1212
Anal. Chem. 1992, 64, 1212-1216
Isotope Dilution Liquid Chromatography/Mass Spectrometry Using a Particle Beam Interface Daniel R. Doerge,'J Mike W. Burger$ and Steve Bajicll Departments of Environmental Biochemistry and Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, and VG BioTech, Altrincham, Cheshire, U.K.
The use of a particle beam (PB) interface for quantltatlon by Irotope dllutlon LC/MS was Investigated. Coelutlon of slnglelabeled Internalstandards (IS)wlth natlve compounds caused enhancement of the ISslgnal. The magnitude of enhancement for [3-1aCllcaffelne was affected by several experlmental parameters, but no differences were observed In the 12C/13C response ratlos under these condltlons or upon anaiyte lntroductlon vla a gas chromatography (GC) Interface. No coelutlon enhancement was observed wlth [1,3,7-13Calcaff elne, d e " t r a t l n g that mass transfer eff ects and chemkal complex formation do not affect PB transmission efficiency. Spectral overlap between natlve analyte and IS peaks and nonlinear detector responses cause the observed coelutlon enhancement. These results confirm that PB/LC/MS does not have Inherent limitations for use in isotope dllutlon experiments as they have been performed by GCIMS. An equation was derived that permlts accurate calculatlon of Isotope dilutlon results using a single- or multiple-labeled IS. Application of thls equatlon could allow expanslon of the Isotope dllutlon technique performed by PB/LC/MS or GC/MS to Include singlelabeled IScompounds wtthout the need for nonlinear regresslon analysis of calibratlon curves.
INTRODUCTION The introduction of particle beam (PB) interfaces to mass spectrometry' (MS) has held great promise since the electron impact (EI)spectra obtained contain the fragmentation necessary for matching with library spectra of reference compounds obtained from gas chromatography (GC) or direct insertion probe interfaces.2-4 In addition, the analytical sensitivity is compatible with many biomedical and environmental analyses of polar, nonvolatile, thermally labile compounds by liquid chromatography (LC).2-4 The use of PB/LC/MS as a tool for quantitative analysis of organic compounds has been limited by the fluctuations in absolute response observed in intra- and interday The ideal way to avoid this problem would be the use of
* Address correspondence to this author at his present address: National Center for Toxicological Research, Jefferson, AR 72079. Department of Environmental Biochemistry, University of Hawaii at Manoa. Department of Chemistry, University of Hawaii at Manoa. 11 VG BioTech. (1) Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984, 56, 2626-
*
2631. (2) Doerge, D. R.; Miles, C. J. Anal. Chem. 1991, 63, 1999-2001. (3) Behymer, T. D.; Bellar, T. A.; Budde, W. L. Anal. Chem. 1990,62, 1- fi~lfi-i - -- -fiw - - -. (4) Kim, I. S.; Sassinos, F. I.; Stephens, R. D.; Wang, J.; Brown, M. A. Anal. Chem. 1991, 63, 819-823.
(5) Bellar, T. A.; Behymer, T. D.; Budde, W. L. Am. SOC.Mass Soectrom. 1990, 1, 92-98. (6) Brown, F. R.; Draper, W. M. Biol. Mass Spectrom. 1991,20,515521. (7) Doerge, D. R.; Cooray, N. M.; Yee, A. B. K.; Niemczura, W. P. J. Labelled Compd. Radiopharm. 1990,28, 739-742.
isotopically labeled internal standards.6 However, a putative deficiency of PB/LC/MS comes from reports that it may not be applicable to quantitative studies which employ isotopically labeled internal standards.5 Using two different commercial PB/LC/MS systems, Bellar et al. described the enhancement of analyte signals upon coelution with either isotopically labeled internal standards or mobile-phase additives (e.g., ammonium acetate). The proposed mechanism of signal enhancement was chemical complex formation leading to larger particle size and, hence, increased efficiency transmission through the PB interface. A recent study demonstrated that reliable isotope dilution MS waa possible using a PB interfaceeand coelution enhancement wm observed but its origin was not defined. The purpose of the present study was to investigate the origin of the reported coelution enhancement effects in order to further develop quantitative isotope dilution techniques using PB/LC/MS.
EXPERIMENTAL SECTION Materials. 3-Amino-1,2,4-triazole(amitrole, AT) and ammonium acetate (HPLC grade) were purchased from Sigma Chemical Co. (St. Louis, MO); caffeine and ethylenethiourea (ETU) were purchased from Aldrich Chemical Co. (Milwaukee, WI). [2-13C]ETUwas synthesized from 99% atom excess 13CS2 (CambridgeIsotope Labs, Woburn, MA) as previouslydescribed: [5-13C]ATwas synthesized as previouslydescribed8from 99 atom % [13C]formicacid (AldrichChemicalCo.), [3J3Cl]caffeine(99.6 atom % ) was purchased from MSD Isotopes (Los Angeles, CA), [1,3,7-13C31caffeine(99 atom % ) and atrazine-da (98 atom % ) were purchased from Cambrige Isotope Labs. LC solvents, acetonitrile (Optima, Fisher Scientific, Fairlawn, NJ) and Milli-Q water (Millipore Inc., Bedford, MA), were degassed by helium sparging. Mass Spectrometry. Mass spectrometry was performed using a VG Trio 2A equipped with the LINC PB interface (VG BioTech, Altrincham, U.K.) and either a Hildebrand (Leeman Labs) or a concentric pneumatic nebulizer (VG Analytical). Helium flow into the nebulizer was maintained at 40 psi, the temperature of the gas flow through the desolvation chamber was maintained at 30 "C (except where specifically noted and in caffeine analysis where 40 "C was used), a source temperature of 200 "C was maintained, and typical operating pressures were 5,0.8,3X mbar at the first stage momentum separator pump, the second stage momentum separator pump, and the ion source housing, respectively. Positive ion spectra were obtained using E1 (70 eV) and full scan (50-650 mlz in 1 s) conditions. The amount of analyte was determined from the area under the mass chromatogram of the specified ion (usually M+). The mass spectrometer was tuned and calibrated daily using perfluorotributylamine. For determination of caffeine content in coffee, selected ion recording of 108, 109, 110, 111 mlz fragment ions and 193, 194, 195,196miz molecular ions (0.08-s acquisition time per channel, 0.02-s interchannel delay, span = 0.4 amu) and the concentric nebulizer were used. Mass spectra for labeled and unlabeled standards were obtained from 100-ng samples under identical (8) Doerge, D. R.; Niemczura, W. P. Chem. Res. Toxicol. 1989,2,100103.
0003-2700/92/0364-1212$03.00/0 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
conditions (mobilephase, flow rate, acquisition mode, nebulizer type) to those used in the respective isotope dilution analysis. Average ion intensities were obtained from at least four determinations. Liquid Chromatography. LC was performed using a PerkinElmer Series 10 isocratic pump in either flow injection mode (FIA) using 50% acetonitrile/water at 0.5 mL-min-' or using a Novapak C18 column (Millipore, Milford, MA) for atrazine separations(98%acetonitrile/waterat 0.5 ml-min-l)or a PRP-1 column (HamiltonCo., Reno, NV) for caffeine (20% acetonitrile/ water at 0.5 ml-min-'). Mixtures of standards were prepared in mobile phase with a fixed amount of labeled internal standard (IS) and varying amounts of native analyte in ratios from 1:1to 1:100, and 10-pLaliquotswere injected. It was determined that no shift in mass chromatograms occurred for any native/labeled pair either in FIA or on-column modes of operation. Commercially brewed samples of regular and decaffeinated Kona coffeewere passed through 0.45-pm filters (ArcoDisc, Fisher Scientific) and diluted 100-fold with Milli-Q water. Aliquots were analyzed by LC/UV using a Novapak C18 column at a flow rate of 1.5 ml-min-l with detection at 274 nm. The amount of caffeine present in regular and decaffeinated Kona coffee samples was determined from linear regression of the standard curve. For isotope dilution experiments with caffeine and atrazine, 2.5 ng-pl-l of the IS was present along with 1-100 ngpL-1 of the native analyte, and 10-pL aliquots were analyzed. Gas Chromatography. Standards containing 2.5-5 pg/mL [3-W]caffeine and 1.&150 pg/mL native caffeine were dissolved in acetonitrile, and 0.3-1.0-pL aliquots were analyzed by EIGC/MS, and positive ion spectra were obtained from full scans (50-650 m/z in 1s) with a VG Trio 2A using a 25-m DB5 column (0.25 pg, 1 mL.min-' He flow, splitless injection at 50 O C , temperature program 50-200 "C at 25 degmin-l, with transfer line and injector temperatures of 250 "C).
RESULTS AND DISCUSSION The present study used a commercial PB/MS system that is different from those previously evaluated394 in that interchanging nebulizer design is a simple process. In this study, two different pneumatic nebulizers were used a Hildebrand double-grid type and a concentric type that is similar to that used in the two commercial PB/MS systems previously evaluated. The performance characteristics of these and other nebulizers with respect to sensitivity and band spreading parameters have been described elsewhere.9 Qualitatively, the droplet spray emanating from the concentric nebulizer appeared much finer and more homogeneous than that from the Hildebrand nebulizer which exhibited larger variation in droplet size and signal pulsations. However, the magnitude of response to a given amount of most compounds tested was 2-5-fold higher with the Hildebrand nebulizer.9 Other experimental parameters affecting the desolvation process are the temperature of the gas flow through the desolvation chamber and solvent composition. While only small differences in signal intensity for 10 ng of ETU were observed in the temperature range of 25-50 OC, the response increased ca. 4-fold in going from 25 to 100% acetonitrile in water. As previously described,5 addition of ammonium acetate (50 mM) to the mobile phase increased the magnitude of response for all analytes tested ca. 3-5-fold (data not shown). Initial experiments showed that the signal for analytes was also enhanced upon coelution with an isotopic variant incremented by a single mass unit (e.g., [WIETU, [WIAT, [13C11caffeine). This enhancement was mirrored by enhancement of W-labeled IS signal by the native analyte in the reciprocal experiment, although the magnitude was slightly different. This observation was in accord with previous reports of coelution enhancement for [15N21caffeine, benzidine-ds, 3,3-dichlorobenzidine-d6,5and deuterated styrene metabolites.6 The magnitude of the coelution effect on (9) Miles, C. J.; Doerge, D. R.; Bajic, S.Arch. Enuiron. Contam. Toricol. 1992, 22, 247-251.
100
0
200
300
400
1213
500
' ~ - E T U (ng)
Flgure 1. Coelution enhancement for The signal enhancement for 10 ng of
[W]ETU signal by [12C]ETU. [W]ETU (peak area for m/r 103)was determined in the presence of varying amounts of coeluting [WIETU Injected uslng FIA. Enhancement factor = l3C signal wlth coeluting 1%+ 1% signal wkhoutcoelutlng 12C. The computed s e m & order regression plot of the data Is shown. IS was dependent on the amount of native ETU injected over the range of 10-500 ng (see Figure 1). As previously described,2 no signal enhancement was observed when small amounts of IS and native compound coeluted (e.g., 10 ng each [WIETU and [l3C1ETU). The coelution enhancement factor (mlz 103peak area with coeluting [WIETU + m/z 103 peak area for noncoeluting) for 10 ng of 13C-labeled ETU in the presence of a 50-fold amount of nitive ETU was found to vary with (a) nebulizer type (concentric > Hildebrand by 2-fold); (b) mobile-phase composition (ca. 2-fold increase in going from 25 to 100% acetonitrile in water); (c) temperature of the desolvation chamber gas flow (ca. 5-fold increase by changing the temperature from 25 to 50 "C); (d) the presence of ammonium acetate (3-fold greater without 50 mM ammonium acetate); and (e) the compound tested (caffeine > ETU > AT). It should be noted that the magnitude of the coelution enhancement was considerably greater than that expected from contribution of the (M + 1)+ion of the native compound. For example, the enhancement was ca. 15-fold when 500 ng of native ETU and 10 ng of Kl3C1ETUcoeluted vs that predicted from the natural abundance contribution from [WIETU (ca. 3%) to the (M 1)+peak of ca. 2.6-fold. The possibility that self-CI effects were operative in the PB inlet system was investigated. This effect would be concentration-dependent and would have the net effect of increasing the (M + 1)+peak and decreasing the M+ peak. However, over the range of amounts used in this study, no changes within experimental error in the (M + l)+/M+ratio were observed for any analyte tested. An example is shown for labeled and native ETU in Figure 2. Data for a given compound were collected and analyzed in the fashion of an isotope dilution experiment by plotting M+/ (M 1)+peak area ratio (isotope response ratio) vs amount of native analyte with addition of a constant amount of 13Clabeled IS. The isotope response ratio plots for each of the three single-labeled IS/naive pairs showed a curvilinear dependence as shown in Figure 3 for ETU. These plots were compared under the different experimental conditions tested above (a-d). Despite the dramatic effects on the magnitude of the coelution enhancement noted above, no change in the isotope response ratio plot was observed for any of these parameters. An example is shown in Figure 3 for two different desolvation temperatures. These results suggested that the observed coelution enhancement was due to an experimental artifact that equally affected both isotopic forms and canceled when native/IS response ratios were calculated. This suggested some possible origins for the "coelution effect": (1)A mass-dependent transfer efficiencyeffect where transmission of the IS through the PB interface is more efficient in the presence of larger amounts of total analyte
+
+
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
1214
10
5
0
200
100
300 ETU (ns)
0
10
400
500
+
Flgurr 2. Invariance of (M l)+/M+ ratio with total amount of ETU. The intensities for (M 1)+ and M+ ions were determined in triplicate from the mass spectrum of either natlve or [ l%]ETU over the range
+
6 / AGC,high gain
of amounts used in the enhancement studies. The data are plotted as the averages, and the error bars are standard deviations.
'2C/''C
0
EONCEl4lR4nON (UTI0
i 10
20
30
40
''C/'k CONCENTRATION RATIO Figure 4. Isotope response ratio plots for W/MS and PB/LC/MS analysis of caffeine. The response ratios (peak area for m/r 194 peak area for m/r 195)for [3-Wl]caffeinein the presence of varylng
+
amountsof [l2C]caffe~injectedweredeterminedfoWowingintroduction into the mass spectrometer via PB interface using FIA or GC capliary column. Inset: Signal enhancement for a constant amount of [313Cl]caffeine(m/r 195)in the presence of varying amounts of [%Icaffeine for introductlon via PB or t% interface at normal or high detector gain settings. The computedfirst-andsecondorder regression plots of the data are shown.
0
!
50 0
100
12C-ETU 200 (ng) 300
400
500
Flgurr 9. Isotope response ratio plots for ETU at different desolvation temperatures. The lW1%response ratios (peak area for m/z 102 + peak area for m/z 103) for 10 ng of [l%]ETU in the presence of varying amounts of [l%]ETU injected using FIA were determined at desolvatlon gas flow temperatures of 25 and 50 O C . The computed secondorder regression plot of the data is shown.
present in the particle beam. Chemical interactions such as complex formation or solvation, which lead to larger particle sizes, cause an increase in transmission efficiency through the particle beam. This has been proposed as the basis of the ammonium acetate and IS coelution enhancement effect.5~6 (2) The contribution of spectral overlap in the observed MS signals. The effect of spectral overlap is not limited to the natural abundance contribution of native to the (M 1)+ peak and contribution to the M+ peak due to incomplete isotopic substitution of the IS. In addition, fragmentation can yield a significant amount of (M - 1)+peak for native and IS alike as is the case for two compounds tested in this study. Both caffeine and ETU have significant (M - 1)+fragment peaks. In this light, the problem of spectral overlap is not limited to cases where a single isotopic substitution has been introduced. Compoundswhich contain multiple labels often have significant amounts of unlabeled material and spectral overlap due to fragmentation can also occur. These hypotheses were tested experimentally. First, a parallel isotope dilution analysis of [3-Wllcaffeine using both PB and GC interfaces wasperformed. GC is the moat common MS interface used for isotope dilution studies,"' and no mass transfer effects have been reported in a direct capillary inlet system. Indeed, no evidence for a mass-dependent transfer efficiency effect with caffeine was observed with GC/MS (Figure 4, inset). Under conditions where large 'coelution effects" occurred with PB/LC/MS, coelution through the GC capillary interface of identical ratios of labeled and unlabeled compounds had no effect on IS signal. Because of the
+
(10)Colby, B. N.; McCaman, M. W. Biomed. Mass Spectrom. 1979, 6,225-230. (11) Colby, B. N.;Rosecrance, A. E.;Colby, M. E.Anal. Chem. 1981, 53, 1907-1911.
different dynamic ranges fro the two techniques, the total amount of analyte injected onto the GC was 33-fold lower and the relative response greater since the photodynode detector gain was only 67% of that used in the PB experiments. Thus, the presence of large coelution effects with the PB interface could be consistent with a mass-transfereffect where transmission through the PB interface is more efficient as the total amount of analyte passing through the PB interface is increased. It could also be an experimental artifact caused by the 33-fold difference in analyte injected or the difference in detector signals. However, in this case, as with the other experimental parameters affecting coelution enhancement, IS and native analyte are clearly affected equally as the enhancement effects cancel when the isotope response ratios are calculated (seeFigure 4). The isotope response ratio plots obtained from the two interfaces gave essentially identical curvilinear plots. These results confirm that there are no inherent limitations on the use of PB/LC/MS in isotope dilution experiments6as they have been previously performed by GC/MS.'O-12 Secondly,the PB/LC/MS isotope dilution experiment was repeated using [W31caffeine to eliminate spectral overlap of natural abundance and fragment peaks. In this case, essentially no coelution enhancement was observed by PB/LC/ MS (see Figure 51, and the plot of IS response ratio vs the amount of native coeluting analyte was linear over the concentration range tested with a slope near unity (slope = 1.02, see Figure 6). It is deemed unlikely that the small chemical differences induced by one vs three 13Csubstitutions could cause such a large change in the magnitude of the coelution effect observed for caffeine. Therefore, these results are inconsistent with any chemical interaction (i.e., complex formation) between native and labeled caffeine as a causative factor in the coelution effect. To better show the effect of spectral overlap between native and labeled peaks, the data from the same injections shown in Figure 5 were plotted as the enhancement factor observed for the (M - 1)+ fragment ion instead of the M+ for [W3]caffeine. This causes the maximum coelution effect to increase to approximately 18 (data not shown). This demonstrates that spectral overlap is required for the detection of coelution enhancement. (12) Rose,M. E.; Johnstone,R. A. W. Mass SpectrometryforChemists and Biochemists; Cambridge University Press: Cambridge, 1982; pp 98105.
ANALYTICAL CHEMISTRY, VOL. 64. NO. 11, JUNE 1, 1992
Chart I
0
400
200 1
2
600 ~
(ng) -
~
~
~ 0 5 Coelutknenhancementfor . [3-13Cl]-and [ 1,3,7-13C3]caffelne slgnals by ['C]caffeine. The signal enhancementfor 25 ngof labeled caffelne(peak area for mlz 195 or mlz 197)Inthe presence of varying amounEsof [lC]caffelneanalyzedbyPB/MSusingFIA. Thecomputed flrst-order regression plots of the data are shown.
0
0
100
200
'%-CAFFEINE
300
400
LH, * ~ ,I[ i
:jUk+,*
i\
1
1000
800 ~
r
1215
1
500
(ng)
Flguro 6. Isotope response ratlo plot using [ 1,3,7-13C3]caffelne.The response ratlos (peak area for m/z 197 peak area for m/z 194) were determlnedfolbwlng oncolumnseparatknfor samplescontaining 0-50 ng/pL [ICIcaffeine and 2.5 ng/pL [1,3,7-1SC3]caffelne. The computed flrstorder regression plot of the data is shown.
+
These data preclude a mass-transferefficiency effect caused by the PB interface when [W31caffeinecoelutes with native caffeine. This suggeststhat the observed differencesin [13Cllcaffeine signal enhancement seen with the PB and GC interfaces (see above) are caused by differences in detector response due to the different dynamic ranges used. This nonlinear detector response for the PB interface was confirmed by observing the detector response for 10-500 ng of [Wlcaffeine. Similar levels of signal *enhancement" were seen when comparing responses at low w high levels. However, the detector response was linear when caffeinewas introduced via the GC interface. It appeared that the larger signal intensity generated by GUMS, in spite of the larger sample loading used for PB/MS, allowed the detector to operate well within its linear region. The lower ion intensity levels produced by PB/MS could easily fall in the nonlinear toe region of the detector response curve. It was likely that the enhancement of detector response for the ca. 10% natural abundance 195m / z peak from the increasingamounts of ['TIcaffeine could cause the apparent enhancement of IS response in the PB experiment. This hypothesis was confirmed when the GUMS experiment was performed at low sensitivity such that the detector gain required was comparable to that used in the PB experiment. A nonlinear (exponential) response curve (peak area vs nanogram of caffeine) resulted that was similar to that seen in the PB experiment (data not shown). The coelution experiment was repeated under these conditions, and although there was no change in the 12C/13C response ratio plot (see Figure 41, large enhancements of 1% signal were observed upon coelution (see Figure 4, inset). These observations eliminate mass transfer and chemical effects on transmission efficiency through the PB interfaceand establish that spectral overlap of native analyte ions into the labeled
~
~
~
~
~
spectrum in conjunction with detector nonlinearity is the basis of the observed coelution effect. Detector nonlinearity is the probable basis for the exponential calibration curves in previous PB/LC/MS studies.5sgJ3J4 It has also been reported that mobile-phase additives caused linearization of calibration curves in several analyses.5J2J3 This effect has beenattributedto the formation of larger particles containing additive and analyte which are transported more efficientlythrough the PB interface.5 This has the result of increasing total detector response for a given amount of analyte. The observed linearization of calibration plots results because the "enhanced" amounts of analyte push the detector response into its linear range. Even though all spectral overlap and detector nonlinearity effects cancel when isotope response ratios are calculated, the isotope ratio plots using single-labeled IS compounds (see Figures 3 and 4) still deviate from the typical linear responses reported in quantitative isotope dilution studies employing GC/MSlOJlJ4 or PB/LC/MS.s In those previous studies, the recorded spectrum of a mixture of an unknown amount of native analyte in the presence of a known amount of labeled IS represents a mixture of two componentswith characteristic peaks a t m/z M and at n amu higher (M+ n). The ratio of the two peak heights HM and HM+"is used to calculate the ratio of native analyte to IS in the sample. This relationship has proven useful so long as the native analyte has no significantpeaks at maas M nor that the IS has nosignificant peaks at mass M. For this reason, IS reference compounds are typically selected with a mass shift of 3 amu or more and must be synthesized to a high degree of purity.12 This is an attempt to move the inevitable companion peaks associated with the labeled and unlabeled components far enough apart so that no significant overlap occurs. Under these conditione, a h e a r calibration line is obtained from plotting the peak height ratio of HM/HM+,vs the amount of native analyte in the presence of fixed IS.1+12 This relationship was found to hold when [1,3,7-13Cdcaffeine (Figure 6, slope = 1.02, r2 = 0.998) and atrazine-& (data not shown, slope = 1.09, r2 = 0.9998) or deuterated styrene metabolites6 were used as IS compounds in quantitative isotope dilution employing PB/ LC/MS. However, when spectral overlap occurs from either (1) normal isotope patterns, (2) isotopic contamination in the labeled IS, or (3) fragmentation, a very different relationship is observed which appears to be a second-order function (see Figures 3 and 4). Efforts to resolve this nonlinear dependence initially used a computer simulation which revealed that the curvilinear function was predicted by the condition of spectral overlap. The simulation also yielded curves that were nearly exact matches for the observed data. In order to solve the spectral overlap problem, Chart I was prepared. If XHMis the peak height of the observed mixture spectrum at mass M and XHM+,is the observed peak height
+
(13) Kim, I. S.; Sasinos, F. I.; Stephens, R. D.; Brown, M. A. J. Agric. Food Chem. 1990,38,1223-1226. (14) Incorvia Mattina, M. J. Proceedings ofthe 39thASMS Conference on Mass Spectrometry and Allied Topics; 1991; pp 131Cb1311.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
+
of the mixture spectrum at mass M n, the curve representing the ratio of these two peak heights plotted vs the amount of unknown is completely characterized by the amount of unknown, U,the amount of labelled IS, L , the peak heights of the unlabeled and labeled compounds at masses M and M n where UHM,LHM,UHM+,,and LHM+nare the normalized peak heights obtained experimentally from spectra of unlabeled and labeled standards. The mathematical relationship is
+
--
xHM+n
-
and
u/L= xHMLHM+,/xHM+,uHM The plot of UIL vs xHM/xHM+, is a straight line passing through the origin with a slope equal to the ratio of the standard peak heights, UHM and LHM+,. In the case that UHM and LH,, are both 100% peaks in the respective spectra, the slope = 1 and the line makes at 45O angle with the axes. This is the equation of a typical calibration plot widely used in isotope dilution studies employing GC/MS.lOJ1 The presence of spectral overlap between the unlabeled and labeled compounds makes the situation more complicated:
[uH,(U/L)I + LHM XHM+, [ u H ~ + , ( u / L )+l LHM+, Since the standard peak heights and the amount of added IS (L)are known, U is the only variable and the equation is of the form: XHM --
+ b,
The function is a quotient of two equations of the form mX b, a rational function of degree one. As U approaches infinity, the function approaches uHM/ UHM+,. As a result, the plot approaches a limit line parallel to the X-axis at u&/ UHM+,,the peak ratio for unlabeled analyte. This prediction was confirmed experimentally in this study for ETU where UJ UM+,= 18and [13Cllcaffeinewhere U M / UM+,= 9. Figures 3 and 4 show that the isotope response ratio plots approach these limiting values as the amount of unlabeled analyte becomes large. When U = 0, the curve has a y-intercept at LHM/LHM+,, the peak ratio for the labeled IS. The general solution for UIL in the spectral overlap case is
+
UIL =
xHMLHM+n-
xHM+nLHM
xHMuHM+n-
xHM+nuHM
sample decaffeinated + 40 ng duplicate injection decaffeinated + 40 ng duplicate injection av
LC/UV
LC/UV
(UH,&)+OL ou + (LHM+,L)
xHM/xHM+, =: mlU + bl/m,U
~~~
regular duplicate injection regular duplicate injection av
(UHMU + (LHML) xHM+n (UHM+,U) (LHM+,L) In the absence of spectral overlap this becomes XHM
-=
XHM
Analysis of Caffeine Content in Coffee
T a b l e I.
(1)
The results from application of eq 1have been compared to those from the computer simulation and are identical. This equation, however,can be solved using a hand-held calculator. This approach, which uses only the experimentally-derived maas spectral fingerprintsfor labeled and unlabeled standards has been used to calculate the amount of native standards known mixtures containing labeled IS for the followingsinglylabeled compounds: [3-l3C1caffeine,[5-l3C1AT,and [2-W]ETU. The calculated values were found to be linearly correlated to a high degree (r220.999)with the spiked amount of unlabeled analyte. In addition, all calculated values are essentially equal to the spike level (average slope = 1.03 f 0.07). This approach is equally applicable to either the molecular ion or fragments ions that contain an isotopic label
molecular ion data, ng
fragment ion data, ng
40.8 41.0 40.9 40.4 40.8 0.3 40.4 0.1 44.7 45.1 48.4 46.2 46.1 f 1.7 42.9 h 0.6
37.9 38.3 36.7 37.6 37.6 0.7 44.2 46.8 48.4 43.4 45.7 2.3
since the values used in eq 1 are obtained experimentally from spectra determined under conditions identical to those used in each analyticalprocedure. It is also completelygeneral in its applications since no assumptions are made about the relationship between percent enrichment and relative peak heights. This can be especially important in cases where protonation or deprotonation leads to significant amounts of (M + 1)+and (M - 1)+peaks, respectively, in the mass spectra. This PBILCIMS isotope dilution method was validated by analyzing the caffeine content in coffee using on-column PB/ LC/MS and comparingthe values with those determined using LCIUV. Regular coffee samples were spiked with a constant amount of [3-W]caffeine (or [1,3,7-13C31caffeine). In addition, known amounts of [l2C1caffeineand IS were spiked into decaffeinated coffee. Analysis of these samples by oncolumn PBILCIMS and computation of analyte levels according to eq 1 gave accurate values compared with those determined by LCIUV with good precision (see Table I). This method validation on authentic samples gives additional confidencein the use of quantitative PBILCIMS with isotopic internal standards containing either single or multiple labels.
This study c o n f i i that PBILCIMS is a viable quantitative technique6 and describes the basis for the enhancement of IS signal upon coelution. These results extend the isotope dilution technique to include thermally labile, nonvolatile, polar compounds that are not amenable to chromatographic analysis by GC. The equation derived here yields accurate results from isotope dilution analysisusing single-labelIS compoundsand does not require nonlinear regression analysis of calibration curves.1s The application of eq 1could permit an expansion in the number of potential isotope dilution studies that could be performed by either PBILCIMS or GCIMS because many potential IS compounds cannot be synthesized containing multiple isotopic substitutions or to high isotopic purity at reasonable costs. In addition, the use of singly-labeled IS compounds can also be important because their properties more closely resemble those of the unlabeled ones.
ACKNOWLEDGMENT This paper is submitted as Journal Series No. 3656 from the Hawaii Institute for Tropical Agriculture and Human Resources. RECEIVED for review October 10, 1991. Accepted March 16, 1992. (15) Trager, W.F.; Levy, R. H.; Patel, I. H.; Neal, J. N. Anal. Lett. 1978, B l l , 119-133.
