Electrospray tandem mass spectrometric analysis of a dimeric conjugate, salvialeriafone and related compounds

Background Electrospray tandem mass spectrometry approach is widely used for the rapid characterization of natural products. This paper describes the gas-phased ESI-MS/MS fragmentation of abietane-type diterpenoids and their novel dimeric conjugate, salvialeriafone (1) using both positive and negative ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. Diterpenoids are widely distributed throughout the plant kingdom and posses interesting biological activities. Results ESI-QqTOF-MS (positive ion mode) of diterpenoids 1–6 under collision-induced dissociation tandem mass spectrometric analysis (CID-MS/MS) showed the characteristic losses of water, carbonmonoxide and propene molecules, while analysis in negative ion mode showed the characteristic losses of water, carbon monoxide, methane molecules and methyl radical. Results demonstrated the differences in the product ions and base peaks due to the differences in the skeleton. A novel dimeric conjugate, salvialeriafone (1) showed characteristic fragmentation pattern and was found to be more prone to form radical ions, as compared to monomeric diterpenoids. The fragmentation pathways of characteristic fragments were proposed with the aid of HRESIMS. Conclusions Extensive tandem mass spectrometric studies of salvialeriafone (1) and related diterpenoids 2–6 were conducted and their characteristic fragments were identified. The knowledge of the fragmentation pattern of these diterpenoids will be useful for the characterization of new dimers of this class of compounds.

The genus Salvia constitutes the largest of the plant family Labiatea with 900 species wide-spread throughout the world. The genus has yielded various classes of natural products, including the major class of terpenoids, particularly the diterpenoids. Diterpenoids of genus Salvia are abietane and neo-clerodane types [10]. More than 400 diterpenoids with different abietane skeletons have been isolated from Salvia plants [11]. Diterpenoids from Salvia species showed antinflammatory, antidiabetic, ipolipidemic and antiaggregating effects [9]. We have recently isolated salvialeriafone, a dimeric conjugate from Salvia leriaefolia which exhibit in vitro antiproliferative activity against the human cervical cancer cell line (Hela) [12]. S. leriaefolia is used for the treatment of stomach and chronic disorders in Iran.
Tandem mass spectrometric studies of natural products revealed the identification of key fragments which can be helpful for their rapid characterization in the plant extract utilizing LC-MS/MS approach, particularly for the thermally labile compounds. The knowledge of CID-fragmentation pattern of the precursor protonated (or deprotonated) molecule is essentially required prior to the analysis quantification of the desired compounds by LC-MS/MS.
In continuation of our research on the novel characterization and the fragmentation routes of natural product compounds [22][23][24], we report in this manuscript for the first time the ESI-MS and CID-MS/MS (+ andmodes) of salvialeriafone and its related abietane diterpenoids isolated from S. leriaefolia. Knowledge about the characteristic fragments and neutral losses of diterpenoids can be immensely helpful for the rapid identification of these compounds in future phytochemical studies.

Standard and reagents
Chemicals and solvents were of analytical and HPLC grades, respectively, and were purchased from Aldrich-Sigma (USA). Deionized water (Milli-Q) was used in the study. Standard diterpenoids were obtained from the Molecular Bank facility at the Dr. Panjwani Center for Molecular Medicine and Drug Research (International Center for Chemical and Biological Sciences), University of Karachi. The isolation procedure and spectroscopic data of the standard diterpenoids has already been reported [12].

Positive ion mode
The diterpenoids were dissolved in methanol, and working dilution was prepared in 1:1 acetonitrile-water containing 0.1% trifluoroacetic acid and analyzed by electrospray ionization (ESI) and collision-induced dissociation (CID),  positive ion mode, on QqTOF-MS/MS instrument (QSTAR XL mass spectrometer Applied Biosystem/ MDS Sciex, Darmstadt, Germany) at room temperature. Highpurity nitrogen gas was used as the curtain gas and collision gas delivered from Peak Scientific nitrogen generator.
The ESI interface conditions were as follows: ion spray capillary voltage of 5500 V, curtain gas flow rate 20 L min -1 , nebulizer gas flow rate 30 L min -1 , DP1 60 V, DP2 15 V, and focusing potential of 265 V. The collision energy was swept from 05 to 45 eV for MS/MS analysis. Calibration was performed by using internal calibration process. Sample was introduced into the mass spectrometer using a Harvard syringe pump (Holliston, MA) at a flow rate of 5 μL/ min. MS 2 Experiment was conducted by selecting the product ion.

Negative ion mode
The diterpenoids were dissolved in methanol and working dilutions were prepared in 1:1 acetonitrile-water containing 4 mM ammonium acetate (0.2 ng/uL taurochloric acid was used as an internal calibrant) and analyzed by electrospray ionization (ESI) and collision-induced dissociation (CID) negative ion mode on QqTOF-MS/MS instrument (QSTAR XL mass spectrometer Applied Biosystem/ MDS Sciex, Darmstadt, Germany) at room temperature. Highpurity nitrogen gas was used as the curtain gas and collision gas delivered from Peak Scientific nitrogen generator.
The ESI interface conditions were as follows: ion spray capillary voltage of −4200 V, curtain gas flow rate 20 L min -1 , nebulizer gas flow rate 25 L min -1 , DP1 -55 V, DP2 -15 V, and focusing potential of −265 V. The collision energy was swept from 20 to 55 eV for MS/MS analysis. Calibration was performed using internal calibration process. Sample was introduced into the mass spectrometer using a Harvard syringe pump (Holliston, MA) at a flow rate of 5 μL/ min. MS 2 Experiment was conducted by selecting the product ion.

Results and discussion
Salvialeriafone (1)  energy ranging were plotted for salvialeriafone (1) (Figure 2). It was observed that the product ions formation was at best at collision energy 40 eV (Figure 2A) in positive ion mode, whereas in negative ionization mode the product ions were best formed at the optimum energy collision energy 50 eV ( Figure 2B).

Fragmentation pattern in positive ionization
All diterpenoids produced abundant [M+H] + ions which were selected as the precursor ions to produce MS/MS spectra, but the [M+H] + ion is absent in the MS spectra of compound 5 which gives the dehydrated peak [M+H-H 2 O] + , therefore this ion was selected as a precursor ion to produce MS/MS spectra of compound 5. HRESIMS data of all compounds in positive ion mode is presented in Table 1. All the compounds showed similar fragmentation pattern with minor differences and similar losses of H 2 O, CO, C 3 H 6 ( Table 1), but the extent of fragmentation and optimium collision energy vary due to different substituents and presence of double bonds in the ring. All these diterpenoid 2-6 showed intense fragmentation and neutral losses, while compound 1 which is a conjugate of two diterpenoids showed very low fragmentation even at high collision energy. It showed the loss of propene after the water removal as a base peak, i. [M+H-C 3 H 6 -CH 4 ] + . Fragments due to the loss of CO and other consequent lossess did not appear in the MS/ MS spectra of compound 1 ( Figure 3A).
The fragmentation pattern of momeric diterpenoids 2-6 were studied in order to further validate the neutral lossess and fragmentation pattern of the dimeric conjugate, salvialeriafone (1). All diterpenoids showed the removal of water molecule, i.e. [M+H-H 2 O] + except compound 4 which showed the removal of methanol due to the presence of methoxy group followed by the loss of water molecule. A loss of water molecule followed by the loss of CO was observed in compounds 2, 3, 5, and 6, while compounds having OH group in ring B (compounds 3 and 5) showed the loss of two water molecules, i.e. [M+H-2H 2 O] + . All analyzed compounds showed the removal of propene moiety directly from the [M+H] + and/or after the removal of H 2 O and CO groups. These compounds also showed the neutral lossess of ethene and butane molecules. Compounds 3 and 5 having same molecular formula but difference of skeleton in ring B produced differentiated base peak and some other abundant peaks (Table 1). Compound 3 showed the base peak at m/z 283 [M+H-2H 2 O-CO] + , while compound 5 showed the base peak at m/z 281 [M+H-2H 2 O-CH 2 O] + and compound 5 also showed the intense fragments at m/z 217 and 205, while the peak at m/z 319 [M+H-CO] + is absent in compound 5. Like compound 1, compounds 2 and 6 also showed the presence of abundant peaks of radical cations due to the loss of propyl radical. Unlike monomeric diterpenoids the product ion spectra of compound 1 was very simple, having only a few peaks in the range of ≥ m/z 486. However, being a dimer, compound 1 possess two isopropyl and four hydroxyl substituents, but showed the loss of substituents equivalent to the loss which are derived from a monomeric diterpenoid unit. Fragmentation pathway for the fragments formed due to the removal of CO, C 3 H 6 , and H 2 O has also proposed as and shown in Scheme 1 (compound 2 is taken as a representive of monomeric diterpenoids).

Fragmentation pattern in negative ionization
All diterpenoids 1-6 produced abundant [M-H]ions, which were selected as the precursor ions to produce MS/MS spectra. The QTOF-MS/MS the low-energy collision induced dissociation tandem mass spectrometry experiments (CID-MS/MS) were optimized and developed through ramping collision voltage to induce more product ions and the optimum collision energy for recording product ion spectra of diterpenoids in negative ion mode is ranging from 30 to 45 eV HRESIMS data of all compounds in negative ion mode is presented in  showed different fragmentation pattern due to different substituents and the presence of double bonds in the ring. These compounds showed the common losses of H 2 O, CO, CH 4 , and CH 3 radical, similar to positive ion mode, except the loss of CH 4 , and CH 3 radical Table 2.
Compound 1, which is a dimer of two diterpenoids, showed the loss of propene radical as a base peak i.e. The product ion spectra of compound 1 in negative ionization was very simple with only a few peaks in mass range of ≥ m/z 499. Unlike compound 1, compounds 2-6 did not show the lossess of propene and propyl radical in negative ionization. Fragmentation pathway for the fragments formed due to the removal of CO, CH 4 , and CH 3 through negative ionization has also been proposed and shown in Scheme 2 (compound 6 is taken as a representive of monomeric diterpenoids).

Conclusion
In conclusion, fragmentation pattern of six abietane-type diterpenoids and one novel dimeric conjugate diterpenoid, salvialeriafone (1) have been studied by using ESI-QqTOF-MS/MS in both positive and negative ionization mode. It has been observed that many characteristic neutral losses and formation of key fragment ions can provide important structural information about the basic skeleton of abietane-type diterpenoids having dimeric linkages. The dimeric conjugate showed somewhat different pattern and less fragmentation as compared to monomeric diterpenoid analogue. The knowledge of fragmentation pattern is immensely helpful for the rapid characterization of abietane-type diterpenoids through liquid chromatography coupled with mass spectrometry in complex mixtures such as plant extracts or herbal formulations by utilizing their analytical amount.