M51), M110, M115 and M144 (30 Da higher than M51), M7, M19, M62, M76 and M112 (16 Da higher than M51) could be individually identified as glucuronidation, methoxylation and hydroxylation products of M51, while M104 (32 Da higher than M88) was reckoned as dihydroxylation product of M88.

The metabolite M69 exhibited its [M−H]^- ion at m/z 243.06539 (C₁₄H₁₁O₄, 8.29 ppm), which was 26 Da lower than M88. In the ESI-MS² spectrum, the fragmentation behaviors of M69 were in line with that of M88, so it could be deduced that M69 was the product of M88 lost CH = CH. Metabolites M11, M56, M126 and M129, which were separately eluted at 4.08 min, 6.84 min, 9.65 min, and 9.87 min, possessed the same theoretical [M−H]^- ions at m/z 287.09149 (C₁₆H₁₅O₅, error ≤ 9.50 ppm). In their MS/MS spectra, the DPI at m/z 227 and NLFs of 15 Da (m/z 287 → m/z 272 → m/z 257) were both detected, implying that two molecules of methoxy were introduced into the structure. For this reason, M11, M56, M126 and M129 were identified as the de-hydroxylation and dimethoxylation products of M69.

M39 was eluted at 6.29 min and yielded a [M−H]^- ion at m/z 209.05991 (C₁₄H₉O₂, 7.10 ppm). It was 28 Da lower than M140, which revealed that M39 was the dehydrogenation product of M140 lost CH = CH. Metabolites M40, M84, and M100 with the same [M−H]^- ion at m/z 225.05481 (C₁₄H₉O₃, error ≤ 8.00 ppm) were eluted at 6.29 min, 8.06 min, and 8.40 min, respectively. All of them were 16 Da higher than M39, so these three isomers could be characterized as the hydroxylation products of M39. At the same time, M18 and M45 (m/z 255.06532, C₁₅H₁₁O₄) were identified as the hydroxylation and methoxylation products of M39.

M60, M67, M133, M135 and M157 were individually eluted at 7.05 min, 7.49 min, 10.09 min, 10.64 min and 12.13 min with the same [M−H]^- ion at m/z 251.10661 (C₁₇H₁₅O₂, error ≤ 10.00 ppm), which were 14 Da lower than magnolol and its isomers (M156, M163). Hence, it could be presumed that they were the products of M0, M156 and M163 lost CH₂, and they were isomers because the substitution sites of two hydroxyl were different. In the same way, M147 and M174 (C₁₇H₁₅O, 16 Da lower than M60) were inferred as the dehydroxylated products of M60.

The retention times of M74 and M117 were 7.72 min and 8.96 min, respectively, which were 2 Da lower than that of M42, suggesting that they were dehydrogenation products of M42. Metabolites M114 and M143 were separately eluted at 8.84 min and 10.89 min, and produced the same deprotonated molecular ion at m/z 253.12232 (C₁₇H₁₇O₂, error ≤ 9.00 ppm), which were 12 Da lower than that of magnolol. By comparing with the MS/MS spectral data of magnolol, M114 and M143 could be identified as the hydrogenation products of magnolol lost CH₂. In the RP-HPLC system, the compounds with larger Clog P values will be more difficult to be eluted. Therefore, M114 (Clog P, 4.20) and M143 (Clog P, 4.38) were isomers to each other.

3.4.5

3.4.5 Identification of metabolites M52, M81, M90, M137, M152, M168, M170 and their secondary metabolites

The metabolite M137 eluted at 10.64 min generated a [M−H]^- ion at m/z 295.09641 (C₁₈H₁₅O₄, 7.37 ppm), which was 30 Da higher than that of magnolol. In its ESI-MS² spectrum, M137 exhibited a NLF of 44 Da, resulting in the production of [M−CO2−H]^- ion at m/z 251, which was attributed to the presence of carboxyl group in its structure. By comparing the mass fragmentation behaviors of m/z 251 in the MS³ spectra with that of magnolol, M137 could be identified as the carboxylation product of magnolol. The metabolites M52, M81 and M90 were separately eluted at 6.73 min, 7.95 min and 8.17 min, and gave rise to the same [M−H]^- ions at m/z 471.12859 (C₂₄H₂₃O₁₀, error ≤ 6.50 ppm). Their ESI-MSⁿ spectra all showed the DPIs at m/z 295 ([M−GluA−H]^-), which could help us to infer that they were glucuronidation products of M137. With the same inference thinking, M152 (C₁₈H₁₅O₇, 48 Da higher than M137) were identified as the tri-hydroxylated product of M137, while M168 and M170 (C₁₉H₁₇O₄, 14 Da higher than M137) could be presumed to be methylation products of M137.

The metabolite M121 eluted at 9.08 min exhibited its [M−H]^- ion at m/z 427.13877 (C₂₃H₂₃O₈, 7.04 ppm), which was 132 Da higher than M137 or 44 Da lower than M52. In the MS² spectra, M121 produced a DPI at m/z 295 through the neutral loss of 132 Da (C₅H₈O₄), which proved that M121 was the decarboxylation product of M52. M173 displayed a [M−H]^-ion at m/z 584.16969 (C₂₈H₃₀O₉N₃S, −7.92 ppm), which was 289 Da higher than that of M137. The NLF of 305 Da (m/z 584 → m/z 279) suggested that M173 might be the glutathione conjugation product of M137 and that the binding site was at the carboxyl group.

Metabolites M71, M87, and M103, which possessed the same [M−H]^- ions at m/z 271.06029 (C₁₅H₁₁O₅, error ≤ ±9.00 ppm), were eluted at 7.60 min, 8.06 min, and 8.40 min, respectively. By comparing with the molecular formula and atomic occupancy of M137 and combining with the MSⁿ spectral data, M71, M87, and M103 could be surmised as de-propenyl (C₃H₄) and hydroxylation products of M137. The metabolite M176 eluted at 27.75 min showed a [M−H]^- ion at m/z 443.09739 (C₂₂H₁₉O₁₀, −3.26 ppm), and then produced the DPIs at m/z 267 and m/z 223 through the continuous neutral loss of 176 Da (GluA) and 44 Da (CO₂), suggesting that M176 were de-vinyl (C₂H₂) and dehydrogenation product of M52.

Metabolites M10, M22, M49, and M161 were extracted from HREICs at m/z 283.06019 (C₁₆H₁₁O₅, error ≤ 10.00 ppm) in negative ion mode with retention times of 3.97 min, 5.00 min, 6.62 min, and 12.47 min. None of them had fragment ions at m/z 265, so the hydroxyl group in the molecules might be oxidized. In their MSⁿ spectra, the NLFs of 15 Da (m/z 283 → m/z 268), 28 Da (m/z 283 → m/z 255) and 44 Da (m/z 268 → m/z 224) were all observed, which demonstrated that M10, M22, M49 and M161 were oxidation, de-vinyl (C₂H₂) and hydroxylation products of M137. Based on this assertion, M63 (C₁₇H₁₃O₆, 30 Da higher than M10) was identified as the methoxylation product of M10, while M15, M26, M28, M48 and M86 (C₁₅H₉O₅, 14 Da lower than M10) could be reckoned as the demethylation products of M10.

3.4.6

3.4.6 Identification of metabolites M107, M128, M159 and their secondary metabolites

Metabolites M107, M128, and M159 were eluted at 8.61 min, 9.87 min, and 12.31 min, respectively. All of them yielded the same [M−H]^- ions at m/z 279.10151 (C₁₈H₁₅O₃, error ≤ 9.50 ppm), which were 14 Da higher than M0 or 2 Da lower than M61. In their ESI-MS² spectra, the DPI at m/z 261 was also 2 Da lower than the DPI at m/z 263 generated by M61, suggesting that M107, M128, and M159 were dehydrogenation products of M61. In the same way, M92, M105 and M139 (C₁₈H₁₃O₃, 277.08582) could be identified as the dehydrogenation products of M107.

The metabolites M73, M96 and M116, whose retention times were 7.72 min, 8.28 min and 8.96 min, produced the same [M−H]^- ions at m/z 237.09111 (C₁₆H₁₃O₂, error ≤ 9.50 ppm, 28 Da lower than M0). The [M−CH3−H]^- ions at m/z 222 were unreservedly shown in their ESI-MS² spectra, which indicated that M73, M96 and M116 were the de-vinyl (C₂H₂) and dehydrogenation products of M0. The metabolite M164 eluted at 12.91 min possessed a [M−H]^- ion at m/z 271.06029 (C₁₅H₁₁O₅, 8.74 ppm), which was 34 Da higher than that of M73. The NLF of 15 Da (m/z 271 → m/z 256) was observed in its MS² spectrum, so M164 could be speculated as the de-methylene and tri-hydroxylation product of M73.

The metabolite M171 eluted at 17.34 min gave rise to a [M−H]^- ion at m/z 223.07551 (C₁₅H₁₁O₂, 5.31 ppm), which was 14 Da lower than M73. A DPI at m/z 205 was monitored, indicating that M171 was the de-propenyl (C₃H₄) and dehydrogenation product of magnolol. On the basis of the above inference, M158 (C₁₅H₁₁O₄, 255.06532, 32 Da higher than M171) was identified as the di-hydroxylation product of M171.

Metabolites M6, M20, and M41 with retention times of 3.87 min, 4.88 min, and 6.29 min, respectively, showed identical [M−H]^- ions at m/z 253.04971 (C₁₅H₉O₄, error ≤ 10.00 ppm), which were 2 Da lower than M158. In their MS² spectra, the NLFs of 28 Da (m/z 253 → m/z 225 → m/z 197) provided sufficient evidence for inferring that they were oxidation products of M158. Metabolites M3, M8, and M13 owned the same [M−H]^- ions at m/z 415.10249 (C₂₁H₁₉O₉, error ≤ 9.00 ppm). In the ESI-MSⁿ spectra, there were abundant base peak ions at m/z 253 due to the neutral loss of 162 Da (C₆H₁₀O₅), suggesting that they were glucose conjugation products of M6. In the same manner, M9 (C₂₁H₁₇O₁₀, 176 Da higher than M6) was inferred as the glucuronidation product of M6, while M21 (C₁₅H₉O₇S, 80 Da higher than M6) could be identified as the sulfonation product of M6.

3.4.7

3.4.7 Identification of secondary metabolites of metabolites M0, M156 and M163

Metabolite M172, 18 Da lower than M0, exhibited a deprotonated molecular ion at m/z 247.11181 (C₁₈H₁₅O, 5.38 ppm) with the retention time of 17.34 min. The DPIs at m/z 245, 220 and 206 pointed out that fragmentation behaviors of M172 were similar to that of magnolol, and thus M172 was identified as the dehydrated product of magnolol. Conversely, M146 (C₁₈H₁₉O₃, 18 Da higher than M0) was inferred to be the hydrated product of magnolol.

Metabolites M1 and M2 were separately eluted at 2.92 and 3.39 min, and yielded the same [M−H]^- ion at m/z 333.13319 (C₁₈H₂₁O₆, error ≤ 8.00 ppm) in negative ion mode. The consecutive NLFs of 60 Da (m/z 333 → m/z 273 → m/z 213) implied that four methoxy groups were introduced into the molecules. Therefore, M1 and M2 were diagnosed as the tetra-methoxylation products of magnolol lost 2C₂H₂. M72 and M111(C₁₈H₁₇O₄S, 329.08422), whose retention times were 7.60 min and 8.61 min, were 16 Da lower than that of M162, suggesting that both were de-hydroxylation and sulfonation products of M0, M156 and M163.

The metabolites M70, M127, M145 and M150 were individually detected at 7.60 min, 9.76 min, 11.02 min and 11.24 min with the same [M−H]^- ions at m/z 263.10661 (C₁₈H₁₅O₂, error ≤ 9.50 ppm), which were 2 Da lower than M0. By comparing with the mass fragmentation behaviors of M0, M156 and M163, they could be tentatively identified as the dehydrogenation products of magnolol and its isomers.

M34, M109 and M160 gave rise to a deprotonated ion at m/z 261.09107 (C₁₈H₁₃O₂, error ≤ 9.00 ppm), which were eluted at 5.95 min, 8.61 min and 12.36 min, respectively. In the MSⁿ spectra, the DPIs at m/z 233 and m/z 205 were observed owing to continuous neutral loss of CO (28 Da), which validated that the hydroxyl groups in the molecules were oxidized. Accordingly, M34, M109 and M160 were inferred as the oxidation and dehydrogenation products of M0, which were isomers of each other.

The metabolite M134 generated the [M−H]^- ion at m/z 359.09482 (C₁₉H₁₉O₅S, 7.18 ppm) with a retention time of 10.20 min. It was 14 Da higher than M162, and the DPIs at m/z 279 ([M−SO₃−H]^-) and m/z 264 ([M−SO₃−CH₃−H]^-) in its ESI-MS² spectrum further fully confirmed that M134 was the methylation product of M162. In the same way, M166 (C₂₈H₃₂O₁₁N₃S₂, 305 Da higher than M162) was determined as the glutathione conjugation product of M162, while M175 (C₂₉H₃₂O₁₀NS, 145 Da higher than M155) could be deduced as the N-acetylcysteine conjugation product of M155.

3.5

3.5 Proposed biotransformation pathways of magnolol in rats

In the present study, a total of 177 metabolites (prototype compound included) with different molecular structures were monitored and identified in rats after oral administration of magnolol. As a beneficial phenolic compound, the in vivo biotransformation process of magnolol could be summed up as undergoing three fundamental metabolic pathways. The first metabolic pathway (shown in Fig. 6): parent drug and its isomers were bio-transformed into drug metabolism centres(Liu et al., 2020; Mei et al., 2019), such as hydroxylated magnolol, de-methylene magnolol and de-vinyl magnolol, based on which many chemical reactions occurred to generate a series of secondary metabolites. The second metabolic pathway (shown in Fig. 7): magnolol was first metabolized into carboxylated magnolol and dehydrogenated magnolol, both of which served as metabolic centers to gradually produced final metabolites. The third metabolic pathway (shown in Fig. 8): magnolol and its isomers went through intricate biotransformation processes, such as methoxylation, sulfonation, oxidation, and hydration, to yield complicated secondary metabolites. It was noteworthy that while both phase I and phase II metabolic reactions were observed, the vast majority of in vivo metabolites were generated either through binding reactions (phase II) or composite reactions (including various types of biotransformation reactions).

The first metabolic pathway of magnolol in rats in vivo.

The second metabolic pathway of magnolol in rats in vivo.

The third metabolic pathway of magnolol in rats in vivo.

4 Conclusion

It is well known that the identification and structural characterization of relevant metabolites is one of the most challenging tasks in the study of drug component metabolism (also known as xenobiotic metabolism), due to the fact that most of the metabolite signals at low concentrations are masked by complex background noise and matrix interference. Hence, a UHPLC-LTQ-Orbitrap MSⁿ-based multi-channel integrated data-mining method was developed to analyze the in vivo metabolism of magnolol in rats, which combined the online data acquisition approach with off-line data processing and structure elucidation techniques. As a result, based on the characteristic fragmentation patterns, chromatographic behaviors, ESI-MS/MS spectral information, and the corresponding Clog P values, a total of 176 metabolites as well as prototype compound were identified in rat urine and plasma, most of which were discovered for the first time.

On the one hand, our results indicated that magnolol originally underwent hydroxylation, methoxylation, oxidation, and carboxylation in rats to generate the corresponding metabolite cluster centers, followed by sulfonation, glucuronidation, glucose conjugation, glutathione conjugation, and N-acetylcysteine conjugation to produce a large number of secondary metabolites. On the other hand, this data-mining strategy based on ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was validated as a more logical and hierarchical technique for metabolite identification, which could provide a practical reference for future rapid analysis and determination of major-to-trace drug-related components in vivo. In conclusion, the valuable data obtained in our current study not only greatly expanded the understanding of the therapeutic material basis and pharmacological mechanism of magnolol, but also provided new ideas for its toxicity evaluation, safety monitoring and drug delivery forms design.

Author contributions

B. Ma, J. Wang and Z. Wang conceived and designed the research; Y. Guo, Z. Wang and Z. Lin supervised the experimental plan; B. Ma, T. Lou and T. Wang performed UHPLC-LTQ-Orbitrap MS experiment; R. Li, J. Liu, S. Yu, H. Pei, S. Tian and Y. Li performed animal experiment; B. Ma analyzed the data; B. Ma wrote the paper. All authors read and approved the final manuscript.

Acknowledgments

This research was funded by Chinese Pharmacopoeia Commission [grant number, 12] and China National Accreditation Service for Conformity Assessment [grant number, 2020CNAS07].

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Development of a UHPLC-MS/MS-based data-mining method for rapid profiling and characterization of magnolol metabolites in rat urine and plasma

Abstract

Keywords

Magnolol

UHPLC-LTQ-Orbitrap MS

Data-mining techniques

Metabolites

1 Introduction

2 Experiment

2.1 Chemicals and reagents

2.2 Animals

2.3 Drug administration and biological samples preparation

2.4 Instrument and conditions

2.5 Peak selections and data processing

3 Results and discussion

3.1 The establishment of analytical strategy

3.2 Fragmentation behaviors of the magnolol in negative ion mode

3.3 Implement of MMDFs data-mining methods

3.4 Identification of magnolol metabolites

3.4.1 Identification of metabolites M0, M156 and M163

3.4.2 Identification of metabolites M32, M37, M47, M65, M82, M94, M95, M120, M132 and their secondary metabolites

3.4.3 Identification of metabolites M61, M68, M79, M97, M136, M148, M154, M155, M162, M165 and their secondary metabolites

3.4.4 Identification of metabolites M42, M46, M75, M118, M119, M124, M131, M153 and their secondary metabolites

3.4.5 Identification of metabolites M52, M81, M90, M137, M152, M168, M170 and their secondary metabolites

3.4.6 Identification of metabolites M107, M128, M159 and their secondary metabolites

3.4.7 Identification of secondary metabolites of metabolites M0, M156 and M163

3.5 Proposed biotransformation pathways of magnolol in rats

4 Conclusion

Acknowledgments

Declaration of Competing Interest

References

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