2913
Anal. Chem. 1092, $4, 2313-2315
AC RESE-ARCH
Rapid Screening for Taxanes by Tandem Mass Spectrometry Steven H. Hoke 11, Joe M. Wood,+and R. Graham Cooks* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393
Xiao-Hua Li and Ching-jer Chang' Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907-1333
A highly specific and sensitive method Is described for determlnlng taxol, cephaiomannlne, and baccatin I I I in crude plant extracts. Radical anions of the taxanes are formed by d.rorptkn chemkai ionization, and a parent tandem mass spoctrmotrlc scan is wed to recognize these compoundsby thdr characterktlc dissociatkns. The limit of detectionof the IndlvMuai taxanes in typical pianl matrices is less than 500 pg when all thrw species are screened rknutlaneoudy. Becauseof the sensitivity of the method,extractionthe8 can be shortened to 90 min and crude extracts can be examined at the rate of 6/h. Detection of ail three taxanes extracted from a dngk Tax- "#data needle in a combined extraction/ analyrk t h e of less than 1 h is demonstrated.
culturing plant tissues in a laboratory setting has been accomplished on a small scale.* Total synthesis of taxol has not been achieved because of the number of asymmetric centers and the complexity of the diterpene skeleton."" However, a method of partial synthesis of taxol which couples the simpler diterpenoid, baccatin 111, with the synthetic carboxylic acid side chain has been developed.12 Because of the potential usefulness of taxol analogs in the partial synthesis of taxol, methods which screen various plant materials for taxol and other taxanes such as cephalomannine and baccatin I11 are highly desirable. High-performance
INTRODUCT10N The clinical trials of the natural product taxol have shown extremely promising results for treatment of ovarian, breast, and skin cancem'9 Taxol is isolated from the bark of the Pacific yew tree, Taxus brevifolia;4however, the yield of taxol is only approximately 1 g/10 kg of bark6 and it has been estimated that this natural supply of taxol will be consumed within the next 5-10 yearse6 In addition, many of the trees are located in areas where logging is prohibited to protect the habitat of the spotted owl.7 It is this dilemma which led Bruce Chabner of the National Cancer Institute to call the procurement of taxol "the ultimate confrontation between medicine and the envir~nment".~ Aa a result of this natural shortage, alternative methods of taxol production are being explored. Production of taxol by t Present
addrees: Vistakon Johnson and Johnson Vision Products, P.O. Box 10157, Jacksonville, FL 32247. (1) McGuire, W. P.; Rowinsky, E. K.; Donehower, R. C. In Accomplishments in Cancer Research-l9W, Fortner, J. G., Rhoads, J. E., Eds.; J. B. Lippincott: Philadelphia, 1991; pp 276-283. (2) Borman, S. Chem. Eng. Neurs. 1991, Sept 2,ll-18. (3) Suffneee, M.; Cordell, G. A. In The Alkaloids, Chemistry and Pharmacoloau:Brmi, A., Ed.; Academic Press: Orlando, FL, 1985; Vol. xxv, pp s 2 s a . (4) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. SOC.1971,93, 2325-2327. (5) Videneek.N.:Lim.P.:Cam~bell.A.:Carlson,C. J.Nat.Prod. 1990, . . . . - . . 53, '1&09-1610.
(6) Kingston, D. G. I. Pharmucol. Ther. 1991,52, 1-34. (7) Chase, M. A New Cancer Drug May Extend Lives-At Cost of Rare Trees. Wall St. J . [Three Star] East Ed. 1991, April 9, pp Al, A8. 0003-2700/92/0364-2313$03.00/0
R Bz
2
Taxol
BaccaM Dl
Cephalomanninc
liquid chromatography (HPLC) is widely used to determine taxane concentrations,'s's but it is not suited for rapidly screening numerous samples a t low levels. Recently, enzymelinked immunosorbant assays (ELISA) have been used to screen plant extracta for taxanes, but with current technology cephalomanninecross reacts with taxol.'6 Other methods of taxane analysis including multimodal thin-layer chromatog(8) Lansing, A.; Haertel, M.; Gordon, M.; Floas, H. G. Program and Abstracts of the 32nd Annual Meeting of the American Society of Pharmacognosy; Chicago, 1991; p-109. (9) Holton, R. A. J. Am. Chem. SOC.1984,106,5731-5732. (10) Gubritte-Voegelein,F.; Gubnard, D.; Potier, P. J. Nat. Prod. 1987, 50, 9-16.
(11) Holton, R. A.; Juo, R. R.; Kim, H. B.; Williams, A. D.; Haruaawa, S.; Lowenthal, R. E.: Yogai,S. J. Am. Chem. SOC. 1988,110,6558-6560. (12) Denis, J.-N.; Greene, A. E.; GuBnard, D.; GuBritte-Voegelein,F.; Mangatal, L.; Potier, P. J. Am. Chem. SOC. 1988,110,5917-5919. (13) Witherup, K. M.; Look, S. A.; Stasko, M. W.; McCloud, T. G.; Issaq, H. J.; Muschik, G. M. J. Lip. Chromatogr. 1989, 12, 2117-2132. (14) Harvey, S. D.; Campbell, J. A.; Kelsey, R. G.; Vance, N. C. J. Chromatogr. 1991,587, 300-305. (16) Cardellina, J. H., 11. J. Liq. Chromatogr. 1991,14,659-665. (16) Jaziri, M.; Diallo, B. M.; Vanhaelen, M. H.; Vanhaelen-Faatre, R. J.; Zhui, A.; Becu, A. G.; Homes, J. J. Pharm. Belg. 1991,46, 93-99.
0 1992 American Chemlcai Soclety
2314
ANALYTICAL CHEMISTRY, VOL. 84, NO. 20, OCTOBER 15, 1992
'
'"1
e
4
1 d
"
526 1534
'001
I x5
586
I
r
-267 so
(R-H)+AcOH
526
I mass-to-charge
Flguro 1. MSlMS product Ion spectrum of the taxol radlcal anbn produced by colllsion-lnduced dlssoclatbn uslng an argon target and a collision energy of 24 eV. The loss of the neutral species, R. corresponds to cleavage of the acyl-oxygen bond of the C13 side
chain.
raphy (TLC)," fast atom bombardment mass spectrometry,18 and combinations of techniques such as HPLC and thermospray mass spectrometry,'Q do not provide both rapid analysis of crude extracta and low limite of detection. Tandem mass spectrometry (MS/MS) is particularly well suited for trace analysis of target compounds in complex matrices.20-24 This report presenta a highly specific and extremely sensitive parent tandem mass spectrometric method for screening plant extracts for taxol, cephalomannine, and baccatin I11usinga triple quadrupole mass spectrometer. A preliminary account of this result has been given elsewhere.%
m-
500
800
7"
EXPERIMENTAL SECTION Extraction Procedure. Plant needle and barksamples were collected,dried, ground, and extracted in 95 % ethanol overnight. The resulting solution was filtered and evaporated to dryness. After partitioning between methylene chloride and water (l:l), the methylene chloride fraction was dried and partitioned between hexane and (90%) methanol and the methanol fraction was analyzed using the MSIMS parent scan. Analysis Procedure. A Finnigan triple-stage quadrupole (TSQ) 700 was used for the analysis of samples. Radical anions of the taxanes were generated in an ammonia chemicalionization plasma by electron attachment*% after sample molecules had been thermally desorbed from a direct evaporation probe. Under the ionization conditions used during these experiments, some [M- HI-was also produced but was not utilized in the screening procedure. (17) Staeko, M. W.; Witherup, K. M.; Ghiorzi, T. J.; McCloud, T. G.; Look, 5.;Muechik, G. M.; k q , H.J.J. Liq. Chromatogr. 1989,12,21332143. (18) McClure, T. D.; Reimer, M. L. J.; Schrm, K. H. Proceedings of
the 38th ASMS Conference on Mass Spectrometry and Allied Topics; Tucson, 1990, pp 1008-1009. (19) Auriola, 5 . 0 . K.; Lepiet8, A.-M.; Naaranlahti, T.; Lapinjoki, S. P. J. Chromatogr. 1992,594, 153-158. (20) Roush, R. A.; Cooks,R. G. J. Nat. Prod. 1984,47, 197-214. (21) Busch, K. L.; Gliih, G.L.; McLuckey, S. A. Mass Spectrometry/ Mass Spectrometry; V C H New York, 1988. (22) Fetterolf, D. D.; Yost, R. A. Znt. J.Mass Spectrom. Zon Processes 1984,62,33-49. (23)Lau, B. P.-Y.; Scott, P. M. Proceedings of the 32nd ASMS Conference on Mass Spectrometry and Allied Topics;San Antonio, 1984; pp 789-790. (24) Plattner, R. D.; Bennett, G.A.; Stubblefield, R. D. J. Assoc. Off. Anal. Chem. 1984,67,734-738. (25) Wood, J. M.; Hoke, S.H.; Cooks, R. G.;Chang,C.-j.; Heinatein,
P. F. Proceedings of the 39th ASMS Conferenceon Mass Spectrometry and Allied Topics; Nashville, 1991; pp 1679-1680. (26) yon Ardenne, M.; Steinfelder, K.; Tummler, R. Elektronenanlugerungs-massenepektrometric Organischer Substanzen;Springer: Ber-
lin. -1971. ----. - - -(27) Hunt, D. F.; Sethi, S. K. J. Am. Chem. S O ~1980,102,69534963. .
(28)Dillard, J. G. Chem. Rev. 1973, 73, 589-643.
586
J,
"1 20
500
800
7"
eo0
mass-to-charge
Flgwo 2. (a) Fulkcan, slngle-stage,mass spectrum of a plant extract containing less than 10 ng of each of the taxanes. (b) Parent ion spectrumof mlz 528 of the same plant extract. (c)Parent ion sped" of a mixture of taxol, cephalomannlne, and baccatln 111 standards. Spectra In (b) and (c)were produced by cdHskn-lnduced dissociation on an argon target at 40 eV. Taxol (mlz 853), cephalomnnlne (mlz 8311, and baccatln 111 (mlz 588) are represented In all three spectra.
Collisional activation was generally achieved with 30-eV collisions upon an argon target gas at 0.5 mTorr in quadrupole 2 during the parent ion scan of mlz 526. To decrease the limit of detection, multiple reaction monitoringwas performed in which a 10-Da window around the molecular ion regions of taxol, cephalomannine, and baccatin I11 was scanned in 0.05 8. Quantitation data were taken in the profile mode. Analysis of each plant extract required two measurements. First, 1 pL of the plant extract (1 mg/mL) was placed on the filament and the ion current for each of the taxanes was recorded. Second, 1pL of the sample was spiked with taxol, cephalomannine, and baccatin I11 and reexamined. The spike was typically 1, 5, or 10 ng depending on the ion current recorded from the sample alone. This entire process took approximately 10 min. The concentration of each of the taxanes in the samplewas then determined from a plot of the ion abundance verses the amount of taxane added.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
RESULTS AND DISCUSSION The MS/MS product ion spectrum of taxol (Figure 1)is recorded by ma-selecting the radical anion in quadrupole 1, effecting collision-induced dissociation in quadrupole 2, and scanning quadrupole 3 to determine the masses of the fiagment ions. This spectrum shows several neutral losses which are characteristic of taxol including the loss of acetic and benzoic acids. A prominent peak at mlz 526 is observed in this spectrum and also in the MS/MS product ion spectra of cephalomannine and baccatin 111. Because the ion mlz 526 is common to the product ion spectra of these three taxanes, a parent ion scan was utilized to screen for allthree taxanes simultaneously. With this type of scan, the ion current recorded by the electron multiplier results from ions that lose a neutral species and form mlz 628, therefore,the method is selectivefor moleculescontaining the same diterpene skeleton as taxol. To illustrate the selectivity of the method for the target compounds, the parent ion spectrum (Figure 2b) is compared to a conventional single-stage m w spectrum (Figure 2a) of the same plant extract. Note the presence of ions representative of the three species of interest, taxol (mlz 853), cephalomannine (mlz831),and baccatin I11(mlz 586) in both spectra. However, there are many unknown ions present in the mass spectrum and the signal to noise ratio is poor compared to the parent scan. Figure 2c is a parent scan of a mixtureof the three standard compounds. All three taxanes have been recognized in plant extracts at levels less than 500 pg; however, the ion current recorded for the taxanes is expected to decrease in matrices containing high concentrations of species with positive electron affmities. The parent ion scan of mlz 526 was tested as a rapid screening method for texanes by examining needle and bark extracts from a variety of Taxus species. Table I displays the weight percentages of each of the taxanes in various plant samples ea determined by the MS/MS methodology. The Taxusbrevifolia bark extract containedthe largest percentage of taxol, as expeded.6JS A Taxus c u s p i d a t a fresh bark extract was run four times with the concentrations of the taxanes determined as 3.70 f 1.15 ng1pL for taxol, 3.15 f 1.01nglpL for cephalomannine, and 0.56 f 0.11 ng/pL for baccatin 111, with the 90% confidence interval displayed. The reproducibility of the method is adequate, but its main value is in rapidly screeninglarge numbers of plant extracts to determine approximate taxane concentrations.
2915
Table I. Weight Percentages of Taxol, Cephalomannine, and Baccatin I11 in Various Taxus Species As Determined by MS/MS plant part needle twigbark needle twig bark needle bark T.floridana needle old bark T.canadensis needle bark T.baccata needle old bark sample T.cuspidata (dry) T.cuspidata (fresh) T.brevifolia
% taxol
0.0050 0.0065 0.0069 0.0030 0.0036 0.084
0.012 0.0046 0.019 0.0029 0.0070 0.0026
% % cephalomannine baccatin I11
0.0015 0.0023 0.0066 0.0025 0.00054 0.0096 0.0054 0.0013 0.0043 0.0013 0.0020 0.0011
0.00022 0.00042 0.00035 0.00045 0.00078 0.021 0.00067 0.00090 0.00073 0.0022 0.00040 0.00069
Because intense ion signals were recorded for most of the samples analyzed, a T. c u s p i d a t a fresh needle sample was extracted using an abbreviated procedure. The sample was ground and extracted for 30 min in 95 7% ethanol. The taxanes were determined as 0.0046 % (wt % ) for taxol, 0.0017 ?4 for cephalomannine and 0.00031 % for baccatin 111. Using this 30-min extraction procedure, 5.0 mg of extract was obtained from a single T.cuspidata needle. This extract was dissolved in ethanol (1mgImL), and 1pL of the resulting solution was analyzed. The presence of these three taxanes in a single needle was.confirmed in less than 1-h combined extraction and analysis time. These results demonstrate the great potential of tandem mass spectrometry for rapid selection of high taxane producing species from hybrid yews cultivated by numerous nurseries or from plant tissue culture specimens.
ACKNOWLEDGMENT The authors thank Dr. Richard Spjut of World Botanical Associates for collection of the T.breuifolia, T. baccata, T. canadensis, and T.f l o r i d a n a samples and for identification of the Taxus species at Purdue University from which the T. c u s p i d a t a extracts were taken. This work was supported by the National Cancer Institute, R01 CA55118. S.H.H.acknowledges support from BASF Corp. RECEIVED for review July 20, 1992. Accepted August 20, 1992.
Anal. Chem. 1092, $4, 2313-2315
AC RESE-ARCH
Rapid Screening for Taxanes by Tandem Mass Spectrometry Steven H. Hoke 11, Joe M. Wood,+and R. Graham Cooks* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393
Xiao-Hua Li and Ching-jer Chang' Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907-1333
A highly specific and sensitive method Is described for determlnlng taxol, cephaiomannlne, and baccatin I I I in crude plant extracts. Radical anions of the taxanes are formed by d.rorptkn chemkai ionization, and a parent tandem mass spoctrmotrlc scan is wed to recognize these compoundsby thdr characterktlc dissociatkns. The limit of detectionof the IndlvMuai taxanes in typical pianl matrices is less than 500 pg when all thrw species are screened rknutlaneoudy. Becauseof the sensitivity of the method,extractionthe8 can be shortened to 90 min and crude extracts can be examined at the rate of 6/h. Detection of ail three taxanes extracted from a dngk Tax- "#data needle in a combined extraction/ analyrk t h e of less than 1 h is demonstrated.
culturing plant tissues in a laboratory setting has been accomplished on a small scale.* Total synthesis of taxol has not been achieved because of the number of asymmetric centers and the complexity of the diterpene skeleton."" However, a method of partial synthesis of taxol which couples the simpler diterpenoid, baccatin 111, with the synthetic carboxylic acid side chain has been developed.12 Because of the potential usefulness of taxol analogs in the partial synthesis of taxol, methods which screen various plant materials for taxol and other taxanes such as cephalomannine and baccatin I11 are highly desirable. High-performance
INTRODUCT10N The clinical trials of the natural product taxol have shown extremely promising results for treatment of ovarian, breast, and skin cancem'9 Taxol is isolated from the bark of the Pacific yew tree, Taxus brevifolia;4however, the yield of taxol is only approximately 1 g/10 kg of bark6 and it has been estimated that this natural supply of taxol will be consumed within the next 5-10 yearse6 In addition, many of the trees are located in areas where logging is prohibited to protect the habitat of the spotted owl.7 It is this dilemma which led Bruce Chabner of the National Cancer Institute to call the procurement of taxol "the ultimate confrontation between medicine and the envir~nment".~ Aa a result of this natural shortage, alternative methods of taxol production are being explored. Production of taxol by t Present
addrees: Vistakon Johnson and Johnson Vision Products, P.O. Box 10157, Jacksonville, FL 32247. (1) McGuire, W. P.; Rowinsky, E. K.; Donehower, R. C. In Accomplishments in Cancer Research-l9W, Fortner, J. G., Rhoads, J. E., Eds.; J. B. Lippincott: Philadelphia, 1991; pp 276-283. (2) Borman, S. Chem. Eng. Neurs. 1991, Sept 2,ll-18. (3) Suffneee, M.; Cordell, G. A. In The Alkaloids, Chemistry and Pharmacoloau:Brmi, A., Ed.; Academic Press: Orlando, FL, 1985; Vol. xxv, pp s 2 s a . (4) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. SOC.1971,93, 2325-2327. (5) Videneek.N.:Lim.P.:Cam~bell.A.:Carlson,C. J.Nat.Prod. 1990, . . . . - . . 53, '1&09-1610.
(6) Kingston, D. G. I. Pharmucol. Ther. 1991,52, 1-34. (7) Chase, M. A New Cancer Drug May Extend Lives-At Cost of Rare Trees. Wall St. J . [Three Star] East Ed. 1991, April 9, pp Al, A8. 0003-2700/92/0364-2313$03.00/0
R Bz
2
Taxol
BaccaM Dl
Cephalomanninc
liquid chromatography (HPLC) is widely used to determine taxane concentrations,'s's but it is not suited for rapidly screening numerous samples a t low levels. Recently, enzymelinked immunosorbant assays (ELISA) have been used to screen plant extracta for taxanes, but with current technology cephalomanninecross reacts with taxol.'6 Other methods of taxane analysis including multimodal thin-layer chromatog(8) Lansing, A.; Haertel, M.; Gordon, M.; Floas, H. G. Program and Abstracts of the 32nd Annual Meeting of the American Society of Pharmacognosy; Chicago, 1991; p-109. (9) Holton, R. A. J. Am. Chem. SOC.1984,106,5731-5732. (10) Gubritte-Voegelein,F.; Gubnard, D.; Potier, P. J. Nat. Prod. 1987, 50, 9-16.
(11) Holton, R. A.; Juo, R. R.; Kim, H. B.; Williams, A. D.; Haruaawa, S.; Lowenthal, R. E.: Yogai,S. J. Am. Chem. SOC. 1988,110,6558-6560. (12) Denis, J.-N.; Greene, A. E.; GuBnard, D.; GuBritte-Voegelein,F.; Mangatal, L.; Potier, P. J. Am. Chem. SOC. 1988,110,5917-5919. (13) Witherup, K. M.; Look, S. A.; Stasko, M. W.; McCloud, T. G.; Issaq, H. J.; Muschik, G. M. J. Lip. Chromatogr. 1989, 12, 2117-2132. (14) Harvey, S. D.; Campbell, J. A.; Kelsey, R. G.; Vance, N. C. J. Chromatogr. 1991,587, 300-305. (16) Cardellina, J. H., 11. J. Liq. Chromatogr. 1991,14,659-665. (16) Jaziri, M.; Diallo, B. M.; Vanhaelen, M. H.; Vanhaelen-Faatre, R. J.; Zhui, A.; Becu, A. G.; Homes, J. J. Pharm. Belg. 1991,46, 93-99.
0 1992 American Chemlcai Soclety
2314
ANALYTICAL CHEMISTRY, VOL. 84, NO. 20, OCTOBER 15, 1992
'
'"1
e
4
1 d
"
526 1534
'001
I x5
586
I
r
-267 so
(R-H)+AcOH
526
I mass-to-charge
Flguro 1. MSlMS product Ion spectrum of the taxol radlcal anbn produced by colllsion-lnduced dlssoclatbn uslng an argon target and a collision energy of 24 eV. The loss of the neutral species, R. corresponds to cleavage of the acyl-oxygen bond of the C13 side
chain.
raphy (TLC)," fast atom bombardment mass spectrometry,18 and combinations of techniques such as HPLC and thermospray mass spectrometry,'Q do not provide both rapid analysis of crude extracta and low limite of detection. Tandem mass spectrometry (MS/MS) is particularly well suited for trace analysis of target compounds in complex matrices.20-24 This report presenta a highly specific and extremely sensitive parent tandem mass spectrometric method for screening plant extracts for taxol, cephalomannine, and baccatin I11usinga triple quadrupole mass spectrometer. A preliminary account of this result has been given elsewhere.%
m-
500
800
7"
EXPERIMENTAL SECTION Extraction Procedure. Plant needle and barksamples were collected,dried, ground, and extracted in 95 % ethanol overnight. The resulting solution was filtered and evaporated to dryness. After partitioning between methylene chloride and water (l:l), the methylene chloride fraction was dried and partitioned between hexane and (90%) methanol and the methanol fraction was analyzed using the MSIMS parent scan. Analysis Procedure. A Finnigan triple-stage quadrupole (TSQ) 700 was used for the analysis of samples. Radical anions of the taxanes were generated in an ammonia chemicalionization plasma by electron attachment*% after sample molecules had been thermally desorbed from a direct evaporation probe. Under the ionization conditions used during these experiments, some [M- HI-was also produced but was not utilized in the screening procedure. (17) Staeko, M. W.; Witherup, K. M.; Ghiorzi, T. J.; McCloud, T. G.; Look, 5.;Muechik, G. M.; k q , H.J.J. Liq. Chromatogr. 1989,12,21332143. (18) McClure, T. D.; Reimer, M. L. J.; Schrm, K. H. Proceedings of
the 38th ASMS Conference on Mass Spectrometry and Allied Topics; Tucson, 1990, pp 1008-1009. (19) Auriola, 5 . 0 . K.; Lepiet8, A.-M.; Naaranlahti, T.; Lapinjoki, S. P. J. Chromatogr. 1992,594, 153-158. (20) Roush, R. A.; Cooks,R. G. J. Nat. Prod. 1984,47, 197-214. (21) Busch, K. L.; Gliih, G.L.; McLuckey, S. A. Mass Spectrometry/ Mass Spectrometry; V C H New York, 1988. (22) Fetterolf, D. D.; Yost, R. A. Znt. J.Mass Spectrom. Zon Processes 1984,62,33-49. (23)Lau, B. P.-Y.; Scott, P. M. Proceedings of the 32nd ASMS Conference on Mass Spectrometry and Allied Topics;San Antonio, 1984; pp 789-790. (24) Plattner, R. D.; Bennett, G.A.; Stubblefield, R. D. J. Assoc. Off. Anal. Chem. 1984,67,734-738. (25) Wood, J. M.; Hoke, S.H.; Cooks, R. G.;Chang,C.-j.; Heinatein,
P. F. Proceedings of the 39th ASMS Conferenceon Mass Spectrometry and Allied Topics; Nashville, 1991; pp 1679-1680. (26) yon Ardenne, M.; Steinfelder, K.; Tummler, R. Elektronenanlugerungs-massenepektrometric Organischer Substanzen;Springer: Ber-
lin. -1971. ----. - - -(27) Hunt, D. F.; Sethi, S. K. J. Am. Chem. S O ~1980,102,69534963. .
(28)Dillard, J. G. Chem. Rev. 1973, 73, 589-643.
586
J,
"1 20
500
800
7"
eo0
mass-to-charge
Flgwo 2. (a) Fulkcan, slngle-stage,mass spectrum of a plant extract containing less than 10 ng of each of the taxanes. (b) Parent ion spectrumof mlz 528 of the same plant extract. (c)Parent ion sped" of a mixture of taxol, cephalomannlne, and baccatln 111 standards. Spectra In (b) and (c)were produced by cdHskn-lnduced dissociation on an argon target at 40 eV. Taxol (mlz 853), cephalomnnlne (mlz 8311, and baccatln 111 (mlz 588) are represented In all three spectra.
Collisional activation was generally achieved with 30-eV collisions upon an argon target gas at 0.5 mTorr in quadrupole 2 during the parent ion scan of mlz 526. To decrease the limit of detection, multiple reaction monitoringwas performed in which a 10-Da window around the molecular ion regions of taxol, cephalomannine, and baccatin I11 was scanned in 0.05 8. Quantitation data were taken in the profile mode. Analysis of each plant extract required two measurements. First, 1 pL of the plant extract (1 mg/mL) was placed on the filament and the ion current for each of the taxanes was recorded. Second, 1pL of the sample was spiked with taxol, cephalomannine, and baccatin I11 and reexamined. The spike was typically 1, 5, or 10 ng depending on the ion current recorded from the sample alone. This entire process took approximately 10 min. The concentration of each of the taxanes in the samplewas then determined from a plot of the ion abundance verses the amount of taxane added.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
RESULTS AND DISCUSSION The MS/MS product ion spectrum of taxol (Figure 1)is recorded by ma-selecting the radical anion in quadrupole 1, effecting collision-induced dissociation in quadrupole 2, and scanning quadrupole 3 to determine the masses of the fiagment ions. This spectrum shows several neutral losses which are characteristic of taxol including the loss of acetic and benzoic acids. A prominent peak at mlz 526 is observed in this spectrum and also in the MS/MS product ion spectra of cephalomannine and baccatin 111. Because the ion mlz 526 is common to the product ion spectra of these three taxanes, a parent ion scan was utilized to screen for allthree taxanes simultaneously. With this type of scan, the ion current recorded by the electron multiplier results from ions that lose a neutral species and form mlz 628, therefore,the method is selectivefor moleculescontaining the same diterpene skeleton as taxol. To illustrate the selectivity of the method for the target compounds, the parent ion spectrum (Figure 2b) is compared to a conventional single-stage m w spectrum (Figure 2a) of the same plant extract. Note the presence of ions representative of the three species of interest, taxol (mlz 853), cephalomannine (mlz831),and baccatin I11(mlz 586) in both spectra. However, there are many unknown ions present in the mass spectrum and the signal to noise ratio is poor compared to the parent scan. Figure 2c is a parent scan of a mixtureof the three standard compounds. All three taxanes have been recognized in plant extracts at levels less than 500 pg; however, the ion current recorded for the taxanes is expected to decrease in matrices containing high concentrations of species with positive electron affmities. The parent ion scan of mlz 526 was tested as a rapid screening method for texanes by examining needle and bark extracts from a variety of Taxus species. Table I displays the weight percentages of each of the taxanes in various plant samples ea determined by the MS/MS methodology. The Taxusbrevifolia bark extract containedthe largest percentage of taxol, as expeded.6JS A Taxus c u s p i d a t a fresh bark extract was run four times with the concentrations of the taxanes determined as 3.70 f 1.15 ng1pL for taxol, 3.15 f 1.01nglpL for cephalomannine, and 0.56 f 0.11 ng/pL for baccatin 111, with the 90% confidence interval displayed. The reproducibility of the method is adequate, but its main value is in rapidly screeninglarge numbers of plant extracts to determine approximate taxane concentrations.
2915
Table I. Weight Percentages of Taxol, Cephalomannine, and Baccatin I11 in Various Taxus Species As Determined by MS/MS plant part needle twigbark needle twig bark needle bark T.floridana needle old bark T.canadensis needle bark T.baccata needle old bark sample T.cuspidata (dry) T.cuspidata (fresh) T.brevifolia
% taxol
0.0050 0.0065 0.0069 0.0030 0.0036 0.084
0.012 0.0046 0.019 0.0029 0.0070 0.0026
% % cephalomannine baccatin I11
0.0015 0.0023 0.0066 0.0025 0.00054 0.0096 0.0054 0.0013 0.0043 0.0013 0.0020 0.0011
0.00022 0.00042 0.00035 0.00045 0.00078 0.021 0.00067 0.00090 0.00073 0.0022 0.00040 0.00069
Because intense ion signals were recorded for most of the samples analyzed, a T. c u s p i d a t a fresh needle sample was extracted using an abbreviated procedure. The sample was ground and extracted for 30 min in 95 7% ethanol. The taxanes were determined as 0.0046 % (wt % ) for taxol, 0.0017 ?4 for cephalomannine and 0.00031 % for baccatin 111. Using this 30-min extraction procedure, 5.0 mg of extract was obtained from a single T.cuspidata needle. This extract was dissolved in ethanol (1mgImL), and 1pL of the resulting solution was analyzed. The presence of these three taxanes in a single needle was.confirmed in less than 1-h combined extraction and analysis time. These results demonstrate the great potential of tandem mass spectrometry for rapid selection of high taxane producing species from hybrid yews cultivated by numerous nurseries or from plant tissue culture specimens.
ACKNOWLEDGMENT The authors thank Dr. Richard Spjut of World Botanical Associates for collection of the T.breuifolia, T. baccata, T. canadensis, and T.f l o r i d a n a samples and for identification of the Taxus species at Purdue University from which the T. c u s p i d a t a extracts were taken. This work was supported by the National Cancer Institute, R01 CA55118. S.H.H.acknowledges support from BASF Corp. RECEIVED for review July 20, 1992. Accepted August 20, 1992.
