SSa (β-OH at position C₁₆) and SSd (α-OH at position C₁₆) were a pair of epimers, with the same molecular ion ([M−H]⁻) at m/z 779; their fragmentation behaviors were similar. In the ESI^–mode, the fragment ions of the MS/MS spectrum (Supplementary Table S1) were analyzed, and the Y₁ (m/z 617) ion corresponding to the [M–H]^– lost a fragment of 162 Da, indicating that a glucosyl group (Glu, 162 Da, C₆H₁₀O₅) at the end of the saccharide chain was lost. The X₀ (m/z 541) ion was produced by the breakage of the Y₁ (m/z 617) ion, corresponding to the Y₁ ion with a lost fragment of 76 Da (C₃H₈O₂), which represented the intracyclic breakage of furanose (0, 3-bond breakage). The [Y₁−76−H]^– (m/z 541) fragment ion was produced when the furanose (Fuc, 146 Da, C₆H₁₀O₄) was directly attached to the aglycone in the structure of saikosaponins; conversely, the presence of the [Y₁−76−H]^– fragment ion was not detected for saikosaponins without furanose. Thus, a fragment of 76 Da (C₃H₈O₂) was lost as the specific fragmentation pathway of furanose, which was consistent with previous reports (Huang et al., 2008). Among the characteristic fragment ions of SSa/SSd was m/z 471 ([M−Glu−Fuc−H]^–), which corresponded to the molecular ion without the complete saccharide chain and corresponded to the sapogenin ion ([aglycone-H]⁻); the [aglycone−H]⁻ lost a fragment of 32 Da to produce the m/z 439 ([aglycone−CH₃OH−H]⁻) fragment ion, corresponding to the aglycone with the loss of one molecule of CH₃OH at position C₄, which was consistent with the available literature (Zhao et al., 1996). The fragmentation pathway of SSa/SSd is shown in Figure 2A. Similarly, the fragmentation pathways of SSe, Prosaikogenin f, Prosaikogenin g, and other type I saikosaponins are similar to those of SSa/SSd. For SSc, the aglycone structure is different from that of SSa; the group at position C₂₃ is methyl (CH₃), and its fragmentation pathway is shown in Figure 2B.

23-Hydroxy-13β,28β-epoxyolean-11-ene-16-one 3-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranoside presents a carbonyl group (C=O) in the aglycone moiety and its characteristic fragment ions appeared at m/z 407 ([aglycone−CH₃OH−30−H]⁻) in the ESI^–mode, corresponding to the [aglycone−H]⁻, and lost a fragment of 30 Da. It was speculated that the C₁₃,C₂₈-epoxy ether bond was broken first and was followed by a rearrangement reaction of the C=O group at the C₁₆ position and breakage. The fragmentation pathway is shown in Supplementary Figure S2.

Retention times of type I saikosaponins showed (Supplementary Table S1) that for a pair of epimers, the retention time of β-OH at position C₁₆ was earlier than that of the α-OH epimer (such as SSa/SSd and Prosaikogenin g/Prosaikogenin f). For the type I saikosaponins, their aglycones present only one double bond (no conjugation), so their maximum UV wavelengths were generally around at the terminal ends (190–220 nm); the representative UV spectra are shown in Figure 8A.

Epimers SSb₁ (β-OH at position C₁₆) and SSb₂ (α-OH at position C₁₆) are converted from SSa and SSd, respectively. In the ESI^–mode, the fragmentation behavior of the two epimers was similar to that of SSa/SSd, and they also produced the following fragment ions: m/z 617 ([M−Glu−H]^–), m/z 541 ([M−Glu−C₃H₈O₂−H]^–), m/z 471 ([M−Glu−Fuc−H]^–, corresponding to [aglycone−H]⁻), and m/z 439 ([aglycone−CH₃OH−H]⁻). Compared with SSa/SSd (type I), SSb₁/SSb₂ lost a CH₂OH group at position C₁₇ and a hydroxyl (OH) group at position C₁₆ in the aglycone, simultaneously, and generated the m/z 423 ([aglycone−48−H]⁻) fragment ion, which was consistent with the fragmentation behavior found by Liu et al. (Liu et al., 2019). Because SSf presented a single double bond of aglycone and did not produce a m/z 409 ([aglycone−48−H]⁻) fragment ion, it can be inferred that this fragmentation pathway only occurred when two double bonds were conjugated. Interestingly, it was worth noting that this experiment found that the abundance of m/z 423 ([aglycone−CH₂OH−OH−H]⁻) fragment ion produced by SSb₁ having the β-OH at the C₁₆ position was greater than that of the m/z 439 ([aglycone−CH₃OH−H]⁻) fragment ion, while the abundance of the m/z 423 fragment ion produced by the SSb₂ with the α-OH at position C₁₆ was lower than that of the m/z 439 fragment ion produced. As shown in Supplementary Figure S3, it is helpful to distinguish between the same pair of epimers in type II saikosaponin. These rules and findings were also applicable to other type II saikosaponins such as Prosaikogenin a, Prosaikogenin d, SSh, etc. The fragmentation pathway of SSb₁/SSb₂ is shown in Figure 3. The fragmentation pathway of SSh (the group at the C₂₃ position is CH₃) is shown in Supplementary Figure S4 and also produced a m/z 407 ([aglycone−CH₂OH−OH−H]⁻) fragment ion. Thus, the [aglycone−48−H]⁻ could be considered diagnostic fragment ions of type II saikosaponins, while the loss of 48 Da (neutral loss of CH₂OH and OH groups) may also be considered a characteristic loss of type II saikosaponins.

Retention times of type II saikosaponins are reported in Supplementary Table S1. For the pair of epimers, the retention time of β-OH at the C₁₆ position was later than that of the α-OH epimer (such as SSb₁/SSb₂ and Prosaikogenin a/Prosaikogenin d), which was contrary to that of type I saikosaponins. For the type II saikosaponins, the aglycones presented two double bonds (isocyclic diene conjugation), and the maximum UV wavelengths were generally around 250 nm; the representative UV spectra are shown in Figure 8B.

SSb₃ (β-OH at position C₁₆) and SSb₄ (α-OH at position C₁₆) with the aglycone structure of C₁₁ -OCH₃ are epimers. According to the fragment ions produced in the ESI^–mode, the aglycone was released at the C₁₁ position, one molecule of CH₃OH (32Da) was lost and produced an aglycone with C₈-C₁₁ double bonds, and the m/z 471 ([aglycone−CH₃OH−H]⁻) fragment ion was produced. Thus, there were two double bonds (Δ^8,11 and Δ^12,13) in the aglycone due to the diene bond conjugation, the CH₂OH group at position C₁₇ and the OH group at position C₁₆, which could be lost simultaneously (loss of 48 Da), which produced the m/z 423 ([aglycone−CH₃OH−CH₂OH−OH−H]⁻) fragment ion. The loss of C₄-CH₃OH produced the fragment ion m/z 391 ([aglycone−2CH₃OH−CH₂OH−OH−H]⁻). Based on the m/z 439 ([aglycone −2CH₃OH−H]⁻), another fragmentation pathway of the aglycone produced the loss of C₁₁-CH₃OH and C₄-CH₃OH in turn; the fragmentation pathway of SSb₃/SSb₄ is shown in Figure 4. Similarly, for SSf, Ne-SSk, and 11α-methoxy-SSf, the fragmentation pathways were similar and are shown in Supplementary Figures S5–S7. Thus, the fragment ions produced by loss of a 48-Da fragment ([aglycone−CH₂OH−OH−H]⁻) were also a diagnostic fragment ion of type III saikosaponins in the ESI⁻ mode.

The fragmentation behavior of Tibesaikosaponin V with the carbonyl (C=O) group was similar to that of SSb₃/SSb₄, but the characteristic fragment ions were produced by the loss of 30 Da (neutral loss of CH₂O), such as m/z 473 ([aglycone–30–H]⁻). This behavior was the same for 23-hydroxy-13β,28β-epoxyolean-11-ene-16-one 3-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranoside (type I), and all had a carbonyl (C=O) group in the aglycone moiety. The fragmentation pathway is shown in Figure 5. Thus, the characteristic fragment ions produced by loss of 30 Da could be considered the diagnostic fragment ions of saikosaponins containing carbonyl groups in the ESI⁻ mode. This study analyzed and summarized the characteristic fragment ions and fragmentation pathways of saikosaponins containing the C=O group for the first time, which is of great significance and benefit for the identification of these types of saikosaponins.

Retention times of type III saikosaponins revealed (Supplementary Table S1) that in the pair of epimers, the retention time of β-OH at position C₁₆ was earlier than that of the α-OH epimer. For type III saikosaponins, similar to type I saikosaponins, the aglycones also had a single double bond (no conjugation), and the respective maximum UV wavelengths were also approximately 190–220 nm. The representative UV spectra are shown in Figure 8C.

SSg was compared with the type II saikosaponin SSb₁ (isomer of SSg) generated fragment ions that were essentially the same in ESI^–, which could produce m/z 423 ([aglycone−CH₂OH−OH−H]⁻) and other fragment ions of SSb₁. However, this is the first study to observe the m/z 453 ([aglycone–18–H]⁻) fragment ion from SSg, while the isomer SSb₁ (type II) or SSa (type I) did not produce the same fragment. It was speculated that due to the homocyclic diene conjugation effect, the OH group can be lost alone; the fragmentation pathway was shown in Figure 6. Thus, in addition to the [aglycone−CH₂OH−OH−H]⁻, the characteristic fragment ions such as [aglycone−H₂O−H]⁻ produced by loss of 18 Da could be considered a diagnostic fragment ion of type IV saikosaponins in the ESI⁻ mode.

For type IV saikosaponins, the aglycone moieties present two double bonds (homocyclic diene conjugation), so their maximum UV wavelengths were generally around 282 nm, and the representative UV spectra are shown in Figure 8D.

The maximum UV wavelengths, characteristic fragment ions, and fragmentation pathways of type IV saikosaponins were analyzed and summarized for the first time, and we compared differences in fragmentation patterns with other types of saikosaponins, which is of great significance and contributes to the identification of type IV saikosaponins.

In the ESI^– mode, acetylated saikosaponins such as 6″-O-acetyl-SSb₃, 2″-O-acetyl-SSa, 6″-O-acetyl-SSa, and 6″-O-acetyl-SSd generally produced [M–42–H]⁻ and [M–60–H]⁻ fragment ions (Supplementary Table S1), corresponding to the loss of one acetyl (CH₂CO) group and the loss of one CH₃COOH group, respectively. Subsequently, a series of fragment ions of the prototype saikosaponins mentioned above were produced. As an example, 6″-O-acetyl-SSa fragmentation is shown in Supplementary Figure S8. Prior studies have reported (Huang et al., 2008) that the malonylated saikosaponins produced characteristic [M–H–44]^– and [M–H–86]^– fragment ions in the ESI⁻ mode. As an example, the fragmentation pathway of 6″-O-malonyl-SSa is shown in Supplementary Figure S9.

For acetylated/malonylated saikosaponins in the ESI^– mode, the fragment ions of the aglycone-substituted and saccharide-substituted saikosaponins were the same, which made them difficult to distinguish; but in the ESI⁺ mode, their fragment ions differed so that they could easily be distinguished. In the ESI⁺ mode, 6″-O-acetyl-SSb₃, 2″-O-acetyl-SSa, 6″-O-acetyl-SSa, and 6″-O-acetyl-SSd, whose acetyl groups are substituted by the saccharide chain, were analyzed. The fragment ions produced are shown in Supplementary Table S2. The OH group in the aglycone was easily lost as were all produced [aglycone-nH₂O + H]⁺ (n = 1, 2, or 3) fragment ions. For the 2″-O- acetyl-SSa, 6″-O-acetyl-SSa, and 6″-O-acetyl-SSd, the OH group at position C₁₆ was lost first, which produced the m/z 805 ([M–H₂O + H]⁺) fragment ion, and then OH groups were lost at other positions and produced the fragment ions m/z 455 ([aglycone–H₂O + H]⁺), m/z 437 ([aglycone–2H₂O + H]⁺), and m/z 419 ([aglycone–3H₂O + H]⁺. As a representative example, the fragmentation pathway of 6″-O-acetyl-SSa is shown in Figure 7, while that of 6″-O-acetyl-SSb₃ in the ESI⁺ mode is shown in Supplementary Figure S10. These rules and findings were also applicable to malonylated saikosaponins (the malonyl group is substituted by the saccharide chain) according to the related literature (Liu et al., 2019) and also produce [aglycone-nH₂O + H]⁺ (n = 1 or 2 or 3) in the ESI⁺ mode, while the fragment ions m/z 497 ([aglycone+42–H₂O + H]⁺) and m/z 479 ([aglycone+42–2H₂O + H]⁺ indicate that the acetyl group is substituted by the aglycone. For example, the fragmentation pathway in the ESI⁺ mode of 23-O-acetyl-SSa (the malonyl group is substituted by the aglycone) (Liu et al., 2019) is shown in Supplementary Figure S11.

Retention times were analyzed according to standard substances and according to that reported in the literature (Zhao et al., 1996; Huang et al., 2008). The retention time of acetylated saikosaponins is later than that of the prototype saikosaponins. The acetyl substituent at different positions of the saccharide chain can affect the retention time; the retention time of the acetyl substituent at the beginning of the saccharide chain is earlier than that of the acetyl substituent at the rear position. For acetylated-SSa, the acetyl group can be substituted at different positions on the terminal glucose of the saccharide chain, so that the retention times for 2″-O-acetyl-SSa, 3″-O-acetyl-SSa, 4″-O-acetyl-SSa, and 6″-O-acetyl-SSa range from earlier to later.

Thus, the fragment ions [M–42–H]⁻ and [M–60–H]⁻(or [M–H–44]^– and [M–H–86]^–) are diagnostic fragment ions for acetylated (or malonylated) saikosaponins in the ESI^–mode, combined with the analysis of their respective m/z values for [aglycone-nH₂O + H]⁺ in the ESI⁺ mode, and the order of retention times can be combined to speculate the specific position of their acetyl/malonyl group.

Among the isomers, several pairs of epimeric saikosaponins such as SSa/SSd, SSb₃/SSb₄, SSb₁/SSb₂, Prosaikogenin a/Prosaikogenin d, and Prosaikogenin g/Prosaikogenin f differed only in the configuration of the OH group at position C₁₆. Of these, saikosaponins with only one double bond in the aglycone moiety (types I and III), and the retention time of β-OH at the C₁₆ position was earlier than that of the α-OH epimer, such as SSa/SSd, SSb₃/SSb₄, and Prosaikogenin g/Prosaikogenin f. In contrast, for type II saikosaponins (whose aglycone is an isocyclic diene), the retention time of β-OH at position C₁₆ was later than that of the α-OH epimer, such as SSb₁/SSb₂ and Prosaikogenin a/Prosaikogenin d. For acetylated (or malonylated) saikosaponins, the combination of retention times is helpful to infer the specific position of acetyl (or malonyl) substituents of the saccharide chain.

The maximum UV absorption wavelengths of saikosaponins can be summarized as follows: saikosaponins (such asSSa/SSd/SSc/SSb₃/SSb₄/SSe/SSf) with the aglycone as the monoene (types I and III) generally located around the terminal ends, ranged 190–220 nm; saikosaponins (such as SSb₁/SSb₂/SSc/SSh) with the aglycone as the isocyclic diene (type II), approximately 250 nm; saikosaponins (such as SSg/SSi) with the aglycone as the homocyclic diene (type IV), approximately 282 nm. The representative UV spectra are shown in Figure 8.

The characteristic mass losses and typical fragmentation pathways for different types of saikosaponins can be summarized as follows: all compounds produced a base peak at [M–H]⁻, and the molecular formula of each compound could be determined based on its exact mass. In addition to maximum UV absorption wavelengths, the type of aglycones could be inferred through their characteristic neutral losses of the aglycone moiety, such as type II and III (characteristic loss of 48 Da) and type IV (characteristic loss of 48 and 18 Da). The number and sequences of saccharide chains could be inferred through the characteristic neutral losses of sugar moieties, such as rhamnose (Rha, 146 Da), furanose (Fuc, 146 and 76 Da), xylose (Xyl, 132 Da), and glucose (Glu, 162 Da). Other typical losses were those such as OCH₄ (32 Da), H₂O (18 Da), CH₃OH (32 Da), CH₂O (30 Da), AcOH/C₂H₂O (60 Da/42 Da), and C₃H₂O₃/CO₂ (80 Da/44 Da), which corresponded to the presence of an OCH₃ group, an OH group, a CH₂OH group, a C=O group, an acetyl group, and a malonyl group, respectively. Figure 9 illustrates the strategy for identification of saikosaponins.

According to the fragment ions, retention times, and maximum UV wavelength of compounds 40, 42, 58, and 71 (Supplementary Datasheet S1), their characteristic mass losses and UV maximum absorption wavelengths (190–220 nm) are consistent with those of type I saikosaponins, so it was inferred that these compounds had the same aglycones or similar aglycones (with the morehydroxyl substituents at different positions) as type I saikosaponins. 11 type I saikosaponins were identified in this study, including saikosaponin compounds compared with type I related standard substances (acetylated/malonylated type I saikosaponins were described separately below and not included).

In the ESI⁻ mode, the fragment ions of compound 42 had fragment ions m/z 633 ([M− C₆H₁₀O₅−H]^–), m/z 557 ([633−C₃H₈O₂−H]^–, the specific fragmentation pathway of Fuc), and m/z 487 ([633−C₆H₁₀O₄−H]^–, aglycone ion), revealing the number and sequence of its saccharide chain, and based on the fragment ions m/z 455 ([aglycone−CH₃OH−H]⁻) and m/z 437 ([455−H₂O−H]⁻), which indicated that it comprised one more hydroxyl substituent at the aglycone moiety compared with SSa, compound 42 was identified as clinoposaponin XIV based on a literature review (Miyase and Matsushima, 1997); the fragmentation pathway is shown in Supplementary Figure S12. The same analytical procedure was employed to identify compounds 40, 58, and 71. Compounds 40 and 58 were identified as Sandrosaponin VII (Sánchez-Contreras et al., 2000) and Clinoposaponin XII (Cui et al., 2014). Compound 71 was initially identified as Bupleruoside I or Buddlejasaponin IV (Barrero et al., 2000; Zhu et al., 2018); the two differed only in the order of the saccharide chain and could not be distinguished temporarily. The structures of compounds 40, 58, and 71 are shown in Supplementary Figure S13.

According to the fragment ions, retention times, and maximum UV wavelength (254 nm around), for compounds 13, 38, 31, 33, 2, 14, 7, 50, 76, 17, 77, and 79 (Supplementary Datasheet S1), their characteristic mass losses (characteristic loss of 48 Da) and UV maximum absorption wavelengths (approximately 250 nm) are consistent with those of type II saikosaponins, so they have the same aglycones or similar aglycone structures (probably with the more OH or C=O substituents at different positions) as type II saikosaponins. 15 type II saikosaponins were identified in this study, including saikosaponin compounds compared with related type II standard substances (acetylated/malonylated type II saikosaponins were described separately below and not included).

In the ESI⁻ mode, compounds 31 and 33 shared the same molecular formula. Their fragment ions all showed m/z 779 ([M−C₆H₁₀O₅−H]^–), m/z 795 ([M−C₆H₁₀O₄−H]^–), m/z 633 ([M−C₆H₁₀O₅−C₆H₁₀O₄−H]^–), and m/z 471 ([M−2C₆H₁₀O₅−C₆H₁₀O₄−H]^–, aglycone ion), which revealed the number and sequences of its saccharide chain. Furthermore, based on the diagnostic fragment ions m/z 439 ([aglycone−CH₃OH−H]⁻) and m/z 423 ([aglycone−CH₂OH−OH−H]⁻, produced by the loss of 48 Da (characteristic loss of type II saikosaponins), it was concluded that these compounds had the same aglycone moieties as SSb₁/SSb₂. Based on the combined order of their retention times and the abundance of the m/z 423 fragment ion of compound 31, which was smaller than the m/z 439 fragment ion, and compound 33 showing opposite fragments, it was inferred that compound 31 was SSs (α-OH at position C₁₆) and compound 33 was SSn (β-OH at position C₁₆) (Fang et al., 2017). The fragmentation pathway is shown in Supplementary Figure S14. The same analytical procedure was employed to identify compounds 13 and 38 (having the same molecular formula), and compounds 13 and 38 were identified as SSl (α-OH at position C₁₆) and Tibesaikosaponin IV (β-OH at position C₁₆), respectively (Yu et al., 2013; Fang et al., 2017); their fragmentation pathways are shown in Supplementary Figure S15.

Employing the same analytical procedure to analyze the characteristic fragment ions and formulas of compounds 2, 14, 7, 50, and 76, their saccharide chains and aglycone structures could be deduced. Through a literature review, compounds 2 and 14 were identified as SSq or its isomer (Huang et al., 2008), and the fragmentation pathway of SSq is shown in Supplementary Figure S16. Compound 7 was identified as SSr (Barrero et al., 2000). The fragment ions and formula of compound 50 were the same as those of SSh, which was identified as Saponin BK1 (isomer of SSh) (Sinha et al., 2021). As for compound 76, its formula and fragment ions were the same as those of SSe (Compound 90, confirmed by the standard substance) and was identified as SSm (Shan et al., 2018). The structures of compounds 7, 50, and 76 are shown in Supplementary Figure S17.

Compounds 17 and 77 shared the same molecular formula and fragment ions, and the diagnostic fragment ions produced from the loss of a 30-Da fragment (the neutral loss of CH₂O) included m/z 763 ([M–30−H]⁻), m/z 601 ([M–C₆H₁₀O₅–30−H]⁻), and m/z 455 ([aglycone–30–H]⁻); it was inferred that compounds 17 and 77 had a C=O group on the aglycone moiety. Based on other fragment ions, their saccharide chains and aglycone structures could be deduced, and through a review of the related literature, they were identified as Tibesaikosaponin II or its isomer (Fang et al., 2017); the fragmentation pathway is shown in Supplementary Figure S18. For compound 79, its formula and fragment ions were the same as those of compound 99 (23-hydroxy-13β,28β-epoxyolean-11-ene-16-one 3-O-β-D-glucopyranosyl-(1-3)-β-D-fucopyranoside, confirmed by the standard substance), combined with the maximum UV wavelength (250 nm) and a review of the related literature (Li et al., 2015); it was identified as 3β,23,28-trihydroxyolean-11,13 (18)-diene-16-one 3-O-β-D-glucopyranosyl-(1-3)-β-D-fucopyranoside, and its structure is shown in Supplementary Figure S19.

According to the fragment ions, retention times, and maximum UV wavelength of compounds 8, 30, 3, 51, 54, 25, and 27 (Supplementary Datasheet S1), their characteristic mass losses (characteristic loss of 48 Da) and UV maximum absorption wavelengths (190–220 nm) are consistent with those of type III saikosaponins, and it was inferred that these compounds have the same aglycones or similar aglycones as type III saikosaponins. 13 type III saikosaponins were identified in this study, including saikosaponin compounds compared with related type III standard substances (acetylated/malonylated type III saikosaponins were described separately below and not included).

Compounds 8 and 30 shared the same molecular formula as C₄₂H₆₈O₁₄, and the fragment ions of compound 8 produced m/z 633 ([M−C₆H₁₀O₅−H]^–), m/z 557 ([633−C₃H₈O₂−H]^–, the specific fragmentation pathway of Fuc), and m/z 487 ([633−C₆H₁₀O₄−H]^–, aglycone ion), which revealed the number and sequences of its saccharide chain, and based on the diagnostic fragment ions m/z 455 ([aglycone−CH₃OH−H]⁻) and m/z 407 ([455−CH₂OH−OH−H]⁻), its aglycone moiety could be deduced. Thus, compound 8 was identified as SSt (or Bupleuroside IX) by reviewing the related literature (Yu et al., 2014). The fragmentation pathway of SSt is shown in Supplementary Figure S20. For compound 30, in addition to the above fragment ions of compound 8, additional fragment ions produced from the loss of 30 Da (neutral loss of CH₂O) appeared, which included fragment ions m/z 765 ([M−30−H]⁻), m/z 603 ([M−C₆H₁₀O₅−30−H]⁻), and m/z 457 ([aglycone−30−H]⁻), which indicated the presence of a C=O group on the aglycone moiety and was identified as Bupleuroside VI (Liang et al., 2014). The structure is shown in Supplementary Figure S21.

Compound 3, based on the fragment ions and formula, was identified as Rotundioside P by reviewing the related literature (Fujioka et al., 2006), and its fragmentation pathway is shown in Supplementary Figure S22. The same analytical procedure was employed to identify compounds 25 and 27, which presented an additional hydroxyl substituent at the aglycone moiety compared with compounds SSa/SSd, and combined with the order of the respective retention times, it was speculated that compound 25 was hydroxysaikosaponin a and compound 27 was hydroxysaikosaponin d (Huang et al., 2008); both structures are shown in Supplementary Figure S23. Compounds 51 and 54 shared the same molecular formula and fragment ion pattern as SSf (compound 52, confirmed by the standard substance) and were identified as the isomer of SSf, in which we speculated that the position of the double bond was different from that of SSf.

For compound 6, the fragment ions m/z 647 ([M−C₆H₁₀O₅−H]^–), m/z 571 ([647−C₃H₈O₂−H]^–, the specific fragmentation pathway of Fuc), and m/z 501 ([647−C₆H₁₀O₄−H]^–, aglycone ion) revealed the number and sequences of the saccharide chain. According to the diagnostic fragment ions m/z 453 ([aglycone−CH₂OH−OH−H]⁻) and m/z 483 ([aglycone−H₂O−H]⁻) and combined with the maximum UV wavelength (282 nm), it was inferred that compound 6 had a similar aglycone moiety to type IV saikosaponins and was speculated to be an isomer of Bupleuroside V, with the following structure, 3β, 16α, 23,28-Tetrahydroxy-olean-9,12 (13)-dien-29-oic acid 3-O-β- D-glucopyranosyl-(1-3)-β-D-fucopyranoside. Its fragmentation pathway is shown in Supplementary Figure S24. 3 type IV saikosaponins were identified in this study, including saikosaponin compounds compared with related type IV standard substances.

For compound 10, the number and sequence of its saccharide chain was deduced from the fragment ions (Supplementary Datasheet S1) m/z 453 ([aglycone−CH₂OH−OH−H]⁻) and m/z 469 ([aglycone−CH₃OH−H]⁻), but without m/z 483 ([aglycone−H₂O−H]⁻), and combined with its maximum UV wavelength (251 nm) and formula, it was identified as Bupleuroside V using a literature review (Yoshikawa, 1997). Its fragmentation pathway is shown in Supplementary Figure S25. The same analytical procedure was employed to identify compound 9, which was identified as 3β,16α,23,28-tetrahydroxy-olean-11,13 (18)-dien-30-oicacid-3-O-β-D-glucopyranosyl-(1-2)-β-D-glucopyranosyl-(1-3)-β-D-fucopyranoside (Liang et al., 2013); its structure is shown in Supplementary Figure S26. Compounds 9 and 10 belonged to the type VI saikosaponins.

According to the formula, fragment ions, retention times, and maximum UV wavelength of compounds 1, 5, 11–12, 15-16, 19–23, 26, 29, 32, 35, 41, 44–45, 49, 60, 68, 72, 74, and 94–96 (Supplementary Datasheet S1), the number and sequences of the saccharide chain can be deduced. In addition, the structures of the aglycone can be identified through further analysis of their fragment ions.

For compound 1, its molecular ion and fragment ions were 18 Da more than those of compound 8 (SSt), and combined with its formula, this suggested it had an additional hydroxyl substituent at the aglycone moiety compared with SSt; thus, compound 1 was tentatively identified as hydroxysaikosaponin t. Its fragmentation pathway is shown in Supplementary Figure S27. The same analytical procedure was employed to identify compounds 23, 11, 35, 44, 60, and 49. Compound 23 had an additional hydroxyl substituent at the aglycone moiety compared with SSh (compound 56, confirmed by the standard substance); thus, compound 23 was tentatively identified as hydroxysaikosaponin h (11α-hydroxy-SSh). Similarly, compounds 11, 35, 44, and 60 had an additional hydroxyl substituent at the aglycone moiety compared with SSc (compound 47, confirmed by the standard substance); thus, they were tentatively identified as hydroxysaikosaponin c (11α-hydroxy-SSc) or its isomer (Ebata et al., 1996); for compound 49, its retention time followed that of hydroxysaikosaponin a and hydroxysaikosaponin d (compounds 25 and 27). The diagnostic fragment ions m/z 439 ([aglycone−CH₃OH−H]⁻) and m/z 423 ([aglycone−CH₂OH−OH−H]⁻) and the abundance of the fragment ion m/z 423 were smaller than the fragment m/z 439; thus, it was speculated to be hydroxysaikosaponin b₂ (α-OH at position C₁₆). The structures of hydroxysaikosaponin c, hydroxysaikosaponin h, and hydroxysaikosaponin b₂ are shown in Supplementary Figure S28.

For compound 32, its formula and fragment ions indicated that it had an additional OC₂H₅ substituent at the aglycone moiety compared with SSs/SSn (compound 31/33), based on the abundance of the m/z 423 ([aglycone−OC₂H₅−48−H]⁻) fragment ion, which was smaller than m/z 439 ([aglycone−OC₂H₅−32−H]⁻) when, and combined with its retention time, it was identified as 11α-ethoxyl-SSs (α-OH at position C₁₆). Similarly, compounds 72 and 74 were identified as 11α-ethoxyl-SSb₂ and 11α-ethoxyl-SSb₁, respectively, while compounds 96 and 45 were identified as 11α-butoxyl-SSb₂ and 11α-ethoxyl-SSh, respectively. Their fragmentation pathways are shown in Supplementary Figure S29–S32.

Compounds 4, 12, 16, 19, and 20 shared the same molecular formula and fragment ions, and they could be deduced to have one less double bond than SSq (compounds 2 and 14), so they were identified as dihydro-SSq or its isomer (such as 11,12-dihydro-SSq, 13,18-dihydro-SSq, and other possible isomers); their possible structures are shown in Supplementary Figure S33.

Using the same approach, compound 41 was deduced to have one less double bond than Tibesaikosaponin I (compound 29), and combined with the maximum UV wavelength (254 nm), it was tentatively identified as Δ^21,22-Tibesaikosaponin I; the structure is shown in Supplementary Figure S34. For compounds 29 and 94, it was speculated that their fragmentation pathways were similar to that of Tibesaikosaponin II, but there were no fragment ions produced by the loss of 30 Da, and thus, they were identified as Tibesaikosaponin I or its isomer (Fang et al., 2017), and presumably the C=O group was conjugated with the adjacent olefinic bond, which stabilized the C=O group and was not easy to lose; its fragmentation pathway is shown in Supplementary Figure S35.

Based on the analysis of the formulas, fragment ions, retention times, and maximum UV wavelengths, combined with a review of the literature, compound 21 was identified as Magnoside B (Haddad et al., 2012), and its fragmentation pathway is shown in Supplementary Figure S36. Compound 22 was identified as 3-O-[α-L-Rhamnopyranosyl (1–4)-β-D-glucopyranosyl] oleanolic acid 28-O-β-D-glucopyranosyl ester (Achouri et al., 2017). Compounds 5 and 15 were identified as (3β,21β,22α)-28-[[2-O-(6-Deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-21,22-dihydroxyolean-12-en-3-yl 6-O-β-D-glucopyranosyl-β-D-glucopyranoside or its isomers (Xiao et al., 2013). Compound 68 was identified as (3β,4α,16α)-3,16,23-trihydroxyoleanan-28-yl O-6-deoxy-α-L-mannopyranosyl-(1–4)-O-[β-D-glucopyranosyl-(1–6)]-β-D-glucopyranoside (Luo and Jin, 1991). Compound 95 was identified as Bupleuroside XI (Yoshikawa, 1997). All structures are shown in Supplementary Figure S37.

The mass spectra fragmentation information of the acetylated/malonylated saikosaponins in the ESI⁺ mode is shown in Supplementary Table S3. 35 acetylated/malonylated saikosaponins were identified in this study, including saikosaponin compounds compared with related standard substances.

Compounds 18, 53, 55, 57, 59, 61–63, 66–67, 69,78, 80–83, 85, 87–88, 92–93, 98, 100, 102–104, and 109 all produced [M–42–H]⁻ and [M–60–H]⁻ fragment ions as diagnostic ions (Supplementary Datasheet S1); thus, they were identified as acetylated saikosaponins.

The compounds 81, 87, 88, 92, 55, 62, 66, 102, 103, 104, 109, 78, 83, 93, and 98 were analyzed, and according to Supplementary Table S3, all fragment ions in the ESI⁺ mode were m/z 455 ([aglycone−H₂O + H]⁺), m/z 437 ([aglycone−2H₂O + H]⁺), and m/z 419 ([aglycone−3H₂O + H]⁺), indicating that their acetyl groups were all substituted by the saccharide chain. In the ESI⁻ mode, in addition to m/z 779 ([M–42–H]⁻) and m/z 761 ([M–60–H]⁻), the remaining fragment ions of compounds 81, 87, 88, and 92 were the same as those of SSa/SSd, and their retention times followed those of SSa but were before SSd; thus, they were identified as acetylated derivatives of SSa. For SSa, SSd, SSb₁, and SSb₂, the OH group at the 1″ position of the terminal glucose of the saccharide chain was attached to rhamnose, and only four OH groups at positions 2″, 3″, 4″, and 6″ were substituted by an acetyl group. Based on the order of their respective retention times, compounds 81, 87, 88, and 92 were identified as 2″-O-acetyl-SSa, 3″-O-acetyl-SSa, 4″-O-acetyl-SSa, and 6″-O-acetyl-SSa, respectively. Of these, compounds 81 and 92 were confirmed by the standard substance. Similarly, the retention times of compounds 102, 103, 104, and 109 followed SSd, and compound 109 was 6″-O-acetyl-SSd by comparison with the standard substance; according to the order of retention times, the compounds 102, 103, and 104 were inferred to be 2″-O- acetyl-SSd, 3″-O-acetyl-SSd, and 4″-O-acetyl-SSd, respectively. For compounds 78, 83, 93, 98, and 100, the abundance of the m/z 423 fragment ion was smaller than that of the m/z 439 fragment ion, indicating that they were acetylated derivatives of SSb₂. Combined with the order of their retention times, compounds 78, 83, 98, and 100 were 2″-O-acetyl-SSb₂, 3″-O-acetyl-SSb₂, 4″-O-acetyl-SSb₂, and 6″-O-acetyl-SSb₂, respectively. The abundance of the m/z 423 fragment ion of compound 93 was slightly higher than that of m/z 439, which indicated that it was an acetylated derivative of SSb₁ (O-acetyl-SSb₁). The structures of these acetylated saikosaponins are shown in Supplementary Figure S38.

For compounds 53, 55, 62, and 66 in the ESI^–mode, in addition to fragment ions such as m/z 925 ([M−42−H]^–) and m/z 907 ([M−60−H]^–), the other fragment ions were the same as those of SSc; thus, these were acetylated derivatives of SSc. Their fragment ions in the ESI⁺ mode are shown in Supplementary Table S3 and in combination with the fragment ion in the ESI^–mode m/z 779 ([M−42−C₆H₁₀O₄−H]^–) and m/z 761 ([M−60−C₆H₁₀O₄−H]^–), it was deduced that the acetyl group was substituted by the OH group of terminal glucose of the saccharide chain, rather than that of the aglycone. For SSc, the OH group at the 1‴ position of the terminal glucose of the saccharide chain was attached to rhamnose, and only four OH groups at positions 2‴, 3‴, 4‴, and 6'" could be substituted by an acetyl group. Based on the order of their retention times, the 4 compounds were identified as 2‴-O-acetyl-SSc, 3‴-O-acetyl-SSc, 4‴-O-acetyl-SSc, and 6‴-O-acetyl-SSc, respectively. The fragmentation pathway in the ESI^–mode for 6‴-O-acetyl-SSc is shown as a representative example in Supplementary Figure S39. Similarly, compounds 57, 59, 63, and 69 were identified as 2‴-O-acetyl-SSf, 3‴-O-acetyl-SSf, 4‴-O-acetyl-SSf, and 6‴-O- acetyl-SSf, respectively. Their structures are shown in Supplementary Figure S40.

Based on the fragment ions, compounds 67, 70, 80, and 85 were acetylated derivatives of SSb₃/SSb₄, and according to their fragment ions in the ESI⁺ mode (Supplementary Table S3), their acetyl groups were substituted by saccharide chains. Of these, compound 67 had the shortest retention time, which was 2″-O-acetyl-SSb₃. After comparison with the standard substance, compound 80 was deduced to be 6″-O-acetyl-SSb₃, while compound 85 had a retention time that followed that of compound 80 and was thus deduced to be 6″-O-acetyl-SSb₄; both structures are shown in Supplementary Figure S41.

The same analytical procedure was employed to identify compounds 36, 39, and 46 in the ESI^–mode, and the abundance of the m/z 423 ([aglycone−48−H]⁻) fragment ion was smaller than that of the fragment ion m/z 439 ([aglycone −32−H]⁻) for all compounds; thus, they were identified as acetylated derivatives of SSs (α-OH at position C₁₆). Combined with their fragment ions in the ESI⁺ mode (Supplementary Figure S28), the acetyl groups were all substituted by the terminal glucose of the saccharide chain. According to the order of the respective retention times, compounds 36, 39, and 46 were identified as three of 2‴-O-acetyl-SSs, 3‴-O-acetyl-SSs, 4‴-O-acetyl-SSs, and 6‴-O-acetyl-SSs, and the respective structures are shown in Supplementary Figure S42. With 6‴-O-acetyl-SSs as a representative example, the fragmentation pathway in ESI^–is shown in Supplementary Figure S43. Similarly, compound 18 was identified as O-acetyl-Bupleuroside V. For compound 61, based on its formula and fragment ions in the ESI^–mode, it was speculated that the C₁₁ position had an additional OCH₃ substituent at the aglycone compared with O-acetyl-SSs, according to its fragment ion pattern in ESI⁺ (Supplementary Table S3 ), which indicated that an acetyl group was substituted by the aglycone; it was identified as 23-O-acetyl-(11α-methoxyl-SSs), and its fragmentation pathway in the ESI^–mode is shown in Supplementary Figure S44.

Compounds 106 and 108 were analyzed in ESI^–, their fragment ions all appeared to be m/z 821 ([M−42−H]^–), m/z 779 ([M−84−H]^–), and m/z 761 ([M−60−42−H]^–), and the remaining fragment ions were the same as those of SSa/SSd, which indicated that they were diacetyl derivatization products of SSa/SSd. Supplementary Table S3 indicated that their acetyl groups were all substituted by the saccharide chain; according to their retention times, compounds 106 and 108 all followed the SSd; thus, they were identified as O-diacetyl-SSd.

For compounds 82 and 101 in the ESI^–mode, m/z 821 ([M−44−H]^–) and m/z 779 ([M−86−H]^–) fragment ions were produced, and the remaining fragment ions were the same as those of SSa/SSd, which indicated that they were malonylated derivatives of SSa/SSd. As shown in Supplementary Table S3, the malonyl groups were all substituted by the saccharide chain, and after assessing their retention time, compound 82 followed SSa and preceded SSd, and compound 101 followed SSd; thus, compound 82 was O-malonyl-SSa and compound 101 was O-malonyl-SSd. Similarly, compound 43 was identified as O-malony-hydroxysaikosaponin c.

Compounds 91 and 107 were analyzed in the ESI^– mode, their fragment ions all appeared at m/z 863 ([M−44−H]^–), m/z 821 ([M−86−H]^–), and m/z 779 ([M−42−86−H]^–), and the remaining fragment ions were the same as those of SSa/SSd, which indicated that their structures contained an additional malonyl substituent and an additional acetyl substituent compared with SSa/SSd. Supplementary Table S3 indicated that their malonyl and acetyl groups were substituted by the saccharide chain. Analyzing their retention times, compound 91 followed SSa and preceded SSd, and compound 107 followed SSd; thus, the former was O-malonyl-acetyl-SSa, and the latter was O-malonyl-acetyl-SSd.