Identification of Alkaloids from Hippeastrum aulicum ( Ker Gawl . ) Herb . ( Amaryllidaceae ) Using CGC-MS and Ambient Ionization Mass Spectrometry ( PSMS and LS-MS )

Amaryllidaceae alkaloids are well-known isoquinolines which have demonstrated a wide range of biological activities such as antiviral, anticancer, acetylcholinesterase inhibition, antimalarial, among others. Mass spectrometry (MS) studies based on capillary gas chromatography (CGC), paper spray (PS), and leaf spray (LS) ionization were carried out for alkaloid investigation of the native Brazilian species Hippeastrum aulicum, along with nuclear magnetic resonance (NMR) techniques. Thirty-one alkaloids were identified including the new compound haemanthamine N-oxide. The results from PSand LS-MS techniques were consistent with those observed in CGC-MS analysis. To the best of our knowledge, it is the first study combining NMR, CGC-MS and the ambient ionization-mass spectrometry (PSand LS-MS) on Amaryllidaceae plants.


Introduction
Amaryllidaceae is a well-known family of monocotyledons which are distributed widely over the temperate and warm regions of the world. 1 Amaryllidaceae plants are able to synthesize a specific group of isoquinoline alkaloids, which have demonstrated remarkable biological activities, such as antitumoral, antiviral, antiparasitic, acetylcholinesterase inhibitory, among others. 1The outstanding feature of Amaryllidaceae plants is a consistent presence of a unique group of alkaloids, which have been isolated from all the genera of this family. 1The current investigation on Amaryllidaceae alkaloids has focused on hyphenated techniques, which exploit the advantages of both chromatographic and spectral methods. 2,3This strategy can be understood as a kind of dereplication process, 4 which is very attractive in that it avoids labor-intensive chromatographic steps and analysis of worthless components.
Capillary gas chromatography-mass spectrometry (CGC-MS) has become the most successful technique for a dereplication approach to Amaryllidaceae alkaloids since these compounds have shown accurate detection under CGC-MS conditions.The construction of an in-home alkaloidal database based on electron impactmass spectrometry fragmentation (EI-MS) and retention index allows a quick identification of known compounds. 5lthough most Amaryllidaceae alkaloids are suitable for CGC-MS analysis, even as underivatized compounds, there are exceptions like alkaloids in the form of salts or N-oxide, 6 which therefore require the support of other methodologies for correct identification and/or quantification.
An easier way of generating ions in mass spectrometry was introduced with the new ionization techniques, such as ambient mass spectrometry (or ambient MS). 7,8These techniques constitute simplified and efficient alternatives for the detection and quantification of analytes directly from their natural environments (the "real world") or when placed on auxiliary surfaces. 9n important ambient ionization technique is paper spray (PS) ionization.PS analysis is performed by placing the sample in the middle of a triangular piece of paper, held by a metallic clip attached to a high voltage source that is positioned in front of the inlet orifice of the mass spectrometer (MS).The paper is moistened with a solvent and a high voltage is applied to the paper through the metal clip.Consequently, in the paper tip is formed a spray with charged droplets that go towards the entrance of the MS and are analyzed. 9,10ecently, a variant technique of PS was introduced, the leaf spray (LS).The difference between paper spray and leaf spray is that the latter uses the sample itself (a plant tissue) for generating ions in gaseous phase.Ions can be generated in plant tissue without adding a solvent, 11 due to the natural juice present in fruit and vegetables.However, mass spectra with more intense signals and an improved signal/noise ratio can be obtained when a solvent is added.
The PS-MS and LS-MS have been applied in chemical identification of natural products present in coffee, 12 fruits, 13 extra-virgin olive oils 14 and herbal teas. 15Furthermore, LS-MS has also demonstrated excellent sensitivity for the direct identification of chemical species on the surface of leaves of plants such as Populus deltoids, Populus grandidentata, 11 Hibiscus moscheutos, Hibiscus syriacus 16 and Illicium anisatum. 17n the present work, indigenous Brazilian Hippeastrum aulicum was submitted to a classical phytochemical procedure assisted by CGC-MS, PS-MS and LS-MS.Thirty-one compounds were identified, including the new compound haemanthamine N-oxide, which was completely characterized by mono (1D) and bidimensional (2D) nuclear magnetic resonance (NMR) experiments.The results obtained via CGC-MS were compared with ambient mass spectrometry techniques (PS-MS and LS-MS).

General experimental procedures
Column chromatography (CC) and vacuum liquid chromatography (VLC) were carried out using silica gel 60 (70-230 mesh, Merck) and silica gel 60 ACC (6-35 µm, Chromagel-SDS), respectively.For thin layer chromatography (TLC), commercial plates with silica gel F 254 as the stationary phase and dimensions of 20 cm × 20 cm × 0.20 mm and 20 cm × 20 cm × 0.25 mm were used for analytical and semi-preparative TLC (SPTLC), respectively.High performance liquid chromatography (HPLC) was performed on an Agilent G1311C-1260 quaternary pump coupled to a UV-Vis diode array (DAD), model G1315D-1260, using the semi-preparative column Zorbax RX-Sil (9.4 × 250 mm, 5 µm) and HPLC grade solvents.NMR spectra (nuclear magnetic resonance) were recovered on a Varian 400 MHz instrument using deuterated chloroform (CDCl 3 ) or deuterated methanol (CD 3 OD) as solvents and tetramethylsilane (TMS) as the internal standard.The CGC-MS spectra (capillary gas chromatography-mass spectrometry) were obtained on a GC-17A Shimadzu CG-MS QP 5000 operating in the EI mode at 70 eV using a DB5 MS column (30 m × 0.25 mm × 0.25 µm).The temperature program was as follows: 100-180 °C at 15 °C min -1 , 1 min hold at 180 °C and 180-300 °C at 5 °C min -1 and 10 min hold at 300 °C.The injector temperature was 280 °C.The flow rate of carrier gas (helium) was 0.8 mL min -1 , and the split ratio was 1:20.HRESIMS (high-resolution electrospray ionization mass spectrometry) was performed on 9.4 T FT-ICRMS (Solarix) by direct injection of the compound dissolved in methanol (MeOH).A Jasco-J-810 Spectrophotometer (Easton, MD, USA) was used to run CD (circular dichroism) spectra, all recorded in MeOH.Infrared (IR) spectrum was recorded on a PerkinElmer Spectrum 400 FT-IR/FT-NIR Spectrometer.UV (ultraviolet) spectrum was obtained on a UV-PerkinElmer, Lambda 45, UV-Vis.

Plant material
Approximately 1.7 kg of fresh bulbs and 1.0 kg of fresh leaves of Hippeastrum aulicum (Ker Gawl.)Herb.were collected in Biritiba-Mirim City of São Paulo State, in September 2013.A voucher specimen was deposited in the Herbarium UEC (Campinas-SP, Brazil), under the reference number 114.The species was identified by Dr Renata S. de Oliveira.A new specimen was collected at the same location in February 2016 and again identified as H. aulicum (Ker Gawl.)Herb.by Dr Renata S. de Oliveira.This specimen was used for ambient MS analysis.

Extract procedure
Fresh bulbs and leaves from H. aulicum were crushed and extracted with MeOH and the mixture was immediately filtered and the solvent evaporated under reduced pressure.The plant material was then twice extracted with MeOH (48 hours each), filtered and the solvent evaporated under reduced pressure.The remaining crude extract was finally combined.
The extract IIIA showed a negligible alkaloid content by CGC-MS and analytical TLC analysis.

Bulbs
The bulb extract was submitted to the same acid-base extraction as outlined previously.The n-Hex extract (IB) afforded 506.7 mg, while the EtOAc extract (IIB) yielded 2.65 g.Finally, 2.06 g was provided by the EtOAc:MeOH (3:1) extract (IIIB).
Like extract IIIA, extract IIIB showed a negligible alkaloid content by CGC-MS and analytical TLC analysis.

Identification of alkaloids by CGC-MS
The alkaloids were identified by comparing their CGC-MS spectra and Kovats retention indices (RI) with our library database.This library has been regularly updated with alkaloids isolated and unequivocally identified via physical and spectroscopic methods. 189][20][21][22][23][24][25][26] Mass spectra were deconvoluted using AMDIS 2.64 software (NIST) (WA, USA) and RIs recorded using a standard n-hydrocarbon calibration mixture (C9-C36).The proportion of individual components in the alkaloid fractions are expressed as a percentage of total alkaloid content.CGC-MS peak areas are dependent on the concentration of the injected alkaloid as well as the intensity of its mass spectral fragmentation.Although the data given in Table 2 are not representative of a validated alkaloid quantification method, they can be used for relative comparison purposes.

PS-MS and LS-MS
For LS-MS analysis, freshly collected leaves and bulbs of H. aulicum were cut into a triangle (base and height of 1 cm each) and held by a metal clip at a distance of 5-7 mm from the mass spectrometer inlet (Figure 1a). 9Approximately 10 µL of MeOH (HPLC grade, JTBaker) and a high voltage (3 kV) supplied by the mass spectrometer were applied to the leaf or bulb to generate the LS mass spectra.
For PS-MS analysis, extracts of bulbs and leaves were dissolved in MeOH at 2 mg mL -1 .Then, 10 µL of solution was applied on the surface of a triangular paper (Whatman Grade 1, GE Healthcare) 27,28 with base and height of 1 cm each.The triangular paper was fixed with a metal clip, connected to 0.5 mm wire linked to the mass spectrometer (Figure 1b).Then, 20 µL of MeOH was applied with a high voltage (3 kV) to the triangular paper to generate the PS mass spectra.
LS and PS-MS experiments were performed in positive ion mode (LS(+) and PS(+)) using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS, model 9.4 T Solarix, Bruker Daltonics Bremen). 27,29Ion time accumulation was 0.010 s.LS(+) and PS(+)-FT-ICR mass spectra were acquired by accumulating 32 scans of timedomain transient signals in 16 mega-point time-domain data sets.All mass spectra were externally calibrated using NaTFA (m/z from 200 to 1200).A resolving power, m /Dm 50% = 78000 (in which Dm 50% is the full peak width at the half-maximum peak height of m/z 300) and a mass accuracy of < 2 ppm provided the unambiguous molecular formula assignments for singly charged molecular ions.The proposed structures for each formula were assigned using the chemspider (www.chemspider.com)database.The degree of unsaturation for each molecule can be deduced directly from its double bond equivalent (DBE) value according to equation DBE = c -h / 2 + n / 2 + 1, where c, h, and n are the numbers of carbon, hydrogen, and nitrogen atoms, respectively, in the molecular formula. 29

Alkaloid comparison
The phytochemical procedure assisted by MS and NMR approaches identified thirty-one compounds in H. aulicum (Figure 2 and Table 2).Thirteen alkaloids are reported here for the first time in H. aulicum, although some chemical similarities with previous studies have also been found. 18,30he H. aulicum studied here, from Biritiba-Mirim City (São Paulo, Brazil), displayed aulicine (15), haemanthamine (16), and lycorine (25) as the main compounds, as does H. aulicum from Cunha City (São Paulo, Brazil). 18Both Brazilian cities are relatively close, which may explain the presence of the same major components.Lycorine (25) was found to be one of the main alkaloids (see Experimental section) even though its low solubility in MeOH 31 covered the correct relative quantification by CGC-MS (Table 2).A former H. aulicum investigation 30 also revealed alkaloids such as norpluviine (14) and galanthine (21), which were observed in the present work.No information about the collection of the plant species in this previous study is available.In contrast, while ambelline, anhydrolycorine, chlidanthine, montanine, narcissidine and pseudolycorine have been previously reported, 18,30 they were not found in this work.Notably, the identification of the alkaloids incartine (26) and buphanisine (6), which are uncommon in the Hippeastrum genus, is reported here for the first time in H. aulicum. 32

CGC-MS dereplication
The CGC-MS results are shown in Table 2.The specific EI-MS fragmentation mechanisms for the distinct skeleton types together with retention indices are the key for alkaloid identification in Amaryllidaceae research.Concerning the skeleton types found in H. aulicum, the EI mass fragmentation of homolycorine-type alkaloid D 3,4 -derivatives features a dominant retro-Diels Alder process and cleavage of ring C, yielding a very abundant ion peak characterized by the pyrrolidine ring (m/z 109). 33Consequently, the alkaloids 9, 10, 12, 27, 29 and 30 show the base peak at m/z 109 (100%), with all remaining ion peaks displaying less than 10% of abundance.Otherwise, alkaloid 31 possesses a methoxyl group at C-2 and so displays the base peak at m/z 139, which is in agreement with the pyrrolidine residue along with the methoxyl substituent at C-2. 33 Lycorinetype compounds, which are biogenetically related to the homolycorine skeleton, also suffer a retro-Diels Alder process followed by the loss of C-1 and C-2, along with their substituents, yielding the base peak at m/z 228, 242 and 226 for compounds 14, 21 and 25, respectively.Alkaloid 26, which lacks the 3,4-unsaturation, possesses the base peak at m/z 332 (M-1) instead of the typical base peak after the retro-Diels Alder process. 34he EI-MS fragmentation of the tazettine-type skeleton, represented by compounds 18, 19 and 28, is strongly supported by the stereochemistry of the substituent at C-3.The b-orientation of the C-3 substituent induces a retro-Diels Alder process at ring C followed by the loss of the neutral fragment [C 5 H 8 O], which yields the base peaks at m/z 247 and 245 for compounds 18 and 28, respectively (Table 2).Compound 18 is an artefact of 19 under CGC-MS. 31onsidering the high temperature of the CGC-MS method, the haemanthamine-type skeleton also suffers a thermal decomposition, particularly those compounds with a substitution at C-11, as in the case of 13, 16, 17, 20, 22, 23 and 24. 6,35Conversely, the EI-MS fragmentation of galanthamine-type compounds under CGC and direct insertion probe (DIP) conditions are very similar and feature abundant [M] + and [M -H] + ion peaks, similarly to lycorine-type alkaloids. 36The galanthamine-type compounds also show typical fragmentation, with domination of one or another pathway depending on the substituents of the skeleton.In summary, the 4,4a-unsaturation induces the loss of the substituent at C-3 followed by the elimination of the nitrogen [C 3 H 7 N], as in galanthamine (4) and narwedine (8).In the EI-MS spectrum of lycoramine (5), a dihydro derivative, this induction is weaker and the remaining ion peaks are considerably less abundant than those observed for galanthamine (4) and narwedine (8) (Table 2).
Phytochemical procedure and NMR data of haemanthamine N-oxide (1)   In the course of the phytochemical procedure, the known Amaryllidaceae alkaloids haemanthamine (16), 19,23  Figure 3 displays the LS(+) mass spectra for bulb and leaf analyses of H. aulicum, which had a similar chemical profile.Signals varying from m/z 280 to 400 were detected as protonated molecules, [M + H] + .A similar m/z distribution has been observed for Hibiscus species. 16able 3 shows measured m/z values, mass error (ppm), DBE and molecular formula of the main compounds detected by the LS(+)-MS technique, where 10 and 8 species were identified in bulb and leaf, respectively.The ions of m/z 302, 318, 320, 332 and 348 are the most abundant species found, having double bond equivalents (DBEs) of 7-9.These species correspond to alkaloids, presenting odd molecular weight values from molecular formula of neutral species (M).As a consequence, an odd number of nitrogen is detected in their chemical structure, N 1 O x class, where x = 4-6 (Table 3).16) and/or its isomers and aulicine (15), respectively (Figure 1).The high relative intensity detected for these species is in good agreement with the CGC-MS data (  (24) or hamayne (20).The galanthamine (4) isobar, M = C 17 H 21 NO 3 and M w 287 Da, was not identified, as indicated in Table 2. Another alkaloid was detected from LS(+)-MS as a [C 18 H 25 NO 5 + H] + ion of m/z 336 (Table 3), although no chemical structure has been proposed so far.The only species not classified as an alkaloid was detected by LS(+)-MS as a sugar: an adduct sucrose, [C 12 H 22 O 11 + K] + ion of m/z 381.0801.
Figure 4 shows PS(+)-FT-ICR mass spectra from n-Hex and EtOAc extracts of bulbs and leaves for H. aulicum.The chemical profiles for extracts of bulbs and leaves are quite similar.However, the PS(+) technique promotes a selective ionization for compounds of m/z ≥ 332 for bulb extracts (n-Hex and EtOAc), while in leaf extracts, two alkaloids are selectively identified: ions of m/z 362 ([C 20 H 27 NO 5 + H] + ), and 376 ([C 20 H 25 NO 6 + H] + ).Among them, the ion of m/z 362 (7-methoxy-O-methyllycorenine (12)) is also listed in  5a) and PS(+)-MS (Figure 5b).In general, a good agreement is observed between the ambient MS techniques.LS(+)-MS has proved to be a promising approach for the identification of alkaloids from Hippeastrum aulicum (Ker Gawl.)Herb.and should be explored in future work with other species, since it is a fast and easy analysis requiring no prior step of sample preparation.

Conclusions
The phytochemical investigation of Hippeastrum aulicum resulted in the identification of thirty-one  alkaloids and the CGC-MS dereplication proved to be very useful for the fast identification of a great number of compounds from an alkaloid-rich H. aulicum extract.The phytochemical fractionation assisted by CGC-MS analysis allowed the isolation of the new compound haemanthamine N-oxide (1), reported for the first time from a natural source.
Paper and leaf spray ionization mass spectrometry constitute a new family of ionization techniques able to identify alkaloids directly from their natural environments, i.e., in the "real world" of analytes (as demonstrated by LS-MS) or when placed on auxiliary surfaces such as PS-MS.A total of 13 species were identified with m/z ranging from 200 to 400, DBEs of 7-10 and exact mass lower than 2 ppm.Regarding the chemical structure of alkaloids, their carbon number varied from C 16 to C 20 , containing NO x as the heteroatom class, where x = 4-6.Among the main species detected were compounds with m/z 302, 318, 320, 332 and 348, which correspond to haemanthamine (16) and/or its isomers (17 and 30), haemanthamine N-oxide (1) and its isomers (22 and 23), aulicine (15), tazettine/pretazettine (18  and 19), and nerinine (10), respectively.Similar LS(+)-MS and PS(+)-MS results were obtained from leaf and bulb surfaces.Taken together, the results obtained from ambient ionization MS demonstrated a notable agreement with the CGC-MS analysis.

Figure 4 .
Figure 4. PS(+)-FT-ICR mass spectra from n-Hex and EtOAc extracts of bulbs and leaves for H. aulicum.

Figure 5 .
Figure 5.Comparison of relative intensity of main ions detected from LS(+) and PS(+)-FT-ICR MS of bulbs and leaves for H. aulicum.

Table 2 .
CGC-MS data for H. aulicum.Values are expressed as a relative percentage of total ion current (TIC) a Alkaloid percentage in the total mixture of alkaloids from n-Hex and EtOAc fractions of bulbs (IB and IIB, respectively); b alkaloid percentage in the total mixture of alkaloids from n-Hex and EtOAc fractions of leaves (IA and IIA, respectively); c tazettine detection by CGC-MS mean identification of both alkaloids tazettine(18)and pretazettine (19); 31 d traces < 0.05 of TIC.The alkaloid Haemanthamine N-oxide (1) is not suitable to CGC-MS detection; d alkaloids identified using NIST 05 database; recursive procedure, HR-MS and literature data.RI: Kovats retention indices.