Assay of Retinoids in Biological Samples by Reverse-Phase High-Pressure
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[CANCER RESEARCH 38. 3327-3332, October 1978]
Assay of Retinoids in Biological Samples by Reverse-Phase
High-Pressure Liquid Chromatography
Anita B. Roberts,1 Margaret D. Nichols, Charles A. Frolik, Dianne L. Newton, and Michael B. Sporn
Lung Cancer Branch, National Cancer Institute, Bethesda, Maryland 20014
ABSTRACT the routine bioassay of the liver storage of retinoids (3), as
well as the biochemical investigation of the metabolism of
A separation procedure for retinoids based on reverse- various retinoids (7, 10,13, 15).
phase high-pressure liquid Chromatography with solvent In the past the only simple and rapid procedures available
mixtures of acetonitrile and water is described. The for retinoid determination have been based on either the
method may be applied to the screening of synthetic Carr-Price antimony trichloride colorimetrie assay (3) or the
retinoids, which have potential for use in the prevention trifluoroacetic acid modification of Dugan ef al. (6), both of
of cancer. It is easily adapted to a variety of biological which confound retinol and retinyl esters. A separate esti
samples and can be applied to other conventional retinoid mation of retinol and its esters has required lengthy Chro
assays in liver and plasma, detecting as little as 1 nmol matographie procedures (10-13, 15, 19). The method pro
retinyl esters and less than 0.3 nmol retinol per g tissue. posed here is based on the reverse-phase HPLC2 system
The one-step Chromatography results in separation and recently described by Frolik ef al. (9), which can rapidly and
simultaneous determination of many of the synthetic reti reproducibly separate and simultaneously quantitate a va
noids and all of the natural retinoids, including the retinyl riety of retinoids with little or no degradation. The use of a
esters that are separated into their major fatty acid com 2-step elution system results in a clean separation of retinol
ponents. The method has been applied to the analysis of from many closely related synthetic derivatives and from
retinoid levels in the liver and intestine of vitamin A- other physiological forms of the vitamin such as retinoic
deficient hamsters following a p.o. dose (0.5 mg/day for 2 acid, retinal, and retinyl esters. In addition, the retinyl
days) of retinyl acetate or of a synthetic vitamin A analog esters are separated into their major fatty acid components,
and is predictive of the degree to which various synthetic which by previous methods required 2 extra Chromato
retinoids can be converted to retinol and stored in the graphie steps (12).
liver as retinyl esters. Because of its speed, excellent With this method it has been shown that 2 synthetic
recoveries, and high resolution, the method offers signifi retinoids, N-acetyl retinyl amine (14) and retinal acetylhy-
cant advantages over previous, more lengthy procedures. drazone (18) (Chart 1), each undergo conversion to retinol
and retinyl esters in the vitamin A-deficient hamster. The
INTRODUCTION splitting of these derivatives to a physiological form of the
Increasing attention is being drawn to the chemopreven- vitamin could account for the reported vitamin A activity of
tive role of retinoids in the development of cancers of the the compounds in the reversal of keratinization in trachéal
epithelial tissues. As recently reviewed (22) this family of organ cultures (23).
compounds with "vitamin A activity" has been shown to be
effective at pharmacological levels in preventing or delaying MATERIALS AND METHODS
the development of cancers of the bladder, lung, skin, and
Chemicals. Retinol was obtained from Eastman Organic
breast of experimental animals, possibly by an enhance
ment of its physiological role in the maintenance of normal Chemicals, Rochester, N. Y.; retinyl palmitate was from
Sigma Chemical Corporation, St. Louis, Mo.; and axeroph-
epithelial cell differentiation. The natural retinoids, how
thene was from BASF/AG, Ludwigshafen am Rhein, Ger
ever, have several undesirable properties that severely limit many. All other retinoids were a generous gift of Hoffmann-
their clinical application. In addition to their intrinsic toxic-
ity (5), their conversion to long-chain retinyl esters can La Roche Inc., Nutley, N. J. Solvents used for HPLC were
obtained from Burdick & Jackson Laboratories, Muskegon,
result in excessive liver storage and damage to that tissue
(21). For circumvention of these properties, synthetic reti Mich., and other reagents used were analytical grade. The
water used for HPLC was redistilled over glass.
noids are being developed. It was therefore desirable to
develop a method that could quickly and accurately deter Dosing of Animals. Weanling male and female Syrian
golden hamsters raised on a vitamin A-deficient regimen (4,
mine the amounts of retinoids in tissue samples and the
degree to which synthetic retinoids might be converted to 9) were used at 30 to 33 days of age. At this time the livers
retinyl esters and stored in the liver. Furthermore, the contained no detectable retinol and very small amounts of
limited amounts of the trial compounds required that the retinyl esters (average concentration of 1 nmol/g).
assay be more sensitive than were previous methods. Other Hamsters were dosed daily with either retinoids or vehicle
applications of the assay include the estimation of plasma for 1 or 2 days by stomach intubation of 0.2 ml of a solution
retinol and retinyl esters in vitamin A toxicity (17, 21) and of ethanohtrioctanoin (1:3) containing 0.025% butylated
hydroxytoluene.
1To whom requests tor reprints should be addressed.
Received May 1. 1978; accepted July 18, 1978. ! The abbreviation used is: HPLC, high-pressure liquid Chromatography.
OCTOBER 1978 3327
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Research.A. B. Roberts et al.
approximately 10 min following the change of the eluant
composition to 98% acetonitrile.
The identification of the various peaks was established by
chromatography of standard compounds as well as by
cochromatography of standard retinoid solutions with the
tissue extracts.
The integrated peak areas were converted to nmol of
retinoid by comparing the integrator number for the peak
area at 325 nm of the retinoid of interest with an average
retinal acetylhydrazone standard integrator number/nmol of that retinoid, obtained
from the chromatography and'peak integration of several
Chart 1. Structure of synthetic retinoids.
reference samples in the range of 0.1 to 2 /xg.
Extraction of Tissues. Animals were killed by CO2 nar
RESULTS
cosis 24 hr after receiving the last p.o. dose. Duplicate 1-g
samples of the liver and a single 1-g sample of the intestine The separation of retinol, retinal acetylhydrazone, N-ace-
(the first 15 cm washed free of its contents) were weighed tyl retinyl amine, retinyl acetate, axerophthene (the vitamin
and stored for up to 3 weeks in a nitrogen freezer. Storage A hydrocarbon), and retinyl palmitate by reverse-phase
for longer periods resulted in a gradual decrease in the HPLC is shown in Chart 2. Depending on which retinoids
amount of retinol detected in tissue samples, although the are present in a particular tissue sample, the percentage of
esters appeared to be stable for at least 5 weeks. acetonitrile in the initial eluting solvent can be modified to
Preparation of the tissue for extraction was by 1 of 2 give separation of the more polar retinoids in the minimal
previously described procedures, the method of Ames ef al. time. For example, retinol and retinyl acetate are eluted at
(2) or that of Ito ef a/. (13). Ether containing 0.1% butylated 3.3 and 4.6 min, respectively, when the initial solvent is 84%
hydroxytoluene was used for extraction. An aliquot of the acetonitrile in water.
extract was evaporated to dryness under a stream of nitro With anhydrous ether as the solvent, the sodium sulfate
gen, and the residue was redissolved in 100% chloroform, powder procedure of Ames ef al. (2) and the lyophilization
followed by the addition of 0.5 volume of methanol to give method of Itoef al. (13) were shown to be equivalent for the
a final composition of 2:1. This solution was centrifuged to determination of tissue retinoids, although the lyophiliza
clarity and analyzed directly by HPLC. All procedures were tion procedure has distinct advantages when water-misci-
carried out in minimal light or under red lights, and sample ble solvents are used for extraction. The relative efficiency
solutions were stored in the dark at -20°prior to chroma-
of ether, ethyl acetate, methanol, or chloroform:methanol
tography. For the trifluoroacetic acid determinations, ali-
quots of the same HPLC solution as above were analyzed
by a modification (8) of the procedure of Dugan ef al. (6).
For determination of the efficiency of various solvents in .448 -
extracting the retinoids, the lyophilization procedure of Ito
ef al. (13) was used. Retinyl acetate was added to each
extraction as an internal standard. All calculations on the
retinol and retinyl esters extracted from these livers were
adjusted relative to a constant amount of retinyl acetate to
eliminate variance due to evaporative losses.
HPLC and Retinoid Determination. HPLC analysis was
carried out on a Spectra Physics Model 3500B unit fitted
with a Valco sample injection valve. Dual wavelength detec
tion was accomplished with a Spectra Physics 8200 UV
detector (maximal sensitivity, 0.0025 A unit, full scale) set
at 365 nm and a Schoeffel Model 770 variable wavelength
detector (maximal sensitivity, 0.01 A unit, full scale) set at
325 nm. The 770 detector was coupled to a Spectra Physics
Autolab System I computing integrator to determine peak
area. A Spectra Physics 5-fj.m Spherisorb ODS column (5%
loading, 3 x 250 mm) was used for all separations. 02468 10 12 14 16 18 20 22 24 26 28
The sample was eluted isocratically at ambient tempera TIME (min)
ture with a solvent flow of 1.2 ml/min and a solvent Chart 2. HPLC of retinoid standards. A 50-¿ilsample containing 0.65 ¿ig
composition in the range of 70 to 90% acetonitrile in water retinol, 0.69 /jg W-acetyl retinyl amine, 0.77 ^g retinyl acetate, 0.88 (¿g
axerophthene, and 1.58 ,ig retinyl palmitate in methanol was injected onto
until all compounds more polar than the retinyl esters had the reverse-phase column and eluted at a flow of 1.2 ml/min with 79%
eluted, followed by a step change to 98% acetonitrile in acetonitrile in water, followed at Point A by 98% acetonitrile in water.
water until all esters were eluted from the column (total Chromatography under identical conditions of a mixture of retinol, retinal
acetylhydrazone (0.99 ^g), and retinyl acetate was used to establish the
time, approximately 20 to 30 min). The esters were very elution time of the acetylhydrazone derivative ( ), which under these
strongly retained on the column and were not eluted for conditions is not separated from N-acetyl retinyl amine.
3328 CANCER RESEARCH VOL. 38
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Research.Bioassay of Retinoids by HPLC
(1:1) in extracting retinol and retinyl esters from livers was tively, when dried onto intestinal samples and ground with
compared. Differences among solvents were not signifi sodium sulfate. These findings are not inconsistent with the
cant. The ease of evaporation of the ether made it the data of Ames ef al. (2), as their reported 99% recovery for
solvent of choice for this analysis. However, the effective retinyl acetate was quantitated by the Carr-Price reaction,
extraction of certain retinoids may require the use of a which cannot distinguish between retinol and retinyl esters.
different solvent. For example, retinoic acid, which is more It follows, however, that the lyophilization procedure, which
polar than is retinol, was extracted by ether only 50 to 60% minimizes these effects, is the method of choice for this
as well as by methanol. analysis.
With the sodium sulfate powder method with ether as the The choice of a solvent for redissolving the sample
extracting solvent, the recovery of 261 nmol retinol and 378 following evaporation of an aliquot of the extracting solvent
nmol retinyl palmitate was determined. A 100-^1 aliquot of was particularly critical, as shown in Table 1. Both 100%
a chloroform solution of each retinoid was either applied methanol and 100% acetonitrile, while adequately dissolv
directly onto the liver or added to the ether in the first ing the retinol in the sample, were poor solvent choices for
extraction step. Drying of the chloroform solutions onto a the solution of the very nonpolar retinyl esters. Increasing
1-g sample of liver from a control hamster prior to grinding amounts of chloroform in mixture with methanol resulted in
the liver with sodium sulfate resulted in 98.5 and 94.5% maximal solubilization of both retinol and retinyl esters.
recovery of the retinol and retinyl palmitate, respectively, The limited solubility of chloroform mixtures in acetoni-
compared to the recovery of the same amount of standard trile:water solutions, however, necessitated a compromise
added to the ether at the time of the first extraction step. between the percentage of acetonitrile in the initial eluant
Absolute recoveries in the latter procedure were 109 and (and therefore the degree of resolution desired) and the
111%, respectively. percentage of chloroform in the sample solution. With 90%
Unexpectedly, chloroform solutions (100 ¿ilof a 1-mg/ml acetonitrile as eluant, 10-/xl samples could be injected in
solution) of retinyl esters underwent a tissue-dependent 100% chloroform, but if 70% acetonitrile was used (as is
hydrolysis to retinol in both the sodium sulfate powder necessary to resolve closely related retinoids) the high
method and the lyophilization procedure. Aliquots of a degree of immiscibility of the 100% chloroform sample
retinyl acetate solution dried onto the liver prior to grinding solution and the eluant caused a loss of resolution of the
with sodium sulfate were hydrolyzed 55 to 92%, whereas an early peaks. The ester peaks, however, were unaffected.
aliquot added to the medium prior to homogenization in the Selection of the percentage of acetonitrile in the initial
lyophilization method resulted in a lesser extent of hydroly eluant and of the sample solvent composition must there
sis (17 to 24%). Further investigation of the hydrolysis of fore depend on the particular array of retinoids present and
retinyl esters by the sodium sulfate powder method showed the degree of resolution necessary to quantitate them
that aliquots of a retinyl acetate solution that had been accurately.
added either to a liver sample that had been boiled in 0.9% A linear relationship was found between the total dose
NaCI solution for 15 min or directly to sodium sulfate with administered to the hamster and the liver retinoid concen
no liver tissue present were recovered unchanged. Retinyl tration in the dose range tested of 0.2 to 1 mg retinyl acetate
palmitate solutions, on the other hand, were hydrolyzed to per day for 1 or 2 days (Chart 3). As can be seen, liver
retinol to only a minor degree by either procedure (grinding, storage of retinoids was increased by a multiple dosing
3.1%; lyophilization, 0.5%), and the synthetic retinoid, reti schedule. For example, the liver storage of retinyl esters
nal acetylhydrazone, was not hydrolyzed at all by the same was increased approximately 60% by the administration of
procedures. Retinyl acetate and retinyl palmitate solutions 0.5 mg retinyl acetate for each of 2 successive days, as
were hydrolyzed to retinol approximately 5 and 2%, respec compared to a single 1-mgdose. Whether this phenomenon
Table 1
Effect of solvent composition of the solution of retinol and retinyl esters for HPLC
Miscibility6 in parts/100 in acetoni-
Relative solubility"
trile:water mixtures at:
Sample solvent
composition100% esters1.0
methanol
100%CH3CN 0.96 0.91 00>60 00 00X>60
CHCI.,:methanol 0.940.97 1.951.99 0033
(1:1)
CHCI.,:methanol 9 13
(2:1)
100%CHCI.1Retinol1.0 0.98Retinyl 2.0560%X0013A. B. Roberts et al
lOOOr mitted the collection of samples in the regions identified as
Ester 1. Ester 2 + 3, and Ester 4. Each of these samples had
a UV absorption spectrum characteristic of retinyl esters
(Xmax 325 nm). Furthermore, hydrolysis of each of these
fractions yielded retinol as the only 325-nm-absorbing ma-
0.056
0.048
0.040
0.032
O 0.024
OÃ-
0.016
0.008
0.4 0.8 1.2 1.6 2.0 2.4
CUMULATIVE DOSE RETINYL ACETATE (mgl O 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Chart 3. Total retinoid (nmol) per g liver versus cumulative dose of retinyl TIME Iminl
acetate. Vitamin A-deficient hamsters (3 in each group) were given daily p.o.
doses of either 0.2, 0.5, or 1.0 mg retinyl acetate in 0.2 ml ethanohtrioctanoin
(1:3) containing 0.025% butylated hydroxytoluene for either 1 (---- ) or 2
(- ) days prior to sacrifice 24 hr after administration of the last dose.
Tissues were extracted with ether by the method of Ames ef a/. (2). The total
nmol retinoid is the sum of the values for retinol and all retinyl esters as
determined by HPLC (•)or the value derived from the Irifluoroacetic acid
method (A) of Dugan et al. (6) determined on an aliquot of the same sample.
0.010
results from a decreased ability of the deficient animal to
store the initial dose of retinyl acetate or from an increase
in liver storage with time, as reported by Ames and Harris 0.008
(1), is unknown. The supplementation schedule chosen for
synthetic retinoids was a p.o. dose of 0.5 mg/day for 2 days 0006
followed by sacrifice 24 hr after administration of the final
dose.
O 0004
Since the colorimetrie determination of vitamin A has
formed the basis of many assays (1 , 2, 17), it was desirable
0002
to compare that method with the present HPLC method.
The amount of sample used for the trifluoroacetic acid
determination was on the average 20 times the amount
used for the HPLC. As seen in Chart 3, the values were quite
O 24 6 8 10 12 14 16 18 20 22 24 26 28 30
consistent with those for the HPLC, averaging about 91% of
TIME (mini
the HPLC value. Although the trifluoroacetic acid method is
Chart 4. HPLC of ether extracts of liver (A) and small intestine (B) of a
faster, the HPLC method provides significantly more infor vitamin A-deficient hamster fed 0.5 mg retinal acetylhydrazone per day for 2
mation about the nature of the retinoids involved. days. A 10-jil sample representing '/75th of the ether extract of 1 g of liver or
2/7sth'sof the extract of 1 g of small intestine was injected onto the reverse-
The Chromatographie profile of the ether extract of the
phase column in chlorofomvmethanol (2:1). The sample was eluted with a
liver and small intestine of a vitamin A-deficient hamster fed solvent flow of 1.2 ml/min with 80% acetonitrile in water (1240 psi) for the
2 p.o. doses of 0.5 mg retinal acetylhydrazone per day is first 8 min, followed at Point A by 98% acetonitrile in water (880 psi) until all
shown in Chart 4. The parent compound (A,,,ax380 nm) is esters were eluted from the column. The peak at 9 to 14 min (approximately,
0.005 absorbance unit) was caused by a refractive index change resulting
easily distinguished from retinol (Xrmix 325 nm) by the from the change to a new solvent at Point A. It is most apparent at the lower
relative peak heights of the 365- and the 325-nm detector. absorbance units, full scale, setting (B). The peak immediately preceding
Ester 1 in 8 is an artifact present in all intestinal samples. Ester 3 is retinyl
In the retinyl ester portion of the chromatogram, only Ester palmitate, and Esters 1, 2, and 4 can be tentatively identified as retinyl
3 was positively identified by cochromatography as retinyl linoleate, oleate, and stéarate,respectively. Dual wavelength detection was
palmitate. The other peaks in the retinyl palmitate portion performed with a Spectra Physics 8200 UV detector equipped with a 365 nm
filter ( ) and with a Schoeffel Model 770 variable wavelength detector
of the chromatogram were determined to be retinyl esters set at 325 nm ( ) to aid further in the identification of retinol (X,,,a,.325
by several criteria. Chromatography of larger samples per nm) and retinal acetyl hydrazone (xm;lx,380 nm).
3330 CANCER RESEARCH VOL. 38
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Research.Bioassay of Retinoids by HPLC
terial on HPLC. Comparison of the elution order of the ester sents an application of the reverse-phase HPLC system of
peaks with known elution patterns from reverse-phase pa Frolik ef al. (9) to the bioassay of tissue retinoids. The
per Chromatographie systems (8, 10, 12) permits tentative reverse-phase system was chosen because of the excellent
identification of Esters 1, 2, and 4 as retinyl linoleate, recoveries attainable, the reproducibility, and the mild na
oleate, and stéarate,respectively. Whereas the parent reti- ture of the Chromatographie process, which minimizes the
noid represents over 40% of the total in the intestine, it production of artifacts. The method as described in this
represents only about 2% of the total retinoid in the liver, paper is capable of quantitating retinoids at less than 1
possibly implying that hydrolysis occurs prior to absorption. nmol/g tissue, which represents an approximately 20-fold
The retinoid concentrations, in nmol/g liver or small increase in sensitivity over previous methods (1).
intestine, of hamsters given either retinyl acetate, retinal, or The use of vitamin A-deficient animals is important when
the synthetic retinoids, retinal acetylhydrazone or A/-acetyl working within the limits of the sensitivity of this assay, a
retinyl aminé,on a dose schedule of 0.5 mg/day for 2 days feature that was also included in the liver storage assay
are tabulated in Table 2. The 2 synthetic retinoids are procedure of Ames and Harris (1). The vitamin A-deficient
converted to retinol and retinyl esters, although consider hamsters have on the average less than 1 nmol retinyl esters
ably less efficiently than is retinyl acetate or retinal. Neither per g liver and therefore make the detection of ester levels
retinal nor retinyl acetate was detected in the tissues of greater than 2 nmol/g significant compared to controls.
hamsters dosed with those retinoids. This presumably re This permits the conservative use of synthetic retinoids in
sults from their rapid conversion to other retinoid species. the assay.
In the case of retinyl acetate, hydrolysis during the work-up A major feature of this Chromatographie system is the
procedure could account for this, in part. However, the resolution of the long-chain fatty acid esters of retinol.
partial hydrolysis in liver extracted by the lyophilization Although absolute identification of the ester peaks other
procedure (17 to 24%), coupled with the fact that no retinyl than retinyl palmitate would require cochromatography
acetate was detected by either extraction procedure, makes with standard compounds, comparison of the elution pat
it unlikely that this artifactual hydrolysis must be taken into tern with published R, values from a reverse-phase paper
consideration in the interpretation of the data. Only for the chromatography system (16) allows for tentative identifica
synthetic analogs, which seem to have a longer half-life in tion as stated in the legend for Chart 4. Supporting this
vivo, is the parent compound still detected 1 day after the tentative identification is the close agreement of the relative
last dose. Regardless of the retinoid dosed, the major proportions of the different ester components in the liver
retinoid species in the liver are retinyl esters with varying and intestine with that reported by Goodman ef al. (10).
smaller amounts of retinol. The slightly different retinyl Most notable is the decrease in the proportion of palmitate
ester distribution in the intestine as compared to the liver is and the increase in the proportion of stéaratein the intes
tine as compared to the liver. The "critical pair" (20) of the
consistent with that reported previously (10).
oleate (18:1) and palmitate (16:0) esters could not be sepa
DISCUSSION
rated completely even under conditions resulting in large
separations of this peak from the other ester peaks.
HPLC is increasingly becoming the method of choice for A useful refinement of this assay would be the addition of
various biochemical assays because of both the high reso an internal reference standard to the extracting solvent to
lution attainable and the short elution times as compared to eliminate sample variance due to evaporation and other
conventional Chromatographie systems. This work repre losses. The choice of a standard should be limited to
Table 2
Relative retinoid levels in liver and small intestine of vitamin A-deficient hamsters following p.o. administration of 0.5
mg retinoid per day for 2 days
esters*
esters)S (% of total
givenRetinyl
Retinoid esters44133751.160.78.77.3A. B. Roberts et al.
compounds not found in biological samples and intermedi REFERENCES
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3332 CANCER RESEARCH VOL. 38
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High-Pressure Liquid Chromatography
Anita B. Roberts, Margaret D. Nichols, Charles A. Frolik, et al.
Cancer Res 1978;38:3327-3332.
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