Research Article Received: 21 August 2010 Revised: 19 October 2010 Accepted: 26 October 2010 Published online in Wiley Online Library: 00 Month 2010 Rapid Commun. Mass Spectrom. 2011, 25, 104–114 (wileyonlinelibrary.com) DOI: 10.1002/rcm.4835 Analysis and development of structure-fragmentation relationships in withanolides using an electrospray ionization quadropole time-of-flight tandem mass spectrometry hybrid instrument Syed Ghulam Musharraf*, Arslan Ali, Rahat Azher Ali, Sammer Yousuf, Atta-ur- Rahman and M. Iqbal Choudhary H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi-75270, Pakistan Structural elucidation and gas-phase fragmentation of ten withanolides (steroidal lactones) were studied using a positive ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. Withanolides form an important class of plant secondary metabolites, known to possess a variety of biological activities. Withanolides which possess hydroxyl groups at C-4, C-5, C-17, C-20, and C-27, and an epoxy group at C-5/C-6, were evaluated to determine the characteristic fragments and their possible pathways. ESI-QqTOF-MS (positive ion mode) showed the presence of the protonated molecules [M R H]R. Low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis of the protonated molecule [M R H]R indicated multiple losses of water and the removal of the C-17-substituted lactone moiety affording the [M R H–Lac]R product ion as the predominant pathways. However, withanolides containing a hydroxyl group at C-24 of the lactone moiety showed a different fragmentation pathway, which include the loss of steroidal part as a neutral molecule, with highly diagnostic ions at m/z 95 and 67 being generated from the cleavage of lactone moiety. Our results also determined the influence of the presence and positions of hydroxyl and epoxy groups on product ion formation and stability. Moreover, the knowledge of the fragmentation pattern was utilized in rapid identification of withanolides by the LC/MS/MS analysis of a Withania somnifera extract. Copyright ß 2010 John Wiley & Sons, Ltd. Lactone rings are found in various classes of natural products and are known to be responsible for interesting biological activities. The withanolides are a series of naturally occurring steroids containing a lactone with a side chain of nine carbons, generally attached to C-17,[1] although a great variation of the lactone moiety is found in various classes of withanolides. They exhibit a wide range of pharmacological activities, including antimicrobial, antitumor, antiinflammatory, hepatoprotective, immunomodulatory, cytotoxic, insecticidal, and insect-antifeedant properties.[1–6] Withanolides have been reported from plants of the families Solanaceae, Taccaceae[6] and Leguminosae,[7] as well as from some marine organisms.[8] The genus Withania is among the richest sources of steroidal lactones in nature. Withania somnifera, commonly known as Aswaghandha, is extensively used in traditional Indian and Ayurvedic medicines.[9] It is an ingredient of many formulations prescribed for a variety of musculoskeletal conditions, and as a general health tonic for elderly persons and lactating mothers. The leaves of this plant are bitter and recommended in fever, painful swellings, inflammation and opthalmitis.[10] The methanolic extracts of different parts of W. somnifera 104 * Correspondence to: S. G. Musharraf, H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi-75270, Pakistan. E-mail: musharraf1977@yahoo.com Rapid Commun. Mass Spectrom. 2011, 25, 104–114 exhibit therapeutic potential against cardiovascular problems, and are also effective against hyperlipidemia, obesity, aging, and copper-induced pathophysiological conditions.[11] Aswaghandaha is reputed to be beneficial for the heart and to act as a blood purifier.[12] Interestingly, most of the biological activities associated with this plant are largely attributed to the presence of withanolide A and its congeners. We have previously reported several new withanolides from various species of Withania, namely Withania somnifera [13,14] and Withania cogulance.[1,2,15] The availability of small quantities of complex mixtures of plant material for investigation of natural products is a major challenge associated with phytochemical methods. Therefore, sensitive and powerful analytical techniques are needed to identify minor constituents of plant extracts. Liquid chromatography (LC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a useful analytical technique for the analysis of complex mixtures in small quantities,[16–18] and for the dereplication of polar and thermally labile compounds. However, structural elucidation by LC/ESI-MS/MS requires a previous knowledge and understanding of the gas-phase fragmentation patterns of the protonated molecules obtained from this class of compounds. Low-energy tandem mass spectrometry studies, combined with accurate mass data, provide an excellent strategy to elucidate the structural elucidation of the protonated molecules and offer a good understanding of the obtained fragmentations. In the Copyright ß 2010 John Wiley & Sons, Ltd. ESI-QqTOF-MS/MS analysis of withanolides case of withanolides and their congeners, its use has not been reported previously. Moreover, there are only a few publications on ESI-MS/MS of substituted five- and six-membered lactones.[19–21] To the best of our knowledge, the mechanistic pathways of fragmentation of withanolide lactones (i.e. six-membered lactones possessing a,bunsaturation and a,b,d-trisubstitution) have not been reported. This manuscript describes the ESI-QqTOF-MS (þ ion mode) and the CID-MS/MS analysis of the protonated ions and their respective product ions selected from the unmodified naturally occurring withanolides. The elucidation of the gas-phase fragmentation pathways and the rapid characterization of the complex structures of the withanolides will allow the prospective elucidation of other compounds isolated from various plant extracts. ion mode on an ESI-QqTOF-LCMS/MS instrument (QSTAR XL; Applied Biosystem/ MDS Sciex, Darmstadt, Germany) at room temperature. High-purity nitrogen gas was used as the curtain gas and collision gas delivered from a nitrogen generator (Peak Scientific). The ESI interface conditions were as follows: ion spray capillary voltage, 5000 V; curtain gas flow rate, 20 L min 1; nebulizer gas flow rate, 5 L min 1; DP1 60 V; DP2 15 V; focusing potential, 265 V. The collision energy was swept from 5 eV to 45 eV for MS/MS analysis. Calibration was performed by using an internal calibration process. Samples were introduced into the mass spectrometer using a syringe pump (Harvard, Holliston, MA, USA) at a flow rate of 5 mL/min. HPLC/QqTOF-MS conditions EXPERIMENTAL Chemicals and reagents Chemicals and solvents used were of analytical and HPLC grades, respectively, and were purchased from Aldrich-Sigma. Deionized water (Milli-Q) was used throughout the study. The withanolides investigated in this study were withaferin A (1), 17-hydroxywithanolide D (2), 12-deoxywithastramonolide (3), 14,15-dehydro-27- deoxywithaferin A (4), sominolide (5), 2,3-dihydrowithaferin A (6), 3-methoxy-2,3-dihydrowithaferin A (7), withanolide A (8), withanone (9), and 27-deoxy-17hydroxywithaferin A (10). Standards 1, 3 and 8 were purchased from Chromadex, while other compounds were obtained from the compound bank of the Dr. Panjwani Center for Molecular Medicine and Drug Research (International Centre for Chemical and Biological Sciences), University of Karachi. The isolation procedures and characterization of these withanolides have already been reported.[8–14] Collection of plant material and extraction of withanolides The whole plant of Withania somnifera was collected within the premises of the University of Karachi in October 2008, and identified by Prof. Dr. Surraiya Khatoon, Taxonomist at the Department of Botany, University of Karachi (voucher specimen no. KUH1550). In order to extract the withanolides, the fresh leaves (4.0 g) were finally powdered in liquid nitrogen with a pestle and mortar and extracted overnight in 20 mL of methanol/water (95:5 v/v) at room temperature on a shaker, and then filtered. The filtrate was collected and the residue was extracted twice with the same volume of extractant. The filtrates were combined and extracted with n-hexane (3  60 mL), chloroform (3  60 mL) and ethyl acetate (3  60 mL). The choloroform fractions were combined and concentrated to a dry powder. A sample (1 mg) of the dry powder was dissolved in HPLC-grade methanol (1 mL), filtered through Millipore filter (0.44 mm), and subjected to LC/MS/MS analysis. Mass spectrometry Rapid Commun. Mass Spectrom. 2011, 25, 104–114 RESULTS AND DISCUSSION Ten standard withanolides (Fig. 1), including withaferin A (1) and withanolide A (8), commonly used as biomarkers in the standardization of Withania somnifera, were investigated by ESI-QqTOF-MS (þve ion mode) and CID-MS/MS analysis for the characterization of the gas-phase fragmention routes of unmodified withanolides. Protonated ions obtained from ESI-QqTOF-MS analyses of standard compounds are presented in Fig. 1. All the product ion scans were recorded from the extracted protonated molecules and are presented in Copyright ß 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm 105 Withanolides were dissolved in 0.1% HCOOH and MeOH (9:1 v/v, 1 mg/mL) and analyzed by electrospray ionization (ESI) and collision-induced dissociation (CID) in the positive Chromatographic separation was performed using a micro-HPLC system (Series 1100, Agilent Technologies, Waldbronn, Germany), and reversed-phase capillary column (ZORBAX XDB-C18, 150 x 4.6 mm, 5 mm, Agilent). Injection volumes were 1 mL. The mobile phases were as follows: eluent A, H2O (0.5% formic acid) and eluent B, ACN (0.5% formic acid). The flow rate was 20 mL min 1, and a gradient elution program was used. The chromatographic procedure was initialized at 10% B, raised to 30% B at 6 min, followed increased up to 35% B at 10 min, 45% B at 20 min and then a linear increase to 90% B in 25 min before returning to initial conditions to 10% B at 30 min. The data were recorded via information-dependent acquisition (IDA) experiments with TOF scan range of m/z 300 to 550 amu at the rate of 1 scan s 1 with the three most abundant peaks. Switch criteria was used for masses between 400 to 500 amu. Precursor ion scans were recorded between 50 to 500 amu on a QSTAR XL mass spectrometer (Applied Biosystem/ MDS Sciex, Darmstadt, Germany), equipped with an ESI spray source running with Analyst QS 1.1 (Applied Biosystems). The system was operated in the positive ion mode and the source and inlet parameters were optimized as: ion spray capillary voltage, 5500 V; curtain gas flow rate, 10 L min 1; nebulizer gas flow rate, 30 L min 1; DP1 60 V; DP2 15 V; focusing potential, 265 V; and collisional energy, of 25 eV for MS/MS analysis. Calibration was performed using reserpine as an internal calibrant. Computational studies were performed using density functional theory (DFT) at the B3LYP level with the 6-31G* basis set in Spartan 08 version 1.2.0 (Wavefunction, CA, USA). Theoretical fragmentation of protonated withanolides was evaluated by using ACD/MS Fragmenter software (ACD Labs). S. G. Musharraf et al. Figure 1. Structures and ESI-QqTOF-MS data (þve ion mode) of standard withanolides 1–10. Table 1. The CID-MS/MS scans of the precursor protonated molecules [M þ H]þ showed that all the withanolides afforded similar product ions, with minor differences, due to the oxygen functionalities on the lactone and the steroidal parts. Relative intensities of selected product ions of [M þ H]þ versus laboratory collision energy ranging from 10 eV to 45 eV (with stepping up of 5eV each time) were plotted for withanolide A (8) (See Supplementary Fig. 1, Supporting Information). It showed that the optimum collision energy (CE) for recording product ion spectra of withanolides was 25 eV. However, the abundance of ions in dehydrated peaks was significantly influenced by the variation in collision energy. Fragmentation pattern in withanolides 106 The diagnostic product ions obtained from the precursors [M þ H]þ were obtained mainly by loss of water and subsequent cleavage/rearrangement of the lactone (Lac) moiety. Characteristic dehydrated fragments were observed in withanolide spectra by the multiple removal of water moieties from both the intact molecular ion [M þ H]þ and ergostane part of the molecule [M þ H–Lac]þ. Compound 1 showed peaks at m/z 453, 435, 417 and 399, corresponding to [M þ H–H2O]þ, [M þ H–2H2O]þ, [M þ H-3H2O]þ and [M þ H–4H2O]þ, respectively. Similar patterns were observed in compounds 3 and 5–10, while compounds 2 wileyonlinelibrary.com/journal/rcm and 4 showed removal of five and three water molecules, respectively. All the CID-MS/MS analyses of protonated molecules obtained from the withanolides showed the removal of at least three molecules of water, two from the ergostane fragment [M þ H–Lac]þ to form [M þ H–Lac–H2O]þ and [M þ H–Lac–2H2O]þ, whereas the loss of one water molecule was at least observed from the lactone functionality to offer [M þ H–H2O]þ in all analyzed compounds. The removal of the rest of the H2O molecules was dependent on the number of hydroxyl substituents. Compound 7 showed the successive removal of four water molecules from [M þ H]þ at m/z 485, 467, 449 and 431, corresponding to [M þ H–H2O]þ, [M þ H–2H2O]þ, [M þ H–3H2O]þ and [M þ H–4H2O]þ, whereas another series of four dehydrated ions were observed from the successive loss of water molecules from [M þ H–CH3OH]þ at m/z 453, 435, 417 and 399, corresponding to [M þ H–CH3OH–H2O]þ, [M þ H–CH3OH–2H2O]þ, [M þ H–CH3OH–3H2O]þ and [M þ H–CH3OH–4H2O]þ. Product ion formation and stability was found to be influenced by the presence and positions of hydroxyl and epoxy groups, and applied CE. Compounds 2, 8, 9 and 10 which possess hydroxyl groups at C-17 and/or C-20 shows dehydration of [M þ H]þ ions even at very low CE (10 to 20 eV). CID-MS/MS analyses of the protonated molecule extracted from compound 2 showed a successive removal of four water molecules from [M þ H]þ to produce product ions at m/z 469 (base peak), 451, 433 and Copyright ß 2010 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 104–114 (1) (1) (2) (22) - (12) (40) - (2) (12) (11)b (17)c (13) (10) (8)b (3)c - (33) (11)b (56) (28)b (15) (44) - (31) (2) (1)b - (13) (37) (2) - (24) (21) (16)b (7)c (4) (22) (25)b (32) (7)b 263 (5) 263 (11) (20)b (27)c 263 (100) 263(6) (7)b 263 (7) 265 (0) (11)b 295 (1) 263 (100) (100)b (100)c 263 (0) (100)b 263 (8) 281 (100) 281 (100) (88)b (68)c 281 (39) 281 (3) (100)b 281 (100) 283 (0) (36)b 313 (64) 281 (12) (13)b (9)c 281 (0) (6)b 281 (4) at 15 eV. - at 20 eV and Relative abundances are given in parentheses; at CE 25 eV, 10 453(0) (46)b 453 (5) 415, corresponding to [M þ H–H2O]þ, [M þ H–2H2O]þ, [M þ H–3H2O]þ and [M þ H–4H2O]þ in significant abundance only at CE 10 eV (Fig. 2). A similar pattern of successive dehydration at low CE was observed in compounds 8, 9 and 10, therefore showing the significant influence of CE on the stability and the abundances of the resulting product ions. The ergostane part [M þ H–Lac]þ of the molecule also showed two subsequent H2O removals due to the presence of the hydroxyl-epoxide pair (Table 1). MS/MS spectral comparison of compounds 1 and 3 at CE 25 eV indicated that the position of the hydroxyl-epoxide pair on the ergostane moiety plays a vital role in the elimination of two water molecules from this portion. Compound 1, possessing 4-hydroxy-5,6-epoxy groups, showed a [M þ H–Lac]þ peak at m/z 299, while the dehydration peaks appeared at m/z 281 as the base peak, and at m/z 263, corresponding to [M þ H–Lac–H2O]þ and [M þ H–Lac–2H2O]þ, respectively. However, in contrast to compound 1, compound 3, which possesses 5-hydroxy-6,7-epoxy groups, showed the base peak of the [M þ H–Lac–2H2O]þ product ion at m/z 263 (Figs. 3(A) and 3(B)). Here simultaneous removal of two water molecules may be favored due to the formation of extended conjugation with the C1-ab-unsaturated keto group. Similarly, compounds 8 and 9 also showed a [M þ H–Lac–2H2O]þ product ion as a base peak. Removal of the lactone moiety was observed in all CID-MS/MS analyses of the protonated molecules extracted from the various withonolides analyzed and it represents the most dominant fragmentation pathway. This appears to be due to the sensitive structure of the lactone part that readily cleaves between C-17/C-20, causing the charged fragments in the ergostane framework to be detected, while the lactone part is removed as a neutral entity. All the CID-MS/MS analyses of the protonated molecules extracted from the various withonolides showed the [M þ H–Lac]þ product ion at m/z 299, while the corresponding [M þ H–Lac]þ product ions for compounds 6 and 7 appeared at m/z 301 and 331, respectively. A very interesting alternative pathway is observed in CID precursor ion scans obtained from the withanolides that possess a hydroxyl group at C-27 at a CE greater than 20 eV. In this case, the ergostane part is removed as a neutral moiety, while the lactone part retains the charge and on cleavage this leads to the formation of two characteristic peaks at m/z 95 and 67. These two diagnostic product ions appeared with high abundance, above CE 20 eV, and were found in all CID analyses of the precursor ions obtained from the withanolides possessing 27-hydroxy groups. Both product ions can therefore serve as the differentiation between withanolides bearing OH at C-27 from other withanolides. CID analysis of the precursor ion obtained from compounds 1, 3, 5, 6, and 7 also showed similar fragment ions (Table 1). Product ions at m/z 171/175 were also observed in all withanolide spectra (except compound 9), but were not characterized. In the light of the above studies, a flow chart is proposed for the rapid identification and structural analysis of withanolides with positive CID experiments (Scheme 1). Elucidation of fragmentation pathway Computational studies and ACD/MS Fragmenter software were used to aid the elucidation of the fragmentation Copyright ß 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm 107 Rapid Commun. Mass Spectrom. 2011, 25, 104–114 9 503 (28) 471(1.2) (4)b 7 8 5 6 3 4 471 (0) (22)b 471 (23) 435 (0) (7)b 435 (5) 417 (0) (49)b 417 (4) b 399 (0) (15)b 399 (3) c - - - 399 (3) 401 (0) (4)b 431 (3) 399 (16) (31)b 299 (46) 299 (55) (100)b (88)c 299 (63) 299 (27) (93)b 299 (49) 301 (0) (100)b 331 (100) 299 (6) (4)b (3)c 299 (0) (5)b 299 (49) 397 (0) (2)b (2)c 399 (3) 415 (2) (5)b (7)c 399 (14) - 417 (5) 433 (3) (7)b (11)c 417 (22) 399 (0) (1)b 417 (5) 419 (0) (5)b 449 (6) 417 (14) (25)b 453 (2) 469 (1) (17)b (100)c 453 (5) 435 (4) (11)b 453 (2) 455 (0) (3)b 485 (5) 453 (2) (13)b 1 2 471 (15) 487 (0) (2)b (8)c 471 (14) 453 (2) (20)b 471 (18) 473 (0) (62)b 435 (8) 451 (3) (7)b (34)c 435 (17) 417 (3) (9)b 435 (7) 437 (0) (8)b 467 (10) 435 (5) (19)b [M þ H–Lac–H2O]þ [M þ H–Lac]þ [M þ H–5H2O]þ [M þ H–4H2O]þ [M þ H–3H2O]þ [M þ H–2H2O]þ [M þ H–H2O]þ [M þ H]þ No. Table 1. Diagnostic precursor and product ions isolated from the standard withanolides 1–10 at collision energy of 25 eV [M þ H–Lac–2H2O]þ 175 171 95 67 ESI-QqTOF-MS/MS analysis of withanolides S. G. Musharraf et al. Figure 2. MS/MS spectra of 17-hydroxywithanolide D (2): at a CE of (A) 10 eV, (B) 15 eV, and (C) 20 eV. 108 pathways. In silico studies were performed to investigate the most probable protonation site in withaferin A (1) (taken as a representative withanolide). Minimum energy conformation of the neutral molecules was first optimized and every possible protonation site was then individually protonated and analyzed after optimization. There are six possible protonation sites: the lactone oxygen (O-1), lactone carbonyl oxygen (O-2), C-27 hydroxyl (O-3), a,b-unsaturated ketone (O-4), C-4 hydroxyl (O-5), and epoxy oxygen (O-6). It was found that the lactone carbonyl oxygen (O-2) showed a minimum energy of –1541.50592 Hartrees, and therefore it was the most favorable site of protonation among the six possible sites along with O-3 which showed an energy of –1541.50283 Hartrees (an energy difference of approximately 2 kcal/mol) (see Supplementary Table S1, Supporting wileyonlinelibrary.com/journal/rcm Information). Therefore, both these forms were considered for elucidating the major fragmentation pathways of withaferin A-type withanolides. Moreover, the bond lengths of protonated and nonprotonated forms of two protonation sites, O-2 and O-3, were also investigated. A comparison of bond lengths between both forms showed considerable bond elongation which took place in the vicinity of the protonation site, thereby leading to the characteristic fragments of this class. Therefore, both forms were considered for the elucidation of fragmentation pathways. The differences in bond lengths in both forms of O-2 and O-3 are summarized in Supplementary Table S2 (Supporting Information). The product ion spectrum of 1 is considered as a representative of other withanolide congeners in predicting Copyright ß 2010 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 104–114 ESI-QqTOF-MS/MS analysis of withanolides Figure 3. CID-MS/MS spectra of the protonated molecules [M þ H]þ isolated from the withanolides using a collision energy of 25 eV: (A) withaferin A (1), (B) 12-deoxywithastramonolide (3), and C) withanolide A (8). Rapid Commun. Mass Spectrom. 2011, 25, 104–114 a,b-conjugation. The secondary carbocation thus formed is readily converted into the more stable tertiary carbocation B through a 1,2-H shift. Another 1,2-H shift converts fragment B into fragment C. Fragment D, i.e. [M þ H–Lac]þ at m/z 299.1659 (C19H23Oþ 3 , calc. 299.1642), may be derived from fragment C by the removal of the lactone moiety through C-17–C-20 bond fission. Another possible route to form Copyright ß 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm 109 fragmentation pathways. As predicted by computational calculations, protonation at the lactone carbonyl oxygen (O-2) causes considerable elongation of bond c along with a decrease in the bond length of bond b (see Supplementary Table S2, Supporting Information), hence facilitating the C-22–O bond fission to form A, an a,b-unsaturated carboxylic acid. Here removal of CO2 was not favorable due to the S. G. Musharraf et al. Figure 4. LC/MS/MS spectra of Withania somnifera. (A) Total ion chromatogram (TIC), (B) extracted ion chromatogram (XIC) of m/z 487, (C) TOF-MS at retention time (Rt) 14.5 min, and (D) MS/MS spectra of m/z 487. 110 fragment D is through formation of ion E due to the shifting of the the C-24 pi-bond to C-25, thereby creating a positive center at C-24. A 1,5-H shift is proposed to convert ion E into ion F, eventually leading to formation of ion D via the above-mentioned route. As molecules may have many resonance forms, therefore the possibility of more than one fragmentation pathway exists. Another protonated form of the [M þ H]þ ion, i.e. O-3, is expected to arise in the system. It wileyonlinelibrary.com/journal/rcm may take a similar route to generate the fragment D. Loss of a water molecule from C-27 along with the shifting of C-24 pi-bond to C-25 converts O-3 into fragment ion G with m/z 453. A 1,5-H shift results in fragment H, which eventually loses a lactone moiety to afford fragment D. In the case of compound 4, fragment D may be stabilized by the shifting of the double bond from D14-15 to D16-17 via an unclear mechanism. Further removal of one or two water molecules is Copyright ß 2010 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 104–114 ESI-QqTOF-MS/MS analysis of withanolides Scheme 1. General strategy for the rapid identification of withanolides by ESI-QqTOF-MS/MS. Rapid Commun. Mass Spectrom. 2011, 25, 104–114 six-membered lactone ring contains substitution on an oxygen-bearing carbon position, which possibly causes some deviation from the reported fragmentation pattern of five- or six-membered cyclic lactones. LC/MS/MS analysis of Withania somnifera The full ESI-QqTOF-MS scan in positive mode showed the presence of withanolides abundantly as protonated [M þ H]þ and ammoniated [M þ NH4]þ molecular ions (see Supplementary Fig. S2, Supporting Information). Analysis showed the presence of six different withanolides at m/z 437.2705, 453.2693, 455.2852, 469.2592, 471.2676 and 487.2722, corresponding to the molecular formulae C28H37O4, C28H37O5, C28H39O5, C28H37O6, C28H39O6 and C28H38O7, respectively (Table 2). All compounds were searched in the updated Dictionary of Natural Products (DNP, version 19.1) on the basis of their deprotonated molecular masses, and respective formulae for the identification of compounds. In the case of more than one match, the search was narrowed down to the Copyright ß 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm 111 also observed from fragment D, giving rise to the [M þ H–Lac–H2O]þ product ion at m/z 281.1547 (C19H21Oþ 2, calc. 281.1536) and the [M þ H–Lac–2H2O]þ product ion at m/z 263.1431 (C19H19Oþ, calc. 263.1432) in the spectrum (Scheme 2). At higher energy (>20 eV or higher), withaferin A follows a second pathway causing charge retention on the lactone part, instead of the steroidal portion, as is characteristically observed in those withanolides that possess a C-27 hydroxyl group. This pathway is initiated by the removal of C-27 OH as water thus converting O-2 into fragment G. This fragment G may undergo rearrangement initiated by the positive centre at C-27 which acts as an electron sink, therefore forming fragment I of m/z 95.0465 (C6H7Oþ, calc. 95.0491) along with the removal of the steroidal part as a neutral moiety. Elimination of CO thus produces fragment M with m/z 67.0518 (C5Hþ 7 , calc. 67.0542). The above-mentioned pathway is a characteristics pathway in withanolides containing OH at C-27. This pathway is usually not observed in reported CID-MS/MS analysis of lactones as in this case the S. G. Musharraf et al. Scheme 2. Proposed CID-MS/MS fragmentation pathway of the precursor ion at m/z 471.2714 that yields the product ion at m/z 299. plant species (Withania somnifera) and to the class of compound (withanolides). Finally, the compounds were identified by utilizing the proposed flow chart (Scheme 1), and the presence of diagnostic fragments from their MS/MS data. Most of the protonated withanolides were isobaric and difficult to identify only by accurate mass data and MS/MS analysis. Complex mixtures of isobaric withanolides were simplified using the LC system, and eluted with different retention times. A total of nine withanolides were separated and detected. The diagnostic fragments which can be used to identify them are summarized in Table 2. Six compounds were identified, while three compounds remained unidentified but classified as 27-dehydroxywithanolides or 27-hydroxywithanolides. Withaferin A was eluted at 14.47 min with the highest intensity (9707 counts per second (cps)). The compound which eluted at 14.47 min showed [M þ H]þ at m/z 487, corresponding to the molecular formula C28H39O7 and afforded three hits in the DNP, while the MS/ MS spectra showed the base peak at m/z 281 corresponding to [M þ H–Lac–H2O]þ (Fig. 4). The absence of diagnostic fragments of 27-OH at m/z 95 and 67 indicated the presence of 4-hydroxy-5,6-epoxide and 27-deoxywithanolide, respectively, as per Scheme 1. Therefore, the compound was identified as 17-hydroxywithanolide D (Table 2). All the other compounds in Table 2 were identified by following the same strategy. The study clearly demonstrated the successful application of ESI-QqTOF-MS/MS for the rapid identification of withanolides in complex mixtures. 112 Scheme 3. Proposed CID-MS/MS fragmentation pathway of the precursor ion at m/z 471.2714 that yields the product ions at m/z 67 and 95. wileyonlinelibrary.com/journal/rcm Copyright ß 2010 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 104–114 17-hydroxywithanolide D 27-dehydroxylated withanolide 27-hydroxylated withanolide withaferin A 27-dehydroxylated withanolide 5,6-epoxy-4-hydroxy-1-oxowitha-2,14,24-trienolide 6,7-epoxy-17-hydroxy-1-oxowitha-4,24-dienolide Somniferanolide withanolide G2 14.47(2928) 16.78(1702) 15.45(1662) 19.02(9707) 16.8(5047) 20.7(7853) 26.8(2060) 14.47(5077) 267(100), 249(7), 171(12), 167(8), 135(12) 283(100), 265(10), 171(17) 299(36), 281(100), 175(21) 267(100), 171(7), 155(6) 299(43), 281(100), 263(14), 197(15), 175(28) 131(17) 435(6), 399(5), 299(45), 281(100), 263(7), 175(29), 131(5), 95(32), 67(17) 283(100), 265(9), 171(8) 471(25), 299(24), 281(100), 263(16), 175(25), 95(59), 67(16) 299(47), 281(100), 263(10), 253(10), 131(8) 26.8(6437) In conclusion, we report here a study on the fragmentation pattern of withanolides using ESI-QqTOF-MS/MS. Structure-fragmentation relationships to study withanolides was carefully developed. The fragmentation pattern in this class of molecules is strongly influenced by the presence of the lactone moiety, and position and number of hydroxyl and epoxide groups. Most of the diagnostic fragments are produced by single or multiple removals of water molecules from [M þ H]þ and [M þ H–Lac]þ, while the presence of hydroxyl at C-27 leads to the formation of another characteristic fragmentation pathway. Identified key fragments are carefully applied to the characterization of withnolides in the complex extract of Withania somnifera. We believe that these results will be useful in assisting in the identification of known and novel withanolides in plant extracts, biological and pharmaceutical formulations and other complex mixtures which may be available in very low quantities for analysis. SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article. Acknowledgements The author is grateful to Ms. Sadia Siddiq, in charge of the Compound Bank (Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi) for providing the standard withanolides. - 488 488 488 470 - REFERENCES 454 MS/MS fragments (ion abundance) Ret. time (Intensity cps) [M þ NH4]þ CONCLUSIONS [1] 6.5046 487.2722 487.2696 C28H39O7 10.7880 18.6388 471.2792 471.2829 471.2747 471.2747 C28H39O6 C28H39O6 13.8261 471.2676 471.2747 C28H39O6 2.9759 12.6831 13.1759 1.5647 453.2649 453.2693 455.2852 469.2592 453.2635 453.2635 455.2792 469.2584 C28H37O5 C28H37O5 C28H39O5 C28H37O6 4.2617 437.2705 437.2692 C28H37O4 Error (ppm) Exact mass Molecular Formula Observed mass [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] 9 7 8 6 2 3 4 5 [12] 1 No Table 2. 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