Investigation of Symphytum cordatum alkaloids by liquid–liquid partitioning, thin-layer chromatography and liquid chromatography–ion-trap mass spectrometry

Analytica Chimica Acta 566 (2006) 157–166

Investigation of Symphytum cordatum alkaloids by liquid–liquid partitioning, thin-layer chromatography and liquid chromatography–ion-trap mass spectrometry Tomasz Mroczek a,∗ , Karine Ndjoko-Ioset b , Kazimierz Głowniak a , Agnieszka Mi˛etkiewicz-Capała a , Kurt Hostettmann b a

Department of Pharmacognosy with Medicinal Plants Laboratory, Medical University, 1 Chod´zki St., 20-093 Lublin, Poland b Laboratoire de Pharmacognosie et Phytochimie, Ecole de Pharmacie Geneve-Lausanne, Universit´ e de Geneve, Quai Ernest-Ansermet 30, CH-1211 Geneve 4, Switzerland Received 27 January 2006; received in revised form 2 March 2006; accepted 5 March 2006 Available online 14 March 2006

Abstract From the alkalised crude extract of Symphytum cordatum (L.) W.K. roots, pyrrolizidine alkaloids (PAs) were extracted as free tertiary bases and polar N-oxides in a merely one-step liquid–liquid partitioning (LLP) in separation funnel and subsequently pre-fractionated by preparative multiple-development (MD) thin-layer chromatography (TLC) on silica gel plates. In this way three alkaloid fractions of different polarities and retention on silica gel plates were obtained as: the most polar N-oxides of the highest retention, the tertiary bases of medium retention, and diesterified N-oxides of the lowest retention. The former fraction was reduced into free bases by sodium hydrosulfite and purified by LLP on Extrelut-NT3 cartridge. It was further analysed together with the two other fractions by high-performance liquid chromatography (HPLC)–ion-trap mass spectrometry with atmospheric pressure chemical ionization (APCI) interface on XTerra C18 column using a gradient elution. Based on MSn spectra, 18 various alkaloids have been tentatively determined for the first time in this plant as the following types of structure: echimidine-N-oxide (three diasteroisomers), 7-sarracinyl-9-viridiflorylretronecine (two diasteroisomers), echimidine (two diasteroisomers), lycopsamine (two diasteroisomers), dihydroechinatine-N-oxide, dihydroheliospathuline-N-oxide, lycopsamine-N-oxide (three diasteroisomers), 7-acetyllycopsamine-N-oxide, symphytine-N-oxide (two diasteroisomers) and 2 ,3 -epoxyechiumine-N-oxide. © 2006 Elsevier B.V. All rights reserved. Keywords: Pyrrolizidine alkaloids; Symphytum cordatum (L.) W.K.; Preparative thin-layer chromatography; Liquid–liquid partitioning; High-performance liquid chromatography; Ion-trap mass spectrometry

1. Introduction Pyrrolizidine alkaloids (PAs) with unsaturated necine moiety exhibit carcinogenic and hepatotoxic properties [1,2,27]. They have been found in Boraginaceae, Compositae and Fabaceae plant families [1,2,27]. The methods of their monitoring in plant samples or in food products are still expected. For toxic PAs determination, visual (vis) spectrophotometry was elaborated based on the reaction of 3,4-dehydro-1,2-

∗

Corresponding author. Tel.: +48 81 74103 51; fax: +48 81 74103 51. E-mail address: [email protected] (T. Mroczek).

0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.03.016

unsaturated necine with Ehrlich reagent [3–5]. The same conditions can be straightforward applied to detect PAs on thin-layer chromatography (TLC) plates [6,7]. Due to their sensitivity, gas chromatography (GC) and high-performance liquid chromatography (HPLC) methods are mainly used for the analysis of PAs in natural samples. Capillary GC methods with flame-ionization (FID) [8,9] and more often quadrupole mass spectrometry (MS) detectors [10–16] in tandem MS/MS mode were described [10–16]. For the identification of PAs in comfrey roots, another GC-hyphenated method so called: gas chromatography–matrix-isolation Fourier transform infrared spectroscopy (GC–MI-FT-IR) was efficiently used [15]. However, in GC analysis the thermal decomposition of

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labile N-oxides and sometimes also of diesterified PAs is frequently observed. For these reasons and purposes, HPLC are often considered. Various stationary phases in HPLC analysis were applied and comprised: C8 [17], C18 [18–20], cyano-bonded [21] and aminobonded ones [20]. Ion-pair RP–PLC procedure was sometimes advised due to the problem of peak asymmetry [22,23]. Liquid chromatography (LC)–MS analysis of macrocyclic PAs in Senecio sp. using thermospray interface was previously reported [17,24]. Lin et al. [18] analysed various types of macrocyclic PAs with both HPLC–MS with in-source collision induced dissociation in the electrospray ionisation source (CID) or HPLC–MS–MS (CID in the collision cell) using electrospray interface on a quadrupole instrument. Atmospheric pressure chemical ionization (APCI) HPLC–MS method was also reported for the determination of macrocyclic PAs in honey [25]. RP-HPLC-DAD-EI-MS with thermabeam interface using single ion monitoring (SIM) mode was recently described for rapid screening of toxic PAs in new plant species [26]. Ndjoko et al. [24] applied LC–MR technique to distinguish E and Z isomers of senecionine in plant extracts. Combination of cation-exchange solid-phase extraction and LC ion-trap mass spectrometry has been efficiently applied in on-line structural determination of different kinds of PAs present merely in small amounts in Onosma stellulatum and Emilia coccinea plants [27]. Symphytum cordatum (L.) W.K. is a comfrey species naturally occurring in Beskidy and Karpaty mountains with hart-shaped leaves and white flowers [34]. As its alkaloids composition has not been investigated till now, these studies could give more information about chemotaxonomy of comfrey alkaloids. This paper proposed a new methodology for efficient separation and structural investigation of PAs in S. cordatum (L.) W.K. Liquid–liquid partitioning (LLP) was applied to the crude extract. Multiple TLC development allows pre-fractionation of pre-purified extracts which were further analysed by RP-HPLC ion-trap MS.

sine and retrorsine-N-oxide were from Carl Roth (Karlsruhe, Germany). Double-distilled water was used in the experiments. Roots of S. cordatum (L.) W.K. (voucher no. 0125) and also Symphytum tuberosum L. (voucher no. 0122) were harvested from the Botanical Garden of the Maria Curie-Skłodowska University of Lublin and also from the Podhale district (S. cordatum roots). The plant material was identified by botanist (Ms Maria By´c). A voucher specimen of each plant is deposited at the herbarium of the Department of Pharmacognosy with Medicinal Plants Laboratory, Medical University of Lublin. The material was dried at room temperature and powdered.

2. Experimental

About 23 mg of alkaloids complex (N-oxides and tertiary bases) dissolved in 2.5 ml volume of chloroform–methanol mixture (1:1, v/v) was being applied onto each TLC plate by Camag (Muttenz, Switzerland) TLC III v. 2.12 autosampler controlled by WinCats software and separated by multiple development preparative TLC on silica gel 60 F254 , 0.5 mm thickness plates (five plates were used for the separation of the whole complex and the suitable fractions collected together). The mobile phase consisted of: chloroform–methanol–25% ammonia (100:10:2, v/v/v) which was applied three times over a distance of 9 cm. The compounds were thus separated into three bands with the following Rf values: (a) 0.03–0.2 (12 mg); (b) 0.25–0.35 (29 mg); and (c) 0.35–0.40 (22.5 mg) detectable under UV light according to positive reaction with Dragendorff’s reagent. These bands were scrapped off the plates and extracted with methanol. The PA fractions obtained (excluding the first band of the highest retention) were dissolved in 2 ml of methanol and analysed by RP-HPLC ion-trap MS method. Densitometry of the crude unpurified extract was done according to our previous procedure [30] using the preparative 0.5 mm thickness silica gel 60 F254 plate

2.1. Chemicals and reagents Extrelut-NT3 pre-packed glass columns (3 ml) filled with wide-pore kieselguhr with a high pore volume, TLC silica gel 60 F254 , 20 cm × 10 cm (width × height according to development mode), 0.25 mm thickness analytical plates and Dragendorff’s reagent were purchased from E Merck (Darmstadt, Germany). TLC silica gel F254 , 20 cm × 10 cm (width × height), 0.5 mm thickness preparative plates were laboratory prepared using silica gel 60 GF254 sorbent (E Merck) suspended homogeneously with distilled water (60 g sorbent + 90 g water for 10 plates, 10 cm × 20 cm) in round-bottomed flask and spread with a coating machine. Methanol, acetonitrile and 25% ammonia (each solvent was of HPLC gradient grade) were obtained from J.T. Baker (Gross-Gerau, Germany). Tartaric acid, chloroform, nbutanol, 36% hydrochloric acid, sodium sulphate and sodium hydrosulfite were of analytical grade and purchased from the Polish Reagents (POCh) Gliwice, Poland. Standards of retror-

2.2. Extraction of plant material and the sample clean-up by liquid–liquid partitioning Sixty grams of S. cordatum roots were extracted in 5 g portions with 250 ml of 1% tartaric acid in methanol solution on heating mantle under reflux for 2 h. The extracts were collected together and evaporated to dryness under vacuum at temperature of 52 ◦ C. The dry residue was dissolved in 50 ml of 0.05 M hydrochloric acid and transferred into the separation funnel for liquid–liquid partitioning clean up. At first, chloroform extraction was performed (1 × 50 ml) and the organic fraction discarded. The remaining water fraction was alkalised with 25% ammonia (pH = about 10.0) followed by extraction with a mixture of chloroform-n-butanol (2:1, v/v) (5 × 10 ml). Organic fractions were collected and evaporated to dryness under vacuum at 52 ◦ C. An oily alkaloids mixture (114 mg) was obtained and was further pre-fractionated by preparative TLC. Under the same conditions, a S. tuberosum L. roots extract (150 g of material) was prepared in order to study the reproducibility of the extraction procedure using Extrelut. 2.3. Pre-fractionation of alkaloids mixture by multiple-development (MD) preparative thin-layer chromatography

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and the same mobile phase as mentioned above. Scans were recorded at 220 nm using Camag TLC scanner v. 3. 2.4. PAs N-oxides reduction by sodium hydrosulfite and alkaloids purification by liquid–liquid partitioning using Extrelut-NT3 cartridge The N-oxides fraction was dissolved in 10 ml of 0.05 M HCl, transferred into a 100 ml conical flask together with 30 mg of sodium hydrosulfite (Na2 S2 O4 ), put in a wrist-action shaker and shaken for 30 min. After reduction, the alkaloids were extracted as free bases by liquid–liquid partitioning on Extrelut-NT3 cartridge. For this purpose, the acidified fraction was concentrated into 2.5 ml volume and alkalised with 0.5 ml of 25% ammonia into pH about 10 and applied into the cartridge without conditioning. The alkaloids were then eluted with 15 ml of chloroform-n-butanol (2:1, v/v) mixture under atmospheric pressure. The effluent was dried over anhydrous sodium sulphate(VI), evaporated to dryness under vacuum. The residue (7.5 mg) was dissolved in 5 ml of methanol for hyphenated LC–MS analysis. Reproducibility of the Extrelut extraction procedure was explored with crude comfrey (S. tuberosum) root extract (0.75 g/ml). A 1 ml of retrorsine and retrorsine-N-oxide mixture in methanol (0.2 mg/ml of each compound) was added to 2 ml volumes of this extract and evaporated to dryness under vacuum. The residues were dissolved in 2.5 ml of 0.05 M HCl and prepared as described above. The recoveries of the isolated alkaloids (six repeatable injections, each one was a 10 ␮l together with 6-point concentration levels of retrorsine and retrorsine-Noxide standard mixture in the range of 4 ␮g/ml–100 ␮g/ml) were measured by HPTLC-densitometry using the analytical 0.25 mm thickness silica gel 60 F254 plate as previously reported [30]. 2.5. High-performance liquid chromatography–diode array-ion-trap mass spectrometric assay (HPLC–DAD-IT MS) of the alkaloid fractions The HPLC–DAD-IT MS experiments of the fractions (reduced A fraction, B and C, see Fig. 1) after MD TLC separation were performed using Agilent 1100 Series HPLC equipped with a diode array (DAD) detector coupled to an ion-trap MS instrument (Finnigan LCQ). The analytical column was a 5 ␮m XTerra C18 (Waters, U.S.A.), 150 mm × 4.6 mm I.D. held at 25 ◦ C. A two-pump gradient working at 1.0 ml/min was programmed: reservoir A contained 15 mM ammonia water solution, and reservoir B contained 100% acetonitrile. The injection volume was 20 ␮l. The following gradient was used: 0–20 min: linear gradient from 5 to 50% B; 20–25 min: isocratic at 50% B; 25–28 min: linear gradient from 50 to 100% B; 28–33 min: isocratic at 100% B; 33–36 min: linear gradient from 100 to 5% B. The total time of analysis was 36 min. Finnigan LCQ mass spectrometer which uses a quadrupole ion-trap mass analyser and electron multiplier (Cearmax 7545M form K with M Electronics) ions detector was equipped with an atmospheric pressure chemical ionization interface operating under the following conditions: sheath gas, N2 with flow of

Fig. 1. Preparative multiple development thin-layer chromatography (MD TLC) of purified extract of S. cordatum roots. Stationary phase: silica gel 60 F254 , mobile phase: chloroform–methanol–25% ammonia (100:10:2, v/v/v); distance of development: 3 cm × 9 cm. Densitogram was recorded at 220 nm. Abbreviations used: (A) the band of the mostly retained PA-N-oxides; (B) the major band of tertiary bases; (C) the common band of echimidine-N-oxide type of alkaloids.

70 l/min; capillary temperature, 150 ◦ C; APCI vaporizer temperature, 400 ◦ C; source voltage, 6 kV; source current, 5 ␮A; capillary voltage, 46 V; tube lens offset, 55 V; multipole 1 offset, −1.50 V; multipole two offset, −6.50 V; inter-multipole lens voltage, −18 V; trap dc offset voltage, −10 V, source-induced dissociation (SID) 10 V. Full-scan mass spectra (150–500 amu) were recorded every 2 s in the positive ion mode. The protonated molecules [M + H]+ of PAs and N-oxides were chosen as precursor ions for isolation and fragmentation. Isolation width was 1 amu. MSn experiments were recorded under the following conditions: He CID pressure, 0.5 mTorr; relative collision energy, 38%; fragmentation time, 200 ms. The spectra were acquired every second. 3. Results and discussion 3.1. Extraction of plant material and the sample clean-up by liquid–liquid partitioning The extraction of PAs from comfrey samples has been previously optimised [28]. A 1% tartaric acid in methanol as the most suitable extraction solvent was chosen due to the highest recoveries of both N-oxide and tertiary bases under slightly acidic conditions. Purification and concentration of PAs-extracts by cation-exchange solid-phase extraction for analytical scale was also previously reported [23,26,27]. This procedure enabled the purification of 200–500 mg of crude alkaloidal extract containing both polar N-oxides and non-polar tertiary bases in only one simple step omitting Zn reduction. LLP was selected for the isolation of PAs from the whole crude extract (50 g/200 ml) (on the preparative scale). The amount of the whole PAs-fraction was about 0.19%, among others about 54% comprised PAs-Noxides estimated by UV–vis spectrophotometry according to procedure described elsewhere [28]. Thus, LLP procedure with chloroform-n-butanol (2:1, v/v) as extraction mixture permitted efficient and rapid extraction of both N-oxides and tertiary bases, omitting time-consuming with Zn reduction step. The

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purified extract could be further pre-fractionated by preparative MD TLC. 3.2. Pre-fractionation of alkaloids mixture by preparative multiple development thin-layer chromatography Pre-fractionation by MD TLC was applied for investigation of different types of PAs retention on silica gel plates. In Fig. 1 the separation of the extract into three bands on one 0.5 mm thickness silica gel 60 F254 plate is presented. Already simple three-fold development in chloroform–methanol–25% ammonia (100:10:2, v/v/v) was sufficient to isolate common broad band of polar N-oxides mixture (band A, Rf 0.03–0.2) and mainly tertiary bases of medium retention on silica gel (band B, Rf 0.25–0.35). The diesterified N-oxides of the lowest retention were eluted between Rf values 0.35 and 0.40 as sharp band (C). Multiple developments were necessary in order to achieve almost baseline separation of bands B and C which were co-eluted after single development. Although, it was unable

to separate individual compounds among the fractions by MD TLC, and it was unnecessary in this study, the data collected (Rf values, retention times from HPLC profiles, MS1 and MS2 spectra) may surely have an importance in chemotaxonomic studies of complex PAs mixtures in plant samples. 3.3. PAs N-oxides reduction by sodium hydrosulfite and alkaloids purification by LLP using Extrelut-NT3 cartridge Complex of the most polar N-oxides (band A) was reduced with sodium hydrosulfite to obtain free bases that could be easily and rapidly separated by RP-HPLC–DAD-MS. Zn-dust reduction was not performed due to its time-consumption, lack of reproducibility and probably partial degradation, especially of N-oxides as it was noted before [28]. The reduced mixture was purified and concentrated by LLP procedure on Extrelut-NT3 cartridges again with chloroform-n-butanol (2:1, v/v) as elution mixture. Under these conditions about 10 mg of reduced PAs could be isolated in a simple manner. The reproducibility of the

Table 1 Chromatographic and MS date of the alkaloid fractions determined in S. cordatum (L.) W.K. by RP-HPLC IT MS method Compounda

Retention time (min)

MS1 (relative intensity)

Common name

References

A1 A2

8.85 10.81

[M + H]+ 300.30 (100%) [M + H]+ 302.28 (100%)

Intermedine/lycopsamine (or stereoisomer) Dihydroechinatine/dihydrolycopsamine (or stereoisomer) Lycopsamine-N-oxide/intermedine-N-oxide

[27,29–31] [27,29–31] [13,27,30,31]

Lycopsamine-N-oxide/intermedine-N-oxide

[13,27,30,31]

7-Acetylintermedine/7-acetyllycopsamine (or stereoisomer) Symlandine/symphytine/myoscorpine/echiumine (or stereoisomer) Symlandine/symphytine/myoscorpine/echiumine (or stereoisomer) 2 ,3 -Epoxyechiumine

[27,30,31]

Intermedine/lycopsamine (or stereoisomer) Intermedine/lycopsamine (or stereoisomer) Dihydroheliospathuline-N-oxide

[27,29–31] [27,29–31] [27]

7-Sarracinyl-9-viridiflorylretronecine 7-Sarracinyl-9-viridiflorylretronecine Heliosupine/echimidine/hydroxymioscorpine (or stereoisomer) Heliosupine/echimidine/hydroxymioscorpine (or stereoisomer) Echimidine-N-oxide/heliosupine-Noxide/echihumiline-N-oxide (or stereoisomer) Echimidine-N-oxide/heliosupine-Noxide/echihumiline-N-oxide (or stereoisomer) Echimidine-N-oxide/heliosupine-Noxide/echihumiline-N-oxide (or stereoisomer)

[13] [13] [26,27,30,31]

A5

12.72

[M + H]+ 316.28 (100%); [M + H − O]+ 300.20 (2.9%) [M + H]+ 316.28 (100%); [M + H − O]+ 300.20 (2.5%) [M + H]+ 342.25 (100%)

A6

17.79

[M + H]+ 382.30 (100%)

A7

18.26

[M + H]+ 382.39 (100%)

A8

12.41

[M + H]+ 398.17 (100%)

A3 (traces)

5.24

A4 (traces)

5.87

B4 B5 B6

12.53 12.87 15.05

[M + H]+ 300.30 (100%) [M + H]+ 300.27 (100%) [M + H]+ 318.33 (100%) [M + H − O]+ 302.20 (1.4%) [M + H]+ 398.19 (100%) [M + H]+ 398.38 (100%) [M + H]+ 398.12 (100%)

B7

15.34

[M + H]+ 398.22 (100%)

C1

9.84

[M + H]+ 414.24 (100%); [M + H − O]+ 398.15 (1.4%)

C2

10.09

[M + H]+ 414.27 (100%); [M + H − O]+ 398.20 (1.2%)

C3

10.47

[M + H]+ 414.24 (100%); [M + H − O]+ 398.18 (4.5%)

B1 B2 B3 (traces)

7.97 8.52 9.67

[29–31] [29–31] [32]

[26,27,30,31] [27,33]

[27,33]

[27,33]

Stationary phase: Waters XTerra C18 , 250 mm × 4.6 mm I.D.; dp = 5 ␮m. Mobile phase: gradient of acetonitrile in 15 mM ammonia solution (see the text). Flow rate: 1.0 ml/min; column temperature: 25 ◦ C; injection volume: 20 ␮l. APCI-(+) interface was applied. The protonated molecule [M + H]+ of PAs and N-oxides were chosen as the parent ions for isolation and fragmentation. a Compounds A1–A8 (in the reduced fraction in plant occurred as N-oxides); B1–B7 and C1–C3 compounds were in the fractions separated by MD TLC with the following Rf values: 0.25–0.35 and 0.35–0.40, respectively.

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procedure was estimated by quantitative HPTLC-densitometry. In Fig. 2 the separation of the fortified (with retrorsine and its N-oxide) and the Extrelut-NT3 cartridges purified comfrey extract is presented. The recoveries obtained in this procedure were 72.83% (S.D. = 2.44, n = 6) for retrorsine and 117.78% (S.D. = 13.47, n = 6) for its N-oxide. It can be concluded that this method can be efficiently used for both N-oxides and tertiary bases purification step. 3.4. DAD, total ion current and single ion monitoring The PAs protonated molecules (with characteristic maximum at 220 nm) were detected at m/z 300, 302, 316, 318, 342, 382, 398 and 414. In Fig. 3 (schemes a–c) LC–DAD-ion-trap MS separation of the compounds in A (reduced), B and C bands after MD TLC procedure is presented. Only three compounds (C1–C3, see Table 1) occurring in band C with the same UV and MS–MS spectra were closely separated, suggesting diastereoisomeric

Fig. 2. Preparative multiple development thin-layer chromatography (MD TLC) of purified by LLP on Extrelut-NT3 cartridge extract of S. tuberosum roots fortified with retrorsine (Rr) and retrorsine-N-oxide (Rr-NO) standards mixture (see Section 2.4). Stationary phase: silica gel 60 F254 , mobile phase: chloroform–methanol–25% ammonia (100:10:2, v/v/v); distance of development: 2 cm × 9 cm. Densitogram was recorded at 220 nm.

Fig. 3. RP-HPLC-DAD-ion-trap MS analysis of the compounds in A, B and C bands separated by MD TLC method (see Section 2.3). Stationary phase: Waters XTerra C18 , 250 mm × 4.6 mm I.D.; dp = 5 ␮m. Mobile phase: gradient of acetonitrile in 15 mM ammonia solution (see Section 2.5). Flow rate: 1.0 ml/min. APCI-(+) interface was applied; (a) TIC and SIM chromatograms of A1–A8 compounds separated in band A after reduction with sodium hydrosulfite and MD TLC; (b) TIC and SIM chromatograms of B1–B7 compounds separated in band B after MD TLC; (c) TIC, DAD and SIM chromatograms of C1–C3 compounds separated in band C after MD TLC; Abbreviations used: TIC, total ion current; DAD 220 nm, UV track recorded at 220 nm by diode array detector; SIM, single ion monitoring; R.A., relative abundance.

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Fig. 4. Representative MS2 spectra of protonated molecule [M + H]+ of each typical compound and proposed structures of the predominating fragmentation ions; (a) MS2 spectrum of A1, B1 and B2 compounds with [M + H]+ 300 (intermedine/lycopsamine type of PA); (b) MS2 spectrum of A2 compound with [M + H]+ 302 (dihydroechinatine type of PA); (c) MS2 spectrum of B3 compound with [M + H]+ 318 (dihydroheliospathuline-N-oxide type of PA); (d) MS2 spectrum of A5 compound with [M + H]+ 342 (7-acetyllycopsamine type of PA); (e) MS2 spectrum of A6, A7 compounds with [M + H]+ 382 (symphytine type of PA); (f) MS2 spectrum of A8 compound with [M + H]+ 398 (2 ,3 -epoxyechiumine type of PA); (g) MS2 spectrum of B6, B7 compounds with [M + H]+ 398 (echimidine type of PA); (h) MS2 spectrum of B4, B5 compounds with [M + H]+ 398 (7-sarracinyl-9-viridiflorylretronecine type of PA) and (i) MS2 spectrum of C1–C3 compounds with [M + H]+ 414 (echimidine-N-oxide type of PA).

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Fig. 4. (Continued ).

structures. Therefore, the applied MD TLC method turned out to be quite selective in the separation of the alkaloids having the same recorded ion at m/z 414. 3.5. Chromatographic and MS data of the alkaloids determined In Table 1 are summarised the retention times, MS1 spectra establishing molecular weight and proposed types of structures of the alkaloids isolated in three various fractions by MD TLC and analysed by RP-HPLC IT procedure. The following order of elution for different types of PAs has been established according to the increase of retention factor in the LC separation: lycopsamine-N-oxide (two epimers) < lycopsamine (three epimers) < dihydroheliospathuline-N-oxide < echimidine-N-oxide (three epimers) < dihydroechinatine < 2 ,3 -epoxyechiumine < 7-sarracinyl-9-viridiflorylretronecine < 7-acetylycopsamine < 7-sarracinyl-9-viridiflorylretronecine (II epimer) < echi midine (two epimers) < symphytine (two epimers). Usually N-oxides exhibited higher retention compare to corresponding free bases on the charge-transfer sorbent. The alkaloids with

free OH group rather at position C7 than C9 were less retained on this stationary phase. Diesterified unsaturated alkaloids free bases were of the highest retention on XTerra RP-18 column. All the investigated PAs (18 compounds) were rapidly and efficiently separated merely in about 19 min. MSn spectra recorded at high signal to noise ratio permitting tentative structural determination of the complicated mixture. 3.6. MSn fragmentation of PAs observed during LC–MS studies For the diasteroisomeric compounds (A1, B1 and B2) with the same protonated molecules [M + H]+ at m/z 300, the product ion at m/z 138 was the most abundant in MS2 spectrum (Fig. 4a). That is typically encountered in monoesters of unsaturated necine with free OH group at position C7 [26–31]. Loss of C2 H4 O (44 Da) and C2 H6 O2 (62 Da) was characteristic for residues of trachelanthic or viridifloric acid, whereas peaks at m/z 94, 120, 136–137 are observed for an unsaturated necine moiety. Other peaks were similar. Therefore, lycopsamine type of structure has been assumpted.

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For compound A2 with [M + H]+ observed at m/z 302, saturated necine moiety is confirmed by a serie of peaks in MS2 spectrum (Fig. 4b): m/z 96, 122 and 140 [18,26,27]. The product ion with m/z 286 usually noted due to cleavage of Noxide bond was absent, so the N-oxide alkaloid was excluded (i.e. trachelanthamine-N-oxide). The peaks at m/z 258, 240 are observed after fragmentation of viridifloric or trachelanthic acid residues and m/z 140 is the most abundant in saturated necines with free OH groups at C7 . Therefore, this type of PA structure has been assigned as dihydroechinatine or its stereoisomer. Similarily to lycopsamine type of alkaloid (A1, B1 and B2), N-oxide structure was established for compounds A3 and A4 [13,27,31]. Typically product ions at m/z values higher of 16 Da were measured. Peak at m/z 300 was formed after cleavage of N O bond. In the case of compound B3 with [M + H]+ observed at m/z 318, MS2 spectrum (Fig. 4c) was similar to previously reported heliospathuline-N-oxide type of alkaloid [27] but fragmentation peaks at m/z values higher of 2 Da were present. The most abundant product ion with m/z 174 again 2 Da higher compare to the compound mentioned above was measured. Peaks with m/z 274 and 256 were due to loss of C2 H4 O and C2 H6 O2 trachelanthic or viridifloric acid residues. Hence, a dihydroheliospathuline-Noxide type of alkaloid has been assumed for this compound.

In the MS2 spectrum of compound A5 (Fig. 4d) with [M + H]+ observed at m/z 342, peaks at m/z 120 and 180 were predominating. That was due to an esterification with acetic acid at C7 of an unsaturated necine [30,31]. Characteristic was also peak at m/z 282 with low intensity created after cleavage of acetic acid (60 Da) at C7 . Also, product ion at m/z 297 was formed after previously mentioned fragmentation of viridifloric or trachelanthic acid at C9 (loss of C2 H5 O). Therefore, the structure of this compound has been determined as 7-acetyllycopsamine [30,31] or its stereoisomers and it was also previously reported in O. stellulatum [27]. For compounds A6, A7 (Fig. 4e) with [M + H]+ observed at m/z 382, product ions, characteristic for the structure of 7angeloyl (or other geometric isomers), an unsaturated necine were noted at m/z 238, 220 and 120, whereas peaks at 320 and 338 are formed after fragmentation of trachelanthic or viridifloric acid residues [27]. It confirmed the suggested structure of symphytine or its steroisomers. There are three compounds with [M + H]+ observed at m/z 398, with different fragmentation patterns in MS2 spectra. It could be also suggested because of differences in the retention times measured. For two diastereoisomers with echimidine type of structure (B6, B7) (Fig. 4g) previously mentioned product ions with m/z 120, 220 and 238 were due to 7-angeloyl (or other

Fig. 5. Proposed fragmentation pathways of echimidine-N-oxide type of PAs observed in MS2 spectrum.

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geometric isomers) an unsaturated necine. Peaks at m/z 336 and 339–341 were of echimidinic acid residue [26,27,30,31]. Compound A8 (Fig. 4f) besides typical product ions of the unsaturated necine (118, 119, 120, 136) exhibited in MS2 spectrum peak at m/z 236 with high abundance and at 254 with weaker intensity. Other product ions were of trachelanthic or viridifloric acid residues (mainly m/z 354). Based on literature data [32] the structure of 2 ,3 -epoxyechiumine has been assumed. In case of compounds B4, B5 (Fig. 4h), product ion with m/z 254 in MS2 was the most abundant but N-oxide structure has been excluded both in MS1 –MS2 analysis. That structure may be stabilized by hydrogen bonding between two OH groups at position C9 and that of sarracinic acid moiety at C7 . Further fragments: 236 ( H2 O), 220, 218 ( 2H2 O) supported this assumption. That was rather surprising, because the N-oxides of the echimidine types in our previous report showed similar pattern [27]. Therefore, we concluded the acid esterified at C7 should possess an additional oxygen atom (i.e. sarracinic acid or its stereoisomer). Also characteristic echimidic acid fragments were absent (m/z 322, 340), but those from trachelanthic or viridifloric acid were present (354, 336). The structure of these compounds has been tentatively assigned as 7-sarracinyl-9-viridiflorylretronecine (or its stereoisomer).

165

In MS2 spectrum of three diastereoisomeric compounds (Fig. 4i) with [M + H]+ observed at m/z 414 (C1, C2, C3) the following product ions confirmed echimidine-N-oxide (or its stereoisomers) structure [33]: 398 (N O cleavage); 396, 370, 356, 352, 338 (echimidinic acid); 296 [angelic acid (or stereoisomer) esterified at C7 ]; 254 (after McLafferty rearrangement, see Fig. 5), 236, 220, 136–137, 120 [unsaturated necine N-oxide with angelic acid (or stereoisomer) at C7 ]. That fragments relations in MS2 spectrum of this compound are presented in Fig. 5. In general, loss of water gave the major fragment ion in MS2 spectrum, and it was quite different from EI MS study. 3.7. Structural types of the alkaloids determined in S. cordatum roots and chemotaxonomical aspects From roots of S. cordatum 18 various alkaloids possessing 10 different types of structure were pre-fractionated and separated for the first time and their structures tentatively determined (Fig. 6). Three compounds with echimidine-N-oxide type of structure (C1–C3) were predominating. In general, 12 compounds were separated as N-oxides (A1–A8-N-oxides, B3, C1–C3). Other compounds occurring as tertiary bases were in considerably lower amounts in the roots. Besides compounds C1–C3, the N-oxides with the following types of structure

Fig. 6. Type of the alkaloids structures separated and determined in S. cordatum roots on the basis of on-line RP-HPLC ion-trap MS (see Section 2.5). Compounds: A1–A8 in plant occurred as the N-oxides were isolated in band A after reduction with sodium hydrosulfite and MD TLC (see Fig. 1), B1–B5 in band B and C1–C3 in band C (see Table 1).

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were differentiated: lycopsamine (three stereoisomers of the Noxides of A1, A3, A4), dihydroechinatine (the N-oxide of A2), 7-acetyllycopsamine (the N-oxide of A5), symphytine (the Noxides of A6, A7); 2 ,3 -epoxyechiumine (the N-oxide of A8) and dihydroheliospathuline (B3). As the tertiary bases lycopsamine (B1, B2), 7-sarracinyl-9-viridiflorylretronecine (B4, B5) and echimidine (B6, B7) types of PAs were determined. Echimidine-N-oxide type of alkaloids as important constituents of Symphytum officinale L. [1,9], Symphytum caucasicum Bieb. [1], Symphytum asperum Lepech [1], S. tuberosum L. [1] and Symphytum × uplandicum Nyman roots [1] were noted among lycopsamine and 7-acetyllycopsamine-Noxide, characteristic alkaloids now found also in the investigated comfrey species (S. cordatum). Saturated PAs now described in this species probably as dihydroechinatine-N-oxide and dihydroheliospathuline-N-oxide types and also 2 ,3 epoxyechiumine-N-oxide alkaloid were interesting findings among comfrey plant species.

[4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

[16] [17]

4. Conclusions

[18]

Combination of LLP of crude extract, MD TLC prefractionation and structural analysis by hyphenated LC ion-trap MS method can be regarded as a very efficient tool in the determination of PAs in very complex mixtures. In this way multidimensional chromatographic (MD TLC × LC) and spectroscopic data (UV, MSn ) can be easily obtained and may have a great potential in modern chemotaxonomic studies of PAs among the plant kingdom.

[19] [20] [21] [22] [23] [24] [25]

Acknowledgement [26]

We would like to gratefully acknowledge the Polish State Committee for the Scientific Research for financial support (grant No. 6 P05F 021 21).

[27] [28] [29]

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