Systematic screening and characterization of cardamonin metabolites using UHPLC-Q-Exactive Orbitrap MS after oral administration to rats

3.4.1 Simple metabolites

When cardamonin underwent glucurondiation or sulfation, these metabolites didn’t show significant differences in backbone structures. Thus, these metabolites were a homologous series of chalcones with different substituents. The DPIs mentioned above could also be directly utilized for determining their possible structures (He et al., 2009).

Glucuronidation is the common metabolic pathway for polyhydroxy flavonoid (Oliveira and Watson, 2000). M6 eluted at 4.8 min presented [M−H]⁻ ion at m/z 445.11398 (C₂₂H₂₁O₁₀; mass error 2.374 ppm). The product ion at m/z 269 was produced by the neutral loss of glucuronide. The DPIs including m/z 254, m/z 227, m/z 175, m/z 165, m/z 150, m/z 121 and m/z 113, were also observed. M6 also presented [M + H]⁺ ion at 447.12778 (C₂₂H₂₃O₁₀, mass error −1.774 ppm). The product ion at m/z 271 [M + H-glucuronide]⁺ and characteristic product ions at m/z 229, m/z 167, m/z 152 and m/z 124 could also be observed. Similarly, M13 displayed [M−H]⁻ ions at m/z 445.11398 (C₂₂H₂₁O_10, mass error 2.374 ppm), detected in urine, which further yielded the product ion at m/z 269 [M−H-glucuronide]⁻ and DPIs at m/z 254, m/z 253, m/z 241, m/z 226, m/z 139 and m/z 113. M13 also possessed [M + H]⁺ ion at m/z 447.12796 (C₂₂H₂₃O_10, mass errors 1.372 ppm), which produced fragment ions at m/z 271, m/z 167, m/z 152 and m/z 124. The fragmentation pathways of M13 resembled those of M6. Hence, M6 and M13 could be tentatively identified as isomeric monoglucuronidation metabolites of cardamonin (see Fig. 3).

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Fig. 3

High resolution extracted ion chromatograms for 40 cardamonin metabolites in rat plasma and urine and faeces (A-C for negative ion mode and D-F for positive ion mode). Plasma: (A) m/z 289.07217, 445.11398, 473.14517, 621.14612, 289.07245, 431.09839, 289.07208, 349.03870, 349.03867, 351.05463, 349.03922, 349.03879, 243.10207, 273.07846, 351.05429, 319.07932; Urine: (B) m/z 243.10243, 621.14624, 621.14636, 445.11404, 621.14612, 431.09836, 349.03857, 379.04916, 243.10307, 255.06651, 289.07034; Faeces: (C) m/z 289.07285, 307.07184, 307.07141, 225.09175, 243.10271, 243.10207; Plasma: (D) m/z 273.07550, 447.12778, 257.08023, 275.12692, 305.10327; Urine: (E) m/z 447.12784, 255.10115, 255.10117, 447.12796, 285.11209, 433.11273, 257.08032; Faeces: (F) m/z 285.11105, 285.11148.

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M10, M12 and M14, which possessed [M−H]⁻ ions at m/z 621.14624, m/z 621.14636 and m/z 621.14612 (C₂₈H₂₉O₁₆, mass errors 1.978 ppm, 2.172 ppm and 1.785 ppm, orderly), were all detected in urine sample. The product ions at m/z 445 [M−H-glucuronide]⁻ and m/z 269 [M−H-2glucuronide]⁻ were generated by the successive loss of glucuronide (176 Da). The characteristic fragment ions including m/z 254, m/z 253, m/z 241, m/z 226, m/z 165, m/z 139, m/z 113 and m/z 103, were all generated in their ESI-MS/MS spectra. The product ion at m/z 117 could also be generated by loss of H₂O and OCH₂ from the ion at m/z 165. Thus, M10, M12 and M14 were all speculated to be isomeric diglucuronidation metabolites of cardamonin (see Fig. 4).

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Fig. 4

The ESI-MS/MS spectra of M6, M12 and M22 in negative ion mode.

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M7 displayed [M−H]⁻ ion at m/z 473.14517 (C₂₄H₂₅O₁₀, 2.001 ppm) in negative ion mode. It yielded the product ion at m/z 297 [M−H-glucuronide]⁻ when the DPIs at m/z 253, m/z 175, m/z 121, m/z 119 and m/z 113 could also be observed. Thus, we could speculate that M7 was dimethylation and glucuronidation product of cardamonin. However, the exact substitution site could not be confirmed by ESI-MS/MS data only (see Fig. 5).

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Fig. 5

The proposed metabolic pathways of cardamonin in rat.

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M22, M24, M26, M30 and M31 afforded the same theoretical [M−H]⁻ ions at m/z 349.03873 (C₁₆H₁₃O₇S, mass errors within 5 ppm) in negative ion mode. Compared with [M−H]⁻ ion at m/z 349, the product ion at m/z 269 [M−H-SO₃]⁻ was generated, which was decreased by 80 Da. A series of characteristic fragment ions at m/z 254, m/z 253, m/z 227, m/z 226, m/z 165, m/z 150, m/z 149, m/z 121 and m/z 113 were also generated. In the ESI-MS/MS spectrum of M31, the product ion at m/z 331 was generated by loss of H₂O (18 Da). Therefore, M22, M24, M26, M30 and M31 were all determined to be isomeric sulfation metabolites of cardamonin.

M28 displayed [M−H]⁻ ion at m/z 379.04935 (C₁₇H₁₅O₈S, mass error 2.494 ppm), which further generated fragment ion at m/z 299 [M−H-SO₃]⁻. The DPI at m/z 165 was the same as that of cardamonin, suggesting that its ring A was not substituted by hydroxyl or methyl. The characteristic ions at m/z 119 and m/z 103 were also observed. Based on the abovementioned data, M28 was tentatively identified as hydroxylation, methylation and sulfation metabolite of cardamonin.

M23 and M27 generated their [M + H]⁺ ions at m/z 285.11105 and m/z 285.11148 (C₁₇H₁₇O₄, mass errors −3.807 ppm and −2.299 ppm), respectively. Serial neutral losses from the ion at m/z 285 produced another two fragment ions at m/z 225 [M + H-CH₂-CO-H₂O]⁺ and m/z 224 [M + H-Ac-H₂O]⁺. In the ESI-MS/MS spectrum of M27, the DPI at m/z 181 was produced by the cleavage between C8 and C9, which was 14 Da more than that of cardamonin in positive ion mode. The data confirmed that the occurrence of methylation was on ring A and further produced fragment ion at m/z 121 and m/z 107 [167-2CH₂-CO-H₂O]⁺. The ion at m/z 105 also confirmed that ring B was not the substituted site. M15 produced [M + H]⁺ ion at m/z 285.11209 (C₁₇H₁₇O₄, mass errors −0.160 ppm), which yielded ESI-MS/MS product ions at m/z 270 [M + H-CH₃]⁺, m/z 242 [M + H-Ac]⁺ and m/z 229 [M + H-2CO]⁺. The DPI at m/z 167 was also observed, indicating that ring A was not substituted by methyl and further generated product ion at m/z 119. Hence, M15, M23 and M27 were characterized as isomeric methylation metabolites of cardamonin.

M9 and M11 displayed [M + H]⁺ ions at m/z 255.10117 and m/z 255.10115 (C₁₆H₁₅O₃, mass errors −1.650 ppm and −1.571 ppm) in positive ion mode, orderly. Both of them were one oxygen less than that of cardamonin, which implied that dehydroxylation occurred. The DPI at m/z 177 was produced by the cleavage between C1 and C7, which was 16 Da less than that of cardamonin and further yielded fragment ions at m/z 145, m/z 129 and m/z 117. The characteristic ions at m/z 115 and m/z 105 were also observed. On account of the neutral losses of H₂O (18 Da) and CO (28 Da), product ions at m/z 237, m/z 227 and m/z 199 were respectively generated in the ESI-MS/MS spectrum of M11. The DPI at m/z 151 could be generated by the cleavage between C8 and C9 and further produced fragment ions at m/z 137, m/z 133, m/z 119 and m/z 115. From the abovementioned analysis, M9 and M11 could be deduced as isomeric dehydroxylation metabolites of cardamonin.

M2 and M3 presented their [M−H]⁻ ions at m/z 307.07184 and m/z 307.07141 (mass errors 3.904 and 2.476 ppm), respectively. Their molecular formulas were established as C₁₆H₁₃O₆, with two oxygens more than that of cardamonin in negative ion mode. This indicated that dihydroxylation might occur. Due to the presence of hydroxyls in molecule, the [M−H]⁻ ions at m/z 307.07184 and m/z 307.07141 were fragmented into product ions at m/z 283 by neutral loss of H₂O (18 Da). In addition, m/z 123 and m/z 107 were generated by RDA cracking ions. Thus, they were tentatively identified as isomeric dihydroxylation metabolites of cardamonin.

3.4.2 Multiple metabolites based on demethylation reactions

Demethylation is also one of the important metabolic pathways of cardamonin. A total of 12 metabolites were involved in demethylation, which induced some changes in the mass-to-charge ratio of characteristic ions. Due to the loss of methyl, the DPI at m/z 151 (14 Da less than that of cardamonin) could be produced by the cleavage between C8 and C9 in negative ion mode. The DPI at m/z 177 was also generated by the cleavage between C1 and C7. Both of them would further generate other product ions by neutral losses of CH₂, CH₃, O, H₂O, CO, CO₂ and their combinations. Because of the structural similarity, these compounds shared certain fragmentation pathways in ESI-MS/MS spectrum and produced many common fragment ions. Thus, the abovementioned DPIs could be regarded as indicators for demethylation metabolites. The fragmentation behaviors in positive ion mode were in consistent with those in negative ion mode.

Both M21 and M33 afforded [M + H]⁺ ions at m/z 257.08023 and m/z 257.08032 (C₁₅H₁₃O_4, mass errors −2.355 ppm and −2.005 ppm) and the product ion at m/z 229 [M + H-CO]⁺, m/z 189 [M + H-2H₂O-2O]⁺, m/z 187 [M + H-CO-COCH₂]⁺, m/z 175 [M + H-CO-3H₂O]⁺ and m/z 159 [M + H-2CO-COCH₂]⁺ were generated. The characteristic ion at m/z 153 was observed and serious neutral losses from the ion at m/z 179 triggered another three fragment ions at m/z 131, m/z 115 and m/z 103. In addition, M33 presented [M−H]⁻ ions at m/z 255.06651 (C₁₅H₁₁O₄, mass error 3.782 ppm), which generated the product ions at m/z 213 [M−H-CO-CH₂]⁻ and m/z 211 [M−H-CO₂]⁻. The DPI at m/z 151 was also produced in negative ion mode. Consequently, M21 and M33 were both assigned as isomeric demethylation metabolites of cardamonin.

M17 and M20 possessed [M−H]⁻ ions at m/z 431.09839 and m/z 431.09836 (C₂₁H₁₉O₁₀, mass errors 2.591 and 2.521 ppm), and the product ions at m/z 255 [M−H-glucuronide]⁻ and m/z 113 were generated. Due to the loss of methyl on ring A, the DPI at m/z 151 was observed, which further produced fragment ion at m/z 107 [151-CO₂]⁻. M17 also displayed [M + H]⁺ ion at m/z 433.11273 (mass error −0.446 ppm) and the product ion at m/z 257 was derived from the precursor ion at m/z 433. In the ESI-MS/MS spectra, the characteristic product ion at m/z 153 that represented ring A was observed. We also speculated that product ion at m/z 151 could be generated from m/z 179 by loss of CO. According to the above analysis, we could speculate that M17 and M20 were demethylation and monoglucuronidation metabolites of cardamonin.

M4 presented [M + H]⁺ ion at m/z 273.07550 (C₁₅H₁₃O₅, mass error −0.915 ppm), which indicated that cardamonin underwent both demethylation and hydroxylation reactions. In the ESI-MS/MS spectrum, it yielded product ion at m/z 209 [M + H-CO-2H₂O]⁺. The DPI at m/z 153 showed that ring A was not substituted by hydroxyl and product ion at m/z 135 [153-H₂O]⁺ was generated by RDA cracking ions. On the basis of these data, M4 was tentatively deduced as demethylation and hydroxylation metabolite of cardamonin.

3.4.3 Multiple metabolites based on hydrogenation reactions

Due to the existence of C⚌C and C⚌O bonds in the molecular structure, cardamonin was able to be hydrogenated and a large part of metabolites underwent monohydrogenation or even dihydrogenation. In addition, C⚌C bonds hydrogenated more easily than C⚌O bonds. On the condition that the cleavage sites were the same with those in cardamonin, the DPI at m/z 165 could be produced by the cleavage between C8 and C9, when the fragment ion at m/z 193 was generated by the cleavage between C1 and C7. As for the dihydrogenation metabolites, the product ions at m/z 167 and m/z 195 would be generated in the same ways. Similarly, these characteristic fragment ions would further generate the other smaller ions by neutral losses of CH₂, CH₃, O, H₂O, CO and their combinations. Then, the corresponding fragment ions were used to identify hydrogenation metabolites.

M34 afforded [M−H]⁻ ion at m/z 273.07846 (C₁₅H₁₃O₅, mass error 4.924 ppm), Furthermore, the ESI-MS/MS spectrum of M34 showed successive losses of CO to yield the product ions at m/z 245 [M−H-CO]⁻ and m/z 217 [M−H-2CO]⁻. Under the premise that ring A was not substituted by hydroxyl, we speculated that the characteristic product ions at m/z 143 and m/z 115 were formed as follows: the fragment ion at m/z 179 could be produced by the cleavage between C1 and C7 when m/z 151 was generated by the cleavage between C8 and C9. Even though both of them did not appear in the ESI-MS/MS spectrum, they could yield the characteristic product ions at m/z 143 and m/z 115 by neutral losses of 2H₂O (36 Da). And the product ion at m/z 131 was generated from the ion at m/z 179 by loss of 3O. Therefore, it could be deduced that M34 was demethylation, hydrogenation and monohydroxylation metabolite of cardamonin.

M1, M5, M16, M19 and M37 possessed the same theoretical [M−H]⁻ ions at m/z 289.07168 (C₁₅H₁₃O₆, with mass errors within 5 ppm) in negative ion mode, which were one hydroxyl more than that of M34. Similarly, if ring A was not substituted by hydroxyl, they could generate product ions at m/z 179 and m/z 151, both of which were not observed. However, fragment ions at m/z 147 [179-2O]⁻, m/z 131 [179-3O]⁻, m/z 119 [179-2O-CO]⁻ were detected, and m/z 115, m/z 105, m/z 103 were produced from the product ion at m/z 151 by losses of 2H₂O, H₂O + CO and 3O. The fragment ion at m/z 175 [M−H-C₆H₆-2H₂O]⁻ could also be detected. Thus, M1, M5, M16, M19 and M37 were determined to be isomeric demethylation, hydrogenation and dihydroxylation metabolites of cardamonin.

With respect to the identification of M18, the ESI-MS/MS spectrum of [M−H]⁻ ion at m/z 225.09175 (C₁₅H₁₃O₂, mass error 3.304 ppm) showed three characteristic fragment ions of cardamonin at m/z 207 [M−H-H₂O]⁻, m/z 197 [M−H-CO]⁻and m/z 181 [M−H-CO₂]⁻. And thus, we speculated that M18 was demethylation, hydrogenation and didehydroxylation metabolite of cardamonin.

M29 and M35 afforded their respective [M−H]⁻ ions at m/z 351.05463 and m/z 351.05429 (C₁₆H₁₅O₇S, mass errors 3.789 ppm and 2.200 ppm). The typical fragment ion at m/z 271 was produced by loss of SO₃. The fragment ion at m/z 315 [M−H-2H₂O]⁻ was generated from the precursor ion at m/z 351 due to the presence of hydroxyls in the structure. In addition, the characteristic fragment ions at m/z 149 [193-CO₂]⁻, m/z 145 [193-3O]⁻, m/z 119 [165-CH₂-2O]⁻ and m/z 106 [193-CO₂-Ac]⁻ were also observed. On the basis of these summarized fragmentation patterns, M29 and M35 were tentatively identified as isomeric hydrogenation and sulfation metabolites of cardamonin.

M40 presented [M + H]⁺ at m/z 305.10327 and the possible molecular formula was C₁₆H₁₇O₆ (mass error 4.278 ppm) with two oxygens and two hydrogens more than that of cardamonin in positive ion mode. In the ESI-MS/MS spectrum, a series of fragment ions at m/z 135 [167-2O]⁺, m/z 131 [167-2H₂O]⁺, m/z 121 [167-CH₂-2O]⁺ and m/z 107 [167-CO₂-2O]⁺ were generated from the RDA cracking ion at m/z 167. Thus, we speculated that M40 was hydrogenation and dihydroxylation metabolite of cardamonin.

The [M−H]⁻ ion at m/z 319.07932 (C₁₆H₁₅O₇, mass error −4.984 ppm) of M36 yielded three representative fragment ions at m/z 301 [M−H-H₂O]⁻, m/z 291 [M−H-CO]⁻ and m/z 275 [M−H-CO₂]⁻. The DPI at m/z 165 was detected and further indicated that hydroxyls were substituted on ring B. Finally, we finally identified M36 as hydrogenation and trihydroxylation metabolite of cardamonin.

M25 presented [M + H]⁺ ion at m/z 275.12692, whose possible elemental compositions was C₁₆H₁₉O₄. (mass error −3.146 ppm). It showed five major product ions at m/z 257 [M + H-H₂O]⁺, m/z 229 [M + H-H₂O-CO]⁺, m/z 215 [M + H-CO₂-O]⁺, m/z 189 [M + H-2Ac]⁺ and m/z 159 [195-2H₂O]⁺ in the ESI-MS/MS spectrum. Hence, M25 could be identified as dihydrogenation metabolite of cardamonin.

M8, M32, M38 and M39 possessed the same theoretical [M−H]⁻ ions at m/z 243.10273 (C₁₅H₁₅O₃, mass errors within 5 ppm) in negative ion mode, which could gave five significant product ions at m/z 225 [M−H-H₂O]⁻, m/z 199 [M−H-CO₂]⁻, m/z 197 [M−H-CO-H₂O]⁻, m/z 183 [M−H-CO₂-O]⁻ and m/z 181 [M−H-CO₂-H₂O]⁻. Due to the loss of methoxyl in ring A, the characteristic ion at m/z 165 could be generated by the cleavage between C1 and C7, which was not observed in fact. However, the product ions at m/z 149 [165-O]⁻, m/z 147 [165-H₂O]⁻, m/z 131 [165-H₂O-O]⁻, m/z 113 [165-2H₂O-O]⁻ were detected. Based on the abovementioned data, M8, M32, M38 and M39 were tentatively identified as isomeric dehydrogenation, demethylation and dihydroxylation metabolites of cardamonin.