A sensitive and specific method for measurement of multiple retinoids in human serum with UHPLC-MS/MS

Article abstract of DOI:10.1194/jlr.D019745

Retinol (vitamin A) circulates at 1-4 μM concentration and is easily measured in serum. However, retinol is biologically inactive. Its metabolite, retinoic acid (RA), is believed to be responsible for biological effects of vitamin A, and hence the measurement of retinol concentrations is of limited value. A UHPLC-MS/MS method using isotope-labeled internal standards was developed and validated for quantitative analysis of endogenous RA isomers and metabolites. The method was used to measure retinoids in serum samples from 20 healthy men. In the fed state, the measured concentrations were 3.1 ± 0.2 nM for at RA, 0.1 ± 0.02 nM for 9-cisRA, 5.3 ± 1.3 nM for 13-cisRA, 0.4 ± 0.4 nM for 9,13-dicisRA, and 17.2 ± 6.8 nM for 4oxo-13-cisRA. The concentrations of the retinoids were not significantly different when measured after an overnight fast (3.0 ± 0.1 nM for atRA, 0.09 ± 0.01nM for 9-cisRA, 3.9 ± 0.2 nM for 13-cisRA, 0.3 ± 0.1 nM for 9,13-dicisRA, and 11.9 ± 1.6 nM for 4oxo-13-cisRA). 11-cisRA and 4OH-RA were not detected in human serum. The high sensitivity of the MS/MS method combined with the UHPLC separation power allowed detection of endogenous 9-cis RA and 4oxo-atRA for the first time in human serum. Copyright

Full text of DOI:10.1194/jlr.D019745

methods
A sensitive and specific method for measurement of
multiple retinoids in human serum with UHPLC-MS/MS
Samuel L. M. Arnold,* John K. Amory,† Thomas J. Walsh,§ and Nina Isoherranen1,*
Department of Pharmaceutics,* School of Pharmacy, Department of Internal Medicine,† School of
Medicine, and Department of Urology,§ School of Medicine, University of Washington, Seattle, WA
Abstract Retinol (vitamin A) circulates at 1–4 ␮M concen-
tration and is easily measured in serum. However, retinol is
biologically inactive. Its metabolite, retinoic acid (RA), is be-
lieved to be responsible for biological effects of vitamin A,
and hence the measurement of retinol concentrations is
of limited value. A UHPLC-MS/MS method using isotope-
labeled internal standards was developed and validated for
quantitative analysis of endogenous RA isomers and metab-
olites. The method was used to measure retinoids in serum
samples from 20 healthy men. In the fed state, the measured
concentrations were 3.1 0.2 nM for atRA, 0.1 0.02 nM
for 9-cisRA, 5.3 1.3 nM for 13-cisRA, 0.4 0.4 nM for 9,13-
dicisRA, and 17.2 6.8 nM for 4oxo-13-cisRA. The concen-
trations of the retinoids were not significantly different
when measured after an overnight fast (3.0 0.1 nM for
atRA, 0.09 0.01nM for 9-cisRA, 3.9 0.2 nM for 13-cisRA,
0.3 0.1 nM for 9,13-dicisRA, and 11.9 1.6 nM for 4oxo-13-
cisRA). 11-cisRA and 4OH-RA were not detected in human
serum. The high sensitivity of the MS/MS method com-
bined with the UHPLC separation power allowed detection
of endogenous 9-cisRA and 4oxo-atRA for the first time in
human serum.—Arnold, S. L. M., J. K. Amory, T. J. Walsh,
and N. Isoherranen. A sensitive and specific method for
measurement of multiple retinoids in human serum with
UHPLC-MS/MS. J. Lipid Res. 2012. 53: 587–598.
isomers are involved in numerous processes associated
with vision, healthy skin, glucose regulation (3), embryonic
development (4), stem cell differentiation (5), and sper-
matogenesis (6), although the exact role of each isomer is
not fully understood. atRA has been shown to induce
remission in patients with acute promyelocytic leukemia,
and 13-cisRA (isotretinoin) is used for the treatment of
acne and in children with high-risk neuroblastoma (7).
9-cisRA (alitretinoin) has been used to treat severe chronic
hand eczema (8) and has been shown to have a role in
regulating insulin-stimulated glucose secretion (3, 6, 9). In
addition to the RA isomers, the oxidized metabolites of RA,
including 4-hydroxy-RA (4OH-RA), 4oxo-RA, and RA-5,6-
epoxide, possess biological activity in in vitro models (10).
4oxo-RA has also been shown to be teratogenic in animal
models and to inhibit cell proliferation in a number of
cell lines (11–13).
The biological activity of retinoids is mediated predomi-
nantly by their association with nuclear receptors, mainly
the retinoic acid receptors (RAR␣, RAR␤, and RAR␥).
However, binding of retinoids to retinoid X receptors
(RXR␣, RXR␤, and RXR␥) and peroxisome proliferator-
activated receptors (PPAR␤ and PPAR␦) has also been
shown (14). Oxidative metabolism of the RA isomers can
result in deactivation or activation of the parent retinoid,
and isomerization of RA can change the specificity and
activity of RA toward nuclear receptors. For example,
9-cisRA binds to RXR with a significantly higher affinity
than atRA, and 4oxo-atRA and 4OH-atRA have been shown
to bind to RAR with similar affinity as atRA (10, 12, 15).
Supplementary key words retinoic acid • isomers • 4oxo-retinoic
acid • MS/MS • metabolites
Retinoids are acquired from the diet as a proretinoid
carotenoid or as retinol and retinyl esters (1). Retinol (vi-
tamin A) is the main circulating retinoid, but it is biologi-
cally inactive and is metabolized in the body to active
retinoic acid (RA). RA has been reported to exist as at
least five isomers, including all-trans retinoic acid (atRA),
9-cisRA, 13-cisRA, 9,13-dicisRA, and 11-cisRA (2). These RA
Abbreviations: atRA, all-trans-retinoic acid; 9-cisRA, 9-cis-retinoic
acid; 13-cisRA, 13-cis-retinoic acid; 4oxo-13-cisRA, 4oxo-13-cis-retinoic
acid; 4oxo-atRA, 4oxo-all-trans-retinoic acid; 4OH-9-cisRA, 4-hydroxy-9-
cis-retinoic acid; 4OH-atRA, 4-hydroxy-all-trans-retinoic acid; can,
acetonitrile; APCI, atmospheric pressure corona ionization; CE, colli-
sion energy; CV, coefficient of variation; CXP, collision cell exit potential;
DP, declustering potential; EP, entrance potential; UHPLC, Ultra-
high-performance liquid chromatography; LXR, liver X receptor;
LLOQ, lower limit of quantification; LLOD, lower limit of detection;
RAR, retinoic acid receptor; RXR, retinoid X receptor; SRM, selected
reaction monitoring; PPAR, peroxisome proliferator-activated recep-
tor; QC, quality control.
This work was supported in part by an National Institutes of Health grants R01
GM081569 and GM081569-S1 and by the Eunice Kennedy Shriver National
Institute of Child Health and Human Development, a division of the National
Institute of Health through cooperative agreement #U01 HD060408. Its con-
tents are solely the responsibility of the authors and do not necessarily represent
the official views of the National Institutes of Health or other granting agencies.
Manuscript received 10 November 2011 and in revised form 9 December 2011.
1 To whom correspondence should be addressed.
e-mail: ni2@u.washington.edu
Published, JLR Papers in Press, December 21, 2011
DOI 10.1194/jlr.D019745
Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 53, 2012
587

dichloromethane. The 4oxo-RA-methyl ester was hydrolyzed
using 2 M KOH in methanol, and 4oxo-atRA was extracted using
ethyl acetate and crystallized. 4OH-atRA was synthesized from
4oxo-RA using two equivalents of NaBH4 in methanol. The prod-
uct was extracted with 1:1 mixture of ethyl acetate and ether,
evaporated to dryness, and crystallized.
The major limitation in understanding the roles and impor-
tance of 4OH-RA, 4oxo-RA, and specific RA isomers in vivo
has been the lack of knowledge on the concentrations of
the specific isomers and metabolites in human tissues.
Analysis of RA isomers and their metabolites has been
difficult due to their similar structures and low concen-
trations in human tissues. A lack of analytical separation
power may have confounded individual isomer detection,
whereas a lack of good MS/MS sensitivity has prevented
selective quantification of individual retinoids. Previous
methods have been developed using LC-MS/MS (9, 16–21),
GC/MS (22), and LC/diode array detector-atmospheric
pressure chemical ionization/MS/MS (23). However,
none of these methods has incorporated measurement
of hydroxylated RA metabolites at endogenous levels in
serum. Endogenous atRA (4.6–5.8 nM), 13-cisRA (4.7–6 nM),
and 4oxo-13-cisRA (8.1–9.8 nM) were measured in human
serum using HPLC with UV detection (24, 25). However,
9,13-dicisRA and 9-cisRA were not measured, nor was
separation of the main four isomers of RA demonstrated.
Separation and quantification of atRA, 9-cisRA, 9,13-dicisRA,
and 13-cisRA in mouse tissues was shown using LC-MS/MS
analysis (9), but no measurement of the metabolites of RA
was conducted.
The goal of this study was to develop a method for the
simultaneous measurement of biologically active endo-
genous retinoids in human serum. Due to the challenges
in separating the isomers of RA as well as the metabolites,
chromatographic separation was optimized for UHPLC.
The method was validated with spiked charcoal-treated
blank human serum and used to determine retinoid con-
centrations in serum from healthy men. The developed
assay is useful for characterization of retinoid disposition
and endogenous retinoid homeostasis and for following
therapeutic interventions with retinoids. The method can
also be used in preclinical studies of compounds that
target changing retinoid concentrations.
9,13-dicisRA was synthesized as previously published (28). In
brief, 100 mg of 9-cis-retinal was dissolved in 20 ml of methanol.
Eight milliliters of Tollens Reagent (equal volumes of 10% AgNO3
and 10% NaOH mixed and titrated with ammonium hydroxide
until precipitate dissolved) was added to the solution, and the
mixture was stirred at 37°C for 6 h. The reaction was quenched
with 4 N HCl on ice and filtered, and the products were extracted
with hexane and evaporated to dryness. The mixture of 9-cisRA
and 9,13-dicisRA was purified using silica gel chromatography
and ethyl acetate:hexane mobile phase. The fraction containing
9,13-dicisRA was quantified by NMR by calculating the ratio be-
tween the area of the 9-cisRA doublet at 6.09 ppm (H in C10) to
the corresponding doublet from 9,13-dicisRA at 6.2 ppm. The
identity of the 9,13-dicisRA was also confirmed by the ratio of the
9,13-dicisRA doublet at 7.7 ppm (H at C12) to the corresponding
doublet in 9-cisRA at 6.3 ppm. Based on NMR quantification, the
reaction yielded 20% 9,13-dicisRA and 80% 9-cisRA.
Extraction of retinoic acid isomers and 4oxo-retinoic acid
All sample processing, preparation, and extraction was con-
ducted on ice under red light. 9-cisRA, 13-cisRA, atRA, 4oxo-13-
cisRA, 4oxo-atRA, 4OH-atRA, and 4OH-9-cisRA were spiked into
blank serum. Into 500 ␮l of serum, 10 ␮l of a 60:40 ACN:MeOH
mixture with 1 ␮M 13-cisRA-d5 and 2 ␮M 4oxo-13-cisRA-d3 was
added as internal standards. All compounds were extracted
according to previously described method for extraction of RA
isomers (29). In brief, 1 ml ACN and 60 ␮l 4 N HCl were added
to each sample, the samples were vortexed, and retinoids were
extracted twice with 5 ml of Hexanes. The organic phase was
separated by centrifugation at 1000 rpm for 3 min and evapo-
rated to dryness under nitrogen stream at 32°C. The dry residue
was reconstituted in 50 ␮l of 60:40 ACN:H2O and transferred to
an amber autosampler vial.
Serum retinoids could also be measured with no extraction
using a simple protein precipitation with ACN. In this method, 50 ␮l
of ACN is added to 50 ␮l of serum followed by centrifugation at
2000 rpm for 10 min at 4°C to pellet serum proteins. The super-
natant is transferred to a 96-well plate, and 20 ␮l is injected into
the UHPLC/MS/MS for analysis.
MATERIALS AND METHODS
Chemicals and reagents
UHPLC MS/MS analysis
atRA, 9-cisRA, 13-cisRA, and 9-cis-retinal were purchased from
Sigma (St. Louis, MO). 4oxo-atRA, 4OH-atRA, and 9,13-dicisRA
were synthesized as described below. 4oxo-13-cisRA, 4OH-9-cisRA,
4oxo-13-cisRA-d3, 13-cisRA-d5, and 11-cisRA were purchased from
Toronto Research Chemicals (North York, Ontario). All com-
pounds were stored in amber vials in ethanol as 1 mM stocks
at Ϫ80°C. Acetonitrile, methanol, water, and formic acid used
in the UHPLC-MS/MS method were from Fischer Scientific
(Pittsburg, PA), and all were Optima LC/MS grade. Blank
human serum (DC Mass Spect Gold MSG 4000) was purchased
from Golden West Biologics (Temecula, CA). This serum has
a normal range of triglycerides (30–200 mg/dl) and cholesterol
(> 20 mg/dl) to mimic the extraction environment of clinical
samples. This is important due to the matrix effects associated
with lipids during extraction and analyte ionization.
The retinoids were separated using an Agilent 1290 UHPLC
(Santa Clara, CA) equipped with a Sigma (St. Louis, MO) Ascentis
Express RP Amide column (2.7 ␮m; 150 mm × 2.1 mm). Gradient
elution with a flow rate of 0.5 ml/min using water (A) and ace-
tonitrile (B) with 40% methanol and 0.1% formic acid in A and
B was used. The gradient was from initial 60% (A) for 2 min to 45%
(A) over 8 min and then to 10% (A) over 7 min. The column was
then washed with 95% (B) for 3 min and returned to initial con-
ditions. The column heater was set to 40°C. Samples were kept
in the autosampler at 4°C with the light turned off. Ten microli-
ters of sample was injected for analysis. Analytes were detected
using an AB Sciex 5500 qTrap Q-LIT mass spectrometer (Foster
City, CA) operated in positive ion APCI mode. The compound
independent MS parameters were curtain gas: 20; collision gas:
low; nebulizer current: 5; temperature: 350°C; ion source gas
1:80. The final compound dependent parameters used for
analysis are summarized in Table 1. The specific MS/MS transi-
tions for each analyte were optimized using the Analyst software
(Applied Biosystems, Foster City, CA) using direct infusion with
4OH-RA and 4oxo-RA were synthesized according to previ-
ously published methods (26, 27). In brief, the methyl ester of
atRA was generated using trimethylsilyldiazomethane. Methyl
4oxo-retinoate was prepared using activated MnO2 in anhydrous
588
Journal of Lipid Research Volume 53, 2012

TABLE 1. Compound dependent mass spectrometer parameters
used for detection of the retinoids
proteins with acetonitrile, centrifugation of precipitated pro-
teins, and direct analysis of the supernatant as described above
for retinoid extraction.
Analyte
Q1 m/z
Q3 m/z
msec
DP
EP
CE
CXP
To determine whether matrix effects altered the ionization
and detection of retinoids in serum, four serum samples were
extracted and reconstituted in duplicate with 50 ␮l of 60:40
ACN/H2O mixture containing 5 nM and 10 nM 13-cisRA-d5. In
parallel, the same ACN/H2O mixture with 5 nM and 10 nM 13-
cisRA-d5 was added into glass tubes with no extracted samples.
After a quick vortex, all samples were transferred into amber
vials and analyzed by LC-MS/MS. The response was quantified
and compared between the matrix containing samples and the
clean standards.
RA (all isomers)
4OH-RA
4oxo-RA
4oxo-RA-d3
13-cisRA-d5
301
299
315
300
306
205
157
159
226
116
100
100
100
100
100
80
59
66
71
97
10
10
10
10
10
17
16
23
35
97
10
15
16
2
6
CE, collision energy; CXP, collision cell exit potential; DP,
declustering potential; EP, entrance potential.
and without the addition of the LC solvents. Initially, MS/MS
transitions were identified for each analyte using the automatic
optimization features of the Analyst software. These transitions
were compared with those from a manual optimization, and the
five most abundant fragments for each analyte found in both the
automatic and manual process were optimized independently
for their declustering potential (DP), entrance potential (EP),
collision energy (CE), and collision cell exit potential (CXP). All
five identified potential MS/MS transitions for each analyte were
included in a method that was used to analyze blank, spiked, and
normal human serum to determine interference, matrix effects,
and signal-to-noise ratios of each MS/MS transition. The MS/MS
transitions that showed the least amount of matrix interference
in the blank serum and the highest signal-to-noise ratio in spiked
samples were chosen for the final analysis.
Subjects
Serum samples were collected from 20 healthy men between
the ages 18 and 65 who were enrolled in a study examining the
relationship between retinoids and spermatogenesis. The study
was approved by the Institutional Review Board at the University
of Washington, and all subjects gave written informed consent
before any study procedures. All subjects exhibited normal blood
chemistry, liver function, hematology, and hormones. Subjects
were excluded from the study if they used anabolic steroids or
illicit drugs, ingested more than four alcoholic drinks a day, or
were being treated with ketoconazole, finasteride, dutasteride,
methadone, or lithium. The first set of blood was collected
between 8 AM and 12 PM during a fed state within 2–6 h of
breakfast. The second blood draw occurred at least 7 days after
the first one, and blood was collected between 8 AM and 12 PM
after an overnight fast of at least 8 h. Blood samples were collected
from the subject’s antecubital vein, and blood tubes were imme-
diately wrapped in aluminum foil to minimize light exposure
(light-protected samples). The blood was allowed to clot at 4°C
and then spun for 20 min at 3,000 g. Serum was aliquoted into
amber sample tubes and stored at –80° C until analysis. Samples
were also collected without the aluminum foil wrapping and
light-protected vials. Five of these samples were extracted and
analyzed together with the light-protected samples.
Assay validation
The assay was validated according to the published guidelines
for bioanalytical method validation (30, 31). The on-column
lower limit of detection (LLOD) determined as signal-to-noise
ratio >3 and the on-column lower limit of quantification (LLOQ)
determined as signal-to-noise ratio >9 were measured for each
compound as standard solution. The linearity of response was
determined using standard curves generated over four orders of
magnitude (0.0001–1.0 ␮M). For quantification, blank serum
was spiked at six concentrations between 0.1 nM and 20 nM for
RA isomers and between 0.5 nM and 20 nM for the metabolites
to construct standard curves for each analyte. The peak area
ratios of analyte/internal standard were plotted as a function of
concentration. 13-cisRA-d5 was used as the internal standard for
quantification of atRA, 9-cisRA, 13-cisRA, and 9,13-dicisRA, and
4oxo-13-cisRA-d3 was used for metabolite quantification. Each
retinoid, with the exception of 9,13-dicisRA, was quantified using
standard curves of the same compound. 9,13-dicisRA concentra-
tions were measured using the standard curve for 9-cisRA after
confirming that the MS/MS responses were identical for the two
compounds using the NMR quantified standard. Quality control
(QC) samples were extracted along with each standard curve and
run at the beginning, middle, and end of each run. All QC
samples had accuracy and precision within published guidelines
for bioanalytical method validation (30, 31).
To determine the intraday and interday coefficients of varia-
tion (CVs), the samples were extracted on three separate days.
The CV was measured at the LLOQ for serum of each compound
and at 7.5 nM of RA isomers and 10 nM of 4oxo-RA metabolites
spiked into blank serum. The LLOQ in serum was determined
by spiking 20 ml of blank serum with RA isomers and RA me-
tabolites at concentrations of 0.05 nM (RA isomers) or 6 nM
(metabolites). Aliquots of the QC samples were frozen and ana-
lyzed on three separate days. Due to their poor extraction re-
covery, the LLOQ (5 nM spiked in blank serum) of 4OH-atRA
and 4OH-9-cisRA was determined after precipitation of serum
Results are expressed as mean SD. All statistical analysis was
done using GraphPad Prism (La Jolla, CA). Due to nonnormality,
comparisons of retinoids between the fasting and fed states were
performed using a Wilcoxon signed-rank test. Correlation between
data sets was tested with linear regression. For all comparisons, a
P value of 0.05 was considered significant.
Analysis of the human serum samples and confirmation
of analyte identity
All the serum samples were analyzed using the described
method. The identities of the quantified retinoids were con-
firmed by collecting MS/MS spectra of each analyte. For this
analysis, four serum samples were extracted with hexanes as
described above, and the hexane phases were combined. After
drying under nitrogen flow, the sample was reconstituted in 50 ␮l
of 40:60 H2O:ACN, and 20 ␮l was injected into the UHPLC-MS/
MS. The UHPLC conditions were identical to those described for
quantitative analysis, and the same MS/MS parent-fragment pairs
used for quantification were recorded to detect the analytes and
trigger MS/MS spectrum acquisition. Once the signal for the MS/
MS transition exceeded a threshold, a fragment ion scan for the
same parent ion was triggered using positive ion APCI and with
collision energy spread of 15 from a set value of 35. A dynamic
fill time, which allows for the maximum amount of ions to be
collected in the linear ion trap for best sensitivity, was used to
collect the fragment ion spectra.
To confirm that each quantified peak for the detected RA
isomers in serum represented only a single compound, two
Quantification of endogenous retinoids in serum
589

independent MS/MS transitions from the extracted serum
samples were monitored, and the response ratio across the
peak was recorded. If a peak consists of two compounds, the
ratio between the two transitions usually changes across the peak.
Both transitions used parent ion m/z 301, and the fragment ions
monitored were m/z 205 and m/z 123. These fragment ions were
chosen based on their signal-to-noise ratios from spiked serum
and retinoic acid standards. The MS parameters for the m/z 301
> 123 transition were DP:62, CE:23, EP:10, CXP:14. The m/z 301 >
123 MS/MS fragmentation of RA is most likely a result of a
cleavage of the bond between carbons C6 and C7, resulting in the
␤-ionone-ring fragment with an m/z 123 as shown previously (32).
The structure of the m/z 301 > 205 fragment could not be as-
signed due to a likely rearrangement of the retinoid structure
during mass spectrometry. However, a corresponding fragment
at m/z 306 > 210 was detected from RA-d5, showing that this
fragment also retained the ␤-ionone-ring (data not shown). The
corresponding fragments (m/z 301 > 205 and m/z 305 > 209) were
detected previously from atRA and 13C4-atRA, respectively, pro-
viding additional confidence for the reproducibility of this
fragmentation (32).
Despite extensive efforts, 11-cisRA could not be separated
chromatographically from the 9,13-dicisRA. To determine whether
the retinoid detected in human serum was 9,13-dicisRA or 11-cisRA,
palladium(II)nitrate was used to convert 11-cisRA to atRA according
to a published method (33). Four light-protected serum sam-
ples were pooled to obtain a total volume of 2.5 ml of serum.
13-cisRA-d5 was spiked into the sample to a final concentration
of 20 nM as an internal standard. A 1.1 ml aliquot was removed
from the pooled sample, and 5 nM 11-cisRA was spiked into the
sample. Two 500 ␮l aliquots were collected from the 11-cisRA spiked
serum and from the remaining pooled control serum. The ali-
quots were extracted as described for serum samples. All four of
the samples were reconstituted in 500 ␮l ACN. A solution con-
taining a 1:4.8 ratio of palladium(II)nitrate to Triethylamine in
ACN was added to one sample with 11-cisRA spiked in and to one
sample without added 11-cisRA. The final Palladium(II)nitrate to
retinoic acid molar ratio was approximately 50,000:1. The reac-
tion was allowed to proceed at 50°C for 5 h in the dark, after
which the ACN was removed under a stream of dry nitrogen. The
samples were reconstituted in ethyl acetate and washed twice
with water to remove the Palladium reagent. Samples were then
dried again under nitrogen and reconstituted in 50 ␮l of a 60:40
ACN/H2O solvent.
analysis. This was due to the apparent loss of water (Ϫ18)
from the 4OH-RA [M+H]+ protonated molecule (m/z 317)
resulting in a base peak at m/z 299. In negative ion mode,
an [M-H]Ϫ ion could be detected for the 4OH-RA com-
pounds, confirming that they were stable during chroma-
tography and unstable in the mass spectrometer (data
not shown).
To achieve separation of the five RA isomers (atRA,
9-cisRA, 9,13-dicisRA, 11-cisRA, and 13-cisRA), 4OH-RA iso-
mers, and 4oxo-RA isomers, numerous C18 and C8 columns
with UHPLC capability were evaluated as well as several
chiral columns with mobile phases, including methanol,
water, acetonitrile, acetic acid, and formic acid. Significant
differences in the separation capacity between the dif-
ferent columns was observed, and in most columns the
13-cisRA and 9,13-dicisRA were not separated, whereas sepa-
ration between 13-cisRA and atRA was obtained in virtually
all columns tested. Baseline separation of atRA, 9-cisRA,
9,13-dicisRA, and 13-cisRA was achieved only by using the
amide column described, and separation of the oxidized
RA metabolite isomers was satisfactory (Fig. 2). The 11-
cisRA isomer could not be separated from the 9,13-dicisRA
regardless of the analytical conditions (vide infra). Opti-
mal separation of the oxidized metabolite isomers was
achieved using a C18 column, but the separation achieved
using the amide column was sufficient for quantification.
Selectivity of the method for endogenous retinoids
Initially, the five MS/MS parent-fragment pairs with the
greatest signal magnitude were chosen for the assay, and
each of these SRM transitions (MS/MS parent-fragment
pairs monitored) was tested in blank human serum for the
existence of interference from the matrix. For several of
the mass transitions, endogenous interference was de-
tected, including m/z 301 > 161, m/z 301 > 159, m/z 301 > 91,
and m/z 301 > 105 for RA isomers; m/z 299 > 91, m/z 299 >
128, and m/z 299 > 115 for 4OH-RA; and m/z 315 > 120 for
4oxo-RA. The m/z 315 > 297 MS/MS transition was not
considered for quantification of 4oxo-RA due to the lack
of specificity of a loss of water fragment. An alternative
fragment that reduced interference from the matrix was
chosen for quantitative analysis for each analyte as sum-
marized in Table 1. Due to matrix interference, fragments
with the highest abundance were generally not the MS/MS
transitions used for final analysis. Close to the retention
time of atRA-d5, a significant interfering peak was de-
tected (Fig. 2E) at the relevant SRM channels of m/z 306 >
131, m/z 306 > 116, m/z 306 > 210, m/z 306 > 96, and m/z
306 > 154 at 13.9 min. Although the interference some-
what separated from atRA-d5 (Fig. 2C and E), its abun-
dance interfered with the quantification of atRA-d5.
Therefore, 13-cisRA-d5 was chosen as the internal standard
for RA isomers (Fig. 2E). No interference was detected at
the retention time of 13-cisRA-d5.
RESULTS
Optimization of chromatographic separation and MS/MS
detection of retinoids
The ionization and fragmentation of the retinoids were
evaluated using electrospray and APCI in both the positive
and negative ion modes. The negative ion mode provided
approximately 10-fold lower sesitivity than the positive ion
mode (data not shown); hence, the positive ion mode was
chosen for further evaluation. The fragmentation and spe-
cific MS/MS ion transitions (SRM transition) for each RA
isomer, for the isotope-labeled internal standards, and for
the metabolites were optimized for maximum sensitivity. The
MS/MS fragmentation patterns of the protonated molecules
([M+H]+s) for representative RA, 4oxo-RA, and 4OH-RA iso-
form are shown in Fig. 1. The protonated molecules
([M+H]+s) of 4OH-atRA and 4OH-9-cisRA were not stable
enough in the positive ion mode to isolate for MS/MS
Validation of the method for serum samples
The on-column LLOD and LLOQ for clean standards
for each analyte are shown in Fig. 3. Standard curves of
atRA, 9-cisRA, 13-cisRA, 4oxo-atRA, and 4oxo-13-cisRA were
590
Journal of Lipid Research Volume 53, 2012

Fig. 1. Representative MS/MS spectra of RA, 4oxo-RA, and 4OH-RA standards and of RA and 4oxo-RA detected in serum samples. The
MS/MS spectra of RA isomers and metabolites as clean standards and from serum samples were collected as described in Materials and
Methods. The MS/MS spectra representative of all detected isomers are shown for atRA (A), 4oxo-13-cisRA (C), and 4OH-atRA (E) stan-
dards. Similar MS/MS spectra were generated for each quantified retinoid in a sample of pooled human serum, and representative MS/MS
spectra for atRA (B) and for 4oxo-13-cisRA (D) are shown.
generated in blank human serum and extracted along
with the study samples. The generated standard curves
were linear, and the equations with r2 values are shown in
Table 2. The validation data at the LLOQ for serum (0.05 nM
for RA isomers and 6 nM for 4oxo-13-cisRA) and at the mid
to high concentration (7.5 nM for RA isomers and 10 nM for
4oxo-13-cisRA) are shown in Table 2. All of the measured
concentrations were within 15% of the true value (accu-
racy) for all analytes at the mid-high QC and within 20%
of the true value at LLOQ. The LLOD in serum for
4OH-RA isomers was 2 nM when the samples were ana-
lyzed after protein precipitation with acetonitrile. The re-
tention times of the analytes were reproducible between
days and within days with variability in the retention times,
being <0.5% for RA isomers and <0.8% for the metabolites.
A lack of significant ion suppression or other matrix ef-
fects on retinoid detection was determined by comparing
the peak areas for 13-cisRA-d5 standard without matrix and
13-cisRA-d5 in the sample spiked after extraction (100%
recovery) at three different concentrations. The matrix-
containing samples were not different in terms of peak
area or retention time from the samples without matrix.
The effect of light exposure during sample collection to
retinoid measurements was tested by comparing measure-
ments from light-protected samples with measurements in
samples that were not collected in light-protected conditions.
All of the samples showed significant decreases (≈70%) in
the concentrations of RA isomers, demonstrating that pro-
tecting the samples from UV light already during sample
collection is critical. No decrease in the concentrations of
Quantification of endogenous retinoids in serum
591

Fig. 2. Separation of retinoic acid and 4oxo-RA isomers and detection of the retinoids and labeled inter-
nal standards in serum. Separation of atRA, 9,13-dicisRA, 9-cisRA, and 13-cisRA standards (A) and detection
of the same isomers in serum (C). (B) Separation of 4oxo-atRA and 4oxo-13-cisRA and (D) detection of these
compounds in serum. The dotted line in panel (D) also shows the lack of detection of 4OH-RA in extracted
serum. All serum samples were prepared by liquid-liquid extraction and analyzed as described in Materials
and Methods. (E) Detection of 13-cisRA-d5 internal standard (at 13.2 min) extracted from serum together
with the structure of the labeled compound. The peak at 13.9 min is not a retinoid but a matrix peak. (F)
Detection of 4oxo-13-cisRA-d3 internal standard in serum together with the structure of the internal stan-
dard. Panels E and F also show the lack of isomerization of the internal standards in serum. The insets on
each panel specify the MS/MS transition used for the specific analyte.
4oxo-13cisRA was detected in response to light exposure
(data not shown).
The identitiy of each detected retinoid was confirmed by
obtaining an MS/MS spectrum of the compound in serum
(Fig. 1). There were no detectable differences between the
MS/MS spectra of 13-cisRA, 9,13-dicisRA, atRA, and 9-cisRA
or between the 4oxo-13-cisRA and 4oxo-atRA. Analysis of the
human serum samples allowed collection of an MS/MS
spectrum for each RA isomer, confirming the identity of the
detected analytes. A representative MS/MS spectrum from
human serum is shown in Fig. 1B. Additionally, the frag-
mentation for the endogenous compound 4oxo-13-cisRA
(Fig. 1D) was similar to the standard. Peak purity was con-
firmed by monitoring two separate SRM transitions for the
RA isomers. The difference in the ratio between the two
transitions (m/z 301 > 205 and m/z 301 > 123) remained con-
stant across each peak in the standard and analyte in serum.
Detection and identification of endogenous retinoids in
human serum
The detection and separation of the endogenous RA
isomers in serum is shown in Fig. 2C and 2D, with the iden-
tified retinoids indicated. As shown in Fig. 4C with 11-cisRA
spiked serum, 11-cisRA could not be separated from 9,13--
dicisRA. However, treatment of serum with palladium(ii)
nitrate converted the spiked 11-cisRA quantitatively to
atRA (Fig. 4D). In the nonspiked sample (Fig 4A, B), no
decrease in the 9,13-dicisRA peak area or increase in atRA
peak area was detected, confirming that 11-cisRA is not
present in human serum at quantifiable concentrations.
592
Journal of Lipid Research Volume 53, 2012

Fig. 3. Chromatograms of the detected retinoids as standards at determined LLOD. The on-column LLOD
and LLOQ values for each compound are shown in the inset table. All retinoids were measured using the
MS/MS transitions summarized in Table 1, and the corresponding SRM channels (m/z 301 > 205 for RA
isomers, m/z 315 > 159 for 4oxo-RA isomers, and m/z 299 > 157 for 4OH-RA isomers) are shown.
The hydroxylated metabolites 4OH-9-cisRA and 4OH--
atRA were not detected in the samples extracted and ana-
lyzed using the method optimized for RA isomers (Fig. 2).
To determine whether this was due to poor extraction re-
covery of 4OH-RA isomers, serum was analyzed after pre-
cipitation of proteins with ACN followed by centrifugation.
The 10 serum samples analyzed had no quantifiable levels
of 4OH-9-cisRA and 4OH-atRA (Fig. 5), showing that the
concentrations of these metabolites, if they are present in
human serum, are <2 nM. It is possible that the two peaks
detected in the serum (Fig. 5C) are 4OH-RA isomers, but
this could not be confirmed because experiments to ob-
tain MS/MS spectra on these peaks were unsuccessful.
to determine the normal concentrations of endogenous
RA isomers and metabolites in human serum. All four RA
isomers and 4oxo-13-cisRA were detected in all samples.
4oxo-atRA could also be detected in 18 of the 40 samples,
but the concentrations were below LLOQ. The concentra-
tion of 4oxo-13-cisRA could not be quantified in one sub-
ject at the fed state and in another subject in the fasted
state, 9-cisRA could not be quantified in one sample from
fed state, and 9,13-dicisRA could not be quantified in sam-
ples from two subjects in the fasted state because concen-
trations were below the LLOQ. The mean concentrations
of the four RA isomers and 4oxo-13-cisRA are shown in
Table 3, and a box and whiskers plot representing the
measured concentrations for each retinoid in the 20 vol-
unteers is shown in Fig. 6. The most abundant RA isomer
in human serum was 13-cisRA, although atRA concentra-
tions were only about 30–40% lower. The concentra-
tions of atRA and 13-cisRA were about 10-fold higher than
Quantification of retinoic acid isomers and metabolites in
human serum
Serum samples collected after an overnight fast and 2–6 h
after normal breakfast from 20 healthy men were analyzed
Quantification of endogenous retinoids in serum
593

TABLE 2. Validation data for the analyzed retinoids
In the fed state, there were significant correlations be-
tween 9-cisRA and 13-cisRA (P < 0.001), between 9-cisRA
and 9,13-dicisRA (P < 0.001), between 13-cisRA and 9,13--
dicisRA (P < 0.05), and between 13-cisRA and 4oxo-13-cisRA
(P < 0.05). No correlation (P > 0.05) between atRA con-
centrations and any of the other retinoids was found in
the fed state and in the fasted state atRA concentrations
correlated only with 9-cisRA (P < 0.01). In addition, in
the fasted state, similar to the fed state, there were signifi-
cant correlations between 9-cisRA and 13-cisRA (P < 0.001),
between 9-cisRA and 9,13-dicisRA (P < 0.05), and between
13-cisRA and 9,13-dicisRA (P < 0.05) concentrations.
Equation for
Calibration
Curve
LLOQ
Middle LOQ
(intraday,
(intraday,
Analytes
r2
interday CV%)a interday CV%)b
atRA
9-cisRA
0.995
0.996
5.4, 15.0
14.3, 19.6
7.3, 13.7
15.8, 17.9
10.8, 18.8
3.6, 6.9
2.1, 4.9
3.2, 3.3
4.0, 13.8
6.5, 6.7
Y = 1.67x Ϫ 0.12
Y = 1.43x Ϫ 0.18
13-cisRA
4oxo-atRA
4oxo-13cisRA
Y = 1.96x + 0.18 0.994
Y = 2.41x + 0.55 0.982
0.997
Y = 7.64x Ϫ 0.42
a
The LLOQs were determined at 0.05 nM RA isomers and 6 nM
4oxo-RA isomers.
b
The LOQs were determined at 7.5 nM RA isomers and 10 nM
4oxo-RA isomers.
9,13-dicisRA concentrations and 30- to 50-fold higher than
9-cisRA concentrations. Two of the RA metabolites (4oxo-
13-cisRA and 4oxo-atRA) were also detected in human
serum, and the concentrations of 4oxo-13-cisRA exceeded
those of 13-cisRA by 3-fold, making this compound the
most prevalent retinoid measured. No statistically signifi-
cant differences (P > 0.05) were found between the fed
and fasted states for any of the detected analytes (Table 3).
Linear regression analysis was used to determine whether
correlations exist between the serum concentrations of the
detected analytes. There was a significant correlation be-
tween the fed and fasted concentrations of 13-cisRA (P <
0.001), 9,13-dicisRA (P < 0.001), and 4oxo-13-cisRA (P <
0.001), whereas the concentrations of atRA and 9-cisRA did
not correlate between the fed and fasted states (P > 0.05).
DISCUSSION
The described UHPLC MS/MS method is to date the
most sensitive method to selectively monitor bioactive
retinoids. The selectivity for RA isomers and metabolies is
achieved using a combination of UHPLC, specfic solvents,
and an amide column that provides the specific separation
capacity needed for each isomer. Surprisingly, positive ion
APCI ionization produced the best signal-to-noise ratio for
the RA isomers despite the readily ionizable carboxylic
acid group in the molecule. However, the RA metabolites
exhibit better sensitivity using electrospray ionization and
negative ion detection. For the quantification of the
retinoids in serum, positive ion APCI was chosen because
matrix interference was observed using electrospray
Fig. 4. Evaluation of the presence of 11-cisRA in serum. (A) Light-protected serum sample without the
addition of 11-cisRA and palladium(II)nitrate extracted as described in Materials and Methods. (C) Chro-
matogram of an aliquot of the same extracted sample spiked with 11-cisRA. Fractions of both samples were
treated with palladium(II)nitrate. (B and D) Chromatograms of the serum samples after the reaction. No
decrease in 9,13-dicisRA peak between panel A and B is detected, and no increase in atRA peak suggesting
that 11-cisRA was not present in serum. In the control reaction, the spiked 11-cisRA was quantitatively con-
verted to atRA (panel C vs. D).
594
Journal of Lipid Research Volume 53, 2012

Fig. 5. Detection of 4OH-RA isomers in human serum using an ACN protein precipitation and centrifuga-
tion for sample preparation. Blank serum (A), 5 nM 4OH-atRA and 4OH-9-cisRA spiked into blank serum
(B), and a representative human serum sample (C) are shown. The peak at 5.75 min in panel C is likely
4OH-atRA, but the peak area is below LLOQ for this compound.
ionization with negative ion, and limited amount of bio-
logical samples may require a single analysis for all relevant
bioactive retinoids. The best sensitivity for retinoid detec-
tion was obtained using a liquid-liquid extraction with
hexanes, and the method was validated for serum using
this extraction method. However, the recovery of the hy-
droxylated metabolites is poor using the extraction method,
and a direct protein precipitation with acetonitrile is
recommended for analysis of hydroxylated metabolites of
RA. A similar extraction method as shown here for serum
samples has been previously used for detection of atRA,
13-cisRA, and 9,13-dicisRA in mouse tissues, and hence the
sample preparation is expected to be applicable for tissue
analysis (9). However, for any given tissue, especially the
eye, a confirmatory analysis should be conducted to deter-
mine the potential presence of 11-cisRA and 9,13-dicisRA
in the samples. In addition, special attention should be
paid to the potential interfering compounds in the matri-
ces analyzed. As demonstrated here, significant interfer-
ence is present in many of the SRM transitions examined,
and it is expected that abundance and identity of interfer-
ing matrix compounds varies between tissues. Because
blank tissues are generally unavailable for retinoid analy-
sis, simple transfer of a method validated for one tissue to
another should be exercised with caution.
TABLE 3. Concentrations of the quantified retinoids in serum from
20 healthy men
One advantage of the described method is the use of
isotope-labeled internal standards for quantification of
endogenous RA and its metabolites. The use of deuteri-
um-labeled internal standards is important because they
are likely to mimic the extraction efficiency of RA isomers
and metabolites. The results of this study show that the
extraction characteristics of retinoids, even within a closely
chemicallyrelatedgroup(e.g.,RA,4oxo-RA,and4OH-RA),
can be very different, and hence using a chemical analog
as an internal standard may confound quantification re-
sults. In addition, binding of the analytes to cellular retin-
oic acid-binding proteins and fatty acid-binding proteins
may affect recovery and may be corrected by isotope labeled
internal standards. Finally, a significant advantage for the
use of deuterated retinoids as internal standards is that
it allows monitoring of isomerization during sample
Wilcoxon
Signed-Rank
Median Maximum TestFed vs.
Mean SD Minimum
Analyte
(nM)
(nM)
(nM)
(nM)
Fast P value
atRA
Fed
3.1 0.9
3.0 0.6
1.7
1.9
2.9
3.1
5.2
4.4
0.82
Fasted
13-cisRA
Fed
Fasted
9-cisRA
Fed
Fasted
9,13-dicisRA
Fed
Fasted
4oxo-13cisRA
Fed
5.3 5.7
3.9 1.0
2.4
2.7
3.9
3.8
29.0
7.3
0.27
0.14
0.09
0.59
0.1 0.1
0.08 0.02
0.05
0.05
0.09
0.09
0.49
0.15
0.4 0.4
0.3 0.1
0.1
0.1
0.3
0.2
1.8
0.7
17.8 31
12.2 7.2
7.1
6.8
10.2
10.7
146
40.4
Fasted
Quantification of endogenous retinoids in serum
595

increased sensitivity of the UHPLC/MS/MS method.
The identity of the detected 9-cisRA was confirmed by
obtaining an MS/MS spectrum of the detected 9-cisRA.
This detection of 9-cisRA is unlikely to be an artifact of the
assay or a result of isomerization because no isomeriza-
tion of the internal standards was detected and because
the ratio between isomers did not change when samples
were reanalyzed. In addition, 9-cisRA was detected in
the samples after ACN precipitation as well as after extrac-
tion, providing additional support for 9-cisRA being
present in human serum. The 9-cisRA has also been de-
tected in mouse pancreas and has been shown to regu-
late glucose homeostasis (3).
The ratio of RA isomers determined in human serum
was different than what has been previously reported in
mouse serum (9). In mouse serum, atRA was the most
abundant retinoid, with 9,13-dicisRA being present at simi-
lar concentrations. The concentrations of 13-cisRA were
much lower than the two other detected retinoids. In con-
trast, in human serum 9,13-dicisRA was a minor circulating
species, and 13-cisRA was the most abundant retinoid. This
demonstrates a significant species difference between the
isomers. It is unlikely that the observed difference is due to
isomerization in the two studies because the extraction
methods were similar and both studies used LC-MS/MS.
In addition, analysis of the human serum samples using
the ACN precipitation protocol decribed here resulted in
an identical ratio of RA isomers as seen with the extraction
method. The precipitation method eliminates the poten-
tial for isomerization, which may result from the extraction
process and which requires multiple pieces of glassware
and the addition of acid. It is likely that the species dif-
ference is due to differences in the age and/or diet of the
mice versus humans. The biological importance of this in-
terspecies variability is not known, and further studies in
species- and age-dependent changes in retinoid profiles
are warranted.
The results presented show no significant differences in
retinoid serum levels after at least 8 h of fasting despite the
fact that a previous study discovered decreasing concen-
trations of atRA and 13-cisRA during fasting (25). However,
the previous study was conducted after a 5-day fast, suggest-
ing that the length of the fasting period has an effect on the
retinoid concentrations. One of the subjects in this study
showed extremely high levels of 13-cisRA and 4oxo-13-cisRA
compared with the mean. It was confirmed that this in-
dividual was not taking exogenous retinoids during the study.
Although he was administered prostaglandin F2␣ eyedrops,
it is unlikely that these eyedrops affected 13-cisRA con-
centration in serum. The possibility of 13-cisRA and 4oxo-13-
cisRA contamination in the samples is low because his serum
concentration was measured multiple times on different
days. In studies involving isotretinoin treatment (80 mg mϪ2
bd), the average concentration of 4oxo-13-cisRA was calcu-
lated to be approximately 4.7 ␮M (7), which is over 100-fold
higher than the concentrations observed in this study. In a
previous study, the effect of diet on atRA levels in serum
was tested, and atRA levels were shown to increase 100%
after the consumption of carrot juice (34). However, no large
Fig. 6. Box and whiskers plots for endogenous retinoid concen-
trations in serum. The box represents the 25th and 75th percen-
tiles of each group. The whiskers are determined based on 10th
and 90th percentiles, and outliers are calculated as 1.5 times the
mean of the sample set. All of the retinoid concentrations, with the
exception of 4oxo-13-cisRA, are plotted against the left y axis. The
4oxo-13-cisRA concentrations are plotted against the right hand
y axis.
preparation and analysis. When serum samples were ex-
posed to light in this study, isomerization of the internal
standard could be detected (data not shown). Deuterated
standards can also be added into the samples during sam-
ple collection to control for stability during storage and
freeze-thaw. Despite the fact that only one isomer internal
standard was used in this study, one may choose to add
individual isomer standards to samples to confirm the re-
tention times of individual analytes.
The retinoid concentrations reported here are likely to
represent average concentrations in healthy adult humans.
Previous studies monitoring endogenous retinoid concen-
trations in the serum of men and women have not noted
any differences between the two (24, 25). The observed
concentrations of atRA are lower and concentrations of
4oxo-13-cisRA are higher than what has been previously
reported. The endogenous concentrations measured in
this study are approximately 40% lower for atRA and about
80% higher for 4oxo-13-cisRA than previously reported
(24, 25). This may be due to improved assay selectivity in
comparison to previous studies that used LC-UV, which
may not have separated all RA and its metabolite isomers,
and matrix interference. As shown in this study, a signifi-
cant interference was detected eluting close to atRA, but it
was mainly detected at the mass transitions relevant for
atRA-d5. However, this interference would likely confound
quantification in LC-UV assays.
The fact that 9-cisRA can be detected in human serum is
of interest because 9-cisRA activates RAR and RXR recep-
tors and may have specific biological functions. In previ-
ous studies, endogenous 9-cisRA has not been detectable
in human or mouse serum (8, 9, 29), and dosing with alit-
retinoin (9-cisRA) was required to detect 9-cisRA in human
serum (8). However, 9-cisRA was quantifiable in all but
one of the samples in this study, most likely due to the
596
Journal of Lipid Research Volume 53, 2012

3. Kane, M. A., A. E. Folias, A. Pingitore, M. Perri, K. M. Obrochta, C.
R. Krois, E. Cione, J. Y. Ryu, and J. L. Napoli. 2010. Identification
of 9-cis-retinoic acid as a pancreas-specific autacoid that attenuates
glucose-stimulated insulin secretion. Proc. Natl. Acad. Sci. USA. 107:
21884–21889.
4. Duester, G. 2008. Retinoic acid synthesis and signaling during early
organogenesis. Cell. 134: 921–931.
5. Gudas, L. J., and J. A. Wagner. 2011. Retinoids regulate stem cell
differentiation. J. Cell. Physiol. 226: 322–330.
6. Chung, S. S., and D. J. Wolgemuth. 2004. Role of retinoid signaling
in the regulation of spermatogenesis. Cytogenet. Genome Res. 105:
189–202.
increases in 13-cisRA concentrations were reported. The
lack of correlation between atRA concentrations and the
concentrations of other RA isomers suggests that measure-
ment of at least atRA and 13-cisRA separately is necessary
to characterize an individual’s RA status. Because the differ-
ent isomers have different pharmacological activity, sepa-
ration of atRA and 13-cisRA from the other isomers is
important in biological assays.
The 4oxo-atRA was detectable in over half of the human
serum samples, but the concentrations were too low to be
quantified. In agreement with published results (24, 25),
4oxo-13-cisRA was the retinoid with highest concentration
in human serum. 4oxo-atRA has properties that overlap
with atRA, such as activation of RAR␤, but it also shows
weak activation of RXR␣ (35). In addition, treatment of
MCF7 breast cancer cells with 4oxo-atRA results in inhi-
bition of proliferation (12), demonstrating that 4oxo-RA
isomers may contribute to the biological activity of RA.
Due to the activity of the 4oxo-RA compounds, future
studies, including accurate quantification in tissues, are
needed to determine their overall biological importance.
The significantly higher concentrations of 4oxo-13-cis-RA
in comparison to 4oxo-atRA are unexpected, based on the
similar levels of atRA and 13-cisRA in serum. Pharma-
cokinetic studies to determine the clearance of atRA,
4oxo-atRA, 13-cisRA, and 4oxo-13-cisRA are needed to
better understand the reason for the large difference in
the ratios between the RA isomers and their correspond-
ing 4oxo-metabolites. These studies would need to include
monitoring glucuronidation of the retinoids in serum and
urine and the determination of accurate clearance values.
Although the 4OH-RA preferentially is glucuronidated
at the hydroxy position, RA and 4oxo-RA are known to
undergo glucuronidation at the carboxyl function (26).
In conclusion, a UHPLC-MS method of retinoid mea-
surement in serum was developed and validated. The
method enabled quantification of four major RA isomers in
serum as well as quantification of 4oxo-13-cisRA. The use of
isotope labeled internal standards and the careful evalua-
tion of matrix interference provided increased confidence
for the quantification of the important retinoids. The devel-
oped method can be used in future studies to correlate
specific retinoid concentrations in human tissues to phar-
macological effects and in evaluating the relationships
between disease states and retinoid concentrations.
7. Veal, G. J., M. Cole, J. Errington, A. D. Pearson, A. B. Foot, G.
Whyman, and A. V. Boddy. 2007. Pharmacokinetics and metabo-
lism of 13-cis-retinoic acid (isotretinoin) in children with high-risk
neuroblastoma - a study of the United Kingdom Children’s Cancer
Study Group. Br. J. Cancer. 96: 424–431.
8. Schmitt-Hoffmann, A. H., B. Roos, J. Sauer, M. Schleimer, P.
Kovacs, K. Stoeckel, and J. Maares. 2011. Influence of food on the
pharmacokinetics of oral alitretinoin (9-cis retinoic acid). Clin. Exp.
Dermatol. 36(Suppl 2): 18–23.
9. Kane, M. A., A. E. Folias, C. Wang, and J. L. Napoli. 2008.
Quantitative profiling of endogenous retinoic acid in vivo and in
vitro by tandem mass spectrometry. Anal. Chem. 80: 1702–1708.
10. Idres, N., J. Marill, M. A. Flexor, and G. G. Chabot. 2002.
Activation of retinoic acid receptor-dependent transcription by
all-trans-retinoic acid metabolites and isomers. J. Biol. Chem. 277:
31491–31498.
11. Kochhar, D. M., and J. D. Penner. 1987. Developmental effects
of isotretinoin and 4-oxo-isotretinoin: the role of metabolism in
teratogenicity. Teratology. 36: 67–75.
12. Van Heusden, J., W. Wouters, F. C. Ramaekers, M. D. Krekels, L.
Dillen, M. Borgers, and G. Smets. 1998. The antiproliferative activ-
ity of all-trans-retinoic acid catabolites and isomers is differentially
modulated by liarozole-fumarate in MCF-7 human breast cancer
cells. Br. J. Cancer. 77: 1229–1235.
13. Creech Kraft, J., B. Lofberg, I. Chahoud, G. Bochert, and H. Nau.
1989. Teratogenicity and placental transfer of all-trans-, 13-cis-,
4-oxo-all-trans-, and 4-oxo-13-cis-retinoic acid after administration
of a low oral dose during organogenesis in mice. Toxicol. Appl.
Pharmacol. 100: 162–176.
14. Schug, T. T., D. C. Berry, N. S. Shaw, S. N. Travis, and N. Noy.
2007. Opposing effects of retinoic acid on cell growth result from
alternate activation of two different nuclear receptors. Cell. 129:
723–733.
15. Ramp, U., C. D. Gerharz, E. Eifler, H. K. Biesalski, and H. E.
Gabbert. 1994. Effects of retinoic acid metabolites on proliferation
and differentiation of the clonal rhabdomyosarcoma cell line BA-
HAN-1C. Biol. Cell. 81: 31–37.
16. Ranalder, U. B., B. B. Lausecker, and C. Huselton. 1993. Micro
liquid chromatography-mass spectrometry with direct liquid in-
troduction used for separation and quantitation of all-trans- and
13-cis-retinoic acids and their 4-oxo metabolites in human plasma.
J. Chromatogr. 617: 129–135.
17. Van Breemen, R. B., and C. R. Huang. 1996. High-performance liq-
uid chromatography-electrospray mass spectrometry of retinoids.
FASEB J. 10: 1098–1101.
18. van Breemen, R. B., D. Nikolic, X. Xu, Y. Xiong, M. van Lieshout,
C. E. West, and A. B. Schilling. 1998. Development of a method
for quantitation of retinol and retinyl palmitate in human serum
using high-performance liquid chromatography-atmospheric pres-
sure chemical ionization-mass spectrometry. J. Chromatogr. A. 794:
245–251.
19. Wang, Y., W. Y. Chang, G. S. Prins, and R. B. van Breemen. 2001.
Simultaneous determination of all-trans, 9-cis, 13-cis retinoic acid
and retinol in rat prostate using liquid chromatography-mass spec-
trometry. J. Mass Spectrom. 36: 882–888.
The authors thank Drs Hua-Fen Liu and Loren Olson from AB
Sciex, Foster City, CA, for their help in the initial method
development and Dr. Jakob A. Shimshoni for his contribution
to initial method development and compound synthesis.
20. Chithalen, J. V., L. Luu, M. Petkovich, and G. Jones. 2002.
HPLC-MS/MS analysis of the products generated from all-trans-
retinoic acid using recombinant human CYP26A. J. Lipid Res. 43:
1133–1142.
21. McCaffery, P., J. Evans, O. Koul, A. Volpert, K. Reid, and M. D.
Ullman. 2002. Retinoid quantification by HPLC/MS(n). J. Lipid
Res. 43: 1143–1149.
22. Eckhoff, C., and H. Nau. 1990. Identification and quantitation of
all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in
human plasma. J. Lipid Res. 31: 1445–1454.
REFERENCES
1. D’Ambrosio, D. N., R. D. Clugston, and W. S. Blaner. 2011. Vitamin
A metabolism: an update. Nutrients. 3: 63–103.
2. Kunchala, S. R., T. Suzuki, and A. Murayama. 2000. Photoisomer-
ization of retinoic acid and its influence on regulation of human
keratinocyte growth and differentiation. Indian J. Biochem. Biophys.
37: 71–76.
Quantification of endogenous retinoids in serum
597

23. Rühl, R. 2006. Method to determine 4-oxo-retinoic acids, retinoic
acids and retinol in serum and cell extracts by liquid chromatog-
raphy/diode-array detection atmospheric pressure chemical ioni-
sation tandem mass spectrometry. Rapid Commun. Mass Spectrom.
20: 2497–2504.
24. Schmidt, C. K., A. Brouwer, and H. Nau. 2003. Chromatographic
analysis of endogenous retinoids in tissues and serum. Anal.
Biochem. 315: 36–48.
25. Berggren Söderlund, M., G. Fex, and P. Nilsson-Ehle. 2003. Decreasing
serum concentrations of all-trans, 13-cis retinoic acids and retinol
during fasting and caloric restriction. J. Intern. Med. 253: 375–380.
26. Samokyszyn, V. M., W. E. Gall, G. Zawada, M. A. Freyaldenhoven,
G. Chen, P. I. Mackenzie, T. R. Tephly, and A. Radominska-Pandya.
2000. 4-hydroxyretinoic acid, a novel substrate for human liver
microsomal UDP-glucuronosyltransferase(s) and recombinant
UGT2B7. J. Biol. Chem. 275: 6908–6914.
27. Patel, J. B., C. K. Huynh, V. D. Handratta, L. K. Gediya, A. M. Brodie,
O. G. Goloubeva, O. O. Clement, I. P. Nanne, D. R. Soprano, and
V. C. Njar. 2004. Novel retinoic acid metabolism blocking agents
endowed with multiple biological activities are efficient growth
inhibitors of human breast and prostate cancer cells in vitro and
a human breast tumor xenograft in nude mice. J. Med. Chem. 47:
6716–6729.
29. Kane, M. A., and J. L. Napoli. 2010. Quantification of endogenous
retinoids. Methods Mol. Biol. 652: 1–54.
30. Shah, V. P., K. K. Midha, J. W. Findlay, H. M. Hill, J. D. Hulse, I. J.
McGilveray, G. McKay, K. J. Miller, R. N. Patnaik, M. L. Powell, et al.
2000. Bioanalytical method validation–a revisit with a decade of
progress. Pharm. Res. 17: 1551–1557.
31. Viswanathan, C. T., S. Bansal, B. Booth, A. J. DeStefano, M. J. Rose,
J. Sailstad, V. P. Shah, J. P. Skelly, P. G. Swann, and R. Weiner. 2007.
Quantitative bioanalytical methods validation and implementa-
tion: best practices for chromatographic and ligand binding assays.
Pharm. Res. 24: 1962–1973.
32. Gundersen, T. E., N. E. Bastani, and R. Blomhoff. 2007. Quantitative
high-throughput determination of endogenous retinoids in human
plasma using triple-stage liquid chromatography/tandem mass spec-
trometry. Rapid Commun. Mass Spectrom. 21: 1176–1186.
33. Topletz, A. R., J. E. Thatcher, A. Zelter, J. D. Lutz, S. Tay, W. L.
Nelson, and N. Isoherranen. 2012. Comparison of the function
and expression of CYP26A1 and CYP26B1, the two retinoic acid
hydroxylases. Biochem. Pharmacol. 83: 149–163.
34. Rühl, R., A. Bub, and B. Watzl. 2008. Modulation of plasma all-trans
retinoic acid concentrations by the consumption of carotenoid-rich
vegetables. Nutrition. 24: 1224–1226.
35. Pijnappel, W. W., H. F. Hendriks, G. E. Folkers, C. E. van den
Brink, E. J. Dekker, C. Edelenbosch, P. T. van der Saag, and
A. J. Durston. 1993. The retinoid ligand 4-oxo-retinoic acid is a
highly active modulator of positional specification. Nature. 366:
340–344.
28. Horst, R. L., T. A. Reinhardt, J. P. Goff, B. J. Nonnecke, V. K.
Gambhir, P. D. Fiorella, and J. L. Napoli. 1995. Identification of
9-cis,13-cis-retinoic acid as a major circulating retinoid in plasma.
Biochemistry. 34: 1203–1209.
598
Journal of Lipid Research Volume 53, 2012