Anal. Chem. 1995, 67, 1711- 1716
Comparison between High=ResolutionSelected Ion Monitoring, Selected Reaction Monitoring, and Four=SectorTandem Mass Spectrometry in Quantitative Analysis of Gibberellins in Milligram Amounts of Plant Tissue Thomas Moritz* and Jorunn E. Olsent Department of Forest Genetics and Plant Physiology, Swedish Universify of Agricultural Sciences, S-901 83 UmeA, Sweden
Gibberellins (GAS) were quantified in s e m i p d e d plant extracts by combined gas chromatography/mass spectrometry (GC/MS). High-resolution selected ion monitoring (HR-SIM), selected reaction monitoring (SRM),and four sector tandem mass spectrometry (MS/MS) were compared. The best selectivitywas found with four-sector MS/MS analysis, but the sensitivity was too low for analysis in milligram amounts of plant tissue. HR-SIM and SRM had similariy low limits of detection, except for the analyses of G&g, but SRM provided better selectivity. The variations of sensitivity and selectivityfor the Merent MS methods are discussed. Gibberellins (GAS) are a group of about 100 diterpenoic acids which act as endogenous growth regulators in plants. In order to evaluate the role of endogenous GAS in plant growth and development, the ability to analyze and quantify of GAS in small amounts of plant tissue is a necessity. GAS occur in picrogram to nanogram per gram fresh weight concentrationsin most plant tissues, and plant extracts are very complex multicomponent mixtures. As a consequence, accurate analysis of endogenous GAS is dependent upon the use of methodology that offers not only high sensitivity but also high selectivity.' Combined gas chromatography/mass spectrometry (GUMS) has become an important tool for identification and quantification of GAS^-^ and is now, together with immmunoa~says~.~ the most widely utilized technique for quantitative analysis. Low-resolution mass spectrometry (LR-MS) provides an accurate assay for the determination of GAS in HPLC-puriiied plant extracts. However, this method does not give acceptable results with semipurified plant extracts containing high levels of impurities. With doublefocusing high-resolution mass spectrometry (HR-MS), the selectiv+ Present address: Department of Plant Physiology and Microbiology, IBG, University of Tromse, N-9037 Tromss, Norway. (1) Crozier, A; Reeve, D. R Anal. Proc. 1 9 9 2 , 29, 422-425. (2) Crozier, A; Moritz, T. In Physico-chimical methods ofplant hormone analysis; Libbenga, K, Hall, M. A, Eds.; Elsevier: Amsterdam, in press. (3) Hedden, P. In The use of combined gas chromatography-mass spectromety in the analysis ofplant growth substances, Modem methods of Plant analysis; Linskens, H. F., Jackson, J. F., Eds.; Springer Verlag: Heidelberg, 1986, Vol. 3, pp 1-22. (4) Gaskin, P.; MacMillan, J. GC-MS of gibberellins and related compounds: methodology and a library of spectra; Cantock's Enterprises; Bristol, U.K, 1991. (5) Atzom, R; Weiler, E. W. Planta 1983, 159, 7-11. (6) Weiler, E. W.; Wieczorek, U. Planta 1981, 152, 159-167.
0003-2700/95/0367-1711$9.00/0 0 1995 American Chemical Society
R1
GAt GAB GA20 GA20
R2
H
OH OH H H
OH
H OH
GAto Flgure 1. Structures of gibberellins analyzed in this study.
ity of the analysis increases and some of the problems caused by interfering substances are overcome. Another approach is to monitor a metastable decomposition reaction, i.e., selected reaction monitoring (SRM)? which has been shown to be useful for trace analysis of compounds in biological sample^.^^^ A further develop ment of this technique is tandem mass spectrometry with foursector tandem mass spectrometers or hybrid instruments, which have been used in analysis of dioxins and related compounds.10 This paper presents a microscale method for quantification of GAS in milligram amounts of plant tissue and compares the accuracy and precision of HR-MS, SRM-, and four-sector MS/ MSbased analyses. EXPERIMENTAL SECTION Chemicals. Nonlabeled GAs (Figure l), originally from Professor N. Takahashi (Riken, Wako-Shi, Japan), were supplied by Dr. Alan Crozier, University of Glasgow, Glasgow, U.K. [17,(7)Gaskell, S. J.; Millington, D. S. Biomed. Mass Spectrom. 1978,5,557-558. (8) Chess, E. IC; Gross, M. L. Anal. Chem. 1980, 52, 2057-2061. (9) Thome, G. C.; Gaskell, S. J. Biomed. Mass Spectrom. 1 9 8 5 , 12, 19-24. (10) Huang, L. Q.; Eitzer, B.; Moore, C.; McGown, S.; Tomer, K B. Biomed. Mass Spectrom. 1991,20, 161-168.
Analytical Chemisrry, Vol. 67, No. 10, May 15, 1995 1711
17-2H2]-GAswere purchased from Professor L. N. Mander, Research School of Chemistry, Canberra, Australia. Extraction and Purification. Seedlings of Salk pentandra L. ecotype from 69"39' N were grown in fertilized peat at 18 "C in a 24-h photoperiod." A 1-10 mg 50 pg weighed sample (Mettler AE160, AI3 Hugo Tillquist, Spinga, Sweden) of plant material was homogenized in liquid N2 and extracted with continuous shaking in 500 pL of 80%aqueous MeOH for 2 h at 4 "C, with 50 pg of [17,17-2Hz]GA19,[17,17JHzlG&, [17,17JHzlGA1, [17,17-2Hz]G&,and [17,17-2H~lGA~9 added as internal standards. After extraction, the sample was applied to a pre-equilibrated 1W mg aminopropyl Bond Elut cartridge (Varian Associates, Harbor City, CA). The cartridge was washed with 1mL of MeOH before being eluted with 5 mL of 0.2 M formic acid. The formic acid eluate was run directly onto a pre-equilibrated 5Wmg CIS Bond Elut cartridge (Varian) which was washed with 2 mL of HzO, pH 3, and then eluted with 4 mL of MeOH. Mass Spectrometry. The samples were methylated with ethereal diazomethane, and after evaporation under a stream of Ns, they were dissolved in 250 pL of MeOH and loaded onto a 1Wmg aminopropyl Bond Elut cartridge. The methylated GAS were eluted with 3 mL of MeOH, which was reduced to dryness and trimethylsilylated in 10 pL of N-methyl-N-(trimethylsilyl)tritluoroacetamide (MSTFA) at 70 "C for 30 min. The derivatization mixture was then reduced to dryness and dissolved in n-heptane. Samples were injected with a Hewlett Packard 7673 splitless autosampler into a Hewlett Packard 5890 GC equipped with a 25m x 0.25"-i.d. fused silica capillary column with a chemically bonded 0.25" SE-30 stationary phase (Quadrex, New Haven, CT). The injector temperature was 270 "C. The column temperature was held at 60 "C for 2 min and then increased by 20 "C min-' to 200 "C and by 4 "C min-' to 260 "C. The column effluent was introduced into the ion source of a JEOL JMSSX/SXlOBA four-sector tandem mass spectrometer of BIEIB& geometry (JEOL,Tokyo, Japan). The interface and the ion source temperatures were 270 "C and 250 "C, respectively. Ions were generated with 70 eV at an ionization current of 600 pA. Full-scan mass spectra were obtained at a rate of 1 s scan-' for a mass range of 50-800 amu. With each sample, n-alkanes, c23-c2S,'2 were co-injected to determine Kovats retention indices. Tandem mass spectra of [2H~lGA1 and [2H21G&9were obtained by selecting m/z 508 with MS1 and colliding the selected ion in a collision cell at a potential of 3 kV in the third field-free region of the instrument. The daughter ions resulting from collisionactivated dissociations (CADS) were analyzed by scanning the MS2 with a rate of 2 s scan-'. The CAD spectra were recorded by decreasing the main beam attenuation to 70%with helium as the collision gas. The resolution of both parent and daughter ions was about 1000. High-resolution selected ion monitoring (HR-SIM) measurements were performed using acceleratingvoltage switching from 10 kV. Perfluorokerosene was used as reference compound, using a suitable lock mass. The dwell time was 50 ms, and for each GA, two ions and two deuterated analogues were recorded: G&o
*
(11) Olsen, J. E.; Moritz, T.; Jensen, E.; Junttila, 0. Physiol. Plunt. 1994, 90, 378-381. (12) Gaskin, P.; MacMillan, J.; Fim, R D.; Pryce, R J. Phytochemisty 1971,10, 1155-1157.
1712 Analytical Chemistry, Vol. 67,No. 70, May 75, 7995
18-20.0 min; GA19 20.0-21.5 min; GAI and G&9,21.5-23.5 min; and GAS, 23.5-25.5 min. In the SRM mode, the acceleration voltage was 10 kV, and the precursor ions were selected by magnetic switching. The daughter ions formed in the first field-free region were detected by switching the magnetic field and the electrostatic field simultaneously. The dwell time was 100 ms, and the reactions m/z 418 to 375 and 420 to 377 for G&o, m/z 434 to 375 and 436 to 377 for GA19, m/z 506 to 448 and 508 to 450 for GA1 and G&9, m/z 594 to 448 and 596 to 450 for G& were recorded at the time interval described above. Xenon was used as the collision gas. The four-sector MS/MS measurements were performed by using an acceleration voltage of 10 kV, selecting the precursor ions by MS1 at a resolution of 1000 and the daughter ions by switching the magnetic field and the electrostatic field simultaneously by MS2. The resolution of the daughter ions was set to 1000. The dwell time was 100 ms, and the same reactions as for the SRM measurements were registered. The collision cell was at ground potential, and helium was used as the collision gas by decreasing the main beam attenuation to 70%. In all the MS modes, calibration curves were recorded from 2 to 40 pg of GA, with 10 pg of [2H21GA,as the internal standard, with the exception of the four-sector MS/MS measurements, where the calibration curve was from 5 to 100 pg of GA, with 25 pg I2H21GA,,as the internal standard. As nonlabeled G& was not available, no calibration curve was recorded. Instead, the quanti6cation results were calculated after corrections for nonlabeled G& in the internal standard and isotopic contributions of [M 2]+ from endogenous G&. All data were processed on a JEOL MSMWOlOD data system. Analyses of Precision. Precision in GC/MS analyses with the different MS methods was estimated by measuring of 2- and 4Gpg GA samples with 10 pg of [2H21GAsas internal standard. The integrated area ratio between m/z GA, and m/z [2H21GA, and the relative standard deviation (RSD) for (5) replicates were calculated. Precision in GC/MS analyses of plant extracts was investigated using three pooled extracts which were analyzed three times by HR-MS (R = 5000) and SRM. Estimation of the precision of GA analysis in a plant extract was performed by homogenization of a S. pentandra leaf in liquid nitrogen and division of the sample into ten l@mgsamples, which were further extracted and purified by the method described above. The final analyses were made in the HR (R = 5000) and SRM modes. The amount of endogenous GAS in each extract, mean amount, and RSD were calculated. GAS in Milligram Amounts of Elongating Plant Tissues. For a rapid screening of GAS in the apical part of elongating S. pentandra seedlings, the following replicate samples were prepared and analyzed as described above: (1) 5 mm of the upper stem of four plants; (2) 10 mm of the subapical stem; (3) the uppermost three small leaflets from the same four plants; and (4) an individual leaf, about 6 mm from the apex.
+
RESULTS AND DlSCUSSlON
One of the major problems associated with analysis of GAS is the large number of GAS and derivatives of GAS occurring in plant tissues. Many of these are structural isomers, with very small differences in mass spectra, e.g., GAI (3,9-0H) and epi-GAl (3aOH). GC retention indices can, however, be different Crable 11, and therefore retention indices are important criteria for identdying of GAS.^ On the other hand, as about 100 GAS have been
Table 1. Kovats Retention indices for QA-MeTMSi Derivatives
GAI 3-epi-GA1 G-42 GA3 isc-G& GA4 GA5 GA7 GAS GAS GAi2 GA13 GA14 GAl5 GA16 GA17 GA18 GAi9
2674 2801 2758 2698 2642 2519 2486 2539 2818 2330 2354 2603 2501 2627 2642 2584 2648 2606
G4o G-422 GA23 G44 G47 G49 GA31 GA34 GA35 GA36 GA37 GA3a GA39 GA4o GA44 GA5i GA53
2498 2701 2753 2460 2899 2680 2560 2670 2651 2610 2771 2933 2816 2539 2795 2534 2501
identitied, it does not necessarily follow that all GAS can be distinguished on the basis of retention times in individual GC systems. This can be a major drawback in analysis of a complex plant extract that has not been subjected to preparative HPLC purification. It is, therefore, very important that MS analysis of a partially purified extract is specifk in that it offers high selectivity. The development of tandem mass spectrometry, e.g. SRM, has been gaining acceptance as a sensitive and selective MS method for the analysis of trace components in complex biological ~amples.'~J~ An important operational parameter in SRM is the choice of a characteristic CAD reaction. Figure 2 shows the tandem mass spectra of the MeTMSi derivatives of [2H21GA~and [2H~1G&9, with parent ion m/z 508. Both GAS are structural isomers with slightly different retention times on the gas chromatograph and slightly different mass spectra. The tandem mass spectra show that for [2H~1GA~, the decompositionof m/z 508 to 450 is abundant and characteristic, corresponding to a loss of C3H60.4 [2H~1G&9 has abundant and characteristic losses of m/z 508 to 449, probably corresponding to a loss of CzH3O2; 377, and 305. For selectivity and very sensitive detection of GA1 and G&g by SRM, different CAD reactions should be registered for GA1 and G&g. However, because of software problems with the data system which are unlikely to be resolved by the manufacturer in the near future, in the present study only one of the reactions was detected together with the native GA reactions. Since GA1 is regarded as the biologically active GA in the regulation of shoot el~ngation,'~ and therefore the most important GA to analyze in a plant extract, the reactions m/z 506 to 448 and m/z 508 to 450 were monitored. It should, however, be emphazised that G&g is still of interest when estimating GA turnover rates. Another factor that can be of importance when considering the SRM and four-sector measurements presented below is that different collision gases were used. SRM utilized Xe from the FAB gas supply, while He was used for four-sector MS/MS. Although it is well known that different gases can have an effect (13)Dobson, R L. M.; Kelm, G. R ; Neal, D. M. Biol. Mass Spectrom. 1994,23, 75-81. (14)Rossi, S.A;Johnson, J. V.; Yost, R A Biol. Mass Spectrom. 1994,23,131139. (15)Phinney, B.0.In Gibberellin A I , d w a f i m and the control ofshoot elongation in higher plants; Crozier, A, Hillman, J. R, Eds.; Cambridge University Press: Cambridge, 1984;pp 17-41.
100-,rx5
i"
50
1
0
450
1
209 100
200
300
400
500 m/z
'OO'B i 209
50
0
I00
200
305 377 ~
300
1 400
;
500 m/z
Figure 2. GC/MS tandem mass spectra of [zH~]GA1-MeTMSi(A) and [2H2]GA29-MeTMSi (6).
on the fragmentation pattern, it was considered to be of minor importance in this instance as linked scan analyses of GA1 carried out on a single MS instrument yielded similar fragmentation patterns and comparable levels of sensitivity (data not shown). To evaluate the different MS methods, a series of replicate GA standards were analyzed. The calibration curves for G&o show a hyperbola form (Figure 3). The regression coefficients were in all cases between 0.993 and 0.998. The shape of the calibration curve depends on the number of labeled atoms in the internal standard. Since there are only two deuterium atoms in the internal standard, the effect of the natural isotope abundances on the linearity of calibration curves is rather large.16J7 The reproducibilities of peak ratio response for five replicate analyses corresponding to 2 and 40 pg were between 1and 12%Cable 2). The precision varies with both GA and MS technique. The analyses of GA19 in the four-sector MS/MS mode show high variation in the low picogram region. This is probably because these analyses are near the detection limit. Colby et a1.18 have discussed the importance of the amount of internal standard relative to the native compound for reliable in quantification by isotope dilution. Several parameters influence the reliability, but it is the degree of isotopic substitution in the internal standard and the difference in masses between the native compound and the internal standard that are of particular importance. Although it is possible to calculate the limits of response ratios, in practice, the response of native versus labeled compounds should be within the range of the calibration curve. The sensitivity of the analysis varied considerably with the method used and the GA analyzed. For G&o, the detection limit (16)Pickup, J. F.;McPherson, K. Anal. Chem. 1976,48, 1885-1890. (17)Colby, B. N.;McCaman, M. W. Biomed. Mass Spectrom. 1979, 6, 225230. (18)Colby, B.N.;Rosecrance, A E.; Colby, M. E.Anal. Chem. 1981,53,19071911. Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
1713
3.0
o
E E& a"
2.5 2.0
1.5 1.0
0.5 0 3.0
g
E
t
R=10 OOO
2.5
8
2.0 1.5
=
1.0
8
t
0.5 0 3.0
.Q
e8
2,5 2.0 1.5
a"
1.0
0.5 0
0.5 1 .O 1.5 2.0 2.5 3.03.54.0 Amount ratlo
Figure 3. Calibration curves for GA20-MeTMSi. Table 2. Precision of Response R a t k DetGmInGlon (%) by HR-SIM, SRM, and M S M S
GAzo 40pg
2pg
HR-SIM ( R = 5000) HR-SIM ( R = 10000) SRM MS/MS
5.5 7.3 4.4 4.6
4.1 3.8 0.9 0.8
GAi9
GAi
zpg
40pg
2pg
40pg
5.5 4.2 4.3 12.2
3.1 3.0 4.1 1.2
4.6 4.2 1.7 4.4
3.8 3.8 1.0 2.5
In the MS/MS measurements, 5 and 1Wpg GA samples were
analyzed; 10 pg of [2H2]GA was cc-injected in HR-SIM and SRM analysis, and 25 pg of f2H21GAwas co-injected in MS/MS analysis.
of the analysis, with a signal-to-noise (S/N) ratio of about 5, was about 50 fg in the HR mode (R = 5OOO), 100 fg in HR loo00 and SRM modes, and 1 pg in the four-sector MS/MS mode. SRM was suitable for the analysis of GAI but not for the analysis of the structural isomer G&g due to reasons described above. Although theoretically GA29 should not be detected by SRM, in practice it was possible. This was probably due to the poor resolution of 1714 Analytical Chemistty, Vol. 67, No. 70, May 15, 7995
the parent ion, giving a detection in the reaction m/z 507 to 448 corresponding to [l3CllG&g. The detection limit for GAB was, however, approximately 5 times higher than that of GA1. The sensitivity in the four-sector MS/MS mode was rather low, 0.810 pg depending on which GA, and it was not possible to detect GAB due to the high resolution of the parent ion compared to the SRM analysis. Although the selectivity of the four-sector MS/ MS technique is superior to that of HR-MS, the low sensitivity is a disadvantage because of the very low amounts of GAS in plant tissues. A method for microscale extraction of GAS in milligram amounts of plant tissue was developed. M e r extraction, the sample was applied to a weak anion exchange cartridge. Further purification involved a CU cartridge and, after methylation, a weak anion exchange cartridge that separates the GAS from nonmethylated compounds which may interfere with the G U M S analysis. The specificity of the analysis is shown in Figure 4. One plant extract was analyzed with different MS methods. The mass chromatograms for the HR measurements of G&o show additional peaks near the analyte. These interference peaks were not a problem in the determination of G&o. However, for GA19,GAS, and occasionally several of the internal standards (data not shown), the mass chromatograms often show serious interference. In contrast, the SRM mass chromatogram traces show relatively few additional peaks near the GA peak of interest, with the exception of GA19. This is even more apparent for the four-sector MS/MS analysis, which provides the most specific analysis. Although SRM is more specific than HR-MS and the resolution of the parent ion can be increased by adjusting the entrance slits of the instruments, the high specificity found in the four-sector MS/MS measurements is an advantage for analysis of this kind. Unfortunately, it was not possible to use MS/MS measurements for all GAS of interest, since the sensitivity was too low for several of the GAS. However, preliminary results show that four-sector MS/MS can be used for the analysis of larger plant extracts (data not shown), where sensitivity is a minor problem but the specificity is crucial. It should be emphazised that the pudication method has to be modjfied when using larger extracts, otherwise problems with a overloaded GC column can occur. A major advantage of the HR-SIM measurements is that the accuracy of the analysis can be checked by recording two ions from the native GA and two from the deuterated analogues. Comparing isotope ratios of several fragments is, in practice, not possible in by SRM. Although this disadvantage of the SRM methodology should not be neglected, due to the high specificity of the SRM analysis, reliable results can be obtained only by observing the chromatographic peak shape for detecting interfering substances. In order to estimate accuracy and precision of the GC/MS analysis of a plant extract, three pudied extracts were pooled and analyzed three times by HR-SIM (R = 5000) and SRM (Table 3). The results were identical for most of the GAS analyzed, with approximately 5% RSD. The only exception was the measurements of GAS, with a rather large difference in the estimated amounts of G& between HR-SIM and SRM. It should be emphasized that an arithmetic correction procedure was used instead of a calibration curve. Another approach would be to use an isotope dilution fit program based on a least-squares algorithm, which has been shown to be an accurate method for quantilication
180
18.5
19.0
19.5
20.0
20.0
20.5
21.0
21
21 5
220
225
230
23
23.5
24.0
245
25.0
25.5
20.0
20.0
20.5
21.0
215
21.5
22.0
225
23.0
23.5
23.5
240
24 5
250
255
23 5
240
24.5
25.0
25.5
II
R=10 000
180
18.5
19.0
19.5
SRM
L
%-.+--mW_.+. 180
185
190
195
200
200
205
210
215
215
220
225
230
235
23.5
24.0
24.5
25.0
25.5
Figure 4. Mass chromatogram for GA20-MeTMSi, GA19-MeTMSi, GAI-MeTMSi, GA29-MeTMSi, and GAa-MeTMSi detected in a 10-mg plant tissue sample by (a) HR-SIM ( R = 5000),(b) HR-SIM ( R = 10 000), (c) SRM, and (d) four-sector MS/MS. Table 3. Comparison of Quantification Results. of GAm, QAls, QA1, GAm, and a& in Three Combined Extracts of Sallx pentandra by QC/MS/SiMand QC/MS/SRM
G4o GAi9 GAi GA29 GAS
HR-MS
SRM
6.4 f 0.28 (4.4) 1.8 f 0.13 (8.7) 5.2 f 0.11 (2.1) 3.1 f.0.16 (5.3) 2.8 f 0.12 (4.4)
6.7 f 0.44 (6.5) 1.5 f 0.13 (8.6) 4.8 f 0.15 (3.1) 4.2 f 0.21 (5.0) 1.4 f 0.08 (5.2)
In nanograms of GA per gram fresh weight. The RSDs (%) are given in parentheses. of GAS.l9 Croker et al.I9 showed that an arithmetic calculation procedure gave consistently higher estimates than both the calibration cuve and the isotope dilution fit methods. This reflects the importance of the calibration curve in the present study. The precision of the extraction method was also estimated, by extraction and purification of 10 identical leaf samples Vable 4). The precision was shown to be rather low for most of the GAS analyzed. The analysis was made by GC/HR-SIM (R= 5000) and SRM. The large variation in method error is probably due to weighing, extraction, and pipetting errors. The variation between different MS methods was small, as expected, with little variation for SRM with GA1, GA19 and G&o, but less satifactory with G&s. The method was used for rapid screeening of GAS in the elongating region of two S. pentandra plants. The results (Table (19) Croker, S.
J.; Gaskin, P.; Hedden, P.; Macmillan, J.; Macneil, K. A G.
Phytochem. Anal. 1994,5, 74-80.
Table 4. Precision of GA Analysis (%) in I O independent Leaf Extracts of Sallx pentandra by HR-MS and SRM
GAzo GAi9 GAi G49 GAS
HR-MS
SRM
13.2 24.3 8.4 11.8 18.2
12.9 23.8 7.8 12.1 19.8
Table 5. Gibberellin Levelsmin Dffferent Plant Parts of Sallx pentandra
GAi9
G4o
GAi
G49
GAS
8.2 7.5 9.3 7.6 6.5 4.9 4.9 5.9
6.7 5.8 5.0 5.8 6.9 6.2 4.8 6.4
9.5 9.7 16 15 7.9 6.8 6.2 8.6
7.2 6.5 7.8 7.5 3.9 5.1 2.2 3.4
3.8 4.3 4.4 5.0 2.7 3.6 1.5 1.2
shoot apices shoot apices subapical subapical apical leaves apical leaves leaves leaves (I
In nanograms of GA per gram fresh weight.
5) clearly show large d&erences in GA content in different parts of the plant. The active hormone GA115 was highest in the subapical and apical parts of the stem. This is in agreement with earlier suggestions that GAS act on the subapical meristem. CONCLUSIONS
The ability to analyze a large number of GAS in a plant extract was demonstrated. The methods described facilate the rapid Analytical Chemistv, Vol. 67, No. 10, May 15, 1995
1715
quantification of GAS in plant extracts without the need for timeconsuming purification steps. The MS methods used are sensitive and enable milligram quantities of plant tissue to be analyzed. The MS mode of choice depends on the GAS analyzed. SRM and four-sector MS/MS have the advantage that the amount of impurities interfering with the GA analysis is decreased. However, since there are problems associated with the analysis of some of the GAS by SRM, and especially four-sector MS/MS, HR-SIM was considered to be a better choice when it was necessary to analyze a large number of GAS.
1716 Analytical Chemistiy, Vol. 67,No. 70,May 15, 7995
ACKNOWLEDGMENT Financial support was provided by The Swedish Natural Science Research Council (T.M.) and The Bo Rydins Foundation
U.E.0.). Received for review November 8, 1994. Accepted March 1, 19951.~ AC941090R @
Abstract published in Advance ACS Abstracts, April 1, 1995
