1. Introduction
Isodon excisoides (Y. Z. Sun ex C.
H. Hu) H. Hara (IE) is a perennial medicinal herb belonging to the genus Isodon in the Labiatae family, mainly distributed in the western region of Henan and Yunnan Provinces in China [1].
Isodonis Excisoidis Herba (the aerial parts of IE, IEH) has been listed in the Chinese Materia Medica and prescribed for the treatment of cold headaches, rheumatic arthralgia, bruises, fractures, bleeding wounds, and snake bites [2]. Meanwhile, local residents also use it to make tea, decoctions, and dressings for treating esophageal cancer, chronic pharyngitis, and hepatitis [3].
Ent-kaurane diterpenoids are the primary bioactive constituents of Isodon plants [4]. For instance, the highly anticipated oridonin possesses promising anti-tumor [5] and anti-leukemia properties [6] and serves as a covalent NLRP3 inhibitor with strong anti-inflammasome activity [7]. Notably, several ent-kaurane diterpenoids with anti-tumor activity have been isolated from Isodon excisoides [8,9,10,11,12,13,14].
Consistent with these reports, our laboratory has isolated and identified 12 ent-kaurane diterpenoids from IEH, including oridonin and lasiokaurin, confirming their significant anti-tumor activity [8,9,10]. However, the structures of ent-kaurane diterpenoids are diverse and mostly homologous, making it difficult to separate and purify them through conventional chemical composition separation methods. In particular, the trace components cannot be enriched and identified, substantially restricting research on the anti-tumor substance basis of this plant.
With the rapid development of chromatography and mass spectrometry (MS) techniques, ultra-high-performance liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS) has become an extremely powerful tool for the targeted and untargeted profiling of natural products from complex matrices [15]. Compared with low-resolution mass spectrometry methods, LTQ-Orbitrap-MS not only provides high sensitivity with a mass accuracy of less than 10 ppm but also offers an unequivocal assignment of the product ions to their respective precursor ions in MSn (n ≥ 3) experiments, allowing us to precisely describe the fragmentation pathways [16]. However, to our knowledge, only a limited number of studies have employed liquid chromatography–mass spectrometry (LC-MS) to analyze Isodon species or their isolated ent-kaurane diterpenoid monomers, with reports on Isodon rubescens [17,18,19], Isodon serra [20,21], Isodon japonica [22], Rabdosia rubescens [23,24], and oridonin [25,26].
Furthermore, the existing literature primarily concentrates on the scanning and analysis of ent-kaurane diterpenoids in the negative ion mode [19,21,22,23,25,26] or on the quantitative analysis of known monomers from Isodon species plants in the positive mode [17,18,20,24]. Research dedicated to elucidating their fragmentation pathways in the positive ion mode remains scarce. Therefore, systematically analyzing multiple ent-kaurane diterpenoids using this highly sensitive and rapid method in the positive ion mode is of significant value.
In this study, we focus on a comprehensive study of the MSn spectra on an LTQ-Orbitrap-MS to elucidate the fragmentation behavior of 12 known compounds isolated from IEH, and we also apply characteristic fragmentation patterns to preliminarily identify additional unknown chemical constituents from this herbal medicine. In this study, twelve compounds were divided into three types according to their basic skeletons: bridgehead-unsubstituted 7,20-epoxy-ent-kaurane diterpenoids, bridgehead-substituted 7,20-epoxy-ent-kaurane diterpenoids, and 7,20-non-epoxy-kaurane diterpenoids. Based on the fragmentation patterns, accurate mass measurements, and retention times, a total of 94 compounds were unambiguously identified or tentatively characterized, 48 of which are potentially new ent-kaurane diterpenoids.
This is the first determination of ent-kaurane diterpenoids in IEH using UHPLC-LTQ-Orbitrap-MS in the positive ion mode.
2. Results and Discussion
2.1. Optimization of the LC and MS Conditions
The UHPLC-LTQ-Orbitrap-MS conditions were systemically optimized to improve the resolution and sensitivity of the analysis and obtain appropriate ionization. Key UHPLC parameters, including the chromatographic column (Syncronis-C18, ACQUITY UPLC HSS-T3, and ACQUITY UPLC BEH-C18), mobile phase compositions (deionized water, deionized water containing 0.1% formic acid, and acetonitrile), gradient profile, and flow rate (0.2–0.5 mL/min), were evaluated and refined with peak resolution as the primary criterion. The results indicated that the ACQUITY UPLC BEH-C18 column with a small particle size (1.7 μm) provided better separation with a higher peak resolution, and that a mobile phase consisting of deionized water with 0.1% formic acid and aqueous acetonitrile outperformed the others.
For LTQ-Orbitrap-MS conditions optimization, positive ion mode was selected due to the stronger signal response observed for the twelve known ent-kaurane diterpenoids and other sample components compared with the negative ion mode. The mass spectrometric parameters were initially tuned by direct infusion of reference standards, employing a resolution power of 30,000 in full-scan mode followed by MSn scan in the Orbitrap (also at R = 30,000) with a 10 s exclusion duration. The collision energies for MS2 and MS3 were optimized over a range up to 35% of the maximum collision-induced dissociation (CID) energy to yield informative fragment ion profiles.
It was observed that at the initial test level of 35%, fragment ions in the higher m/z range showed relatively low abundance, complicating the determination of stepwise fragmentation pathways. Therefore, the CID energies were adjusted to levels that produced more informative profiles suitable for pathway elucidation, ultimately set to 25% for MS2 and 20% for MS3 of the maximum CID energy, thereby ensuring appropriate ionization and informative MS abundance data in the positive ion mode.
2.2. Identification of the Ent-Kaurane Diterpenoids of IEH
Based on the structural characteristics of the twelve reference compounds (Figure 1), they were divided into three groups: bridgehead-unsubstituted 7,20-epoxy-ent-kaurane diterpenoids, including oridonin (2), lasiokaurin (5), and 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one (13); bridgehead-substituted 7,20-epoxy-ent-kaurane diterpenoids, including kamebacetal A (21), kamebacetal B (22), and reniformin C (23); and 7,20-non-epoxy-kaurane diterpenoids, consisting of kamebakaurin (44), kamebanin (52), 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one (54), henryin (57), 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one (64), and 1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-16-en-15-one (74). The fragmentation patterns and pathways of these reference compounds are shown in Table 1. The confirmation of known compounds in IEH for which reference standards were available was performed based on coincidence in retention time, precursor ions, fragment ions, and their abundance patterns with respect to the reference standards analyzed under identical LC-MS conditions.
Moreover, the fragmentation patterns and pathways of the standards were investigated in depth to further confirm the tentative structures of their derivatives. For the unavailable standard compounds, the tentative structures were proposed based on the following steps: (1) The molecular formula was established based on high-accuracy protonated precursors, such as [M + H]+, [M + NH4]+, and [M + H − CH4O]+, within a mass error of 10 ppm and the fractional isotope abundance. (2) When there were several matching isomers, the structures were resolved by querying the chemical information database of IEH components—which was compiled from previously reported compounds in Isodon species, particularly IEH [8,9,10,11,12,17,18,19,20,21,22,23,24,25,26,27]—and by comparing their distinct retention times in the total ion chromatogram (TIC), along with characteristic fragment ion abundance patterns.
(3) The fragment ions from MSn mass spectrometry, together with the fragmentation pathways of known compounds and theoretical simulations from Thermo Scientific™ Mass Frontier 7.0 software, were integrated to propose tentative structures. In particular, for the 7,20-non-epoxy-kaurane diterpenoids, kamebakaurin, henryin, etc., the fragmentation rules for the standards were applied to elucidate the tentative structures of the corresponding derivatives with the same basic skeleton. The total ion chromatograms (TICs) of the IEH samples were acquired in the positive mode for further confirmation (shown in Figure 2).
Based on the MSn analysis and accurate mass measurements and retention times, a total of 94 components, including 17 bridgehead-unsubstituted 7,20-epoxy-ent-kaurane diterpenoids, 6 bridgehead-substituted 7,20-epoxy-ent-kaurane diterpenoids, and 71 7,20-non-epoxy-kaurane diterpenoids, were unambiguously or tentatively identified from IEH. The key diagnostic ions and fragmentation pathways are summarized in Table 2, and the complete MSn dataset is provided in the Supplementary Materials (Table S1).
2.2.1. Bridgehead-Unsubstituted 7,20-Epoxy-Ent-Kaurane Diterpenoids
In the present study, three reference compounds—oridonin (2), lasiokaurin (5), and 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one (13)—were first introduced in the positive ion modes of the ESI source. Because of their remarkably higher abundance in the positive ion mode, the [M + H]+ or [M + NH4]+ ions were selected as the precursor ions for CID fragmentation to produce the MS/MS spectra. Then, the prominent MS/MS product ions were selected for further MSn analysis (n = 3–4).
The fragmentation pathways and MSn mass spectra of oridonin (2), lasiokaurin (5), and 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one (13) are shown in Figure 3, Figure 4 and Figure 5.
Oridonin (2) exhibits the [M + H]+ ion at m/z 365.1955 (C20H29O6) and the [2M + H]+ ion at m/z 729.3835 (C40H57O12) in the MS1 spectra. The [M + H]+ ion at m/z 365.1955 (C20H29O6) produced a prominent product ion at m/z 347.1850 (C20H28O5) through the typical loss of H2O. The ion at m/z 347.1850 was further fragmented to produce the prominent ions at m/z 329.1745 (C20H25O4), 311.1639 (C20H23O3), 283.1690 (C19H23O2), and 265.1585 (C19H21O) by typical losses of 18 Da (H2O), 18 Da (H2O), 28 Da (CO), and 18 Da (H2O) in the MS3 spectrum.
Notably, the loss of CO was apparent at m/z 329.1745 (m/z 329.1745 → 301.1796), m/z 311.1639 (m/z 311.1639 → 283.1690), and m/z 293.1535 (m/z 293.1535 → 265.1585), and the loss of CH2O was apparent at m/z 329.1745 (m/z 329.1745 → 299.1639) in the MS3 spectra, suggesting that the methyleneoxy between C-7 and C-10 was rearranged on the B-ring. The loss of H2O was also apparent in the MS3 spectra at m/z 347.1850 (m/z 347.1850 → 329.1745), m/z 329.1745 (m/z 329.1745 → 311.1639), m/z 311.1639 (m/z 311.1639 → 293.1535), and m/z 283.1690 (m/z 283.1690 → 265.1585), suggesting the cleavage of the hydroxyl groups of oridonin. Due to the existence of the anacetoxy group at C-1, the characteristic fragment ions of lasiokaurin (5) were m/z 407.2052 (C22H31O7), m/z 389.1949 (C22H29O6), and m/z 371.1843 (C22H27O5).
The characteristic ions of lasiokaurin (5) were 42 Da (C2H2O) larger than those of oridonin. The other ions at m/z 329.1739 (C20H25O4), 311.1634 (C20H23O3), 299.1634 (C19H23O3), 283.1684 (C19H23O2), and 265.1578 (C19H21O) were from the fragment ions at m/z 389.1949 (C22H29O6, [M + H‒H2O]+) via typical losses of 60 Da (C2H4O2), 18 Da (H2O), 30 Da (CH2O), 28 Da (CO), and 18 Da (H2O) in the fragmentation pathways.
Compounds 1, 4, and 6 are oridonin derivatives, and they showed similar fragmentation behaviors as oridonin (2). Compound 1 generated the same [M + H]+ ions at m/z 365.1951 (C20H29O6) and the [M + NH4]+ ion at m/z 382.2213 (C20H32O6N) in the positive mode, and a dominant ion is generated at m/z 347.1846 (C20H28O, [M + NH4 − NH3 − H2O]+) in the MS3 spectrum. Therefore, it is tentatively identified as an isomer of oridonin.
Compound 4 generated [M + H]+ precursor ions at m/z 349.2002 (C20H29O5), which displayed common fragment ions at m/z 331.1897 (C20H27O4), 313.1792 (C20H25O3), 295.1686 (C20H23O2), 285.1844 (C19H25O2), 267.1736 (C19H23O), and m/z 249.1617 (C19H21). All were 16 Da smaller than the characteristic ions of oridonin (2), suggesting that Compound 4 represents oridonin (2) lacking OH elements. Compound 6 had [M + H]+ ions at m/z 347.1845 (C20H27O5) and the [M + NH4]+ ion at m/z 364.2108 (C20H30O5N), and it had fragment ions at m/z 329.1740 (C20H25O4), 311.1631 (C20H23O3), 283.1688 (C19H23O2), and 265.1581 (C19H21O).
The characteristic ions of Compound 6 were 18 Da (H2O) smaller than oridonin, suggesting that it represents the reference oridonin compound lacking one hydroxyl group and a double bond addition.
1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one (13) produced the [M + H]+ ion at m/z 375.2150 (C22H31O5) and the [2M + H]+ ion at m/z 749.4241 (C44H61O10) in the MS1 spectra. The [M + H]+ ion at m/z 375.2150 (C22H31O5) produced prominent product ions at m/z 357.2051 (C22H29O4) through a typical loss of H2O and at m/z 315.1946 (C20H27O3) through a typical loss of C2H4O2. The ion at m/z 315.1946 was further fragmented to produce prominent ions at m/z 297.1839 (C20H25O2), 269.1890 (C19H25O), and 251.1785 (C19H23) by typical losses of 18 Da (H2O), 28 Da (CO), and 18 Da (H2O) in the MS3 spectrum.
In addition, the loss of CO was apparent at m/z 315.1946 (m/z 315.1946 → 287.1998) and at m/z 297.1839 (m/z 297.1839 → 269.1890), and the loss of CH2O was apparent at m/z 315.1946 (m/z 315.1946 → 285.1840) in the MS3 spectra, suggesting that the methyleneoxy between C-7 and C-10 was also rearranged on the B-ring. The loss of H2O was also apparent at m/z 375.2150 (m/z 375.2150 → 357.2051), m/z 315.1946 (m/z 315.1946 → 297.1839), and m/z 287.1998 (m/z 287.1998 → 269.1890), proposing the cleavage of the hydroxyl group of 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one.
Compounds 3 and 7 are isomers of 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one and showed fragmentation behaviors similar to those of 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one (13). The typical losses of 18 Da (H2O), 60 Da (C2H4O2), 18 Da (H2O), 28 Da (CO), and 18 Da (H2O) were commonly observed, and most produced ions at m/z 357.2050 (C22H29O4), 315.1949 (C20H27O3), 297.1844 (C20H25O2), 269.1895 (C19H25O), and 251.1785 (C19H23).
Compounds 10 and 14 exhibited [M + H]+ at 317.2104 (C20H29O3), and both presented MS/MS characteristic ions at m/z 299.2000 (C20H27O2), 281.1896 (C20H25O), 271.2052 (C19H27O), and 253.1946 (C19H25). All were 58 Da smaller than the characteristic ions of 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one, suggesting that Compounds 10 and 14 represent 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one lacking the terminal acetoxy group (C2H2O2). Compounds 11 and 12 shared the common ion [M + H]+ at m/z 317.2101 (C20H29O3).
The presence of ions at m/z 299.2000 and m/z 281.1899 indicates that both shared the same skeleton with Compound 10 and were isomers of Compound 10. Notably, the same characteristic fragment ion at m/z 275.1637 (C17H21O3) indicates that the A-ring of Compounds 11 and 12 is prone to cleavage. Compounds 8 and 17 showed [M + H]+ precursor ions at m/z 315.1945 (C20H27O3) and common fragmentation behaviors, such as the ions at m/z 297.1842 (C20H25O2), 273.1484 (C17H21O3), and 269.1893 (C19H25O).
All were 2 Da smaller than Compound 10 during their ESI-MSn fragmentations, suggesting that they represent Compound 10 with an additional double bond. Compound 9 gave rise to an [M + H]+ ion at m/z 331.1897 (C20H27O4) and fragment ions at m/z 313.1794 (C20H25O3), 289.1429 (C17H21O4), and 285.1844 (C19H25O2). All of them were 16 Da larger than Compound 8, suggesting that it represents Compound 8 with a hydroxyl addition.
Compound 15 generated [M + H]+ ions at 357.2055 (C22H29O4) and fragment ions at m/z 297.1844 (C20H25O2), 255.1375 (C17H19O2), and 241.1218 (C16H17O2). The characteristic ions of Compound 15 were 18 Da (H2O) smaller than 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one, suggesting that it represents the reference Compound 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one lacking a hydroxyl group and double bond addition. Compound 16 had [M + H]+ at 359.2209 (C22H31O4), and it presented MS/MS characteristic ions at m/z 299.1996 (C20H27O2) and m/z 281.1897 (C20H25O).
All were 16 Da smaller than the characteristic ions of 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one, suggesting that Compound 16 represents 1α-acetoxy-14β-hydroxyl-7,20-epoxy-ent-kaur-16-en-15-one lacking OH elements.
2.2.2. Bridgehead-Substituted 7,20-Epoxy-Ent-Kaurane Diterpenoids
The bridgehead-substituted 7,20-epoxy-ent-kaurane diterpenoids consisted of kamebacetal A (21), kamebacetal B (22), and reniformin C (23). The MSn mass spectrometry data and the fragmentation pathways of kamebacetal A (21), kamebacetal B (22), and reniformin C (23) are shown in Figure 6, Figure 7 and Figure 8.
Kamebacetal A (21) and kamebacetal B (22) are isomers, and both feature a methoxy group at the C-20 position. They generated the same [M + NH4 − NH3 − CH4O]+ precursor ions at 331.1895 (C20H27O4), very few [M + NH4]+ ions at m/z 380.2448 (C21H34O5N), and [2M + NH4]+ ions at m/z 742.4508 (C42H64O10N) in the MS1 spectra. The precursor ion at m/z 331.1895 could be fragmented to produce ions at m/z 313.1792 (C20H25O3), m/z 295.1686 (C20H23O2), m/z 267.1737 (C19H23O), and m/z 249.1632 (C19H21) through typical losses of 18 Da (H2O), 18 Da (H2O), 28 Da (CO), and 18 Da (H2O).
Remarkably, the loss of CO was apparent at m/z 313.1792 (m/z 313.1792 → 285.1842), m/z 295.1686 (m/z 295.1686 → 267.1737), and m/z 277.1581 (m/z 277.1581 → 249.1632), suggesting that the methyleneoxy between C-7 and C-10 was rearranged on the B-ring. The loss of H2O was also apparent at m/z 331.1895 (m/z 331.1895 → 313.1792), m/z 313.1792 (m/z 313.1792 → 295.1686), and m/z 285.1842 (m/z 285.1842 → 267.1737), suggesting the cleavage of the hydroxyl groups of kamebacetal A and kamebacetal B.
Due to the existence of an ethoxy group at C-20, reniformin C (23) produced the [M + NH4 − NH3 − CH3CH2OH]+ precursor ions at 331.1898 (C20H27O4), very few [M + NH4]+ ions at m/z 394.2577 (C22H36O5N), and [2M + NH4]+ ions at m/z 770.4820 (C44H68O10N) in the MS1 spectra. Then, the precursor ion at m/z 331.1895 also further fragmented to produce ions at m/z 313.1792, 295.1686, 285.1842, 277.1581, 267.1737, and 249.1632, which are the same as those of kamebacetal A. Therefore, the [M + NH4 − NH3 − R]+ (R is the substituent group at the C-20 position) precursor ions at 331.1895 (C20H27O4), and the characteristic ions at m/z 313.1792, 295.1686, 285.1842, 277.1581, 267.1737, and 249.1632 were the most possible fragmentation pathways for bridgehead-substituted 7,20-epoxy-ent-kaurane diterpenoids.
Compounds 18, 19, and 20 generated the [M + NH4 − NH3 − H2O]+ precursor ions at 331.1896 (C20H27O4), [M + NH4]+ ions at m/z 366.2248 (C20H32O5N), and [2M + NH4]+ ions at m/z 714.4156 (C40H60O10N) in the MS1 spectra. Then, the precursor ion at m/z 331.1896 further fragmented to produce the characteristic ions at m/z 313.1792, 295.1688, 285.1844, 277.1582, 267.1738, and 249.1633, which are the same as those of kamebacetal A, indicating that the methoxy group at the C-20 position of kamebacetal A was replaced by the hydroxyl group. Based on previous reports [27], they were tentatively characterized as dimethyl kamebacetal A and its isomers.
2.2.3. 7,20-Non-Epoxy-Kaurane Diterpenoids
Kamebakaurin (44), kamebanin (52), 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one (54), henryin (57), 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one (64), and 1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-16-en-15-one (74) are important 7,20-non-epoxy-kaurane diterpenoid components in IEH. The MSn mass spectrometry data and their fragmentation pathways are shown in Figure 9, Figure 10, Figure 11, Figure 12, Figure 13 and Figure 14.
Henryin (57) generated [M + H]+ precursor ions at 393.2258 (C22H33O6), [M + NH4]+ ions at m/z 410.2522 (C22H36O6N), and [2M + Na]+ ions at m/z 807.4265 (C44H64O12Na) in the MS1 spectra. Due to the existence of three hydroxyl groups at C-1, C-7, and C-14, an acetoxy group at C-20, and a carbonyl group at C-15 of henryin, the precursor ion at m/z 393.2258 further fragmented to produce the characteristic ions at m/z 375.2165 ([M + H − H2O]+), m/z 357.2060 ([M + H − 2H2O]+), m/z 333.2060 ([M + H − C2H4O2]+), m/z 315.1954 ([M + H − H2O − C2H4O2]+), m/z 297.1848 ([M + H − H2O − C2H4O2 − H2O]+), m/z 279.1742 ([M + H − H2O − C2H4O2 − 2H2O]+), m/z 269.1902 ([M + H − H2O − C2H4O2 − H2O − CO]+), m/z 267.1737 ([M + H − H2O − C2H4O2 − H2O − CH2O]+), and m/z 251.1790 ([M + H − H2O − C2H4O2 − 2H2O − CO]+).
Compounds 27, 30, 51, 59–60, and 66 are isomers of henryin and exhibited fragmentation behaviors similar to those of henryin (57). The typical losses of 18 Da (H2O), 60 Da (C2H4O2), and 28 Da (CO) were commonly observed, and most produced ions at m/z 375.2159 (C22H31O5), 357.2054 (C22H29O4), 315.1948 (C20H27O3), 297.1843 (C20H25O2), 279.1740 (C20H23O), 269.1893 (C19H25O), and 251.1789 (C19H23). Compound 89 showed an [M + NH4]+ precursor ion at 408.2370 (C22H34O6N) and then produced an [M + NH4 − NH3 − H2O]+ ion at 373.2001 (C22H29O5) through the loss of H2O. [M + NH4 − NH3 − H2O]+ could be further fragmented to produce the ions at m/z 355.1898 (C22H27O4), 331.1898 (C20H27O4), 313.1793 (C20H25O3), 295.1688 (C20H23O2), 277.1583 (C20H21O), 267.1739 (C19H23O), and 249.1634 (C19H21) via typical losses of 18 Da (H2O), 60 Da (C2H4O2), and 28 Da (CO) in the MS3 spectrum, suggesting that it represents the reference compound henryin lacking 2H, and it was tentatively identified as dehydro-henryin.
Compounds 33 and 49 gave rise to the [M + NH4]+ ions at 426.2475 (C22H36O7N), and both presented MS/MS characteristic ions at m/z 391.2108 (C22H31O6), 373.2003 (C22H29O5), 331.1899 (C20H27O4), 313.1803 (C20H25O3), 295.1686 (C20H23O2), 285.1839 (C19H25O2), and 267.1743 (C19H23O). These characteristic ions were 16 Da (O) larger than henryin, suggesting that they represent henryin upon adding an OH group. Compound 82 had [M + NH4]+ ions at m/z 392.2422 (C22H34O5N) and an [M + H]+ ion at m/z 375.2156 (C22H31O5), which were 18 Da (H2O) smaller than henryin, and then, it produced the same characteristic ions of henryin at m/z 357.2053 (C22H29O4), 333.2054 (C20H29O4), 315.1949 (C20H27O3), 297.1843 (C20H25O2), 279.1737 (C20H23O), 269.11894 (C19H25O), and 251.1788 (C19H23), suggesting that it represents henryin lacking one hydroxyl group and double bond additions.
Compound 92 gave rise to the [M + H]+ ion at m/z 785.4455 (C44H65O12), which was 392 Da (C22H32O6) larger than henryin (C22H32O6), and the MSn spectrum showed the characteristic fragment ions at m/z 767.4342 (C44H63O11, [M + H − H2O]+), 749.4239 (C44H61O10, [M + H − 2H2O]+), 707.4133 (C42H59O9, [M + H − H2O − C2H4O2]+), 689.4028 (C42H57O8, [M + H − H2O − C2H4O2 − H2O]+), 671.3923 (C42H55O7, [M + H − H2O − C2H4O2 − 2H2O]+), 661.4083 (C41H57O7, [M + H − H2O − C2H4O2 − H2O − CO]+), 643.3975 (C41H55O6, [M + H − H2O − C2H4O2 − H2O − CO − H2O]+), 393.2250 (C22H33O6, [M + H − C22H32O6]+), 375.2160 (C22H31O5, [M + H − C22H32O6 − H2O]+), 315.1948 (C20H27O3, [M + H − C22H32O6 − H2O − C2H4O2]+), 297.1840 (C20H25O2, [M + H − C22H32O6 − H2O − C2H4O2 − H2O]+), and 279.1733 (C20H23O, [M + H − C22H32O6 − H2O − C2H4O2 − 2H2O]+) via the successive loss of multiple 18 Da (H2O) and 60 Da (C2H4O2), suggesting that it represents the diploid of henryin; it was tentatively identified as dihenryin. Compound 29 produced the [M + NH4]+ ion at m/z 438.2841 (C24H40O6N), which was 28 Da (C2H4) larger than henryin, and the MSn spectrum showed the characteristic fragment ions at m/z 315.1945 (C20H27O3, [M + NH4 − NH3 − C2H5OH − C2H4O2]+), 297.1844 (C20H25O2, [M + NH4 − NH3 − C2H5OH − C2H4O2 − H2O]+), 279.1743 (C20H23O, [M + NH4 − NH3 − C2H5OH − C2H4O2 − 2H2O]+), and 251.17872 (C19H23O, [M + NH4 − NH3 − C2H5OH − C2H4O2 − 2H2O − CO]+), suggesting that the OH group at C-1 of henryin became the CH3CH2O group. Compound 87 produced the [M + H]+ ion at m/z 421.2570 (C24H37O6), which was also 28 Da (C2H4) larger than henryin.
Notably, the MSn spectrum showed the characteristic fragment ions at m/z 403.2464 (C24H35O5, [M + H − H2O]+), 361.2367 (C22H33O4, [M + H − C2H4O2]+), 343.2255 (C22H31O3, [M + H − H2O − C2H4O2]+), 283.2046 (C20H27O, [M + H − H2O − C2H4O2 − C2H4O2]+), and 265.1947 (C20H25, [M + H − H2O − C2H4O2 − C2H4O2 − H2O]+) through the typical loss of the H2O and C2H4O2 groups, suggesting that the OH group at C-7 of henryin was replaced by a CH3CH2O group.
The only difference between henryin (57) and kamebakaurin (44) regarding chemical structure is that henryin has an OAc group at the C-20 position, while kamebakaurin has an OH group at the C-20 position. Therefore, kamebakaurin (44) produced an [M + NH4]+ ion at m/z 368.2425 (C20H34O5N), an [M + H]+ ion at m/z 351.2163 (C20H31O5), an [M + NH4 − NH3 − H2O]+ ion at m/z 333.2058 (C20H29O4), and an [M + NH4 − NH3 − 2H2O]+ ion at m/z 315.1952 (C20H27O3). All were 42 Da larger than henryin.
The ion at m/z 333.2058 was further fragmented to produce prominent ions at m/z 315.1952 (C20H27O3), 297.1846 (C20H25O2), and 279.1740 (C20H23O) via successive losses of 18 Da (H2O) in the MS3 spectrum.
Compounds 24, 32, 35–37, 40, 43, 50, 53, 62, and 83 are isomers of kamebakaurin and showed fragmentation behaviors similar to those of kamebakaurin (44). The successive losses of 18 Da (H2O) were commonly observed, and most produced the characteristic ions at m/z 351.2161 (C20H31O5), 333.2056 (C20H29O4), 315.1951 (C20H27O3), 297.1846 (C20H25O2), and 279.1743 (C20H23O). Compounds 41, 76, and 81 have the same [M + NH4]+ ions at 366.2266 (C20H32O5N), with an [M + NH4 − NH3 − H2O]+ ion produced at 331.1897 (C20H27O4) through the loss of H2O. [M + NH4 − NH3 − H2O]+ could be further fragmented to produce ions at m/z 331.1897 (C20H27O4), 313.1792 (C20H25O3), and 295.1688 (C20H23O2) via successive losses of 18 Da (H2O) in the MS3 spectrum, suggesting that it represents the reference compound kamebakaurin lacking 2H, and it was tentatively identified as dehydro-kamebakaurin.
Compound 48 had [M + NH4]+ ions at m/z 370.2580 (C20H36O5N) and presented MSn characteristic ions at m/z 335.2211 (C20H31O4), 317.2104 (C20H29O3), and 299.2000 (C20H27O2) through successive losses of 18 Da (H2O), suggesting that it represents kamebakaurin upon adding 2H, and it was tentatively identified as dihydro-kamebakaurin. Compounds 25 and 38 gave rise to an [M + NH4]+ ion at m/z 386.2529 (C20H36O6N) and the fragment ions at m/z 351.2159 (C20H31O5, [M + NH4 − NH3 − H2O]+), 333.2055 (C20H29O4, [M + NH4 − NH3 − 2H2O]+), 315.1951 (C20H27O3, [M + NH4 − NH3 − 3H2O]+), and 297.1838 (C20H25O2, [M + NH4 − NH3 − 4H2O]+). The characteristic ions of Compounds 25 and 38 were 18 Da (H2O) larger than kamebakaurin, suggesting that they represent the reference compound kamebakaurin with a double bond opening and hydroxyl addition.
Compound 61 generated [M + NH4]+ ions at 398.2525 (C21H36O6N) and then produced the [M + NH4 − NH3]+ ion at 381.2261 (C21H33O6), [M + NH4 − NH3 − H2O]+ ion at 363.2157 (C21H31O5), and [M + NH4 − NH3 − 2H2O]+ ion at 345.2057 (C21H29O4). All were 30 Da (CH2O) larger than kamebakaurin. Furthermore, the characteristic fragmentations at m/z 331.1894 (C20H27O4) were from the ions at m/z 363.2157 (C21H31O5) through the loss of CH4O in the fragmentation pathways, indicating that Compound 61 represents the reference compound kamebakaurin upon adding a terminal methoxy group.
Compared with kamebakaurin, kamebanin (52) has no substituent at the C-20 position. Therefore, kamebanin (52) produced an [M + NH4]+ ion at m/z 352.2491 (C20H34O4N), an [M + H]+ ion at m/z 335.2223 (C20H31O4), an [M + NH4 − NH3 − H2O]+ ion at m/z 317.2118 (C20H29O3), and an [M + NH4 − NH3 − 2H2O]+ ion at m/z 299.2011 (C20H27O2). All were 16 Da smaller than kamebakaurin.
The ion at m/z 317.2118 (C20H29O3) was further fragmented to produce prominent ions at m/z 299.2011 (C20H27O2) and 281.1906 (C20H25O) via successive losses of 18 Da (H2O) in the MS3 spectrum, suggesting the cleavage of hydroxyl groups. Significantly, both fragment ions at m/z 299.2011 (m/z 299.2011 → 271.2058) and at m/z 281.1906 (m/z 281.1906 → 253.1953) showed a small CO loss in the MS3 spectra, suggesting the cleavage of the carbonyl group.
Compounds 39, 46, 75, and 84 are isomers of kamebanin and showed fragmentation behaviors similar to those of kamebanin (52). The typical losses of 18 Da (H2O) and 28 Da (CO) were commonly observed, and most produced characteristic ions at m/z 335.2193 (C20H31O4), 317.2092 (C20H29O3), 299.1987 (C20H27O2), 281.1877 (C20H25O), 271.2038 (C19H27O), and 253.1935 (C20H25O). Based on previous reports [11], Compound 39 was tentatively characterized as Wangzaozin A.
Compound 28 has an [M + NH4]+ ion at m/z 532.3102 (C26H46O10N) and [M + H]+ ion at m/z 515.2832 (C26H43O10) through the typical loss of the NH3 group. The MS2 spectrum showed that the ion at m/z 515.2832 (C26H43O10) was cleaved to produce three prominent product ions at m/z 335.2213 (C20H31O4), 317.2100 (C20H29O3), and 299.1994 (C20H27O2) through typical losses of C6H12O6 and H2O, indicating that it represents kamebanin upon adding a terminal glucose monohydrate group (C6H13O6). Compounds 55 and 78 had [M + NH4]+ ions at m/z 354.2633 (C20H36O4N) and presented characteristic MSn ions at m/z 337.2369 (C20H33O4), 319.2263 (C20H31O3), 301.2157 (C20H29O2), and 283.2052 (C20H27O) through successive losses of 18 Da (H2O).
All were 2 Da larger than kamebanin, suggesting that it represents kamebanin upon adding 2H and was tentatively identified as dihydro-kamebanin. Compound 80 gave rise to the [M + NH4]+ ions at 350.2318 (C20H32O4N) and then produced an [M + NH4−NH3]+ ion at 333.2054 (C20H29O4) through the loss of NH3. [M + NH4 − NH3]+ could be further fragmented to produce ions at m/z 315.1949 (C20H27O3) and 297.1849 (C20H25O2) via successive losses of 18 Da (H2O) in the MS3 spectrum, suggesting that it represents the reference compound kamebanin lacking 2H and was tentatively identified as dehydro-kamebanin. Compounds 31, 47, and 70 showed the same [M + H]+ ions at 333.2035 (C20H29O4) and then fragmented to produce ions at m/z 315.1934 (C20H27O3) and 297.1830 (C20H25O2) via the successive losses of 18 Da (H2O), similarly to Compound 80 in the MS3 spectrum, which indicates that these compounds were isomers of Compound 80.
Compound 71 generated [M + H]+ at 303.2312 (C20H31O2), and presented MS/MS characteristic ions at m/z 285.2208 (C20H29O) and m/z 267.2101 (C20H27). All were 32 Da smaller than the characteristic ions of kamebanin, suggesting that Compound 71 represents kamebanin lacking two OH elements. In addition, due to the fewer hydroxyl groups of Compound 71, it was more likely to produce the fragment ions at m/z 239.1784 (C18H23), 225.1632 (C17H21), 211.1478 (C16H19), and 197.1320 (C15H27) through a series of cleavages and hydrogen rearrangements on the C-ring and D-ring, which result in successive losses of CH2.
1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one (64) produced the [M + NH4]+ precursor ion at m/z 452.2635 (C24H38O7N), very few [M + H]+ ions at m/z 435.2369 (C24H35O7), and the [2M + NH4]+ ion at m/z 886.4935 (C48H72O14N) in the MS1 spectra. The precursor ion at m/z 452.2635 (C24H38O7N) produced the [M + NH4 − NH3]+ ion at m/z 435.2369 (C24H35O7) through the typical loss of the NH3. Due to the existence of two hydroxyl groups at C-1 and C-7 and two acetoxy groups at C-14 and C-20, the [M + NH4 − NH3]+ ion at m/z 435.2369 was further fragmented to produce prominent ions at m/z 417.2269 (C24H33O6), m/z 375.2166 (C22H31O5), m/z 357.2052 (C22H29O4), m/z 315.1955 (C20H27O3), m/z 297.1848 (C20H25O2), and m/z 279.1742 (C20H23O) via typical losses of 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), and 18 Da (H2O) in the MS3 spectrum.
Compounds 68, 69, and 77 are isomers of 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one and showed fragmentation behaviors similar to those of 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one (64). The typical losses of 17 Da (NH3), 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), and 18 Da (H2O) were commonly observed, and most produced ions at m/z 435.2367 (C24H35O7), 417.2268 (C24H33O6),375.2157 (C22H31O5), 357.2053 (C22H29O4), 315.1947 (C20H27O3), 297.1842 (C20H25O2), and 279.1736 (C20H23O). Based on previous reports [9], they were tentatively characterized as 1α,14β-diacetoxy-7α,20-dihydroxy-ent-kaur-16-en-15-one, 1α,7α-diacetoxy-14β,20-dihydroxy-ent-kaur-16-en-15-one, and 1α,20-diacetoxy-7α,14β-dihydroxy-ent-kaur-16-en-15-one.
Compound 85 exhibited [M + NH4]+ ions at 450.2474 (C24H36O7N) and then produced an [M + NH4 − NH3]+ ion at 433.2211 (C24H33O7) through the loss of NH3. [M + NH4 − NH3]+ could be further fragmented to produce ions at m/z 373.2002 (C22H29O5), 355.1894 (C22H27O4), 313.1794 (C20H25O3), 295.1689 (C20H23O2), and 277.1575 (C20H21O) via successive losses of 60 Da (C2H4O2), 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), and 18 Da (H2O), suggesting that it represents the reference compound 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one lacking 2H, and it was tentatively identified as dehydro-1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one. Moreover, the fragment ions at m/z 391.2106 (C22H31O6), 373.2000 (C22H29O5), 331.1897 (C20H27O4), and 313.1794 (C20H25O3) were produced from 433.2211 (C24H33O7) through the successive losses of 42 Da (C2H2O), 18 Da (H2O), 42 Da (C2H2O), and 18 Da (H2O), indicating that two acetoxy groups in Compound 85 were more prone to cleavage. Compound 58 generated [M + NH4]+ ions at m/z 454.2785 (C24H40O7N) and presented characteristic MSn ions at m/z 437.2524 (C24H37O7), 419.2414 (C24H35O6), m/z 377.2318 (C22H33O5), m/z 359.2211 (C22H31O4), m/z 317.2110 (C20H29O3), m/z 299.2001 (C20H27O2), and m/z 281.1896 (C20H25O) via typical losses of 17 Da (NH3), 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), and 18 Da (H2O).
All were 2 Da larger than 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one, suggesting that it represents 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one upon adding 2H, and it was tentatively identified as dihydro-1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one. Compound 73 exhibited the same [M + NH4]+ ions at m/z 454.2788 (C24H40O7N) and characteristic ions at m/z 437.2518 (C24H37O7), 419.2415 (C24H35O6), and 359.2209 (C22H31O4), indicating that it had similar fragmentation behaviors as Compound 58. Therefore, this compound was identified as the isomer of Compound 58 in the extracted ion chromatogram.
Compounds 45 and 56 gave rise to the [M + NH4]+ ion at m/z 470.2733 (C24H40O8N) and fragment ions at m/z 453.2468 (C24H37O8, [M + NH4 − NH3]+) and 393.2257 (C22H33O6, [M + NH4 − NH3 − C2H4O2]+). All were 18 Da (H2O) larger than 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one, suggesting that they represent reference compound 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one with a double bond opening and hydroxyl addition. However, Compound 45 also produced a relatively prominent characteristic ion at m/z 435.2366 (C24H35O7, [M + NH4 − NH3 − H2O]+), while Compound 56 generated relatively prominent ions at m/z 393.2262 (C22H33O6, [M + NH4 − NH3 − C2H4O2]+), 333.2053 (C20H29O4, [M + NH4 − NH3 − 2C2H4O2]+), and 315.1949 (C20H27O3, [M + NH4 − NH3 − 2C2H4O2 − H2O]+), indicating that the hydroxyl group of Compound 45 was more likely to cleave first, whereas the acetoxy groups of Compound 56 were more prone to cleavage.
Compound 63 generated [M + NH4]+ ions at 484.2890 (C25H42O8N) and then produced [M + NH4 − NH3]+ ions at 467.2625 (C25H39O8) and [M + NH4 − NH3 − H2O]+ ions at 449.2519 (C25H37O7). All were 14 Da (CH2) larger than Compound 45, indicating that Compound 63 represents Compound 45 upon adding a methyl group. Compound 86 gave rise to the [M + NH4]+ ion at m/z 496.2890 (C26H42O8N) and presented characteristic MSn ions at m/z 479.2627 (C26H39O8, [M + NH4 − NH3]+), m/z 461.2522 (C26H37O7, [M + NH4 − NH3 − H2O]+), m/z 419.2418 (C24H35O6, [M + NH4 − NH3 − H2O − C2H2O]+), m/z 401.2310 (C24H33O5, [M + NH4 − NH3 − H2O − C2H2O − H2O]+), m/z 377.2313 (C22H33O5, [M + NH4 − NH3 − H2O − 2C2H2O]+), m/z 359.2211 (C22H31O4, [M + NH4 − NH3 − H2O − 2C2H2O − H2O]+), m/z 341.2103 (C22H29O3, [M + NH4 − NH3 − H2O − 2C2H2O − H2O − H2O]+), m/z 317.2104 (C20H29O3, [M + NH4 − NH3 − H2O − 2C2H2O − H2O − C2H2O]+), m/z 299.1998 (C20H27O2, [M + NH4 − NH3 − H2O − 2C2H2O − H2O − C2H2O − H2O]+), and m/z 281.1893 (C20H25O, [M + NH4 − NH3 − H2O − 2C2H2O − H2O − C2H2O − 2H2O]+), suggesting that it represents 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one upon adding a terminal OAc group (CH3CH2O).
Compounds 79 and 88 showed the same [M + NH4]+ ions at m/z 496.2890 (C26H42O8N) in the positive mode and dominant ions at m/z 479.2623 (C26H39O8), m/z 461.2522 (C26H37O7), m/z 419.2415 (C24H35O6), m/z 401.2312 (C24H33O5), and m/z 359.2206 (C22H31O4). Therefore, they were tentatively identified as isomers of Compound 86. Compound 94 gave rise to precursor ions at m/z 538.2992 (C28H44O9N, [M + NH4]+), which were 42 Da (C2H2O) larger than Compound 86, and product ions at m/z 461.2521 (C26H37O7, [M + NH4 − NH3 − C2H4O2]+), m/z 419.2416 (C24H35O6, [M + NH4 − NH3 − C2H4O2 − C2H2O]+), m/z 401.2310 (C24H33O5, [M + NH4 − NH3 − C2H4O2 − C2H2O − H2O]+), m/z 359.2208 (C22H31O4, [M + NH4 − NH3 − C2H4O2 − 2C2H2O − H2O]+), m/z 341.2104 (C22H29O3, [M + NH4 − NH3 − C2H4O2 − 2C2H2O − H2O − H2O]+), m/z 299.1995 (C20H27O2, [M + NH4 − NH3 − C2H4O2 − 2C2H2O − H2O − C2H2O − H2O]+), and m/z 281.1893 (C20H25O, [M + NH4 − NH3 − C2H4O2 − 2C2H2O − H2O − C2H2O − 2H2O]+) through the typical loss of the C2H4O2, C2H2O and H2O groups, suggesting that one OH group on Compound 86 was converted to an OAc group (CH3CH2O).
1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-16-en-15-one (74) and 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one (64) are isomers. While 1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-16-en-15-one (74) produced an [M + H]+ precursor ion at m/z 435.2373 (C24H35O7), very few [M + NH4]+ ions were observed at m/z 452.2635 (C24H38O7N), and a [2M + H]+ ion was observed at m/z 869.4676 (C48H69O14) in the MS1 spectra. Due to the existence of two hydroxyl groups at C-1 and C-14 and two acetoxy groups at C-7 and C-20, the [M + H]+ precursor ion at m/z 435.2373 was also further fragmented to produce prominent ions at m/z 417.2270 (C24H33O6), m/z 375.2164 (C22H31O5), m/z 357.2060 (C22H29O4), m/z 315.1954 (C20H27O3), m/z 297.1849 (C20H25O2), and m/z 279.1743 (C20H23O) via typical losses of 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), and 18 Da (H2O).
Compound 72 showed the same [M + H]+ precursor ions at m/z 435.2365 (C24H35O7) and displayed common fragmentations, such as the ions at m/z 417.2261 (C24H33O6), m/z 357.2060 (C22H29O4), m/z 297.1849 (C20H25O2), and m/z 279.1738 (C20H23O), indicating that it was an isomer of 1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-16-en-15-one. Compound 90 gave rise to the [M + H]+ ions at m/z 479.2621 (C26H39O8), which were 44 Da (C2H4O) larger than 1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-16-en-15-one, and presented MSn characteristic ions at m/z 461.2519 (C26H37O7, [M + H − H2O]+), m/z 401.2308 (C24H33O5, [M + H − H2O − C2H4O2]+), m/z 341.2100 (C22H29O3, [M + H − H2O − 2C2H4O2]+), m/z 323.2003 (C22H27O2, [M + H − H2O − 2C2H4O2 − H2O]+), m/z 281.1892 (C20H25O, [M + H − H2O − 2C2H4O2 − H2O − C2H2O]+), and m/z 263.1795 (C20H23, [M + H − H2O − 2C2H4O2 − H2O − C2H2O − H2O]+), suggesting that it represents 1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-16-en-15-one upon adding a terminal OAc group (CH3CH2O).
The full-scan mass spectrum of 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one (54) showed the [M + NH4]+ precursor ion at m/z 412.2683 (C22H38O7N), very few [M + H]+ ions at m/z 395.2417 (C24H35O7), and the [2M + H]+ ion at m/z 789.4762 (C44H69O12) in the MS1 spectra. The precursor ion at m/z 412.2683 (C22H38O7N) produced an [M + NH4 − NH3]+ ion at m/z 395.2425 (C22H35O6) through the typical loss of the NH3. Due to the existence of three hydroxyl groups at C-1, C-7, and C-14 and one acetoxy group at C-20, the [M + NH4 − NH3]+ ion at m/z 395.2425 was further fragmented to produce prominent ions at m/z 377.2313 (C22H33O5), m/z 359.2208 (C22H31O4), m/z 317.2103 (C20H29O3), m/z 299.1998 (C20H27O2), and m/z 281.1890 (C20H25O) via typical losses of 18 Da (H2O), 18 Da (H2O), 42 Da (C2H2O), 18 Da (H2O), and 18 Da (H2O).
Compound 67 is an isomer of 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one and showed fragmentation behaviors similar to those of 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one (54). The typical losses of 17 Da (NH3), 18 Da (H2O), 60 Da (C2H4O2), 18 Da (H2O), and 18 Da (H2O) were commonly observed, and this produced ions at m/z 395.2418 (C22H35O6), m/z 377.2297 (C22H33O5), m/z 317.2088 (C20H29O3), m/z 299.1983 (C20H27O2), and m/z 281.1871 (C20H25O). Compounds 26, 34, and 42 gave rise to the [M + NH4]+ ions at 428.2633 (C22H38O7N), and both presented MS/MS characteristic ions at m/z 411.2373 (C22H35O7), 393.2266 (C22H33O6), m/z 375.2161 (C22H31O5), m/z 333.2056 (C20H29O4), m/z 315.1949 (C20H27O3), and m/z 297.1842 (C20H25O2).
These characteristic ions were 16 Da (O) larger than 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one, suggesting that they represent 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one upon adding an OH group. Compounds 65 and 93 had [M + NH4]+ ions at m/z 394.2559 (C22H36O5N) and [M + H]+ ions at m/z 377.2302 (C22H33O5), which were 18 Da (H2O) smaller than 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one; then, the same characteristic ions of 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one were produced at m/z 359.2192 (C22H31O4), 317.2090 (C20H29O3), and 299.1986 (C20H27O2), suggesting that they represent 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one lacking one hydroxyl group with double bond additions. Based on previous reports [3,12], they were tentatively characterized as leukamenin E and inflexanin A.
Compound 91 gave rise to an [M + H]+ ion at m/z 771.4655 (C44H67O11), which was 18 Da (H2O) smaller than the [2M + H]+ ion of 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-oneat m/z 789.4762 (C44H69O12), and the MSn spectrum showed characteristic fragment ions at m/z 753.4544 (C44H65O10, [M + H − H2O]+), 735.4453 (C44H63O9, [M + H − 2H2O]+), 717.4346 (C44H61O8, [M + H − H2O − 3H2O]+), 675.4242 (C42H59O7, [M + H − 3H2O − C2H2O]+), 657.4134 (C42H57O6, [M + H − 3H2O − C2H4O2]+), 639.4025 (C42H55O5, [M + H − 3H2O − C2H4O2 − H2O]+), 597.3923 (C40H53O4, [M + H − 3H2O − 2C2H4O2]+), 579.3824 (C40H51O3, [M + H − 3H2O − 2C2H4O2 − H2O]+), 561.3722 (C40H49O2, [M + H − 3H2O − 2C2H4O2 − 2H2O]+), and 393.2262 (C22H33O6, [M + H − C22H34O5]+) via the successive loss of multiple 18 Da (H2O), 60 Da (C2H4O2), and 378 Da (C22H34O5), suggesting that it represents the diploid of 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one lacking H2O.
3. Materials and Methods
3.1. Chemicals and Reagents
The standards—including the bridgehead-unsubstituted 7,20-epoxy-ent-kaurane diterpenoids of oridonin (2), lasiokaurin (5), and 1α-acetoxy-14β-hydroxyl-7,20epoxy-ent-kaur-16-en-15-one (13); bridgehead-substituted 7,20-epoxy-ent-kaurane diterpenoids of kamebacetal A (21), kamebacetal B (22), and reniformin C (23); and 7,20-non-epoxy-kaurane diterpenoids of kamebakaurin (44), kamebanin (52), 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one (54), henryin (57), 1α,7α-dihydroxy-14β,20-diacetoxy-ent-kaur-16-en-15-one (64), and 1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-16-en-15-one (74)—were isolated from the IEH in our laboratory. Their structures were unequivocally elucidated via spectroscopic methods (IR, MS, 1H NMR, and 13C NMR). The purity of the standards was determined to be higher than 98% by normalizing the peak area using HPLC-DAD (diode array detector).
Stock solutions (0.1 mg/mL) of the 12 standards were prepared individually in methanol. Acetonitrile (ACN) and formic acid (both HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Methanol (HPLC grade) was purchased from Merck (Darmstadt, Germany).
Deionized water was prepared by passing distilled water through a Milli-Q water purification system (Millipore, Milford, MA, USA).
3.2. Plant Material
Isodonis Excisoidis Herba were collected from Luanchuan, Henan, China (GPS coordinates 33°46′19.6″ N 111°44′59.2″ E) in July 2022 and authenticated by one of the authors, Prof. Liping Dai (Department of Pharmacognosy, Henan University of Chinese Medicine), according to morphological characteristics. A voucher specimen (voucher number 20220721) was deposited at the Pharmacognosy Lab of the Pharmacy College, Henan University of Chinese Medicine, Henan, China.
3.3. Sample Preparation
Isodonis Excisoidis Herba samples were pulverized into powder (1.0 g, 60 mesh), weighed, soaked in 10 mL of 80% ethanol at room temperature, and ultrasonically extracted for 1 h. The extracts were filtered through filter paper, and 5 mL of the successive filtrate was evaporated to dryness. The dried residue was redissolved in 1ml of methanol in a volumetric flask.
All samples were filtered through a 0.22 μm membrane and directly analyzed using UHPLC-LTQ-Orbitrap-MS (Thermo Fisher Scientific, Bremen, Germany).
3.4. UHPLC Separation Conditions
The chromatographic analysis was performed on a Dionex UltiMate 3000 UHPLC system (Thermo Scientific, Germering, Bavaria, Germany) equipped with a binary pump, an online degasser, a thermostatted autosampler, a thermostatically controlled column compartment, and a diode array detector (DAD). Chromatographic separation was performed on a reverse-phase ACQUITY UPLC BEH-C18 column (2.1 × 100 mm, 1.7 μm, Waters) and maintained at 30 °C. The mobile phases consisted of deionized water containing 0.1% formic acid (A) and acetonitrile (B), and the elution gradient was set as follows: 0.0–10.0 min, 10.0% B; 10.0–15.0 min, 10.0–20.0% B; 15.0–20.0 min, 20.0% B; 20.0–36.0 min, 20.0–21.0% B; 36.0–37.0 min, 21.0–29.0% B; 37.0–43.0 min, 29% B; 43.0–48.0 min, 29.0–35.0% B; 48.0–52.0 min, 35.0–40.0% B; 52.0–62.0 min, 40.0% B; 62.0–72.0 min, 40.0–55.0% B; 72.0–75.0 min, 55.0–10.0% B; 75.0–80.0 min, 10% B.
The flow rate was set at 0.3 mL/min, and the injection volume was 2 μL.
3.5. Mass Spectrometry and Data Processing
For the LC-ESI-MSn experiments, a Thermo Fisher LTQ-Orbitrap XL Hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray ionization (ESI) source was connected to the UHPLC instrument. The ESI source parameters were set as follows: ion spray voltage, 4.2 kV; capillary temperature, 300 °C; capillary voltage, 36 V; tube lens voltage, 110 V; and sheath (N2) and auxiliary gas (He) flow rates of 40 and 10 arbitrary units, respectively. The Orbitrap mass analyzer was operated in the positive ion mode, with a mass range of 100 to 2000.
Tuning methods were developed using a multi-objective optimization experiment, similarly to the technique described for ESI-MS. Accurate masses were calibrated according to the manufacturer’s guidelines using a standard mixture of caffeine, MRFA, and Ultramark 1621 in the positive ion mode.
The Fourier transform resolutions were set at 30,000 (full width at half maximum, as defined at m/z 400) for MS and MSn (n = 3). The MS and MSn data were recorded in profile and centroid formats, respectively. The average acquisition time required to finish a scan cycle (containing four scan events) was 3.6 s.
The most intense ions detected in the full-scan spectrum were selected for the data-dependent scan. The normalized collision energy for CID was adjusted to 25% of the maximum in MS2 and 20% of the maximum in MS3, the isolation width of the precursor ions was m/z 2.0, and the default values were used for other CID parameters. To simultaneously obtain fragment information for co-eluting minor peaks, dynamic exclusion was enabled using the following optimal parameters: repeat count, 2; repeat duration, 10 s; exclusion duration, 10 s.
The data were recorded and processed using the Xcalibur 3.0 software (Thermo Fisher Scientific, Waltham, MA, USA) and Mass Frontier 7.0 software (Thermo Fisher Scientific, Waltham, MA, USA). Considering the possible elemental compositions of the potential components present in the IEH samples, the elements in use (C: 0–60; H: 0–100; O: 0–25; N: 0–2), ring double-bond equivalent (RDB equivalent value: −1.0–100.0), and mass tolerance (<10 ppm) were set to reduce the number of options used to determine the elemental compositions of both the precursor and product ions.
4. Conclusions
An efficient and sensitive method employing UHPLC-LTQ-Orbitrap-MS was developed for the qualitative profiling of the chemical constituents of IEH in the positive ion mode. By combining a wide range of information—including the exact mass from LTQ-Orbitrap-MS, fragmentation patterns, and the retention time on UHPLC—and comparing the results with reference substances and the literature, a total of 94 compounds, including 17 bridgehead-unsubstituted 7,20-epoxy-ent-kaurane diterpenoids, 6 bridgehead-substituted 7,20-epoxy-ent-kaurane diterpenoids, and 71 7,20-non-epoxy-kaurane diterpenoids, 48 of which are potentially new ent-kaurane diterpenoids, were detected from IEH. This is the first report on ent-kaurane diterpenoids in IEH using UHPLC-LTQ-Orbitrap-MS.
The results not only provide abundant information for the identification and better understanding of the chemical constituents of IEH but also offer valuable insights for target-guided preparation, bioactivity-guided research, quality control, pharmacokinetic investigations, and metabolic studies of this Chinese medicine. This study also suggested that UHPLC-LTQ-Orbitrap-MS would be a powerful and reliable analytical tool for the characterization of chemical profiles in complex chemical systems, such as herbal medicines and TCM preparations. However, it must be emphasized that this technique has inherent limitations in resolving stereochemistry and regioisomerism.
Consequently, the proposed structures, especially those of potentially new ent-kaurane diterpenoids, require definitive structural confirmation through isolation and comprehensive NMR analysis.