Anal. Chem. 1998, 70, 3084-3087
Technical Notes
HPLC-Electrospray Ionization Mass Spectrometric Analysis of Antimalarial Drug Artemisinin Parmeshwari Sahai and Ram A. Vishwakarma*
Bio-organic Chemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, JNU Complex, New Delhi 110 067, India Shalini Bharel, Anamika Gulati, Malik Z. Abdin, Prem S. Srivastava, and Swatantra K. Jain
Department of Biochemistry, Hamdard University, New Delhi 110 062, India
A new analytical method for high-sensitivity direct detection, quantitation, and isotopomer analysis of antimalarial drug artemisinin (qinghaosu) is described using HPLCelectrospray ionization mass spectrometry, without preor postcolumn derivatization. The method has been shown to be particularly suitable for analysis of artemisinin in biosynthetic stable isotope labeling experiments and quantitative analysis in the nanogram range in crude biological samples such as Artemisia annua plant extracts. The method enabled isotopomer analysis in biosynthetic 18O-labeling experiments with plant cell-free enzyme preparation. Artemisinin (qinghaosu), an unusual sesquiterpene-lactone endoperoxide from Artemisia annua L., is an effective antimalarial drug, particularly against chloroquine-resistant Plasmodium falciparum infection and cerebral malaria.1-4 Artemisinin and its semisynthetic analogues have undergone clinical trials as new lifesaving antimalarials under the auspices of the World Health Organization and have been intensively studied due to their unique structure (1, Figure 1), with an endoperoxide (1,2,4-trioxane) linkage, their novel mechanism of action as the first non-nitrogenous antimalarial, and the worldwide resurgence of drug-resistant falciparum infections. The state-of-the-art production of 1 by chemical and biotechnological methods and analytical aspects have recently been reviewed.5 In our biosynthetic studies on artemisinin toward purification of endoperoxidase enzyme from A. annua, and determination of the source of the endoperoxide oxygen bridge by 18O-isotope labeling in plant cell-free and tissue culture, we required an experimental method suitable for direct detection, quantitation, and isotopomeric analysis. Since 1 is unstable and lacks a chromophore for UV detection in HPLC, it
Figure 1. Structures of artemisinin (1) and arteannuin-B (2).
was not possible to analyze it in biological samples without precolumn chemical or electrochemical derivatization. Several methods successfully used for analysis of 1 in biological samples5 include RP-HPLC after precolumn UV-active transformation to Q260,6 reductive electrochemical HPLC detection,7 GC/MS,8 thermospray MS-MS of fragment ions,9 and the ELISA technique.10 However, these were not suitable for analysis of intact 1 in stable isotope labeling experiments. We now report a new, sensitive method for direct detection and quantitative estimation of artemisinin by HPLC-ESMS and its application for nanogram range analysis of 1 in crude biological samples and isotopomer analysis of stable isotope-labeled [18O]artemisinin from biosynthetic experiments. The success of the method was derived from observation of abundant [M + H]+ of 1 in positive-ion ESMS from HPLC-ESMS of crude plant samples. In earlier MS studies,11 the molecular ion of 1 could not be detected in standard EI, CI, and FABMS. Later, Fales et al.12 used nine different ionization modes, and under MS-MS (positive CI (6) (7) (8) (9)
* Corresponding author. Fax: 091-11-6162125. Phone: 091-11-6174899. Email:
[email protected]. (1) Haynes, R. K.; Vonwiller, S. C. Acc. Chem. Res. 1997, 30, 73-79. (2) Jung, M. Curr. Med. Chem. 1994, 1, 35-49. (3) Zhou, W. S.; Xu, X. X. Acc. Chem. Res. 1994, 27, 211-216. (4) Klayman, D. L. Science 1985, 288, 1049-1055. (5) Geldre, E. V.; Vergauwe, A.; Van den Eeckhout, E. Plant Mol. Biol. 1997, 33, 199-209.
Zhao, S. S.; Zeng, M. Y. Anal. Chem. 1986, 58, 289-292. Acton, N.; Klayman, D. L.; Rollman, I. J. Planta Med. 1985, 51, 445-446. Nair, M. S. R.; Basile, D. V. J. Nat. Prod. 1993, 56, 1559-1566. Ranasinghe, A.; Sweatlock, J. D.; Cooks, R. G. J. Nat. Prod. 1993, 56, 552563. (10) Ferreira, J. F. S.; Janicks, J. Phytochemistry 1996, 41, 97-104. (11) Madhusudanan, K. P.; Vishwakarma, R. A.; Popli, S. P. Indian J. Chem. 1989, 28B, 751-754. (12) Fales, H. M.; Sokoloski, E. A.; Pannell, L. K.; Pu, Q. L.; Klayman, D. L.; Lin, A. J.; Brossi, A.; Kelly, J. A. Anal. Chem. 1990, 62, 2494-2501.
3084 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
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ion-trap, positive ion FABMS, and 252Cf PDMS), M+ and [M + H]+ of 1 could be obtained. In a recent electrospray MS-MS study, Stefansson et al.13 reported extensive multimer formation for small uncharged molecules, including artemisinin due to concentration-dependent aggregation in the gas phase, and showed that addition of cationic primary amines led to complete suppression of such multimer formation. However, application of these MS-MS studies was not shown for detection and analysis of 1 in crude biological samples. In the present method, abundant [M + H]+ of 1 was obtained in a single-stage quadrupole ESMS interfaced with HPLC. A related sesquiterpene, arteannuin-B (2, Figure 1), was used as internal reference for quantitation of 1. EXPERIMENTAL SECTION Materials. The seeds of Artemisia annua, obtained from Walter Reed Army Institute of Research, Washington, DC, were raised in the herbal garden of Hamdard University, New Delhi. The standard 1 was isolated14 from the leaves and characterized by 1H, 13C, and 2D NMR. For analysis of extracts from leaves collected at different stages of plant growth, plant material (1 g, air-dried leaves) of each sample was extracted with hexane (15 mL × 3) at 40 °C, and extracts were concentrated. The residue was dissolved in CH3CN (1 mL), precipitated fatty material was removed by centrifugation, and the supernatant was concentrated under reduced pressure and dissolved in MeOH (1 mL) for LCESMS. Electrospray MS. ESMS data were obtained on a VG Platform II (VG BioTech, Fisons Instruments, Altrincham, UK) quadrupole MS equipped with MassLynx software, a Dynolite detector, and a pneumatic nebulizer-assisted electrospray LCMS interface, Jasco PU-980 intelligent gradient HPLC. The analyzer vacuum was maintained at 3 × 10-5 mbar, and unit resolution was used for all measurements. The source temperature was maintained at 100 °C, and nitrogen at a pressure of 60 psi was used for nebulization of carrier solvent (acetonitrile-water 1:1) and also as drying gas. The nebulizing and drying gas flow rates were 20 and 250 L/h, respectively. The positive ion ESMS was used for detection of 1 (Figure 2a) and internal reference 2 (Figure 2b). Total ion current (TIC) was measured over the range m/z 150-400 in scan function mode, and continuum spectra were obtained at a cone voltage of 40 V and capillary voltage of 3.5 kV. For detection of [M + H]+ of standard 1, 10 µL of solution (300 ng/mL in MeOH) was infused directly into the carrier solvent through a Rheodyne injector without column. For all analytical experiments, on-line HPLC-ESMS was used, and just after the LC column (NovaPack C-18, Waters, 3.9 mm × 300 mm, 1 mL/ min, 1:1 acetonitrile-water) a 97:3 flow split was used to divert 97% of the column eluate to waste and 3% of eluate to the mass spectrometer. In all LC-MS analysis, a 10-µL injection was made into the LC column through a Rheodyne injector. For quantitation, a calibration curve of 1 was prepared using 2 as internal standard. For this purpose, [M + H]+ peaks at m/z 283 for 1 and at m/z 249 for 2 were chosen. The peak areas under the ion current at m/z 283 for various detected amounts of 1 (3-150 ng), with reference to peak area under the ion current at m/z 249 for a (13) Stefansson, M.; Sjo ¨berg, P. J. R.; Markides, K. E. Anal. Chem. 1996, 68, 1792-1797. (14) Klayman, D. L.; Lin, A. J.; Acton, N.; Scovill, J. P.; Hoch, J. M.; Milhous, W. K.; Theodarides, A. D.; Dobek, A. S. J. Nat. Prod. 1984, 47, 715-717.
fixed amount (150 ng) of 2 in single ion recording (SIR) mode, were plotted against detected amounts to generate a second-order calibration curve (Figure 3). Analysis of Artemisinin in Plant Extract. On-line HPLC separation of 1 from plant extract was carried out on the same column. The plant extract in MeOH (10 µL) was injected, and 3% of column eluate was guided to the MS detector using a 97:3 flow split. For quantitation of 1 in plant samples, SIR of m/z 283 were used, and the peak ensuing at tR of standard artemisinin through the column was used for integration and area calculation. To confirm the identity of 1 being eluted from column, the waste eluants (97%) from flow split at tR of 1 were pooled, freeze-dried, dissolved in CDCl3, and analyzed by 1H NMR using high-sensitivity inverse gradient probe. Analysis of Artemisinin in Biosynthetic Experiments. Young leaves of A. annua (2 g) were homogenized with chilled buffer (2 mL Hepes, 100 mM, and β-mercaptoethanol, 2mM; pH 7.2). The extract was centrifuged at 100 000g at 4 °C for 15 min, and the resultant supernatant was used as endoperoxidase enzyme source. This cell-free enzyme preparation (500 µL) was divided in equal volumes in two vials, the first for control experiment for incubation in normal H2O and the second for incubation with labeled H218O. The first vial was diluted with 150 µL of normal H2O, whereas the second was diluted with 150 µL of H218O (95 atom % of 18O; Aldrich) to bring the heavy isotope enrichment of the second incubation mixture (H2O + H218O) to 35.62 18O atom %. The required cofactors ATP, NADPH+, MnSO4, and MgSO4 (0.1 mM each) were added to incubation mixtures. The substrate artemisinic acid (50 µg dissolved in 10% Triton X-100) was added to both mixtures. These were incubated at 30 °C for 2.5 h in a shaking water bath and were then extracted with diethyl ether (10 mL). The organic layer was concentrated with a stream of N2, and the residue dissolved in MeOH (1 mL) was used for HPLC-ESMS. RESULTS AND DISCUSSION In scan function mode, the novel sesquiterpene antimalarial artemisinin (1) gave prominent [M + H]+ at m/z 283 (Figure 2a) in positive ion ESMS; other ions at m/z 300, 305, and 321 were assigned to [M + NH4]+, [M + Na]+, and [M + K]+, respectively, and this direct detection led to development of the present HPLC-ESMS method for analysis of 1 in plant extracts and biosynthetic samples. To minimize experimental errors in quantitation of 1, a related sesquiterpene, arteannuin-B (2), of A. annua was used as internal reference, which gave [M + H]+ at m/z 249 (Figure 2b) and other adduct ions [M + NH4]+, [M + Na]+, and [M + K]+ at m/z 266, 271, and 287, respectively, in ESMS. The retention times of 1 and 2 from the HPLC column were 11.53 (inset of Figure 2a) and 7.56 min (inset of Figure 2b), respectively. The LC-MS of 1 from plant extract showed [M + H]+ at m/z 283, and to confirm that the ion was generated from 1 and not from any other coeluting material at same tR, the waste from the flow split was used for 1H NMR analysis. In SIR mode of LC-ESMS of 1 at m/z 283, the various detected amounts were 3, 15, 30, 75, and 150 ng. Keeping in view the reported13 concentration-dependent multimer ([2M + Na]+, [3M + Na]+) formation, LC-ESMS of 1 and 2 were recorded over the m/z 200-2000 range in scan mode at various concentrations, and only 3-4% multimer formation was observed in the range used for the Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
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Figure 2. ESI mass spectra in continuum mode: (a) positive ion ESMS of artemisinin, inset shows LC-MS chromatogram; (b) positive ion ESMS of arteannuin-B, inset shows LC-MS chromatogram. 3086 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
Figure 3. Calibration curve for artemisinin. A second-order calibration curve was prepared by plotting areas under ion current at m/z at 283 for various detected amounts (3, 15, 30, 75, and 150 ng) of 1 (tR ) 11.53 min); 150 ng of 2 (tR ) 7.56 min) was used as internal reference.
calibration curve. However, significant multimer formation appeared at 420 ng and higher amounts under the MS conditions. Analysis of Artemisinin in Plant Extract. A large number of plant extract samples prepared from leaves of A. annua at different growth phases were analyzed, showing artemisinin content variation from 34 to 90 µg/g dried leaf material by the present LC-ESMS method. The results were in good agreement with values determined by us by the Q260 HPLC method6 (data not shown). The extractability of the analyte 1 and standard 2 by the hexane extraction method was confirmed by TLC (solvent system, EtOAc-hexane 4:6) and HPLC (UV detection of Q260 at 260 nm). This extraction procedure has also been followed in previous analytical methods.5 Analysis of Artemisinin in Biosynthetic Experiments. To understand the mechanism of action of the putative endoperoxidase enzyme of A. annua during biotransformation of precursor artemisinic acid to artemisinin and the source of oxygen atoms
in endoperoxide bridge formation, biosynthetic incorporation experiments were carried out using partially purified plant cell-free enzyme preparation. The incubations were carried out in the presence of stable isotope-labeled H218O (35.62%) and normal H2O (64.38%), the reaction mixture of biosynthesis was analyzed, and isotopic ratios of m/z 283, 284, and 285 peaks were measured. In a number of incubation experiments, an average of 12% isotopic enrichment at m/z 285 was observed, in comparison with that of 1 obtained from control experiment: the isotopic mass ratios for standard 1 were 283 (100%), 284 (16.42%), and 285 (2.95%), whereas these ratios for labeled 1 were 283 (100%), 284 (16.24%), and 285 (15.11%). The dilution of 18O atom % from the initial 35.62% to 12% was due to the presence of endogenous artemisinin and/or intermediates in in vitro cell-free enzyme system. This result indicated that one of the endoperoxide oxygens was derived from H218O of the incubation medium. However, due to the low level of incorporation, the isotopic enrichment in m/z 287 could not be measured accurately; therefore, it is not certain whether the second oxygen atom of endoperoxide comes from H218O or is derived from air by enzymatic trapping of singlet oxygen. The present study provides an efficient procedure for analysis of 1 (minimum 3 ng) without any pre- or postcolumn reaction, with its structure and isotopic ratio intact. The utility has been demonstrated by direct detection, quantitation, and isotopomer analysis of artemisinin in crude plant extracts and biosynthetic samples. ACKNOWLEDGMENT The authors thank Ms. Archana Sinha for technical help and the Council of Scientific and Industrial Research and the Department of Biotechnology (government of India) for financial support. Received for review August 26, 1997. Accepted April 8, 1998. AC970944F
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