Ion trap mass spectrometry for kinetic studies of stable isotope labeled vitamin A at low enrichments

Article abstract of DOI:10.1021/ac970816r

The role of β-carotene in chemoprevention of cancers and other chronic diseases generated controversy when sub-populations taking β-carotene supplements showed increased mortality in clinical trails. Determination of the dynamics of β-carotene in individual human subjects has emerged as a high priority. Stable isotope labeled β-carotene tracers can be employed to determine rates of conversion to retinol (vitamin A), but tracer doses must be small to minimize perturbation of endogenous retinoid and carotenoid pools. In such cases, ratios of labeled tracer/endogenous retinol are often low, and quantitative analysis at enrichments of <1 mol % are unreliable owing to ion-molecule reactions that generate ions at the same mass as the labeled tracer even when no tracer is present. The current study demonstrates improved gas chromatography/mass spectrometry quantification of retinol-d₄ and unlabeled retinol, as their tert-butyldimethylsilyl ethers, at low enrichments using an ion trap mass spectrometer operated in selected ion storage mode. Electron ionization of analyte takes place in the ion trap using conditions that eject ions outside the range m/z 390-420, and molecular ions at m/z 400 and 404 from retinol and retinol-d₄ are quantified. Using this approach, unlabeled retinol yields a signal close to values calculated from natural isotopic abundances (~0.13%), whereas several quadrupole instruments operated using selected ion monitoring yielded 2-5 times greater signal when no labeled retinol was present.

Full text of DOI:10.1021/ac970816r

Anal. Chem. 1998, 70, 1369-1374
Ion Trap Mass Spectrometry for Kinetic Studies of
Stable Isotope Labeled Vitamin A at Low
Enrichments
Stephen R. Dueker,† Roger S. Mercer,‡ A. Daniel Jones,‡ and Andrew J. Clifford*,†
Department of Nutrition and Facility for Advanced Instrumentation, University of California, Davis, California 95616
supplements showed increased incidences of cancer and mortal-
ity.4 Other studies suggested â-carotene supplements were
neither beneficial nor harmful.5 These conflicting observations
highlight a need for an improved understanding of the conversion
of â-carotene to its metabolites and delivery of bioactive caro-
tenoids and retinoids to various tissues and organs in humans.
To establish the role of dietary â-carotene and vitamin A in
human health, stable isotope labeled â-carotene is administered
orally and the resulting time-dependent concentrations of plasma
metabolites such as retinol are determined using gas chromatog-
raphy/ mass spectrometry (GC/ MS). Such investigations are
facilitated by use of â-carotene-d8, which is metabolized to retinol-
d4.6,7 The use of multiple heavy isotopes minimizes interferences
from isotopomers containing naturally occurring 13C, 2H, 18O, and
29Si and 30Si (in silyl derivatives) and improves measurements at
low enrichments.
In retinol-d4/ retinol GC/ MS analyses, the deuterated tracer
(retinol-d4) must be measured in the presence of substantial excess
of endogenous unlabeled retinol. Thus, the critical analytical
performance characteristics are instrument dynamic range and
measurement precision at low enrichments. For retinol measure-
ments, the sensitivity of the method is rarely limiting because
blood concentrations of retinol are significant and relatively
constant (∼1 µM), and sufficiently large blood samples may be
drawn and processed. Even in the case of low retinol-d4 enrich-
ment, precision and accuracy are more limited by interference
from unlabeled retinol.
The role of â-carotene in chemoprevention of cancers and
other chronic diseases generated controversy when sub-
populations taking â-carotene supplements showed in-
creased mortality in clinical trails. Determination of the
dynamics of â-carotene in individual human subjects has
emerged as a high priority. Stable isotope labeled â-caro-
tene tracers can be employed to determine rates of
conversion to retinol (vitamin A), but tracer doses must
be small to minimize perturbation of endogenous retinoid
and carotenoid pools. In such cases, ratios of labeled
tracer/ endogenous retinol are often low, and quantitative
analysis at enrichments of <1 mol % are unreliable owing
to ion-molecule reactions that generate ions at the same
mass as the labeled tracer even when no tracer is present.
The current study demonstrates improved gas chroma-
tography/ mass spectrometry quantification of retinol-d 4
and unlabeled retinol, as their ter t-butyldimethylsilyl
ethers, at low enrichments using an ion trap mass
spectrometer operated in selected ion storage mode.
Electron ionization of analyte takes place in the ion trap
using conditions that eject ions outside the range m / z
3 9 0 -4 2 0 , and molecular ions at m / z 4 0 0 and 4 0 4 from
retinol and retinol-d 4 are quantified. Using this approach,
unlabeled retinol yields a signal close to values calculated
from natural isotopic abundances (∼0 .1 3 %), whereas
several quadrupole instruments operated using selected
ion monitoring yielded 2 -5 times greater signal when no
labeled retinol was present.
The ability to accurately measure vitamin A isotopomer ratios
at low enrichments has been hampered using linear-beam quad-
rupole instruments by high and variable artifactual signal at the
mass of the deuterated isotopomer when nonlabeled retinol is
measured.8-10 Analyses performed on unlabeled retinol showed
more abundant signal at the mass of the deuterated isotopomer
than predicted by natural isotope abundances. Deuterio/ protio
â-Carotene is a phytochemical polyene and essential micro-
nutrient that is cleaved enzymatically to vitamin A (retinol) and
other metabolites including retinoic acid. Many of these metabo-
lites are potent modulators of cellular differentiation and prolifera-
tion. Lower incidences of some diseases including cancers have
been associated with consumption of diets rich in carotenoid-rich
vegetables.1-3 As a result, carotenoids have attracted attention
as potential chemopreventive agents and antioxidants. However,
in recent clinical trials, cigarette smokers adminstered â-carotene
(4) Albanes, D.; Heinonen, O. P.; Huttunen, J. K.; Taylor, P. R.; Virtamo, J.;
Edwards, B. K.; Haapakoski, J.; Rautalahti, M.; Hartman, A. M.; Palmgren,
J.; et al. Am. J. Clin. Nutr. 1 9 9 5 , 62, 1427S-30S.
(5) Hennekens, C. H.; Buring, J. E.; Manson, J. E.; Stampfer, M.; Rosner, B.;
Cook, N. R.; Belanger, C.; LaMotte, F.; Gaziano, J. M.; Ridker, P. M.; Willett,
W.; and Peto, R. N. Engl. J. Med. 1 9 9 6 , 334, 1145-9.
(6) Novotny, J. A.; Dueker, S. R.; Zech, L. A.; Clifford, A. J. J. Lipid Res. 1 9 9 5 ,
36, 1825-38.
* To whom correspondence should be addressed: (e-mail) ajclifford@
ucdavis.edu; (fax) (916) 752-8966.
† Department of Nutrition.
‡ Facility for Advanced Instrumentation.
(1) Peto, R.; Buckley, J. D.; Sporn, M. B. Nature 1 9 8 1 , 290, 201-8.
(2) Ziegler, R. G. J. Nutr. 1 9 8 9 , 119, 116-22.
(3) Mayne, S. FASEB J. 1 9 9 6 , 10, 690-701.
(7) Dueker, S. R.; Jones, A. D.; Smith G. M.; Clifford A. J. Anal. Chem. 1 9 9 4 ,
66, 4177-85.
(8) Handelman, G. J.; Haskell, M. J.; Jones, A. D.; Clifford A. J. Anal. Chem.
1 9 9 3 , 65, 2024-8.
(9) Dueker, S. R.; Lunetta, J. M.; Jones, A. D.; Clifford, A. J. Clin. Chem. 1 9 9 3 ,
39, 2318-22.
S0003-2700(97)00816-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/04/1998
Analytical Chemistry, Vol. 70, No. 7, April 1, 1998 1369

signal ratios of 0.4-1.0% are typical for unlabeled retinol. This
phenomenon has been documented with several linear-beam
single-quadrupole instruments and has been attributed to ion-
molecule reactions occurring in the mass spectrometer ion
source.8,9
It was our hypothesis that by sweeping out nontarget mol-
ecules from the ionization chamber using a complex broad-band
wave form, ion-molecule reactions would be minimized and
undesirable chemical noise eliminated. This reduction in noise
would expand the analytic range of the method to isotopic
enrichment levels lower than those previously obtainable on linear-
beam instruments. We applied this technique to measure retinol-
d4/ retinol ratios in human plasma from an individual administered
an oral dose of 30 mg of â-carotene-d8.
While many studies employing labeled vitamin A and â-caro-
tene tracers have used pharmacological doses of retinyl-d4 acetate
13
(20-45 mg),8-11 [8,9,19- C]retinyl palmitate (55 mg),12 or â-carotene-
d8 (30-120 mg),6,7,13 smaller doses more typical of daily dietary
intake are desirable for quantitating rate processes and evaluating
nutritional status by isotope dilution techniques; physiological-
sized doses will minimize perturbations of endogenous retinoid
and carotenoid pools and limit departure from steady-state kinetics
during the metabolic course of the tracer. Furthermore, admin-
istration of smaller doses reduces experimental costs and improves
the feasibility of epidemiological studies of larger populations.
Smaller doses, however, require increased instrument dynamic
range and precision at lower enrichments. The modeling of
â-carotene is further complicated by a low rate of bioconversion
where as little as one-sixth of a â-carotene dose is converted to
retinol when taken orally.14
EXPERIMENTAL SECTION
Chemicals. trans-â-Carotene-10,10′,19,19,19,19′,19′,19′-d8 (â-
carotene-d8) and retinyl-10,19,19,19-d4 acetate were purchased from
Cambridge Isotope Laboratories (Woburn, MA). Isotopic purity
for the deuterated retinol was 83% retinol-d4, 17% retinol-d3;
â-carotene-d8 was 80% â-carotene-d8, 16% â-carotene-d7, and 4%
â-carotene-d6. N-Methyl-N-(tert-butyldimethylsilyl)trifluoracetamide
(MTBSTFA) was purchased from Regis (Morton Grove, IL) in
glass ampules.
Sample P reparation. Serial blood specimens were drawn
over a 16-day period from a healthy 22-year-old female volunteer
(55 kg) who ingested 30 mg of â-carotene-d8 followed by a low
carotenoid breakfast consisting of a bagel with cream cheese and
a glass of low fat milk. Blood was collected in glass tubes
containing K3EDTA and the plasma separated by centrifugation
and stored at -20 °C prior to analysis. Retinol isotopomers were
extracted using a single-column solid-phase extraction (SPE)
method previously described,7 converted to their tert-butyldi-
methylsilyl ethers (tBDMS-retinol and -retinol-d4) with MTBSTFA,8
and stored at -20 °C until analysis by GC/ MS.
GC/ MS Conditions. The GC/ MS experiments were carried
out using a Varian Saturn 4D quadrupole ion trap mass spectrom-
eter (Varian Associates, Walnut Creek, CA) running Saturn
Revision 5.2 software. SIS experiments were created using SIS
Version 1.0 software. The system was equipped with a Varian
Star 3400CX gas chromatograph and a 1078 universal capillary
injector. The GC was fitted with a DB-23 column (50% cyanopro-
pylpoly(dimethylsiloxane); J&W Scientific, Folsom, CA) 15 m in
length, with an internal diameter of 0.25 mm and a phase thickness
of 0.15 µm. A helium head pressure of 12 psi was used; the carrier
gas also acted as the buffer gas within the ion trap. The injector
and MS transfer line were maintained at 250 °C. The GC oven
temperature was 140 °C (initial hold 1 min), then 25 °C/ min to
195 °C, then 12 °C/ min to 218 °C, and then 25 °C/ min to 255 °C
(final hold 2 min). The ion trap manifold was held at 200 °C.
The molecular masses of tBDMS-retinol and -retinol-d4 are 400
and 404 D, respectively. Analyses were performed using electron
impact ionization in the SIS mode to store ions between m/ z 390
and 420. The voltage adjustment factor (VAF) was set at 175%.
This factor controls the amplitude of the applied rf (radio
frequency) wave form. The manufacturer does not document the
actual value of the voltage controlled by this factor. The
background mass was set to m/ z 48 during the SIS experiments;
this corresponds to a qz value of ∼0.11 for the molecular ions.
Spectra were recorded using an m/ z range of 240-420 using the
AGC feature and an ion current target value set at 8000.
Instrument Calibration. The normal physiological concen-
tration of retinol ranges from 1 to 1.5 µM (286-429 ng/ mL) in
The search for improved precision at low enrichments led to
our examination of ion trap mass spectrometry (ITMS). Ion trap
mass spectrometers have the ability to selectively accumulate ions
and then eject them in a rapid burst to a detector. This
concentrating effect creates higher effective concentrations seen
by the detector, leading to high sensitivity. Traditionally, they
have had a limited dynamic range because of the requirement
that total ion density in the trap does not exceed ∼105 ions.15-17
Without other developments, ITMS instrumentation would prob-
ably not offer many advantages over linear-beam instruments for
retinol isotopomer analyses. However, two developments, the
selected ion storage (SIS) scan functions and automatic gain
control (AGC) have significantly improved the versatility of these
instruments for quantitative analysis.17,18 The SIS scan function
enables the selective storage and/ or ejection of single ion species
using multifrequency wave forms. This feature can increase
instrument sensitivity through the removal of unwanted ions from
the sample matrix and column bleed (gas chromatography
applications). Automatic gain control prevents the buildup of
excessive space charge by maintaining the correct number of ions
in the trap through the adjustment of ionization time, leading to
a greatly improved dynamic range.
(10) Haskell, M. J.; Handelman, G. J.; Peerson, J. M.; Jones, A. J.; Rabbi, M. A.;
Awal, M. A.; Wahed, M. A.; Mahalanabis, D.; Brown, K. H. Am. J. Clin.
Nutr. 1 9 9 7 , 66, 67-74.
(11) Furr, H. C.; Amedee-Manesme, O.; Clifford, A. J.; Bergen, H. R.; Jones, A.
D.; Anderson, D. P.; Olson, J. A. Am. J. Clin. Nutr. 1 9 8 9 , 49, 713-6.
(12) Reinersdorff, D. v.; Bush, E.; Leverato, D. J. J. Lipid Res. 1 9 9 6 , 37, 1875-
85.
(13) Tang, G.; Andrien, B.; Dolnikowski, G. FASEB J. 1 9 9 6 , 10, 3 (Astract 1386).
(14) World Health Organization. Handbook on Human Nutritional Requirements;
WHO: Geneva, Switzerland, 1974; No. 61, p 66.
(15) March, R. E.; Hughes, R. J. Quadrupole Storage Mass Spectrometry; Chemical
Analysis Series 102; Wiley: New York, 1989.
(16) March, R. E., Todd, J. F. J., Eds. Practical Aspects of Ion Trap Mass
Spectrometry; CRC Press: New York, 1996; Vol. I-III.
(17) Stafford, G. C.; Taylor, D. M.; Bradshaw, S. C.; Syka, S. C. 35th ASMS
Conference on Mass Spectrometry and Allied Topics, Denver, CO, 1987; p
775-6.
(18) Buttrill, S. E., Jr.; Shaffer, B.; Karnicky, J.; Arnold, J. T. 40th ASMS
Conference on Mass Spectrometry and Allied Topics, Washington, DC, 1992;
p 1015-6.
1370 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998

human plasma.14 Calibration standards were prepared by adding
8670 ng of retinol from a spectrophotometrically calibrated solution
to 0.00, 19.7, 42.3, 84.6, 141.0, 423.0, 1692.0, and 8670.0 ng of
retinol-d4. These solutions were concentrated to dryness under
argon and derivatized in 100 µL of MTBSTFA to produce molar
ratios of 0.000, 0.0023, 0.0049, 0.0098, 0.0163, and 0.0488 of retinol-
d4/ retinol. Molar ratio standards of 0.1952 and 1.0000 of retinol-
d4/ retinol were also prepared though it was necessary to dilute
these standards by a factor of 10 with isooctane for MS analysis
(see Results section). Typically, 0.5 µL of standard was injected
onto the chromatograph. The best-fit line was determined by
least-squares linear regression of the peak area retinol-d4/ retinol
ratios and measured ratios.
Other Instrumentation. For comparative purposes, the
following linear quadrupole instruments were also tested using
the same standards and samples: VG Trio-2 mass spectrometer
(VG Masslab, Altrincham, U.K.), Hewlett-Packard MSD model
6890 mass spectrometer (Hewlett-Packard, Palo Alto, CA), and
Shimadzu QP-5000 mass spectrometer (Kyoto, Japan). Data were
acquired using 70-eV electron ionization and selected ion monitor-
ing (SIM) of fragments at m/ z 255 and 259, which were chosen
because molecular ion abundances were lower with the quadru-
pole instruments than the ion trap.
RESULTS
Ion trap mass spectra of tBDMS-retinol and tBDMS-retinol-d4
from m/ z 200-420 are presented in Figure 1. Molecular ions
are present at m/ z 400 and 404 for tBDMS-retinol and -retinol-d4
respectively. Major fragment ions are generated from the loss
of C4H9 [M - 57]+on the silyl group and cleavage between the
C-14 and C-15 [M - 145]+ carbons on the side chain, producing
fragment ions of m/ z 343 and 347, and 255 and 259, from tBDMS-
retinol and tBDMS-retinol-d4, respectively.
Figure 1. Electron ionization impact spectra from retinol and retinol-
d4 tBDMS silyl ethers. Molecular ions appear at m/z 400 and 404
and are sufficiently abundant for quantitative analysis. Major fragment
ions appear from mass losses of [M - 57]+and [M - 145]+. The mole
fraction of the deuterated compound was 83% retinol-d4, 17% retinol-
d3.
The molecular and main fragment [M - 145]+ ions are present
at approximately equal magnitudes in the ITMS-generated spectra
(Figure 1), and both were tested for their ability to serve as
quantitative analytes. The utility of the analytes was assessed by
the magnitude and reproducibility of the signal ratio (A404/ A400,
selectivity provided by the SIS method is apparent from the minor
abundance of ions outside the specified mass storage range in
the spectra (bottom panel) and the absence of closely coeluting
interferences in the chromatograms as evidenced by the sym-
metrical peak shape of the ion channel chromatograms.
Selected ion chromatograms of tBDMS-retinol from a VG
Trio-2 quadrupole mass spectrometer are shown in Figure 3. The
observed integrated ratio (A259/ A255) was 0.5% instead of the
theoretical value of 0.006% (based on natural abundances). The
observed ratio varied from 0.4 to 1.0% among the quadrupole
instruments tested (VG Trio-2, Hewlett-Packard MSD model 6890
and Shimadzu QP-5000 mass spectrometers) under conditions of
optimal tuning and mass resolution. Molecular ions were insuf-
ficiently abundant for reliable quantitative analysis though sub-
stantial signal at m/ z 404 was detectable during tBDMS-retinol
analysis.
A259/ A255) obtained from unlabeled retinol. The ratio (A259/ A255
)
obtained from analysis of the tBDMS-retinol fragment (SIS mass
window from m/ z 250 to 265, VAF 150%) was 0.33 ( 0.038%,
relative standard deviation (RSD) 11.5% (n ) 5), with a limit of
quantification of 0.44 mol % enrichment (mean + 3 SD). Quan-
titation at the molecular ion provided superior analytical results
owing to reduced background signal and improved precision.
Replicate ITMS analysis of tBDMS-retinol generated an observed
ratio (A404/ A400) of 0.13 ( 0.0095%, RSD 7.4% (n ) 5). This is
below the value predicted from natural abundances (0.21%). This
small discrepancy is attributed to truncation of weak signal during
signal digitization. The limit of quantification was determined to
be 0.16 mol % enrichment.
Selected ion storage chromatograms showing the dilution of
the retinol-d4 isotope in serial human plasma collected over 16
days following a 30-mg oral dose of â-carotene-d8 are presented
in Figure 2. The broad-band wave form was constructed so that
all ions between m/ z 390 and 420 were retained, while matrix
and fragment ions outside this m/ z range were ejected. The
observed signal ratio (A404/ A400) is 0.64%. Triplicate analyses
showed excellent precision with an RSD < 4% (n ) 3). The
The ITMS method was tested for linearity over molar concen-
tration ratios ranging from 0.0023 to 1.0000 (A404/ A400). Plots
generated over a 4-day period showed good linearity with r2
>
0.99 (n ) 5) though there was difficulty with the 0.1952 and 1.0000
standards. These samples required a 10-fold dilution to provide
accurate and consistent values. The dilution minimized mass
shifts that are attributed to ion-coupling interactions in the trap,
a concentration-dependent phenomenon that is observed when
ions of close secular frequencies are stored (see Discussion). In
Analytical Chemistry, Vol. 70, No. 7, April 1, 1998 1371

Figure 3. Selected ion chromatograms of a tBDMS-retinol standard
(m/z 255) acquired on a VG Trio-2 quadrupole mass spectrometer
illustrating the unexpectedly high background signal at the mass of
the deuterated isotopomer (m/z 259). The observed ratio of 0.5%
(A259/A255) exceeds the value of 0.02% predicted by natural abun-
dances. Background values of 0.4-1% were observed in the spectra
generated on a VG Trio-2, Hewlett-Packard 5972, and Shimadzu QP-
5000 quadrupole instrument.
Figure 2. Selected ion storage chromatograms showing dilution of
the retinol-d4 isotope in human plasma drawn 16 days following a
30-mg oral dose of â-carotene-d8. The calculated ratio (A404/A400
)
shows the plasma sample to contain 0.64% isotope (top panel).
Triplicate analysis showed excellent precision with an RSD < 4% (n
) 3). The efficiency of unwanted ion removal is apparent from the
minor abundance of ions outside the mass storage range (bottom
panel).
practice, these calibration points were not important in our
analyses since levels of enrichment of >5% were not exceeded.
Maximum labeling at day 1 after tracer administration was <5%
and declined rapidly thereafter. For this reason, the standard
curve is presented in Figure 4 over the range of 0.0023-0.0488
(y ) 0.679x + 5.374 × 10-4). As determined from the integrated
areas (A404/ A400), the observed relative molar response of retinol-
d4 centered on 0.68. The magnitude of this value can only partially
be explained by the isotopic profile of the standard (83% retinol-
d4 ). The RSD at retinol-d4/ retinol ratio 0.00488 was ∼3% (n )
5). Approximately 0.5 µL aliquots of the samples and standards
were injected onto the GC/ MS. Analysis of 197 pg of tBDMS-
retinol standard (injected) yielded a signal-to-noise ratio of 20 from
the chromatogram for m/ z 400, suggesting detection limits in the
low-picogram range.
The isotopic purity of the plasma retinol (that originates from
â-carotene-d8) and the synthesized retinol standard are expected
to differ since the retinol standard does not originate from the
â-carotene-d8 molecule. This difference is adjusted for as follows:
The â-carotene-d8 (â-carotene-d8 was 80% â-carotene-d8, 16%
â-carotene-d7, and 4% â-carotene-d6) is predicted to yield retinol
of 88% mole fraction retinol-d4 based upon central cleavage in the
intestine (80% â-carotene-d8 f 80% retinol-d4; 16% â-carotene-d7
f 8% retinol-d4 and 8% retinol-d3; the other â-carotene isotopomers
will not produce a tetradeuterated retinol); the standard is 83%
retinol-d4. Thus, the calculated value for the plasma ratio is
overestimated by 6% when determined from the calibration curve
Figure 4. Expanded view of the instrument response plots for
determination of retinol-d4/retinol generated using standard mixtures
of the isotopomers 0.0023-0.0488 (A404/A400). Mixtures were pre-
pared by adding varying amounts of retinol-d4 to fixed amounts of
retinol as described in experimental Section.
(constructed with synthesized retinol-d4) and all values must be
multiplied by 0.94 to obtain a truer estimate of the actual ratio.
Application of the SIS-ITMS method to plasma specimens
from an adult female who had ingested 30 mg of â-carotene-d8 is
shown in Figure 5. Following the point of maximum label
concentration at 4.6%, the ratio declines exponentially until a point
of maximum concavity, whereafter, the label concentration de-
1372 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998

extensive hydrogenation of retinol when hydrogen was used as a
carrier gas during GC/ MS analysis (unpublished observations).
These findings illustrate the unusual, but not unexpected, sus-
ceptibility of retinol to undergo addition reactions when hydrogen
atom concentrations become significant.
The molecular and main fragment (m/ z 400 and 255, respec-
tively, for the endogenous compounds) ions were tested for their
ability to serve as quantitative analytes. Natural abundances
predict a substantially lower background signal at the mass of
the deuterated isotopomer for the main fragment relative to the
molecular ion due to the absence of the silyl group (A259/ A255
0.006%; A404/ A400 0.21% for unlabeled tBDMS-retinol) though,
experimentally, the molecular ion showed lower background and
provided superior analytical results. The background ratio for
the fragment (A259/ A255) ion was 0.33 ( 0.038% enrichment vs 0.13
( 0.0095% for the molecular ion (A404/ A400). The high value
associated with the fragment ion is attributed to two processes:
(1) the SIS mode does not precisely isolate single mass, but rather
a mass range; this inability to precisely retain only the analyte
ions increases the potential for background signal due to the
coisolation of reactive fragments in the lower mass regions; (2)
intramolecular hydrogen transfers from the silyl group to the
polyene prior to fragmentation and similar intermolecular pro-
Figure 5. Changes in the ratio of retinol-d4/retinol in human plasma
following a 30-mg oral dose of â-carotene-d8. Dramatic differences
among individuals were observed in their relative abilities to metabo-
lize â-carotene to retinol (vitamin A).
clines linearly. Because of the low rate of bioconversion of
â-carotene to retinol, retinol-d4/ retinol ratios rarely exceed 5%.
Significantly higher ratios are seen in retinyl-d4 acetate experi-
ments due to the efficient absorption of this compound. The large
range encountered between the points of maximum labeling and
latter time points illustrates the importance of instrument linearity
over wide range of concentrations.
cesses. Even though a higher than expected signal (A259/ A255
)
was obtained with ITMS analysis of the fragment ion, the signal
was more reproducible than values ranging from 0.4 to 1% obtained
on linear-beam instruments.
Improved signal-to-noise ratio for the molecular ions was
associated with decreasing the frequency notch for the storage
conditions and increasing the modulation amplitude of the rf
storage setting using the VAF. It was observed that higher VAF
settings (range 50-200%) required wider mass windows to avoid
partial ejection of the molecular ions: a VAF setting of 75% with
a m/ z 398-408 mass window produced reproducible calibration
curves; this same window at 175% VAF produced nonlinearity in
the curves and a notable reduction in signal in the 404 channel,
leading to a plateau in the measured ratios at increasing retinol-
d4 concentrations. Large amplitudes are known to distort the
frequency domain cutoffs, causing the pulse to have a wider
frequency range than desired.23 Some loss of total signal was
associated with increasing VAF settings due to increased collision-
induced dissociation (CID) in addition to ion instability. A 175%
setting was selected with the 390-420 mass storage window
because it provided better precision, particularly with biological
samples, due to superior peak shape and an improved signal-to-
noise ratio.
Success with SIS depends on the avoidance of excess space
charge within the trap which appears when the total ion density
in the trap exceeds ∼105 ions. Above this ion concentration, space
charge effects negatively affect mass resolution and ion-molecule
reactions lead to degradation of mass spectral quality.15 Total ion
concentration is controlled though adjustment of ionization times
using the AGC. This feature is critical for GC/ MS applications
where the concentration of the analyte can vary by several orders
of magnitude across the eluting peak. In our analyses of biologic
samples, setting the target value above 14 000 occasionally
produced a loss of mass resolution and peak shape, presumably
DISCUSSION
This study shows that ion trap mass spectrometry operated in
the selected ion storage mode can expand the dynamic range of
retinol isotopomer analyses to enrichments lower than previously
obtainable using linear-beam quadrupole instruments. The im-
provement comes by minmizing the background signal at the
mass of the deuterated isotopomer which arises from proton- and
hydrogen-transfer reaction (ion-molecule reactions) to unlabeled
retinol following ionization. A similar phenomenon was previously
observed in a study of fatty acid methyl esters where the ratios
of signals for labeled isotopomers were shown to be concentration-
dependent;19 this phenomenon was also attributed to ion-
molecule reactions in the ion source. The significance of ion-
molecule reactions can be expected to depend on sample amount
and ion residence times in the source, and with the Saturn ion
trap, ionization occurs within the source. This configuration leads
to reduced ion residence times (compared to external ionization)
and limits the magnitude of facile ion-molecule reaction.
Retinoids are known to be easily protonated as shown from
the reactions of anhydroretinol with anhydrous hydrogen chlo-
ride20 and among several retinyl derivatives with hydrogen ions
released radiolytically in solution.21 Similar behavior has been
observed with other highly conjugated polyenes.22 The EI mass
spectra of retinol derivatives contains an abundance of odd-
electron fragment ions that can serve as reactive donors of protons
or hydrogens. Preliminary evidence in our laboratory showed
(19) Patterson, B. W.; Wolfe, R. R. Biol. Mass Spectrom. 1 9 9 3 , 22, 481-6.
(20) Bulgrin, V. C.; Lookhart, G. L. J. Am. Chem. Soc. 1 9 7 4 , 96, 6077-80.
(21) Bobrowsi, K.; Das, P. K. J. Am. Chem. Soc. 1 9 8 2 , 104, p 1704-9.
(22) Wasserman, A. J. Am. Chem. Soc. 1 9 5 4 , 90, 1296.
(23) Julian, R. K.; Cooks, R. G. Anal. Chem. 1 9 9 3 , 65, 1827-33.
Analytical Chemistry, Vol. 70, No. 7, April 1, 1998 1373

from space charge effects. Standards could be analyzed at higher
AGC settings before space charge effects appeared. To provide
an adequate margin of safety, the method used a target value of
8000 for both standards and biologic samples.
fractions that are sufficiently free from interferences for GC/ MS
analysis using a DB-23 (50% cyanopropyl methylpolysiloxane)
phase. A DB-1 (100% dimethylpolysiloxane) column phase is
suitable following further sample purification of the retinol
containing fraction, such as the reversed-phase high-performance
liquid chromatographic (RP-HPLC) technique described by Han-
dleman et al.8 In cases where very low retinol-d4 incorporation
into endogenous pools is expected, RP-HPLC purification will
increase the accuracy of the measurement at lower levels of
enrichment (∼1/ 200) due to reduced chemical noise in the
deuterated channel. Chromatographic background signal appears
to originate from lipophilic compounds present at high concentra-
tions in biological extracts. Our experience is that the DB-23
phase delivers better selectivity from interferences than the DB-1
even following RP-HPLC purification. The complete removal of
lipophilic contaminants from HPLC systems is difficult and
requires rigorous cleaning of the injector, system tubing, and
column. The presence of lipophilic interferences necessitates the
use of highly selective ion monitoring modes to minimize signal
from nontarget analyte ions.
Calibrating standards were prepared with varying retinol-d4
concentrations in the presence of constant retinol concentrations.
Aliquots of approximately 0.5 µL were injected onto the instru-
ment. For the 0.1952 and 1.0 ratio standards, a 10-fold dilution
with isooctane was necessary to obtain accurate mass assign-
ments. At higher concentration, a 1 unit mass shift was observed
for the protio species, inaccurately producing a m/ z of 401 for
this species, particularly at the apex of the GC peak (highest
concentration). This shift was not observed at lower levels of
enrichment. Such mass shifts have been described by Cox et
al.24 as arising from ‘local space charge effects’ and further
explained in an ion-coupling model proposed by Mo and Todd.25
The ion-coupling phenomenon arises when more than one
species of ion is trapped and is most pronounced when ions of
close secular frequencies are stored. The frequency shift of one
species of ion is directly proportional to the concentration of
neighboring ion species and the inverse of their mass difference.
The net field formed by the sum of the ion-ion interactions
produces delayed ion ejection, and a higher apparent mass is
observed. When experiments are performed with forward rf
amplitude scans, the mass shift is only observed in the lower m/ z
ion as ejection of the high-massed ion would have been preceded
by ejection of the lower massed ion. In our experiments, the
magnitude of the shift was concentration-dependent, and at low
ion concentrations, the ion-ion net field is minimized, reducing
the magnitude of the effect. For this reason, a 10-fold dilution of
the 0.1952 and 1.0 ratio standards was prepared. While none of
our biological samples approached this level of enrichment, in
such circumstances a similar level of dilution is recommended.
Samples were prepared as previously described using amino-
propyl solid-phase fractionation of the hexane extracts of depro-
teinized plasma.7 This method permits the separation of intact
â-carotene-d8 from its retinol-d4 metabolite and provides retinol
In conclusion, ITMS offers the advantage of removing reactive
ions when ionization occurs inside the ion trap. Using this
approach, unlabeled retinol yields a background signal at the mass
of the labeled tracer close to values calculated from natural isotopic
abundances. This will expand the scope of vitamin A/ â-carotene
kinetic studies by permitting the reliable quantitation of labeled
vitamin A at low enrichments.
ACKNOWLEDGMENT
This research has been supported by NIH Grant DK-48307.
The Varian Saturn mass spectrometer was purchased in part with
funds from NIH Grant ES04699 and ES05707. The authors thank
Profs. Susan Ebeler and Kenneth Brown for use of the Hewlett-
Packard MSD and Shimadzu QP-5000 mass spectrometers,
respectively. We also thank Yumei Lin for technical assistance in
the preparation of the samples.
Received for review July 29, 1997. Accepted January 16,
1998.
(24) Cox, K. A.; Cleven, R. G.; Cooks, R. G. Int. J. Mass Spectrom. Ion Processes
1 9 9 5 , 144, 47-65.
(25) Mo, W.; Todd, J. F. J. Rapid Commun. Mass Spectrom. 1 9 9 6 , 10, 424-8.
AC970816R
1374 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998