Chemicals and Reagents
Acetonitrile was HPLC certified solvent (Honeywell Burdick and Jackson, Muskegon, MI, USA). Water for the HPLC mobile phase was Milli-Q-purified (18.2 MΩ/cm) (Millipore, USA). Methanol was reagent ACS/USP/NF grade (Pharmaco Products, Brookfield, CT, USA). The mobile phase additives; ammonium acetate, formic acid, acetic acid and trifluoroacetic acid were each the highest grade available. Junceine (1
), trichodesmine (2
), supinine (3
), amabiline (4
) and lasiocarpine (5
), 7-angelylheliotridine (6
) and 9-angelylheliotridine (7
) () were sourced from the stocks of extracted and purified (ca. 80–95% pure based on HPLC-ESI/MS analysis) pyrrolizidine alkaloids kept by the USDA/ARS Poisonous Plant Research Laboratory. Lycopsamine (8
) was purchased from Planta Analytica (Danbury, CT, USA). The archived seed extract of C. juncea
, sourced from India and previously analyzed using HPLC,5
was supplied by Dr R. J. Molyneux (University of Hawaii at Hilo). Unless otherwise stated, all solid phase extraction cartridges and HPLC columns and guard cartridges were sourced from Phenomenex (Torrance, CA, USA).
Extraction of Plant Samples
The plant samples were supplied by the Natural Resources Conservation Service, Plant Materials Center, US Department of Agriculture from cultivated, seed production fields in Hawaii in April 2011. Composite samples of roots, stems and leaves were each derived from about 10 immature (90 days from planting) plants. The seeds, that are harvested approximately 150 days after planting, were derived from a general seed bank. The air-dried samples were milled to a fine powder.
For the quantitative comparison, an aliquot (ca. 400 – 500 mg) of each composite sample was extracted 5 times with methanol (10 mL) at room temperature and with gentle, inversion mixing for 16 h. Each successive extract was monitored for dehydropyrrolizidine alkaloid content using HPLC-ESI/MS and then combined to yield the four total extracts i.e., roots, stems, leaves and seeds, which were each concentrated to almost dryness in vacuo. The resultant residues were extracted with 0.05 M sulfuric acid (ca. 10 mL) to provide the alkaloid (N-oxides as well as free base alkaloids) solution for further cation exchange enrichment.
For the structural elucidation studies, milled seed (160 g) was Soxhlet-extracted with methanol (1.2 L) for 3 periods of 16 h each. Fresh methanol was used for each extraction period to lessen or avoid any degradation of dehydropyrrolizidine alkaloids following extended periods in the boiling methanol. Again, the yield of dehydropyrrolizidine alkaloids after each extraction was monitored using HPLC-ESI/MS to ensure complete extraction. The combined methanol extracts were evaporated to almost dryness and re-extracted with 0.05 M sulfuric acid (3 × 50 mL) to yield the aqueous acid solution of the alkaloids for further purification.
Strong Cation Exchange, Solid Phase Extraction (SCX SPE)
The filtered 0.05 M sulfuric acid-soluble fraction from each of the concentrated methanolic extracts was applied to a separate stack of two conditioned (according to the manufacturer’s guidelines) 500 mg/3 mL, Strata SCX 55 μm 70 Å SCX SPE columns. After loading, the stacked columns were separated and each was washed with water, methanol and, finally, w ith ammoniated methanol (a 10% solution of saturated ammoniated methanol in methanol) to elute the pyrrolizidine alkaloids and their N-oxides. The ammoniated methanol fractions were immediately evaporated to dryness under a stream of nitrogen and the residues reconstituted in methanol (1 mL). Efficiency of alkaloid capture and elution was monitored using HPLC-ESI/MS. The preparative extract of seeds was processed in a similar way except on a more appropriate larger scale 25 mm × 100 mm cation exchange column using Sepra SCX, 50 μm, 65 Å bulk media.
Chromatographic separation of the dehydropyrrolizidine alkaloids and their N-oxides () was achieved using a Finnigan Surveyor HPLC system, comprising an Autosampler Plus and a MS Pump Plus (Thermo-Finnigan, San Jose, CA, USA), equipped with a 150 mm × 2 mm i.d., 4 μ, Synergi Hydro Reversed Phase column, with a 4 mm × 2 mm i.d. AQ C18 guard column. Sample injections (2 μL) were eluted with a gradient flow (400 μL/min) in which the initial mobile phase composition of 5% acetonitrile in aqueous 0.05% trifluoroacetic acid/0.5% acetic acid was held for 2 min and then linearly increased to 50% acetonitrile over 13 min. This was held for a further 10 min before returning to the initial mobile phase composition and re-equilibration of the column. The column effluent was monitored using a LCQ Advantage ion trap mass spectrometer (Thermo-Finnigan, San Jose, CA, USA) in the electrospray ionization (ESI), positive ion mode. The mass spectrometer response was tuned to a solution of heliotrine (acquired from stocks held by the USDA/ARS Poisonous Plant Research Laboratory) in methanol. The capillary temperature was 275 °C with a capillary voltage of 46 V. The source voltage was 4.5 kV at a source current of 80 μA. The sheath and auxiliary gas flows were set at a ratio of 60:10. Data-dependent MS/MS spectra were acquired in the second scan of a two scan sequence in which ions identified in the first total ion scan were isolated a nd fragmented using 32% applied dissociation energy.
Figure 2 The HPLC-ESI/MS base ion (m/z 200 – 800) chromatograms of the aqueous acid-soluble fraction of the methanolic extract of the stems (A) and seeds (B) of Crotalaria juncea (cv. ‘Tropic Sun’). Peak numbers identify dehydropyrrolizidine (more ...)
An even numbered m/z
for the MH+
ion followed by observation of MS/MS fragment ions characteristic of dehydropyrrolizidine alkaloids eg., m/z
94, 120, 122, 136, 137, 138, 156, 180 highlighted HPLC-ESI/MS peaks as probable dehydropyrrolizidine alkaloids.8, 9
Observation of a significant dimer ion [2M + H]+
indicated potential dehydropyrrolizidine-N
-oxide character under these esiMS conditions.8
treatment, of an analytical sample indicative of N
-oxides, with indigocarmine-based redox resin reduced the N
-oxides to their parent dehydropyrrolizidine alkaloids thereby confirming the N
Standard solutions containing lycopsamine (8) (69, 34.5, 17.25, 8.63, 4.31 and 2.15 μg/mL) and junceine (1) (200, 100, 50, 25, 12.5, 6.25 μg/mL) were made by adding a solution of 8 (200 μL of 138 μg/mL methanol) to a solution of 1 (200 μL of 400 μg/mL methanol) and serially diluting (1:1) an aliquot (200 μL) in methanol. Calibration standard samples were then prepared by adding lasiocarpine (5) solution (10 μL ca. 50μg/mL methanol), to provide a normalizing injection standard (IS), to an aliquot (100 μL) of each of the standard solutions.
The analytical samples were similarly prepared by adding IS (10 μL) to an aliquot (100 μL) of the redox resin-reduced extract solution. Peak areas, generated from reconstructed ion chromatograms displaying the ions of interest, were adjusted within each run by division by the area of the IS for that run.
Preparative Flash Chromatography
The 10% ammoniated m ethanol eluate from the SCX SPE of seed extract was evaporated to dryness to afford a red gum (5 g) that was re-extracted with chloroform (3 × 50 mL). The pooled, orange chloroform extract was filtered, reduced in volume (to about 15 mL) and applied to a Biotage KP silica Samplet (10 g) cartridge of silica gel (Biotage, North America). A narrow orange band was retained on the Samplet cartridge. The loaded Samplet cartridge was fitted to a Biotage Snap KP-silica gel column (50 g) that had been equilibrated with ethyl acetate using the Isolera 1 automated flash chromatography system (Biotage, North America). Elution of retentates from the column assembly was achieved at a flow rate of 50 mL/min, collecting 20 mL fractions, using a linear gradient of 10% ammoniated methanol (0 – 40% over 5 min) into ethyl acetate, hold for 6 min to elute non-dehydropyrrolizidine alkaloid material, then increasing from 40 – 100% ammoniated methanol over 7 min, to elute the dehydropyrrolizidine alkaloids, for a total run time of 18 min. The eluate was simultaneously monitored at 254 and 280 nm that provided an indication, albeit poor due to low UV absorbance, of the alkaloid elution times. The collected fractions were then analyzed for dehydropyrrolizidine alkaloid content using HPLC-ESI/MS.
Preparative and Semi-preparative HPLC
Alkaloid-enriched samples derived using the Biotage flash chromatography system were evaporated to dryness and reconstituted in 0.1% trifluoroacetic acid in water. Aliquots (up to 400 μL) of this solution were applied to a 100 mm × 21.2 mm i.d., 4 μ, 80
, AXIA-packed Synergi Hydro Reversed Phase column with a 15 mm × 21.2 mm i.d. C18 guard column. Using a Waters Prep LC 2000 System (Waters Corporation, Milford, MA, USA), the alkaloids were eluted with an isocratic (0.1% trifluoroacetic acid/acetonitrile: 93/7) flow (20 mL/min) and collected into bulk fractions determined by monitoring the absorbance at 220 nm. The dehydropyrrolizidine alkaloid content of the fractions was then determined using HPLC-ESI/MS.
Using a Finnigan Surveyor HPLC system, comprising a LC Pump Plus and a UV-VIS Plus (Thermo-Finnigan, San Jose, CA, USA), two isocratic, semi-preparative HPLC modes were used to achieve final separation of target analytes in the dehydropyrrolizidine alkaloid concentrates (up to 200 μL injections) derived from the preparative HPLC. The column effluent was monitored at 220 nm. Basic separation conditions were achieved using a 250 mm × 10 mm i.d., 5 μ, 110
Gemini NX C18 column with a 10 mm × 10 mm i.d. guard column of the same material with acetonitrile (7%) into 20 mM ammonium acetate at pH 9.5 (NH4
OH) (93%) at 5 mL/min. Acidic separation conditions were achieved using a 250 mm × 10 mm i.d., 4 μ, 80
Synergi Hydro Reversed Phase column with a 10 mm × 10 mm i.d. Synergi AQ C18 guard column with acetonitrile (7%) into 0.1% trifluoroacetic acid in water (93%) at 5 mL/min.
Low resolution MS and MS/MS data were acquired in the HPLC-ESI/MS mode described in a previous section. The high resolution mass spectrometry (HRMS) measurement was achieved using an Agilent 6220 TOF mass spectrometer in the dual ESI, positive ion mode. One dimensional and 2D (COSY, HSQC and HMBC) 1H (300 MHz) and 13C (75 MHz) NMR data were acquired using a JEOL Eclipse NMR spectrometer using solutions in CDCl3 and the residual proton in the chloroform as the lock signal (except for junceine for which d6-DMSO was used as the solvent and the crude seed extract which was dissolved in d4-methanol).
Junceine (1) (peak 1, ) and Trichodesmine (2) (peak 6, )
The HPLC-ESI/MS data () were the same as for the authenticated standards and the 1
H and 13
C NMR data were consistent with previous reports.12–14
Positive Ion Electrospray Ionization, Ion Trap MS/MS Data for the Dehydropyrrolizidine Alkaloids Detected in Extracts of Crotalaria juncea (cv. ‘Tropic Sun’).
Isohemijunceine A (9) (peak 3, )
Obtained as a colorless oil estimated at > 95% pure using HPLC-ESI/MS and 1H NMR; ESI(+)/MS and MS/MS (); HRMS (found, 283.17842; calcd. for C15H25NO4 283.1784; Δ 0.07 ppm); 1H, 13C, COSY and HMBC NMR ().
1H, 13C, 1H-1H (COSY) and 1H-13C (HMBC) NMR Spectroscopic Data for Isohemijunceine A (9) Extracted from Crotalaria juncea (cv. ‘Tropic Sun’).
Isohemijunceine B (10) (peak 5, )
Obtained as a colorless oil estimated at 85 – 90% pure using HPLC-ESI/MS and 1H NMR; ESI(+)/MS and MS/MS (); HRMS (found, 283.17892; calcd. for C15H25NO4 283.1784; Δ 1.8 ppm); 1H, 13C and COSY NMR ().
1H, 13C, and 1H-1H (COSY) NMR Spectroscopic Data for Isohemijunceine B (10) Extracted from Crotalaria juncea (cv. ‘Tropic Sun’).
Isohemijunceine C (11) (peak 7, )
Obtained as a colorless oil estimated at 85 – 90% pure using HPLC-ESI/MS and 1H NMR; ESI(+)/MS and MS/MS (); HRMS (found, 283.17912; calcd. for C15H25NO4 283.1784; Δ 2.5 ppm); 1H, 13C, COSY and HMBC NMR ().
1H, 13C, 1H-1H (COSY) and 1H-13C (HMBC) NMR Spectroscopic Data for Isohemijunceine C (11) Extracted from Crotalaria juncea (cv. ‘Tropic Sun’).
Combined NMR and HPLC-ESI/MS Analysis of Seed
Seeds (50 g) were milled to a fine powder. Three sub-samples were dried in an oven at 40 °C for 5 days to determine the moisture content of about 5%. A larger sample (40.99 g) was Soxhlet-extracted with methanol (400 mL) for 20 h. The extraction solvent was removed and the extraction continued twice more with fresh solvent (20 h and 48 h) to lessen any degradation that might result from prolonged boiling in methanol. The combined extracts were evaporated to dryness in vacuo to afford a yellow solid (5.7 g, 14%) that was subsequently extracted with 0.05M H2SO4. The aqueous acid solubles were applied to a conditioned 5g/20mL Strata SCX 55 μm 70 Å SCX SPE column that discharged, under gravity, onto a second conditioned 5g/20mL Strata SCX 55 μm 70 Å SCX cartridge. After complete addition of the acid extracts, the two cartridges were separately washed with methanol (causing elution of an orange colour) and then with 10% satd. ammoniated methanol to elute the alkaloids.
The combined ammoniated methanol eluates were evaporated to dryness to yield a dark red oil (0.127 g) that was reconstituted in d4
-methanol (700 μL) and the 1
H NMR spectrum recorded. Then an aliquot of p
-dinitrobenzene (25 μL of 23 mg/mL d4
-methanol) was added and the spectrum re-acquired. A further aliquot (50 μL) of the p
-dinitrobenzene was added and the spectrum again re-acquired to provide an additional check on quantitation. The C2 proton signals in both NMR spectra were integrated relative to the p
-dinitrobenzene singlet, both as a total envelope (ca 6.2 – 5.8 ppm) or as two separate envelopes, about 6.2 ppm for the macrocyclic diesters or about 5.8 ppm for the monoesters, and the amount of dehydropyrrolizidine alkaloids estimated according to Molyneux et al
The same sample, suitably diluted with methanol, was then assessed using the HPLC-ESI/MS method using trichodesmine (2) (isolated from C. juncea in this study; 38.1, 19.05, 9.53, 4.77, 2.38, 1.19 and 0.59 μg/mL) and lycopsamine (8) (34.5, 17.25, 8.63, 4.32, 2.16, 1.08 and 0.54 μg/mL) as the calibration standards for the macrocyclic diesters and monoesters respectively.